U.S. patent application number 09/912020 was filed with the patent office on 2002-04-18 for genes identified as required for proliferation in escherichia coli.
This patent application is currently assigned to ELITRA PHARMACEUTICALS, INC.. Invention is credited to Carr, Grant J., Forsyth, R. Allyn, Froelich, Jamie M., Ohlsen, Kari L., Trawick, John, Xu, H. Howard, Yamamoto, Robert T., Zyskind, Judith.
Application Number | 20020045592 09/912020 |
Document ID | / |
Family ID | 26815257 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045592 |
Kind Code |
A1 |
Zyskind, Judith ; et
al. |
April 18, 2002 |
Genes identified as required for proliferation in escherichia
coli
Abstract
The sequences of nucleic acids encoding proteins required for E.
coli proliferation are disclosed. The nucleic acids can be used to
express proteins or portions thereof, to obtain antibodies capable
of specifically binding to the expressed proteins, and to use those
expressed proteins as a screen to isolate candidate molecules for
rational drug discovery programs. The nucleic acids can also be
used to screen for homologous genes that are required for
proliferation in microorganisms other than E. coli. The nucleic
acids can also be used to design expression vectors and secretion
vectors. The nucleic acids of the present invention can also be
used in various assay systems to screen for proliferation required
genes in other organisms as well as to screen for antimicrobial
agents.
Inventors: |
Zyskind, Judith; (La Jolla,
CA) ; Ohlsen, Kari L.; (San Diego, CA) ;
Trawick, John; (La Mesa, CA) ; Forsyth, R. Allyn;
(San Diego, CA) ; Froelich, Jamie M.; (San Diego,
CA) ; Carr, Grant J.; (Escondido, CA) ;
Yamamoto, Robert T.; (San Diego, CA) ; Xu, H.
Howard; (San Diego, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Assignee: |
ELITRA PHARMACEUTICALS,
INC.
|
Family ID: |
26815257 |
Appl. No.: |
09/912020 |
Filed: |
July 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09912020 |
Jul 23, 2001 |
|
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09492709 |
Jan 27, 2000 |
|
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60117405 |
Jan 27, 1999 |
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Current U.S.
Class: |
514/44A ;
435/476 |
Current CPC
Class: |
C07K 14/245 20130101;
C12Q 1/18 20130101 |
Class at
Publication: |
514/44 ;
435/476 |
International
Class: |
A61K 048/00; C12N
015/74 |
Claims
What is claimed is:
1. A method of inhibiting cellular proliferation comprising
inhibiting the activity or reducing the amount of a polypeptide
comprising a sequence selected from the group consisting of SEQ ID
NOs. 243-357 and SEQ ID NOs. 359-398 or inhibiting the activity or
reducing the amount of a nucleic acid encoding said
polypeptide.
2. The method of claim 1, wherein the cell in which proliferation
is inhibited is selected from the group consisting of Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter
cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus
faecalis, Streptococcus pneumoniae, Haemophilus influenzae,
Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans,
Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella
pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella
cholerasuis, Staphylococcus epidermidis, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus
anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter
jejuni, Chlamydia trachomatus, Chlamydia pneumoniae or any species
falling within the genera of any of the above species.
3. The method of claim 1, wherein the cell in which proliferation
is inhibited is Escherichia coli.
4. A method for inhibiting cellular proliferation comprising
introducing a compound which inhibits the activity or reduces the
amount of a polypeptide comprising a sequence selected from the
group consisting of SEQ ID NOs. 243-357 and SEQ ID NOs. 359-398 or
which inhibits the activity or reduces the amount of a nucleic acid
comprising a nucleotide sequence encoding said polypeptide into a
cell.
5. The method of claim 4, wherein said compound is an antisense
nucleic acid.
6. The method of claim 5, wherein said compound is an antisense
nucleic acid comprising a sequence selected from the group
consisting of SEQ ID NOs.: 405-485, or a proliferation-inhibiting
portion thereof.
7. The method of claim 6, wherein said proliferation inhibiting
portion of one of SEQ ID NOs. 405-485 is a fragment comprising at
least 10, at least 20, at least 25, at least 30, at least 50 or
more than 50 consecutive nucleotides of one of SEQ ID NOs:
405-485.
8. The method of claim 4, wherein said compound is a triple helix
oligonucleotide.
9. The method of claim 4, wherein the cell in which proliferation
is inhibited is selected from the group consisting of Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter
cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus
faecalis, Streptococcus pneumoniae, Haemophilus influenzae,
Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans,
Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella
pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella
cholerasuis, Staphylococcus epidermidis, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus
anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter
jejuni, Chlamydia trachomatus, Chlamydia pneumoniae or any species
falling within the genera of any of the above species.
10. The method of claim 4, wherein the cell in which proliferation
is inhibited is Escherichia coli.
11. A method for inhibiting cellular proliferation comprising
introducing a compound with activity against a gene corresponding
to one of SEQ ID NOs.: 82-242 or with activity against the product
of said gene into a population of cells expressing a gene.
12. The method of claim 11, wherein said compound is an antisense
nucleic acid.
13. The method of claim 12, wherein said compound is an antisense
oligonucleotide comprising a sequence selected from the group
consisting of SEQ ID NOs.: 405-485, or a proliferation-inhibiting
portion thereof.
14. The method of claim 13, wherein said proliferation inhibiting
portion of one of SEQ ID NOs. 405-485 is a fragment comprising at
least 10, at least 20, at least 25, at least 30, at least 50 or
more than 50 consecutive nucleotides of one of SEQ ID NOs:
405-485.
15. The method of claim 11, wherein said compound is a triple helix
oligonucleotide.
16. The method of claim 11, wherein the cell in which proliferation
is inhibited is selected from the group consisting of Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter
cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus
faecalis, Streptococcus pneumoniae, Haemophilus influenzae,
Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans,
Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella
pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella
cholerasuis, Staphylococcus epidermidis, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus
anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter
jejuni, Chlamydia trachomatus, Chlamydia pneumoniae or any species
falling within the genera of any of the above species.
17. The method of claim 11, wherein the cell in which proliferation
is inhibited is Escherichia coli.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application from U.S.
patent application Ser. No. 09/492,709, filed Jan. 27, 2000 which
claims priority to U.S. Provisional Patent Application Serial No.
60/117,405 filed Jan. 27, 1999. The disclosures of U.S. patent
application Ser. No. 09/492,709 and U.S. Provisional Patent
Application Serial No. 60/117,405 are incorporated herein by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Since the discovery of penicillin, the use of antibiotics to
treat the ravages of bacterial infections has saved millions of
lives. With the advent of these "miracle drugs," for a time it was
popularly believed that humanity might, once and for all, be saved
from the scourge of bacterial infections. In fact, during the 1980s
and early 1990s, many large pharmaceutical companies cut back or
eliminated antibiotics research and development. They believed that
infectious disease caused by bacteria finally had been conquered
and that markets for new drugs were limited. Unfortunately, this
belief was overly optimistic.
[0003] The tide is beginning to turn in favor of the bacteria as
reports of drug resistant bacteria become more frequent. The United
States Centers for Disease Control announced that one of the most
powerful known antibiotics, vancomycin, was unable to treat an
infection of the common Staphylococcus aureus (staph). This
organism is commonly found in our environment and is responsible
for many nosocomial infections. The import of this announcement
becomes clear when one considers that vancomycin was used for years
to treat infections caused by stubborn strains of bacteria, like
staph. In short, the bacteria are becoming resistant to our most
powerful antibiotics. If this trend continues, it is conceivable
that we will return to a time when what are presently considered
minor bacterial infections are fatal diseases.
[0004] There are a number of causes for the predicament in which
practitioners of medical arts find themselves. Over-prescription
and improper prescription habits by some physicians have caused an
indiscriminate increase in the availability of antibiotics to the
public. The patient is also partly responsible, for even in
instances where an antibiotic is the appropriate treatment,
patients will often improperly use the drug, the result being yet
another population of bacteria that is resistant, in whole or in
part, to traditional antibiotics.
[0005] The bacterial scourges that have haunted humanity remain, in
spite of the development of modem scientific practices to deal with
the diseases that they cause. Drug resistant bacteria are now
advancing on the health of humanity. A new generation of
antibiotics to once again deal with the pending health threat that
bacteria present is required.
DISCOVERY OF NEW ANTIBIOTICS
[0006] As more and more bacterial strains become resistant to the
panel of available antibiotics, new compounds are required. In the
past, practitioners of pharmacology would have to rely upon
traditional methods of drug discovery to generate novel, safe and
efficacious compounds for the treatment of disease. Traditional
drug discovery methods involve blindly testing potential drug
candidate-molecules, often selected at random, in the hope that one
might prove to be an effective treatment for some disease. The
process is painstaking and laborious, with no guarantee of success.
Today, the average cost to discover and develop a new drug is
nearly US $500 million, and the average time is 15 years from
laboratory to patient. Improving this process, even incrementally,
would represent a huge advance in the generation of novel
antimicrobial agents.
[0007] Newly emerging practices in drug discovery utilize a number
of biochemical techniques to provide for directed approaches to
creating new drugs, rather than discovering them at random. For
example, gene sequences and proteins encoded thereby that are
required for the proliferation of an organism make for excellent
targets since exposure of bacteria to compounds active against
these targets would result in the inactivation of the organism.
Once a target is identified, biochemical analysis of that target
can be used to discover or to design molecules that interact with
and alter the functions of the target. Using physical and
computational techniques, to analyze structural and biochemical
targets in order to derive compounds that interact with a target is
called rational drug design and offers great future potential.
Thus, emerging drug discovery practices use molecular modeling
techniques, combinatorial chemistry approaches, and other means to
produce and screen and/or design large numbers of candidate
compounds.
[0008] Nevertheless, while this approach to drug discovery is
clearly the way of the future, problems remain. For example, the
initial step of identifying molecular targets for investigation can
be an extremely time consuming task. It may also be difficult to
design molecules that interact with the target by using computer
modeling techniques. Furthermore, in cases where the function of
the target is not known or is poorly understood, it may be
difficult to design assays to detect molecules that interact with
and alter the functions of the target. To improve the rate of novel
drug discovery and development, methods of identifying important
molecular targets in pathogenic microorganisms and methods for
identifying molecules that interact with and alter the functions of
such molecular targets are urgently required.
[0009] Escherichia coli represents an excellent model system to
understand bacterial biochemistry and physiology. The estimated
4288 genes scattered along the 4.6.times.10.sup.6 base pairs of the
Escherichia coli (E. coli) chromosome offer tremendous promise for
the understanding of bacterial biochemical processes. In turn, this
knowledge will assist in the development of new tools for the
diagnosis and treatment of bacteria-caused human disease. The
entire E. coli genome has been sequenced, and this body of
information holds a tremendous potential for application to the
discovery and development of new antibiotic compounds. Yet, in
spite of this accomplishment, the general functions or roles of
many of these genes are still unknown. For example, the total
number of proliferation-required genes contained within the E. coli
genome is unknown, but has been variously estimated at around 200
to 700 (Armstrong, K. A. and Fan, D. P. Essential Genes in the
metB-malB Region of Escherichia coli K12, 1975, J. Bacteriol. 126:
48-55).
[0010] Novel, safe and effective antimicrobial compounds are needed
in view of the rapid rise of antibiotic resistant microorganisms.
However, prior to this invention, the characterization of even a
single bacterial gene was a painstaking process, requiring years of
effort. Accordingly, there is an urgent need for more novel methods
to identify and characterize bacterial genomic sequences that
encode gene products required for proliferation and for methods to
identify molecules that interact with and alter the functions of
such genes and gene products.
SUMMARY OF THE INVENTION
[0011] One embodiment of the present invention is a purified or
isolated nucleic acid sequence consisting essentially of one of SEQ
ID NOs: 1-81, 405-485, wherein said nucleic acid inhibits
microorganism proliferation. The nucleic acid sequence may be
complementary to at least a portion of a coding sequence of a gene
whose expression is required for microorganism proliferation. The
nucleic acid sequence may comprise a fragment of one of SEQ ID NOs.
1-81, 405-485, said fragment selected from the group consisting of
fragments comprising at least 10, at least 20, at least 25, at
least 30, at least 50 or more than 50 consecutive bases of one of
SEQ ID NOs: 1-81, 405-485. The nucleic acid sequence may be
complementary to a coding sequence of a gene whose expression is
required for microorganism proliferation.
[0012] Another embodiment of the present invention is a vector
comprising a promoter operably linked to a nucleic acid comprising
a sequence selected from the group consisting of SEQ ID NOs. 1-81,
405-485. The promoter may be active in an organism selected from
the group consisting of Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori,
Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus
pneumoniae, Haemophilus influenzae, Salmonella typhimurium,
Saccharomyces cerevisiae, Candida albicans, Cryptococcus
neoformans, Aspergillus fumigatus, Klebsiella pneumoniae,
Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis,
Staphylococcus epidermidis, Mycobacterium tuberculosis,
Mycobacterium leprae, Treponema pallidum, Bacillus anthracis,
Yersinia pestis, Clostridium botulinum, campylobacter jejuni,
Chlamydia trachomatus, Chlamydia pneumoniae or any species falling
within the genera of any of the above species.
[0013] Another embodiment of the present invention is a host cell
containing the vectors described above.
[0014] Another embodiment of the present invention is a purified or
isolated nucleic acid consisting essentially of the coding sequence
of one of SEQ ID NOs: 82-88, 90-242. One aspect of this embodiment
is a fragment of the nucleic acid comprising at least 10, at least
20, at least 25, at least 30, at least 50 or more than 50
consecutive bases of one of SEQ ID NOs: 82-88, 90-242.
[0015] Another embodiment of the present invention isa vector
comprising a promoter operably linked to the nucleic acids of the
preceding embodiment.
[0016] Another aspect of the present invention is a purified or
isolated nucleic acid comprising a nucleic acid sequence
complementary to at least a portion of an intragenic sequence,
intergenic sequence, sequences spanning at least a portion of two
or more genes, 5' noncoding region, or 3' noncoding region within
an operon encoding a polypeptide comprising a sequence selected
from the group consisting of SEQ ID NOs: 243-357, 359-398.
[0017] Another embodiment of the present invention is a purified or
isolated nucleic acid comprising a nucleic acid having at least 70%
homology to a sequence selected from the group consisting of SEQ ID
NOs 1-81, 405-485, 82-88, 90-242 or the sequences complementary
thereto as determined using BLASTN version 2.0 with the default
parameters. The nucleic acid may be from an organism selected from
the group consisting of Staphylococcus aureus, Pseudomonas
aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria
gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae,
Haemophilus influenzae, Salmonella typhimurium, Saccharomyces
cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus
fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella
paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis,
Mycobacterium tuberculosis, Mycobacterium leprae, Treponema
pallidum, Bacillus anthracis, Yersinia pestis, Clostridium
botulinum, campylobacter jejuni, and Chlamydia trachomatus,
Chlamydia pneumoniae or any species falling within the genera of
any of the above species.
[0018] Another embodiment of the present invention is a purified or
isolated nucleic acid consisting essentially of a nucleic acid
encoding a polypeptide having a sequence selected from the group
consisting of SEQ ID NOs.: 243-357, 359-398.
[0019] Another embodiment of the present invention is a vector
comprising a promoter operably linked to a nucleic acid encoding a
polypeptide having a sequence selected from the group consisting of
SEQ ID NOs.: 243-357, 359-398.
[0020] Another embodiment of the present invention is a host cell
containing the vector of the preceding embodiment.
[0021] Another embodiment of the present invention is purified or
isolated polypeptide comprising the sequence of one of SEQ ID NOs:
243-357, 359-398.
[0022] Another embodiment of the present invention is purified or
isolated polypeptide comprising a fragment of one of the
polypeptides of SEQ ID NOs. 243-357, 359-398, said fragment
selected from the group consisting of fragments comprising at least
5, at least 10, at least 20, at least 30, at least 40, at least 50,
at least 60 or more than 60 consecutive amino acids of one of the
polypeptides of SEQ ID NOs.: 243-357, 359-398.
[0023] Another embodiment of the present invention is an antibody
capable of specifically binding the polypeptide of the preceding
embodiment.
[0024] Another embodiment of the present invention is method of
producing a polypeptide, comprising introducing a vector comprising
a promoter operably linked to a nucleic acid encoding a polypeptide
having a sequence selected from the group consisting of SEQ ID NOs.
243-357, 359-398into a cell. The method may further comprise the
step of isolating said protein.
[0025] Another embodiment of the present invention is a method of
inhibiting proliferation comprising inhibiting the activity or
reducing the amount of a polypeptide having a sequence selected
from the group consisting of SEQ ID NOs. 243-357, 359-398or
inhibiting the activity or reducing the amount of a nucleic acid
encoding said polypeptide.
[0026] Another embodiment of the present invention is method for
identifying compounds which influence the activity of a polypeptide
required for proliferation comprising:
[0027] contacting a polypeptide comprising a sequence selected from
the group consisting of 243-357, 359-398 with a candidate compound;
and
[0028] determining whether said compound influences the activity of
said polypeptide.
[0029] The activity may be an enzymatic activity. The activity may
be a carbon compound catabolism activity. The activity may be a
biosynthetic activity. The activity may be a transporter activity.
The activity may be a transcriptional activity. The activity may be
a DNA replication activity. The activity may be a cell division
activity.
[0030] Another embodiment of the present invention is a compound
identified using the above method.
[0031] Another embodiment of the present invention is method for
assaying compounds for the ability to reduce the activity or level
of a polypeptide required for proliferation, comprising:
[0032] providing a target, wherein said target comprises the coding
sequence of a sequence selected from the group consisting of SEQ ID
NOs. 82-88, 90-242;
[0033] contacting said target with a candidate compound; and
[0034] measuring an activity of said target.
[0035] The target may be a messenger RNA molecule transcribed from
a coding region of one of SEQ ID. NOs.: 82-88, 90-242 and said
activity is translation of said messenger RNA. The target may be a
coding region of one of SEQ ID. NOs. 82-88, 90-242 and said
activity is transcription of said messenger RNA.
[0036] Another embodiment of the present invention is a compound
identified using the method above.
[0037] Another embodiment of the present invention is a method for
identifying compounds which reduce the activity or level of a gene
product required for cell proliferation comprising the steps
of:
[0038] expressing an antisense nucleic acid against a nucleic acid
encoding said gene product in a cell to reduce the activity or
amount of said gene product in said cell, thereby producing a
sensitized cell;
[0039] contacting said sensitized cell with a compound; and
[0040] determining whether said compound inhibits the growth of
said sensitized cell to a greater extent than said compound
inhibits the growth of a nonsensitized cell.
[0041] The cell may be selected from the group consisting of
bacterial cells, fungal cells, plant cells, and animal cells. The
cell may be an E. coli cell. The cell may be from an organism
selected from the group consisting of Staphylococcus aureus,
Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori,
Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus
pneumoniae, Haemophilus influenzae, Salmonella typhimurium,
Saccharomyces cerevisiae, Candida albicans, Cryptococcus
neoformans, Aspergillus fumigatus, Klebsiella pneumoniae,
Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis,
Staphylococcus epidermidis, Mycobacterium tuberculosis,
Mycobacterium leprae, Treponema pallidum, Bacillus anthracis,
Yersinia pestis, Clostridium botulinum, campylobacter jejuni, and
Chlamydia trachomatus, Chlamydia pneumoniae or any species falling
within the genera of any of the above species. The antisense
nucleic acid may be transcribed from an inducible promoter. The
method may, farther comprise the step of contacting said cell with
a concentration of inducer which induces said antisense nucleic
acid to a sublethal level. The sub-lethal concentration of said
inducer may be such that growth inhibition is 8% or more. The
inducer may be isopropyl-1-thio-.beta.-D-galactoside. The growth
inhibition may be measured by monitoring optical density of a
culture growth solution. The gene product may be a polypeptide. The
gene product may be an RNA. The gene product may comprise a
polypeptide having a sequence selected from the group consisting of
SEQ ID NOs.: 243-357, 359-398.
[0042] Another embodiment of the present invention is a compound
identified using the method above.
[0043] Another embodiment of the present invention is a method for
inhibiting cellular proliferation comprising introducing a compound
with activity against a gene corresponding to one of SEQ ID NOs.:
82-88, 90-242 or with activity against the product of said gene
into a population of cells expressing a gene. The compound may be
an antisense oligonucleotide comprising a sequence selected from
the group consisting of SEQ ID NOs.: 1-81, 405-485, or a
proliferation-inhibiting portion thereof. The proliferation
inhibiting portion of one of SEQ ID NOs. 1-81, 405-485 may be a
fragment comprising at least 10, at least 20, at least 25, at least
30, at least 50 or more than 50 consecutive bases of one of SEQ ID
NOs: 1-81, 405-485. The compound may be a triple helix
oligonucleotide.
[0044] Another embodiment of the present invention is a preparation
comprising an effective concentration of an antisense
oligonucleotide comprising a sequence selected from the group
consisting of SEQ ID NOs.: 1-81, 405-485, or a
proliferation-inhibiting portion thereof in a pharmaceutically
acceptable carrier. The proliferation-inhibiting portion of one of
SEQ ID NOs. 1-81, 405-485 may comprise at least 10, at least 20, at
least 25, at least 30, at least 50 or more than 50 consecutive
bases of one of SEQ ID NOs: 1-81, 405-485.
[0045] Another embodiment of the present invention is a method for
inhibiting the expression of a gene in an operon required for
proliferation comprising contacting a cell in a cell population
with an antisense nucleic acid, said cell expressing a gene
corresponding to one of SEQ ID NOs.: 82-88, 90-242, wherein said
antisense nucleic acid comprises at least a
proliferation-inhibiting portion of said operon in an antisense
orientation that is effective in inhibiting expression of said
gene. The antisense nucleic acid may be complementary to a sequence
of a gene comprising one or more of SEQ ID NOs.: 82-88, 90-242. The
antisense nucleic acid may be a sequence of one of SEQ ID NOs.:
1-81, 405-485, or a portion thereof. The cell may be contacted with
said antisense nucleic acid by introducing a plasmid which
expresses said antisense nucleic acid into said cell population.
The cell may be contacted with said antisense nucleic acid by
introducing a phage which expresses said antisense nucleic acid
into said cell population. The cell may be contacted with said
antisense nucleic acid by introducing a sequence encoding said
antisense nucleic acid into the chromosome of said cell into said
cell population. The cell may be contacted with said antisense
nucleic acid by introducing a retron which expresses said antisense
nucleic acid into said cell population. The cell may be contacted
with said antisense nucleic acid by introducing a ribozyme into
said cell-population, wherein a binding portion of said ribozyme is
complementary to said antisense oligonucleotide. The cell may be
contacted with said antisense nucleic acid by introducing a
liposome comprising said antisense oligonucleotide into said cell.
The cell may be contacted with said antisense nucleic acid by
electroporation. The antisense nucleic acid may be a fragment
comprising at least 10, at least 20, at least 25, at least 30, at
least 50 or more than 50 consecutive bases of one of SEQ ID NOs:
82-88, 90-242. The antisense nucleic acid may be an
oligonucleotide.
[0046] Another embodiment of the present invention is a method for
identifying bacterial strains comprising the steps of:
[0047] providing a sample containing a bacterial species; and
[0048] identifying a bacterial species using a species specific
probe having a sequence selected from the group consisting of SEQ
ID NOs. 1-81, 405-485, 82-88, 90-242.
[0049] Another embodiment of the present invention is a method for
identifying a gene in a microorganism required for proliferation
comprising:
[0050] (a) identifying an inhibitory nucleic acid which inhibits
the activity of a gene or gene product required for proliferation
in a first microorganism;
[0051] (b) contacting a second microorganism with said inhibitory
nucleic acid;
[0052] (c) determining whether said inhibitory nucleic acid from
said first microorganism inhibits proliferation of said second
microorganism; and
[0053] (d) identifying the gene in said second microorganism which
is inhibited by said inhibitory nucleic acid.
[0054] Another embodiment of the present invention is a method for
assaying a compound for the ability to inhibit proliferation of a
microorganism comprising:
[0055] (a) identifying a gene or gene product required for
proliferation in a first microorganism;
[0056] (b) identifying a homolog of said gene or gene product in a
second microorganism;
[0057] (c) identifying an inhibitory nucleic acid sequence which
inhibits the activity of said homolog in said second
microorgansim;
[0058] (d) contacting said second microorganism with a
proliferation-inhibiting amount of said inhibitory nucleic acid,
thus sensitizing said second microorganism;
[0059] (e) contacting the sensitized microorganism of step (d) with
a compound; and
[0060] (f) determining whether said compound inhibits proliferation
of said sensitized microorganism to a greater extent than said
compound inhibits proliferation of a nonsensitized
microorganism.
[0061] The step of identifying a gene involved in proliferation in
a first microorganism may comprise:
[0062] introducing a nucleic acid comprising a random genomic
fragment from said first microorganism operably linked to a
promoter wherein said random genomic fragment is in the antisense
orientation; and
[0063] comparing the proliferation of said first microorganism
transcribing a first level of said random genomic fragment to the
proliferation of said first microorganism transcribing a lower
level of said random genomic fragment, wherein a difference in
proliferation indicates that said random genomic fragment comprises
a gene involved in proliferation.
[0064] The step of identifying a homolog of said gene in a second
microorganism may comprise identifying a homologous nucleic acid or
a nucleic acid encoding a homologous polypeptide in a database
using an algorithm selected from the group consisting of BLASTN
version 2.0 with the default parameters and FASTA version 3.0t78
algorithm with the default parameters. The step of identifying a
homolog of said gene in a second microorganism may comprise
identifying a homologous nucleic acid or a nucleic acid encoding a
homologous polypeptide by identifying nucleic acids which hybridize
to said first gene. The step of identifying a homolog of said gene
in a second microorganism may comprise expressing a nucleic acid
which inhibits the proliferation of said first microorganism in
said second microorganism. The inhibitory nucleic acid may be an
antisense nucleic acid. The inhibitory nucleic acid may comprise an
antisense nucleic acid to a portion of said homolog. The inhibitory
nucleic acid may comprise an antisense nucleic acid to a portion of
the operon encoding said homolog. The step of contacting the second
microorganism with a proliferation-inhibiting amount of said
nucleic acid sequence may comprise directly contacting said second
microorganism with said nucleic acid. The step of contacting the
second microorganism with a proliferation-inhibiting amount of said
nucleic acid sequence may comprise expressing an antisense nucleic
acid to said homolog in said second microorganism.
[0065] Another embodiment of the present invention is a compound
identified using the method above.
[0066] Another embodiment of the present invention is a method of
assaying a compound for the ability to inhibit proliferation
comprising:
[0067] (a) identifying an inhibitory nucleic acid sequence which
inhibits the activity of a gene or gene product required for
proliferation in a first microorgansim;
[0068] (b) contacting a second microorganism with a
proliferation-inhibiting amount of said inhibitory nucleic acid,
thus sensitizing said second microorganism;
[0069] (c) contacting the proliferation-inhibited microorganism of
step (b) with a compound; and
[0070] (d) determining whether said compound inhibits proliferation
of said sensitized second microorganism to a greater extent than
said compound inhibits proliferation of a nonsensitized second
microorganism.
[0071] The inhibitory nucleic acid may be an antisense nucleic acid
which inhibits the proliferation of said first microorganism. The
inhibitory nucleic acid may comprise a portion of an antisense
nucleic acid which inhibits the proliferation of said first
microorganism. The inhibitory nucleic acid may comprise an
antisense molecule against the entire coding region of the gene
involved in proliferation of the first microorganism. The
inhibitory nucleic acid may comprise an antisense nucleic acid to a
portion of the operon encoding the gene involved in proliferation
of the first microorganism.
[0072] Another embodiment of the present invention is a compound
identified using the method above.
[0073] Another embodiment of the present invention is a method for
assaying compounds for activity against a biological pathway
required for proliferation comprising:
[0074] sensitizing a cell by expressing an antisense nucleic acid
against a nucleic acid encoding a gene product required for
proliferation in a cell to reduce the activity or amount of said
gene product;
[0075] contacting the sensitized cell with a compound; and
[0076] determining whether said compound inhibits the growth of
said sensitized cell to a greater extent than said compound
inhibits the growth of an nonsensitized cell.
[0077] The cell may be selected from the group consisting of
bacterial cells, fungal cells, plant cells, and animal cells. The
cell may be an E. coli cell. The cell may be an organism selected
from the group consisting of Staphylococcus aureus, Pseudomonas
aeruginosa, Enterobacter cloacae, Helicobacter pylori, Neisseria
gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae,
Haemophilus influenzae, Salmonella typhimurium, Saccharomyces
cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus
fumigatus, Klebsiella pneumoniae, Salmonella typhi, Salmonella
paratyphi, Salmonella cholerasuis, Staphylococcus epidermidis,
Mycobacterium tuberculosis, Mycobacterium leprae, Treponema
pallidum, Bacillus anthracis, Yersinia pestis, Clostridium
botulinum, campylobacter jejuni, and Chlamydia trachomatus,
Chlamydia pneumoniae or any species falling within the genera of
any of the above species. The antisense nucleic acid may be
transcribed from an inducible promoter. The method may further
comprise contacting the cell with an agent which induces expression
of said antisense nucleic acid from said inducible promoter,
wherein said antisense nucleic acid is expressed at a sublethal
level. The sublethal level of said antisense nucleic acid may
inhibit proliferation by 8% or more. The agent may be
isopropyl-1-thio-.beta.-D-g- alactoside (IPTG). The inhibition of
proliferation may be measured by monitoring the optical density of
a liquid culture. The gene product may comprise a polypeptide
having a sequence selected from the group consisting of SEQ ID NOs:
243-357, 359-398.
[0078] Another embodiment of the present invention is a compound
identified using the method above.
[0079] Another embodiment of the present invention is a method for
assaying a compound for the ability to inhibit cellular
proliferation comprising:
[0080] contacting a cell with an agent which reduces the activity
or level of a gene product required for proliferation of said
cell;
[0081] contacting said cell with said compound; and
[0082] determining whether said compound reduces proliferation to a
greater extent than said compound reduces proliferation of cells
which have not been contacted with said agent.
[0083] The agent which reduces the activity or level of a gene
product required for proliferation of said cell may comprise an
antisense nucleic acid to a gene or operon required for
proliferation. The agent which reduces the activity or level of a
gene product required for proliferation of said cell may comprise
an antibiotic. The cell may contain a temperature sensitive
mutation which reduces the activity or level of said gene product
required for proliferation of said cell. The antisense nucleic acid
may be directed against the same functional domain of said gene
product required for proliferation of said cell to which said
antisense nucleic acid is directed. The antisense nucleic acid may
be directed against a different functional domain of said gene
product required for proliferation of said cell than the fucntional
domain to which said antisense nucleic acid is directed.
[0084] Another embodiment of the present invention is a compound
identified using the method above.
[0085] Another embodiment of the present invention is a method for
identifying the pathway in which a proliferation-required nucleic
acid or its gene product lies comprising:
[0086] expressing a sublethal level of an antisense nucleic acid
directed against said proliferation-required nucleic acid in a
cell;
[0087] contacting said cell with an antibiotic, wherein the a
biological pathway on which said antibiotic acts is known; and
[0088] determining whether said cell has a substantially greater
sensitivity to said antibiotic than a cell which does not express
said sublethal level of said antisense nucleic acid.
[0089] Another embodiment of the present invention is a method for
determining the pathway on which a test compound acts
comprising:
[0090] (a) expressing a sublethal level of an antisense nucleic
acid directed against a proliferation-required nucleic acid in a
cell, wherein the biological pathway in which said
proliferation-required nucleic acid lies is known,
[0091] (b) contacting said cell with said test compound; and
[0092] (c) determining whether said cell has a substantially
greater sensitivity to said test compound than a cell which does
not express said sublethal level of said antisense nucleic
acid.
[0093] The method may further comprise:
[0094] (d) expressing a sublethal level of a second antisense
nucleic acid directed against a second proliferation-required
nucleic acid in said cell, wherein said second
proliferation-required nucleic acid is in a different biological
pathway than said proliferation-required nucleic acid in step (a);
and
[0095] (e) determining whether said cell has a substantially
greater sensitivity to said test compound than a cell which does
not express said sublethal level of said second antisense nucleic
acid.
[0096] Another embodiment of the present invention is a purified or
isolated nucleic acid consisting essentially of one of SEQ ID NOs:
358, 399-402.
[0097] Another embodiment of the present invention is a purified or
isolated nucleic acid comprising a sequence selected from the group
consisting of 1-81, 405-485, 82-88, 90-242, 358, 399-402.
[0098] Another embodiment of the present invention is a compound
which interacts with the gene or gene product of a nucleic acid
comprising a sequence of one of SEQ ID NOs: 82-88, 90-242 to
inhibit proliferation.
[0099] Another embodiment of the present invention compound which
interacts with a polypeptide comprising one of SEQ ID NOs. 243-357,
359-398 to inhibit proliferation.
[0100] Another embodiment of the present invention is a compound
which interacts with a nucleic acid comprising one of SEQ ID NOs:
358, 399-402 to inhibit proliferation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0101] FIG. 1 is an IPTG dose response curve in E. coli transformed
with an IPTG-inducible plasmid containing either an antisense clone
to the E. coli ribosomal protein rplW (AS-rplW) which is required
for protein synthesis and essential cell proliferation, or an
antisense clone to the elaD (AS-elaD) gene which is not known to be
involved in protein synthesis and which is also essential for
proliferation.
[0102] FIG. 2A is a tetracycline dose response curve in E. coli
transformed with an IPTG-inducible plasmid containing antisense to
rplW(AS-rplW) in the presence of 0, 20 or 50 .mu.M IPTG.
[0103] FIG. 2B is a tetracycline dose response curve in E. coli
transformed with an IPTG-inducible plasmid containing antisense to
elaD (AS-elaD) in the presence of 0, 20 or 50 .mu.M IPTG.
[0104] FIG. 3 is a graph showing the fold increase in tetracycline
sensitivity of E. coli transfected with antisense clones to
essential ribosomal proteins L23 (AS-rplW) and L7/L12 and L10
(AS-rplLrplJ). Antisense clones to genes known not to be involved
in protein synthesis (atpB/E(AS-atpB/E ), visC (AS-visC, elaD
(AS-elaD), yohH (AS-yohH) are much less sensitive to
tetracycline.
DEFINITIONS
[0105] By "biological pathway" is meant any discrete cell function
or process that is carried out by a gene product or a subset of
gene products. Biological pathways include enzymatic, biochemical
and metabolic pathways as well as pathways involved in the
production of cellular structures such cell walls. Biological
pathways that are usually required for proliferation of
microorganisms include, but are not limited to, cell division, DNA
synthesis & replication, RNA synthesis (transcription), protein
synthesis (translation), protein processing, protein transport,
fatty acid biosynthesis, cell wall synthesis, cell membrane
synthesis & maintenance, etc.
[0106] By "inhibit activity against a gene or gene product" is
meant having the ability to interfere with the function of a gene
or gene product in such a way as to decrease expression of the gene
or to reduce the level or activity of a product of the gene. Agents
which have activity against a gene include agents that inhibit
transcription of the gene and agents that inhibit translation of
the mRNA transcribed from the gene. In microorganisms, agents which
have activity against a gene can act to decrease expression of the
operon in which the gene resides or alter the processing of operon
RNA such as to reduce the level or activity of the gene product.
The gene product can be a non-translated RNA such as ribosomal RNA,
a translated RNA (mRNA) or the protein product resulting from
translation of the gene mRNA. Of particular utility to the present
invention are anti-sense RNAs that have activities against the
operons or genes to which they specifically hybridze.
[0107] By "activity against a gene product" is meant having the
ability to inhibit the function or to reduce the level or activity
of the gene product in a cell.
[0108] By "activity against a protein" is meant having the ability
to inhibit the function or to reduce the level or activity of the
protein in a cell.
[0109] By "activity against nucleic acid" is meant having the
ability to inhibit the function or to reduce the level or activity
of the nucleic acid in a cell.
[0110] As used herein, "sublethal" means a concentration of an
agent below the concentration required to inhibit all cell
growth.
DETAILED DESCRIPTION OF THE INVENTION
[0111] The present invention describes a group of E. coli genes and
gene families required for growth and/or proliferation. A
proliferation-required gene or gene family is one where, in the
absence of a gene transcript and/or gene product, growth or
viability of the microorganism is reduced or eliminated. Thus, as
used herein the terminology "proliferation-required" or "required
for proliferation" encompasses sequences where the absence of a
gene transcript and/or gene product completely eliminates cell
growth as well as sequences where the absence of a gene transcript
and/or gene product merely reduces cell growth. These
proliferation-required genes can be used as potential targets for
the generation of new antimicrobial agents. To achieve that goal,
the present invention also encompasses novel assays for analyzing
proliferation-required genes and for identifying compounds which
interact with the gene products of the proliferation-required
genes. In addition, the present invention contemplates the
expression of genes and the purification of the proteins encoded by
the nucleic acid sequences identified as required proliferation
genes and reported herein. The purified proteins can be used to
generate reagents and screen small molecule libraries or other
candidate compound libraries for compounds that can be further
developed to yield novel antimicrobial compounds. The present
invention also describes methods for identification of homologous
genes in organisms other than E. coli.
[0112] The present invention utilizes a novel method to identify
proliferation-required E. coli sequences. Generally, a library of
nucleic acid sequences from a given source are subcloned or
otherwise inserted into an inducible expression vector, thus
forming an expression library. Although the insert nucleic acids
may be derived from the chromosome of the organism into which the
expression vector is to be introduced, because the insert is not in
its natural chromosomal location, the insert nucleic acid is an
exogenous nucleic acid for the purposes of the discussion herein.
The term expression is defined as the production of an RNA molecule
from a gene, gene fragment, genomic fragment, or operon. Expression
can also be used to refer to the process of peptide or polypeptide
synthesis. An expression vector is defined as a vehicle by which a
ribonucleic acid (RNA) sequence is transcribed from a nucleic acid
sequence carried within the expression vehicle. The expression
vector can also contain features that permit translation of a
protein product from the transcribed RNA message expressed from the
exogenous nucleic acid sequence carried by the expression vector.
Accordingly, an expression vector can produce an RNA molecule as
its sole product or the expression vector can produce a RNA
molecule that is ultimately translated into a protein product.
[0113] Once generated, the expression library containing the
exogenous nucleic acid sequences is introduced into an E. coli
population to search for genes that are required for bacterial
proliferation. Because the library molecules are foreign to the
population of E. coli, the expression vectors and the nucleic acid
segments contained therein are considered exogenous nucleic
acid.
[0114] Expression of the exogenous nucleic acid fragments in the
test population of E. coli containing the expression vector library
is then activated. Activation of the expression vectors consists of
subjecting the cells containing the vectors to conditions that
result in the expression of the exogenous nucleic acid sequences
carried by the expression vector library. The test population of E.
coli cells is then assayed to determine the effect of expressing
the exogenous nucleic acid fragments on the test population of
cells. Those expression vectors that, upon activation and
expression, negatively impact the growth of the E. coli screen
population were identified, isolated, and purified for further
study.
[0115] A variety of assays are contemplated to identify nucleic
acid sequences that negatively impact growth upon expression. In
one embodiment, growth in E. coli cultures expressing exogenous
nucleic acid sequences and growth in cultures not expressing these
sequences is compared. Growth measurements are assayed by examining
the extent of growth by measuring optical densities. Alternatively,
enzymatic assays can be used to measure bacterial growth rates to
identify exogenous nucleic acid sequences of interest. Colony size,
colony morphology, and cell morphology are additional factors used
to evaluate growth of the host cells. Those cultures that failed to
grow or grow with reduced efficiency under expression conditions
are identified as containing an expression vector encoding a
nucleic acid fragment that negatively affects a
proliferation-required gene.
[0116] Once exogenous nucleic acid sequences of interest are
identified, they are analyzed. The first step of the analysis is to
acquire the nucleic acid sequence of the nucleic acid fragment of
interest. To achieve this end, the insert in those expression
vectors identified as containing a sequence of interest is
sequenced, using standard techniques well known in the art. The
next step of the process is to determine the source of the nucleic
acid sequence.
[0117] Determination of sequence source is achieved by comparing
the obtained sequence data with known sequences in various genetic
databases. The sequences identified are used to probe these gene
databases. The result of this procedure is a list of exogenous
nucleic acid sequences corresponding to a list that includeds novel
bacterial genes required for proliferation as well as genes
previously identified as required for proliferation.
[0118] The number of DNA and protein sequences available in
database systems has been growing exponentially for years. For
example, at the end of 1998, the complete sequences of
Caenorhabditis elegans, Saccharomyces cerevisiae and nineteen
bacterial genomes, including E. coli were available. This sequence
information is stored in a number of databanks, such as GenBank
(the National Center for Biotechnology Information (NCBI), and is
publicly available for searching.
[0119] A variety of computer programs are available to assist in
the analysis of the sequences stored within these databases. FastA,
(W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with
FASTP and FASTA" Methods in Enzymology 183:63- 98), Sequence
Retrieval System (SRS), (Etzold & Argos, SRS an indexing and
retrieval tool for flat file data libraries. Comput. Appl. Biosci.
9:49-57, 1993) are two examples of computer programs that can be
used to analyze sequences of interest. In one embodiment of the
present invention, the BLAST family of computer programs, which
includes BLASTN version 2.0 with the default parameters, or BLASTX
version 2.0 with the default parameters, is used to analyze nucleic
acid sequences.
[0120] BLAST, an acronym for "Basic Local Alignment Search Tool,"
is a family of programs for database similarity searching. The
BLAST family of programs includes: BLASTN, a nucleotide sequence
database searching program, BLASTX, a protein database searching
program where the input is a nucleic acid sequence; and BLASTP, a
protein database searching program. BLAST programs embody a fast
algorithm for sequence matching, rigorous statistical methods for
judging the significance of matches, and various options for
tailoring the program for special situations. Assistance in using
the program can be obtained by e-mail at
blast@ncbi.nlm.nih.gov.
[0121] Bacterial genes are often transcribed in polycistronic
groups. These groups comprise operons, which are a collection of
genes and intergenic sequences. The genes of an operon are
co-transcribed and are often related functionally. Given the nature
of the screening protocol, it is possible that the identified
exogenous nucleic acid sequence corresponds to a gene or portion
thereof with or without adjacent noncoding sequences, an intragenic
sequence (i.e. a sequence within a gene), an intergenic sequence
(i.e. a sequence between genes), a sequence spanning at least a
portion of two or more genes, a 5' noncoding region or a 3'
noncoding region located upstream or downstream from the actual
sequence that is required for bacterial proliferation. Accordingly,
determining which of the genes that are encoded within the operons
are individually required for proliferation is often desirable.
[0122] In one embodiment of the present invention, an operon is
dissected to determine which gene or genes are required for
proliferation. For example, the RegulonDB DataBase described by
Huerta et al. (Nucl. Acids Res. 26:55-59, 1998), which may also be
found on the website
http://www.cifn.unam.mx/Computational_Biology/regulondb/, may be
used, to identify the boundaries of operons encoded within
microbial genomes. A number of techniques that are well known in
the art can be used to dissect the operon. In one aspect of this
embodiment, gene disruption by homologous recombination is used to
individually inactivate the genes of an operon that is thought to
contain a gene required for proliferation.
[0123] Several gene disruption techniques have been described for
the replacement of a functional gene with a mutated, non-functional
(null) allele. These techniques generally involve the use of
homologous recombination. The method described by Link et al. (J.
Bacteriol 1997 179:6228; incorporated herein by reference in it's
entirety) serves as an excellent example of these methods as
applicable to disruption of genes in E. coli. This technique uses
crossover PCR to create a null allele with an in-frame deletion of
the coding region of a target gene. The null allele is constructed
in such a way that sequences adjacent to the wild type gene (ca.
500 bp) are retained. These homologous sequences surrounding the
deletion null allele provide targets for homologous recombination
so that the wild type gene on the E. coli chromosome can be
replaced by the constructed null allele.
[0124] The crossover PCR amplification product is subcloned into
the vector pKO3, the features of which include a chloramphenicol
resistance gene, the counter-selectable marker sacB, and a
temperature sensitive autonomous replication function. Following
transformation of an E. coli cell population with such a vector,
selection for cells that have undergone homologous recombination of
the vector into the chromosome is achieved by growth on
chloramphenicol at the non-permissive temperature of 43.degree. C.
Under these conditions, autonomous replication of the plasmid
cannot occur and cell are resistant to chloramphinicol only if the
chloramphenicol resistance gene has been integrated into the
chromosome. Usually a single crossover event is responsible for
this integration event such that the E. coli chromosome now
contains a tandem duplication of the target gene consisting of one
wild type allele and one deletion null allele separated by vector
sequence.
[0125] This new E. coli strain containing the tandem duplication
can be maintained at permissive temperatures in the presence of
drug selection (chloramphenicol). Subsequently, cells of this new
strain are cultured at the permissive temperature 30.degree. C.
without drug selection. Under these conditions, the chromosome of
some of the cells within the population will have undergone an
internal homologous recombination event resulting in removal of the
plasmid sequences. Subsequent culturing of the strain in growth
medium lacking chloramphenicol but containing sucrose is used to
select for such recombinative resolutions. In the presence of the
counter-selectable marker sacB, sucrose is rendered into a toxic
metabolite. Thus, cells that survive this counter-selection have
lost both the plasmid sequences from the chromosome and the
autonomously replicating plasmid that results as a byproduct of
recombinative resolution.
[0126] There are two possible outcomes of the above recombinative
resolution via homologous recombination. Either the wild type copy
of the targeted gene is retained on the chromosome or the mutated
null allele is retained on the chromosome. In the case of an
essential gene, a single copy of the null allele would be lethal
and such cells should not be obtained by the above procedure when
applied to essential genes. In the case of a non-essential gene,
roughly equal numbers of cells containing null alleles and cells
containing wild type alleles should be obtained. Thus, the method
serves as a test for essentiality of the targeted gene: when
applied to essential genes, only cells with a wild type allele on
the chromosome will be obtained.
[0127] Other techniques have also been described for the creation
of disruption mutations in E. coli. For example, Link et al. also
describe inserting an in-frame sequence tag concommitantly with an
in-frame deletion in order to simplify analysis of recombinants
obtained. Further, Link et al. describe disruption of genes with a
drug resistance marker such as a kanamycin resistance gene. Arigoni
et al., (Arigoni, F. et al. A Genome-based Approach for the
Identification of Essential Bacterial Genes, Nature Biotechnology
16: 851-856, the disclosure of which is incorporated herein by
reference in its entirety) describe the use of gene disruption
combined with engineering a second copy of a test gene such that
the expression of the gene is regulated by and inducible promoter
such as the arabinose promoter to test the essentiality of the
gene. Many of these techniques result in the insertion of large
fragments of DNA into the gene of interest, such as a drug
selection marker. An advantage of the technique described by Link
et al. is that it does not rely on an insertion into the gene to
cause a functional defect, but rather results in the precise
removal of the coding region. This insures the lack of polar
effects on the expression of genes downstream from the target
gene.
[0128] Recombinant DNA techniques can be used to express the entire
coding sequences of the gene identified as required for
proliferation, or portions thereof. The over-expressed proteins can
be used as reagents for further study. The identified exogenous
sequences are isolated, purified, and cloned into a suitable
expression vector using methods well known in the art. If desired,
the nucleic acids can contain the sequences encoding a signal
peptide to facilitate secretion of the expressed protein.
[0129] Expression of fragments of the bacterial genes identified as
required for proliferation is also contemplated by the present
invention. The fragments of the identified genes can encode a
polypeptide comprising at least 5, at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, at least 55, at least 60, at least 65, at
least 75, or more than 75 consecutive amino acids of a gene
complementary to one of the identified sequences of the present
invention. The nucleic acids inserted into the expression vectors
can also contain sequences upstream and downstream of the coding
sequence.
[0130] When expressing the coding sequence of an entire gene
identified as required for bacterial proliferation or a fragment
thereof, the nucleic acid sequence to be expressed is operably
linked to a promoter in an expression vector using conventional
cloning technology. The expression vector can be any of the
bacterial, insect, yeast, or mammalian expression systems known in
the art. Commercially available vectors and expression systems are
available from a variety of suppliers including Genetics Institute
(Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega
(Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to
enhance expression and facilitate proper protein folding, the codon
usage and codon bias of the sequence can be optimized for the
particular expression organism in which the expression vector is
introduced, as explained by Hatfield, et al., U.S. Pat. No.
5,082,767, incorporated herein by this reference. Fusion protein
expression systems are also contemplated by the present
invention.
[0131] Following expression of the protein encoded by the
identified exogenous nucleic acid sequence, the protein is
purified. Protein purification techniques are well known in the
art. Proteins encoded and expressed from identified exogenous
nucleic acid sequences can be partially purified using
precipitation techniques, such as precipitation with polyethylene
glycol. Chromatographic methods usable with the present invention
can include ion-exchange chromatography, gel filtration, use of
hydroxyapaptite columns, immobilized reactive dyes,
chromatofocusing, and use of high-performance liquid
chromatography. Electrophoretic methods such one-dimensional gel
electrophoresis, high-resolution two-dimensional polyacrylamide
electrophoresis, isoelectric focusing, and others are contemplated
as purification methods. Also, affinity chromatographic methods,
comprising antibody columns, ligand presenting columns and other
affinity chromatographic matrices are contemplated as purification
methods in the present invention.
[0132] The purified proteins produced from the gene coding
sequences identified as required for proliferation can be used in a
variety of protocols to generate useful antimicrobial reagents. In
one embodiment of the present invention, antibodies are generated
against the proteins expressed from the identified exogenous
nucleic acid sequences. Both monoclonal and polyclonal antibodies
can be generated against the expressed proteins. Methods for
generating monoclonal and polyclonal antibodies are well known in
the art. Also, antibody fragment preparations prepared from the
produced antibodies discussed above are contemplated.
[0133] Another application for the purified proteins of the present
invention is to screen small molecule libraries for candidate
compounds active against the various target proteins of the present
invention. Advances in the field of combinatorial chemistry provide
methods, well known in the art, to produce large numbers of
candidate compounds that can have a binding, or otherwise
inhibitory effect on a target protein. Accordingly, the screening
of small molecule libraries for compounds with binding affinity or
inhibitory activity for a target protein produced from an
identified gene sequence is contemplated by the present
invention.
[0134] The present invention further contemplates utility against a
variety of other pathogenic organisms in addition to E. coli. For
example, the invention has utility in identifying genes required
for proliferation in prokaryotes and eukaryotes. For example, the
invention has utility with protists, such as Plasmodium spp.;
plants; animals, such as Entamoeba spp. and Contracaecum spp; and
fungi including Candida spp., (e.g., Candida albicans),
Saccharomyces cerevisiae, Cryptococcus neoformans, and Aspergillus
fumigatus. In one embodiment of the present invention, monera,
specifically bacteria are probed in search of novel gene sequences
required for proliferation. This embodiment is particularly
important given the rise of drug resistant bacteria.
[0135] The numbers of bacterial species that are becoming resistant
to existing antibiotics are growing. A partial list of these
organisms includes: Staphylococcus spp., such as S. aureus;
Enterococcus spp., such as E. faecalis; Pseudomonas spp., such as
P. aeruginosa, Clostridium spp., such as C. botulinum, Haemophilus
spp., such as H. influenzae, Enterobacter spp., such as E. cloacae,
Vibrio spp., such as V. cholera; Moraxala spp., such as M.
catarrhalis; Streptococcus spp., such as S. pneumoniae, Neisseria
spp., such as N. gonorrhoeae; Mycoplasma spp., such as Mycoplasma
pneumoniae; Salmonella typhimurium; Helicobacter pylori;
Escherichia coli; and Mycobacterium tuberculosis. The sequences
identified as required for proliferation in the present invention
can be used to probe these and other organisms to identify
homologous required proliferation genes contained therein.
[0136] In one embodiment of the present invention, the nucleic acid
sequences disclosed herein are used to screen genomic libraries
generated from bacterial species of interest other than E. coli.
For example, the genomic library may be from Staphylococcus aureus,
Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pylori,
Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus
pneumoniae, Haemophilus influenzae, Salmonella typhimurium,
Saccharomyces cerevisiae, Candida albicans, Cryptococcus
neoformans, Aspergillus fumigatus, Klebsiella pneumoniae,
Salmonella typhi, Salmonella paratyphi, Salmonella cholerasuis,
Staphylococcus epidermidis, Mycobacterium tuberculosis,
Mycobacterium leprae, Treponema pallidum, Bacillus anthracis,
Yersinia pestis, Clostridium botulinum, Campylobacter jejuni,
Chlamydia trachomatus, Chlamydia pneumoniae or any species falling
within the genera of any of the above species. Standard molecular
biology techniques are used to generate genomic libraries from
various microorganisms. In one aspect, the libraries are generated
and bound to nitrocellulose paper. The identified exogenous nucleic
acid sequences of the present invention can then be used as probes
to screen the libraries for homologous sequences. The homologous
sequences identified can then be used as targets for the
identification of new, antimicrobial compounds with activity
against more than one organism.
[0137] For example, the preceding methods may be used to isolate
nucleic acids having a sequence with at least 97%, at least 95%, at
least 90%, at least 85%, at least 80%, or at least 70% identity to
a nucleic acid sequence selected from the group consisting of one
of the sequences of SEQ ID NOS. 1-81, 405-485, 82-88, 90-242,
fragments comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75,
100, 150, 200, 300, 400, or 500 consecutive bases thereof, and the
sequences complementary thereto. Identity may be measured using
BLASTN version 2.0 with the default parameters. (Altschul, S. F. et
al. Gapped BLAST and PSI-BLAST: A New Generation of Protein
Database Search Programs, Nucleic Acid Res. 25: 3389-3402 (1997),
the disclosure of which is incorporated herein by reference in its
entirety). For example, the homologous polynucleotides may have a
coding sequence which is a naturally occurring allelic variant of
one of the coding sequences described herein. Such allelic variants
may have a substitution, deletion or addition of one or more
nucleotides when compared to the nucleic acids of SEQ ID NOs: 1-81,
405-485, 82-88, 90-242 or the sequences complementary thereto.
[0138] Additionally, the above procedures may be used to isolate
nucleic acids which encode polypeptides having at least 99%, 95%,
at least 90%, at least 85%, at least 80%, at least 70%, at least
60%, at least 50%, or at least 40% identity or similarity to a
polypeptide having the sequence of one of SEQ ID NOs: 243-357,
359-398 or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids thereof as
determined using the FASTA version 3.0t78 algorithm with the
default parameters. Alternatively, protein identity or similarity
may be identified using BLASTP with the default parameters, BLASTX
with the default parameters, or TBLASTN with the default
parameters. (Alschul, S. F. et al. Gapped BLAST and PSI-BLAST: A
New Generation of Protein Database Search Programs, Nucleic Acid
Res. 25: 3389-3402 (1997), the disclosure of which is incorporated
herein by reference in its entirety).
[0139] Alternatively, homologous nucleic acids or polypeptides may
be identified by searching a database to identify sequences having
a desired level of homology to a nucleic acid or polypeptide
involved in proliferation or an antisense nucleic acid to a nucleic
acid involved in microbial proliferation. A variety of such
databases are available to those skilled in the art, including
GenBank and GenSeq. In some embodiments, the databases are screened
to identify nucleic acids or polypeptides having at least 97%, at
least 95%, at least 90%, at least 85%, at least 80%, at least 70%,
at least 60%, or at least 50%, at least 40% identity or similarity
to a nucleic acid or polypeptide involved in proliferation or an
antisense nucleic acid involved in proliferation. For example, the
database may be screened to identify nucleic acids homologous to
one of SEQ ID Nos. 1-81, 405-485, 82-88, 90-242 or polypeptides
homologous to SEQ ID NOs. 243-357, 359-398. In some embodiments,
the database may be screened to identify homologous nucleic acids
or polypeptides from organisms other than E. coli, including
organisms such as Staphylococcus aureus, Pseudomonas aeruginosa,
Enterobacter cloacae, Helicobacter pylon, Neisseria gonorrhoeae,
Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus
influenzae, Salmonella typhimurium, Saccharomyces cerevisiae,
Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus,
Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi,
Salmonella cholerasuis, Staphylococcus epidermidis, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus
anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter
jejuni, Chlamydia trachomatus, Chlamydia pneumoniae or any species
falling within the genera of any of the above species.
[0140] In another embodiment, gene expression arrays and
microarrays can be employed. Gene expression arrays are high
density arrays of DNA samples deposited at specific locations on a
glass chip, nylon membrane, or the like. Such arrays can be used by
researchers to quantify relative gene expression under different
conditions. Gene expression arrays are used by researchers to help
identify optimal drug targets, profile new compounds, and determine
disease pathways. An example of this technology is found in U.S.
Pat. No. 5,807,522, which is hereby incorporated by reference.
[0141] It is possible to study the expression of all genes in the
genome of a particular microbial organism using a single array. For
example, the arrays from Genosys consist of 12.times.24 cm nylon
filters containing PCR products corresponding to 4290 ORFs from E.
coli. 10 ngs of each are spotted every 1.5 mm on the filter. Single
stranded labeled cDNAs are prepared for hybridization to the array
(no second strand synthesis or amplification step is done) and
placed in contact with the filter. Thus the labeled cDNAs are of
"antisense" orientation. Quantitative analysis is done by
phosphorimager.
[0142] Hybridization of cDNA made from a sample of total cell mRNA
to such an array followed by detection of binding by one or more of
various techniques known to those in the art results in a signal at
each location on the array to which cDNA hybridized. The intensity
of the hybridization signal obtained at each location in the array
thus reflects the amount of mRNA for that specific gene that was
present in the sample. Comparing the results obtained for mRNA
isolated from cells grown under different conditions thus allows
for a comparison of the relative amount of expression of each
individual gene during growth under the different conditions.
[0143] Gene expression arrays may be used to analyze the total mRNA
expression pattern at various time points after induction of an
antisense nucleic acid against a proliferation-required gene.
Analysis of the expression pattern indicated by hybridization to
the array provides information on whether or not the target gene of
the antisense nucleic acid is being affected by antisense
induction, how quickly the antisense is affecting the target gene,
and for later timepoints, what other genes are affected by
antisense expression. For example, if the antisense is directed
against a gene for ribosomal protein L7/L12 in the 50S subunit, its
targeted mRNA may disappear first and then other mRNAs may be
observed to increase, decrease or stay the same. Similarly, if the
antisense is directed against a different 50S subunit ribosomal
protein mRNA (e.g. L25), that mRNA may disappear first followed by
changes in mRNA expression that are similar to those seen with the
L7/L12 antisense expression. Thus, the mRNA expression pattern
observed with an antinsense nucleic acid against a proliferation
required gene may identify other proliferation-required nucleic
acids in the same pathway as the target of the antisense nucleic
acid. In addition, the mRNA expression patterns observed with
candidate drug compounds may be compared to those observed with
antisense nucleic acids against a proliferation-required nucleic
acid. If the mRNA expression pattern observed with the candidate
drug compound is similar to that observed with the antisense
nucleic acid, the drug compound may be a promising therapeutic
candidate. Thus, the assay would be useful in assisting in the
selection of candidate drug compounds for use in screening methods
such as those described below.
[0144] In cases where the source of nucleic acid deposited on the
array and the source of the nucleic acid being hybridized to the
array are from two different organisms, gene expression arrays can
identify homologous genes in the two organisms.
[0145] The present invention also contemplates additional methods
for screening other microorganisms for proliferation-required
genes. In this embodiment, the conserved portions of sequences
identified as proliferation-required can be used to generate
degenerate primers for use in the polymerase chain reaction (PCR).
The PCR technique is well known in the art. The successful
production of a PCR product using degenerate probes generated from
the sequences identified herein would indicate the presence of a
homologous gene sequence in the species being screened. This
homologous gene is then isolated, expressed, and used as a target
for candidate antibiotic compounds. In another aspect of this
embodiment, the homologous gene is expressed in an autologous
organism or in a heterologous organism in such a way as to alter
the level or activity of a homologous gene required for
proliferation in the autologous or heterologus organism. In still
another aspect of this embodiment, the homologous gene or portion
is expressed in an antisense orientation in such a way as to alter
the level or activity of a nucleic acid required for proliferation
of an autologous or heterologous organism.
[0146] The homologous sequences to proliferation-required genes
identified using the techniques described herein may be used to
identify proliferation-required genes of organisms other than E.
coli, to inhibit the proliferation of organisms other than E. coli
by inhibiting the activity or reducing the amount of the identified
homologous nucleic acid or polypeptide in the organism other than
E. coli, or to identify compounds which inhibit the growth of
organisms other than E. coli as described below.
[0147] In another embodiment of the present invention, E. coli
sequences identified as required for proliferation are transferred
to expression vectors capable of function within non-E coli
species. As would be appreciated by one of ordinary skill in the
art, expression vectors must contain certain elements that are
species specific. These elements can include promoter sequences,
operator sequences, repressor genes, origins of replication,
ribosomal binding sequences, termination sequences, and others. To
use the identified exogenous sequences of the present invention,
one of ordinary skill in the art would know to use standard
molecular biology techniques to isolate vectors containing the
sequences of interest from cultured bacterial cells, isolate and
purify those sequences, and subclone those sequences into an
expression vector adapted for use in the species of bacteria to be
screened.
[0148] Expression vectors for a variety of other species are known
in the art. For example, Cao et al. report the expression of
steroid receptor fragments in Staphylococcus aureus. J. Steroid
Biochem Mol Biol. 44(1):1-11 (1993). Also, Pla et al. have reported
an expression vector that is functional in a number of relevant
hosts including: Salmonella typhimurium, Pseudomonas putida, and
Pseudomonas aeruginosa. J. Bacteriol. 172(8):4448-55 (1990). These
examples demonstrate the existence of molecular biology techniques
capable of constructing expression vectors for the species of
bacteria of interest to the present invention.
[0149] Following the subcloning of the identified nucleic acid
sequences into an expression vector functional in the microorganism
of interest, the identified nucleic acid sequences are
conditionally transcribed to assay for bacterial growth inhibition.
Those expression vectors found to contain sequences that, when
transcribed, inhibit bacterial growth are compared to the known
genomic sequence of the pathogenic microorganism being screened or,
if the homologous sequence from the organism being screened is not
known, it may be identified and isolated by hybridization to the
proliferation-required E. coli sequence interest or by
amplification using primers based on the proliferation-required E.
coli sequence of interest as described above.
[0150] The antisense sequences from the second organism which are
identified as described above may then be operably linked to a
promoter, such as an inducible promoter, and introduced into the
second organism. The techniques described herein for identifying E.
coli genes required for proliferation may thus be employed to
determine whether the identified sequences from a second organism
inhibit the proliferation of the second organism.
[0151] Antisense nucleic acids required for the proliferation of
organisms other than E. coli or the genes corresponding thereto,
may also be hybridized to a microarray containing the E. coli ORFs
to gauge the homology between the E. coli sequences and the
proliferation-required nucleic acids from other organisms. For
example, the proliferation-required nucleic acid may be from
Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter
cloacae, Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus
faecalis, Streptococcus pneumoniae, Haemophilus influenzae,
Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans,
Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella
pneumoniae, Salmonella typhi, Salmonella paratyphi, Salmonella
cholerasuis, Staphylococcus epidermidis, Mycobacterium
tuberculosis, Mycobacterium leprae, Treponema pallidum, bacillus
anthracis, Yersinia pestis, Clostridium botulinum, Campylobacter
jejuni or Chlamydia trachomatus, Chlamydia pneumoniae or any
species falling within the genera of any of the above species. The
proliferation-required nucleic acids from an organism other than E.
coli may be hybridized to the array under a variety of conditions
which permit hybridization to occur when the probe has different
levels of homology to the sequence on the microarray. This would
provide an indication of homology across the organisms as well as
clues to other possible essential genes in these organisms.
[0152] In still another embodiment, the exogenous nucleic acid
sequences of the present invention that are identified as required
for bacterial growth or proliferation can be used as antisense
therapeutics for killing bacteria. The antisense sequences can be
directed against the proliferation-required genes whose sequence
corresponds to the exogenous nucleic acid probes identified here
(i.e. the antisense nucleic acid may hybridize to the gene or a
portion thereof). Alternatively, antisense therapeutics can be
directed against operons in which proliferation-required genes
reside (i.e. the antisense nucleic acid may hybridize to any gene
in the operon in which the proliferation-required genes reside).
Further, antisense therapeutics can be directed against a
proliferation-required gene or portion thereof with or without
adjacent noncoding sequences, an intragenic sequence (i.e. a
sequence within a gene), an intergenic sequence (i.e. a sequence
between genes), a sequence spanning at least a portion of two or
more genes, a 5' noncoding region or a 3' noncoding region located
upstream or downstream from the actual sequence that is required
for bacterial proliferation or an operon containing a
proliferation-required gene.
[0153] In addition to therapeutic applications, the present
invention encompasses the use of nucleic acid sequences
complementary to sequences required for proliferation as diagnostic
tools. For example, nucleic acid probes complementary to
proliferation-required sequences that are specific for particular
species of microorganisms can be used as probes to identify
particular microorganism species in clinical specimens. This
utility provides a rapid and dependable method by which to identify
the causative agent or agents of a bacterial infection. This
utility would provide clinicians the ability to prescribe species
specific antimicrobial compounds to treat such infections. In an
extension of this utility, antibodies generated against proteins
translated from mRNA transcribed from proliferation-required
sequences can also be used to screen for specific microorganisms
that produce such proteins in a species-specific manner.
[0154] The following examples teach the genes of the present
invention and a subset of uses for the E. coli genes identified as
required for proliferation. These examples are illustrative only
and are not intended to limit the scope of the present
invention.
EXAMPLES
[0155] The following examples are directed to the identification
and exploitation of E. coli genes required for proliferation.
Methods of gene identification are discussed as well as a variety
of methods to utilize the identified sequences.
[0156] Genes Identified as Required for Proliferation of E.
coli
[0157] Exogenous nucleic acid sequences were cloned into an
inducible expression vector and assayed for growth inhibition
activity. Example 1 describes the examination of a library of
exogenous nucleic acid sequences cloned into IPTG-inducible
expression vectors. Upon activation or induction, the expression
vectors produced an RNA molecule corresponding to the subcloned
exogenous nucleic acid sequences. The RNA product was in an
antisense orientation with respect to the E. coli genes from which
it was originally derived. This antisense RNA then interacted with
sense mRNA produced from various E. coli genes and interfered with
or inhibited the translation of the sense messenger RNA (mRNA) thus
preventing protein production from these sense mRNA molecules. In
cases where the sense mRNA encoded a protein required for the
proliferation, bacterial cells containing an activated expression
vector failed to grow or grew at a substantially reduced rate.
EXAMPLE 1
Inhibition of Bacterial Proliferation after IPTG Induction
[0158] To study the effects of transcriptional induction in liquid
medium, growth curves were carried out by back diluting cultures
1:200 into fresh media with or without 1 mM IPTG and measuring the
OD.sub.450 every 30 minutes (min). To study the effects of
transcriptional induction on solid medium, 10.sup.2, 10.sup.3,
10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7 and 10.sup.8 fold dilutions
of overnight cultures were prepared. Aliquots of from 0.5 to 3
.mu.l of these dilutions were spotted on selective agar plates with
or without 1 mM IPTG. After overnight incubation, the plates were
compared to assess the sensitivity of the clones to IPTG.
[0159] Of the numerous clones tested, some clones were identified
as a containing sequence that inhibited E. coli growth after IPTG
induction. Accordingly, the gene to which the inserted nucleic acid
sequence corresponds, or a gene within the operon containing the
inserted nucleic acid, may be required for proliferation in E.
coli.
[0160] Characterization of Isolated Clones Negatively Affecting E.
coli Proliferation
[0161] Following the identification of those expression vectors
that, upon expression, negatively impacted E. coli growth or
proliferation, the inserts or nucleic acid fragments contained in
those expression vectors were isolated for subsequent
characterization. Expression vectors of interest were subjected to
nucleic acid sequence determination.
EXAMPLE 2
Nucleic Acid Sequence Determination of Identified Clones Expressing
Nucleic Acid Fragments with Detrimental Effects of E. coli
Proliferation
[0162] The nucleotide sequences for the exogenous identified
sequences were determined using plasmid DNA isolated using QIAPREP
(Qiagen, Valencia, Calif.) and methods supplied by the
manufacturer. The primers used for sequencing the inserts were
5'-TGTTTATCAGACCGCTT-3' (SEQ ID NO: 403) and
5'-ACAATTTCACACAGCCTC-3' (SEQ ID NO: 404). These sequences flank
the polylinker in pLEX5BA. Sequence identification numbers (SEQ ID
NOs) for the identified inserts are listed in Table I and discussed
below.
EXAMPLE 3
Comparison of Isolated Sequences to Known Sequences
[0163] The nucleic acid sequences of the subcloned fragments
obtained from the expression vectors discussed above were compared
to known E. coli sequences in GenBank using BLAST version 1.4 or
version 2.0.6 using the following default parameters: Filtering
off, cost to open a gap=5, cost to extend a gap=2, penalty for a
mismatch in the blast portion of run=-3, reward for a match in the
blast portion of run=1, expectation value (e)=10.0, word size=11,
number of one-line descriptions=100, number of alignments to show
(B)=100. BLAST is described in Altschul, J Mol Biol. 215:403-10
(1990), the disclosure of which is incorporated herein by reference
in its entirety. Expression vectors were found to contain nucleic
acid sequences in both the sense and antisense orientations. The
presence of known genes, open reading frames, and ribosome binding
sites was determined by comparison to public databases holding
genetic information and various computer programs such as the
Genetics Computer Group programs FRAMES and CODONPREFERENCE. Clones
were designated as "antisense" if the cloned fragment was oriented
to the promoter such that the RNA transcript produced was
complementary to the expressed mRNA from a chromosomal locus.
Clones were designated as "sense" if they coded for an RNA fragment
that was identical to a portion of a wild type mRNA from a
chromosomal locus.
[0164] The sequences described in Examples 1-2 that inhibited
bacterial proliferation and contained gene fragments in an
antisense orientation are listed in Table I. This table lists each
identified sequence by: a sequence identification number; a
Molecule Number; a gene to which the identified sequence
corresponds, listed according to the National Center for
Biotechnology Information (NCBI), Blattner (Science
277:1453-1474(1997); also contains the E. coli K-12 genome
sequence), or Rudd (Micro. and Mol. Rev. 62:985-1019 (1998)), (both
papers are hereby incorporated by reference) nomenclatures. The
CONTIG numbers for each identified sequence is shown, as well as
the location of the first and last base pairs located on the E.
coli chromosome. A Molecule Number with a "**" indicates a clone
corresponding to an intergenic sequence.
[0165] The sequences of the nucleic acid inserts of SEQ ID NOs:
1-81 from U.S. Provisional Patent Application No. 60/117,405 which
inhibited proliferation were further analyzed. The reanalyzed
sequences corresponding to SEQ ID NOs. 1-81 of U.S. Provisional
Patent Application No. 60/117,405 have SEQ ID NOs. 405-485 in the
present application.
[0166] SEQ ID NOs: 82-242 in U.S. Provisional Patent Application
No. 60/117,405 are identical to SEQ ID NOs: 82-242 of the present
application with the following exceptions. SEQ ID NO: 148 in the
present application is the complementary strand of SEQ ID NO: 148
in U.S. Provisional Patent Application No. 60/117,405. Accordingly,
the protein of SEQ ID NO: 308 which is encoded by SEQ ID NO: 148
has also been revised. SEQ ID NO: 163 in the present application is
the complementary strand of SEQ ID NO: 163 in U.S. Provisional
Patent Application No. 60/117,405. Accordingly, the protein of SEQ
ID NO: 323 which is encoded by SEQ ID NO: 163 has also been
revised.
[0167] The target gene of SEQ ID NOs. 18 and 19 of U.S. Provisional
Patent Application No. 60/117,405 (SEQ ID NOs. 18, 19, 422, 423 of
the present application) has been revised from dicF to ftsZ to
reflect the fact that these SEQ ID NOs. include natural antisense
molecules which inhibit ftsZ expression.
[0168] The gene products of the nucleic acids of SEQ ID NOs. 198
and 239-242 in U.S. Provisional Patent Application No. 60/117,405
and in the present application (SEQ ID NOs. 358 and 399-402 of the
present application) have been revised to reflect the fact that
these nucleic acids encode nontranslated tRNAs and rRNAs. Tables I
and II have been revised accordingly. The SEQ ID NOs. in Table II
were also revised to reflect the fact that SEQ ID NOs: 89 and 402
were identical in U.S. Provisional Patent Application No.
60/117,405.
1TABLE I Identified Clones with Corresponding Genes and Operons
Molecule Gene Gene Gene SEQ ID NO. No. (NCB1) (Blattner) (Rudd)
CONTIG 1,405 EcXA001 yhhQ b3471 yhhQ AE000423 2,406 EcXA002 lepB
lepB lepB AE000343 3,407 EcXA003 f586 b0955 ycbZ AE000197 4,408
EcXA004 rpsG, rpsL b3341 rpsG, AE0004l0 rpsL 5,409 EcXA005a rplL,
rplJ b3986 rplL, rplJ AE000472 6,410 EcXA005b rplL rplL rplL
AE000472 7,411 EcXA005c rplL, rplJ rplL, rplJ rplL, rplJ AE000472
8,412 EcXA005d rplL, rplJ rplL, rplJ rplL, rplJ AE000472 9,413
EcXA005e rplL rplL rplL AE000472 10,414 EcXA005f rplL rplL rplL
AE000472 11,415 EcXA005g rplL rplL rplL AE000472 12,416 EcXA006 pta
b2297 pta AE000319 13,417 EcXA007 yicP b3666 yicP AE000444 14,418
EcXA008a yhaU b3127 yhaU AE000394 15,419 EcXA008b yhaU yhaU yhaU
AE000394 16,420 EcXA008c yhaU yhaU yhaU AE000394 17,421 EcXA009
ydeY ydeY ydeY AE000249 18,422 EcXA010a dicF b1575 dicF AE000253
(natural as) 19,423 EcXA010b dicF dicF dicF AE000253 20,424 EcXA011
fdnG b1474 fdnG AE000244 21,425 EcXA012a fusA b3340 fusA AE000410
22,426 EcXA012b fusA fusA fusA AE000410 23,427 EcXA012c fusA fusA
fusA AE000410 24,428 EcXA013a o86 b2S62 yfhL AE000342 25,429
EcXA013b o86 b2562 yJhL AE000342 26,430 EcXA013c o86 b2562 yfliL
AE000342 27,431 EcXA014 visC b2906 visC AE000374 28,432 EcXA015
yfdl yfdl yfdl AE000323 29,433 EcXA0O16 yeaQ yeaQ yeaQ AE000274
yoaG yoaG yoaG 30,434 EcXA017a yggE b2922 yggE AE000375 31,435
EcXA017b yggE yggE yggE AE000375 32,436 EcXA018a o464 b2074 yegM
AE000297 33,437 EcXA018b o464 b2074 yegM AE000297 34,438 EcXA019a
yehA yehA yehA AE000300 AE000299 35,439 EcXA019b o172, yehA o172,
yehA o172, AE000299 yehA 36,440 EcXA020 o384, f82 b1794, yeaP,
AE000274 b1795 yeaQ 37,441 EcXA021a fl12 b0218 yafU AE000130 38,442
EcXA021b f112 b0218 yafU AE000130 39,443 EcXA022 o740 b1629 ydgN
AE000258 40,444 EcXA023a f176, f382 b1504, ydeS, AE000247 b1505
ydeT 41,445 EcXA023b f176, f382 b1504, ydeS, AE000247 b1505 ydeT
42,446 EcXA024 ygjM, ygjN b3082 ygiM, A1E000390 ygjN 43,447 EcXA025
O2383 b1878 yeeJ AE000289 44,448 EcXA026 o61 Unpre- Unpre- AE000138
dicted dicted 45,449 EcXA027a yohH yohH yohH AE000303 46,450
EcXA027b yohH yohH yohH AE000303 47,451 EcXA027c yohH yohH yohH
A1E000303 yohl yohl yohl 48,452 EcXA027d yohH yohH yohH AE000303
49,453 EcXA028 f296 b2305 yfcl AE000319 50,454 EcXA029 yjjK b4391
yjjK AE000509 51,455 EcXA030 yi5A b3557 yi5A AE000433 52,456
EcXA031 rplE B3308 rplE AE000408 53,457 EcXA032a ybgD ybgD ybgD
AE000175 54,458 EcXA032b** ybgD ybgD ybgD AE000175 gltA gltA gltA
55,459 EcXA033a f477 (as) b3052 waaE AE000387 AE000386 56,460
EcXA033b f477 b3052 waaE AE000387 57,461 EcXA034a cspA b3SS6 cspA
AE000433 58,462 EcXA034b cspA b3556 cspA AE000433 59,463 EcXA035
yhjU yhjU yhjU AE000431 60,464 EcXA036 yqiF b3101 yqiF AE000392 o99
b3100, yqiK 61,465 EcXAO37 ydeH b1535 ydeH AE000251 62,466 EcXA038
sieB b1353 sieB AE000233 63,467 EcXA039 ybbD ybbD AE000156 64,468
EcXA040 InsB_6 b3445 insB_6 AE000420 65,469 EcXA041 f234 b1138 ymfE
AE000214 66,470 EcXA042a rplY rplY rplY AE000308 67,471 EcXA042b
rplY rplY rplY AE000308 68,472 EcXA043 ybgB ybgB ybgB AE000176 cydA
cydA cydA 69,473 EcXA044 purB b1131 purB AE000213 70,474 EcXA045**
csrA csrA csrA AE000353 serV serV serV 71,475 EcXA046** finE,finA
b4313 finE, AF000502 finA 72,476 EcXA047** f96, cspB f96, cspB
cspB, AE000252 ydfS 73,477 EcXA048 yefE yefE yefE AE000294 74,478
EcXA049 yaiC b0385 yaiC AE000145 75,479 EcXA050 o467, o222
yaiU,yaiV yaiU, AE000144 yaiV 76,480 EcXA051a rplB, rplW rplB, rplW
rplB, AE000408 rplW 77,481 EcXA051b rplW rplW rplW AE000408 78,482
EcXA052 infC infC infC AE000267 AE000266 79,483 EcXA053 gor gor gor
AE000426 80,484 EcXA054 rplF rplF rplF AE000408 81,485 EcXA055 rrlG
rrlG rrlG AE000345
EXAMPLE 4
Identification of Genes and Their Corresponding Operons Affected by
Antisense Inhibition
[0169] The sequencing of the entire E. coli genome is described in
Blattner et al., Science 5 277:1453-1474(1997) the entirety of
which is hereby incorporated by reference and the sequence of the
genome is listed in GenBank Accession No.U00096, the disclosure of
which is incorporated herein by reference in its entirety. The
operons to which the proliferation-inhibiting nucleic acids
correspond were identified using RegulonDB and information in the
literature. The coordinates of the boundaries of these operons on
the E. coli genome are listed in Table III. Table II lists the
molecule numbers of the inserts containing the growth inhibiting
nucleic acid fragments, the genes in the operons corresponding to
the inserts, the SEQ ID NOs of the genes containing the inserts,
the SEQ ID NOs of the proteins encoded by the genes, the start and
stop points of the genes on the E. coli genome, the orientation of
the genes on the genome, whether the operons are predicted or
documented, and the predicted functions of the genes. The
identified operons, their putative functions, and whether or not
the genes are presently thought to be required for proliferation
are discussed below.
[0170] Functions for the identified genes were determined by using
either Blattner functional class designations or by comparing
identified sequence with known sequences in various databases. A
variety of biological functions were noted for the genes to which
the clones of the present invention correspond. The functions for
the genes of interest appear in Table II.
[0171] The proteins that are listed in Table II are involved in a
wide range of biological functions.
2TABLE II All Operon Data with Whole Chromosome Coordinates
Predicted Gene Gene (P) Or Blattner functional Predicted functional
Seq ID Prod. Seq Genes On Left Right Documented class of encoded
class of encoded No. ID No. Mole. No. Operon Coordinate Coordinate
(D) Operon proteins proteins 82 243 EcXA001 yhhQ 3606848 3607513
(P) Hypothetical ORF, Hypothetical outer unclassified, membrane
protein unknown 83 244 dcrB 3607532 3608143 Hypothetical ORF,
Resistance to phage unclassified, C1; periplasmic unknown protein
perhaps anchored to inner membrane 84 245 EcXA002 lepB 2702355
2703329 (P) Transport and Secretion binding proteins 85 246 EcXA003
ycbZ 1015762 1017522 (P) Unknown Protease 86 247 EcXA004 tufA
3467782 3468966 (D) Translation, post- Translation translational
(Elongation factor modification Tu) 87 248 fusA 3469037 3471151
Translation, post- Translation translational (elongation factor
modification efg) 88 249 rpsG 3471179 3471718 Translation, post-
Translation translational modification 89 402 EcXA055 rrsG 2727636
2729178 Translation, post- Translation (rRNA) translational
modification 90 250 rpsL 3471815 3471815 Translation, post-
Translation translational modification 91 251 EcXA005a-g rplJ
4177574 4178071 (D) Translation, post- Translation translational
modification 92 252 rplL 4178138 4178503 Translation, post-
Translation translational modification 93 253 EcXA006 pta 2412767
2414911 (P) Carbon compound Carbon compound catabolism catabolism
94 254 EcXA007 yicP 3841591 3843357 (P) Hypothetical ORF, Probable
adenine unclassified, deaminase unknown 95 255 EcXA008a-c yhaD
3268266 3269492 (P) Hypothetical ORF, unclassified, unknown 96 256
yhaE 3269508 3270407 Putative enzymes 97 257 yhaF 3270428 3271198
Hypothetical ORF, unclassified, unknown 98 258 yhaU 3271214 3272548
Carbon compound Probable integral catabolism membrane protein
Phthalate permease family 99 259 EcXA009 ydeX 1599514 1601049 (P)
Putative transport proteins 100 260 ydeY 1601043 1602071 Putative
transport Putative ABC proteins transporter 101 261 ydeZ 1602071
1603063 Hypothetical ORF, unclassified, unknown 102 262 yneA
1603075 1604097 Hypothetical ORF, unclassified, unknown 103 263
yneB 1604124 1604999 Hypothetical ORF, unclassified, unknown 104
264 yneC 1605023 1605313 Hypothetical ORF, unclassified, unknown
105 265 EcXA010a-b ftsZ 105305 106456 (P) Cell processes (incl.
Regulator of cell Adaptation, division protection) 106 266 EcXA011
fdnG 1545425 1548472 (D) Energy metabolism Anaerobic respiration
(formate dehydro- genase) 107 267 fdnH 1548485 1549369 Energy
metabolism 108 268 fdnl 1549362 1550015 Energy metabolism
EcXA012a-c Same operon as EcXA004 109 269 EcXA013a-c yhfL 2697683
2697943 (P) Hypothetical ORF, No homologues, no unclassified,
motifs unknown 110 270 EcXA014 visC 3049135 3050337 (P)
Hypothetical ORF, Ubiquinone synthesis unclassified, unknown 111
271 ubiH 3050360 3051538 Biosynthesis of cofactors, prosthetic
groups and carriers 112 272 pepP 3051535 3052860 Translation, post-
translational modification 113 273 ygfB 3052886 3053470
Hypothetical ORF, unclassified, unknown 114 274 EcXA015 yfdG
2465875 2466237 (P) Hypothetical ORF, unclassified, unknown 115 275
yfdH 2466234 2467154 Cell structure 116 276 yfdl 2467151 2468482
Hypothetical ORF, Putative membrane unclassified, protein unknown
117 277 EcXA016 yeaQ 1877031 1877279 (P) Hypothetical ORF,
Homologue to unclassified, transgly-cosylase unknown associated
protein 118 278 yoaG 1877427 1877609 (P) Hypothetical ORF, No
homologues unclassified, unknown 119 279 yeaR 1877613 1877972
Hypothetical ORF, unclassified, unknown 120 280 EcXA017a-b yggE
3065360 3066100 (P) Structural proteins Homologues in multiple
bacteria, no motifs 121 281 EcXA018a-b yegM 2151891 2153285 (P)
Putative transport Transport (multiple proteins transferable
resistance) 122 282 yegN 2153285 2156407 Hypothetical ORF,
unclassified, unknown 123 283 yegO 2156408 2159485 Hypothetical
ORF, unclassified, unknown 124 284 yegB 2159486 2160901 Putative
transport proteins 125 285 EcXA019a-b yehA 2185400 2186434 (P) Cell
structure Weak homology to pilin precursor from H. Inf. 126 286
yehB 2186450 2188930 Hypothetical ORF, unclassified, unknown 127
287 yehC 2188946 2189665 Putative chaperones 128 288 yehD 2189700
2190242 Cell structure EcXA020 Same operon as EcXA016 (one of the
two) 129 289 EcXA021a-b yafU 238746 239084 (P) Hypothetical ORF,
Homologues in H. unclassified, Inf. and S. Pombe., no unknown
motifs, transmem- brane region present 130 290 EcXA022 ydgL 1703791
1704372 (P) Hypothetical ORF, unclassified, unknown 131 291 ydgM
1704372 1704950 Hypothetical ORF, unclassified, unknown 132 292
ydgN 1704943 1707165 Hypothetical ORF, unclassified, unknown 133
293 ydgO 1707166 1708224 Hypothetical ORF, unclassified, unknown
134 294 ydgP 1708228 1708848 Hypothetical ORF, unclassified,
unknown 135 295 ydgQ 1708852 1709547 Hypothetical ORF,
unclassified, unknown 136 296 nth 1709547 1710182 Transcription,
RNA processing and degradation 137 297 EcXA023a-b ydeR 1585817
1586320 (P) Hypothetical ORF, unclassified, unknown 138 298 ydeS
1586333 1586863 Hypothetical ORF, fimf-like unclassified, unknown
139 299 ydeT 1586877 1588025 Structural proteins fimd-like 140 300
EcXA024 ygjM 3231369 3231785 (P) Hypothetical ORF, Weak homology to
unclassified, long chain fatty acid unknown coa ligase in
Archaeglobus 141 301 ygjN 3231782 3232096 Hypothetical ORF,
Homologues in unclassified, various bacteria unknown 142 302
EcXA025 yeeJ 2042885 2050036 (P) Hypothetical ORF, Strong
similarity to unclassified, numerous attaching unknown amd effacing
proteins and invasins 143 303 EcXA026 rajA 331001 331184
unpredicted nifm like 144 304 EcXA027a-d yohG 2225343 2226539 (P)
Putative transport proteins 145 305 yohH 2226569 2226859
Hypothetical ORF, Xylose binding unclassified, protein-like unknown
146 306 yohI 2227458 2228405 (P) Putative regulatory protein 147
307 EcXA028 ycfI 2420669 2421559 (P) Hypothetical ORF, Similar to
S. Typhi unclassified, histidine transport unknown gene 148 308
EcXA029 yjjK 4626424 4628091 (P) Hypothetical ORF, Similar to ABC
unclassified, transporter unknown 149 309 EcXA030 yi5A 3718309
3718830 (P) Hypothetical ORF, IS150 orf A unclassified, unknown 150
310 yi5B 3718827 3719678 Phage, transposon, or plasmid 151 311
EcXA031 rpmJ 3440255 3440371 (D) Translation, post- translational
modification 152 312 prlA 3440403 3441734 Putative transport
proteins 153 313 rplO 3441742 3442176 Translation, post-
translational modification 154 314 rpmD 3442180 3442359
Translation, post- translational modification 155 315 rpsE 3442363
3442866 Translation, post- translational modification 156 316 rplR
3442881 3443234 Translation, post translational modification 157
317 rplF 3443244 3443777 Translation, post- Translation
translational modification 158 318 rpsH 3443790 3444182
Translation, post- translational modification 159 319 rpsN 3444216
3444521 Translation, post- translational modification 160 320 rplE
3444536 3445075 Translation, post- Translation translational
modification 161 321 rplX 3445090 3445404 Translation, post-
translational modification 162 322 rplN 3445415 3445786
Translation, post- translational modification 163 323 EcXA032a-b
ybgD 751452 752018 (P) Cell processes (incl. Hypothetical fimbrial
Adaptation, protein protection) 164 324 gltA 752408 753691 (D)
Energy metabolism Glutamine biosynthesis 165 325 EcXA033a-b waaE
3192961 3194394 (P) Putative enzymes ADP heptose
synthase/autotrophic growth protein 166 326 glnE 3194442 3197282
Translation, post- translational modification 167 327 ygiF 3197305
3198606 Hypothetical ORF, unclassified, unknown 168 328 EcXA034a-b
cspA 3717678 3717890 (P) Cell processes (incl. RNA chaperonin
Adaptation, protection) 169 329 EcXA035 yhjS 3694087 3695658 (P)
Translation, post- translational modification 170 330 yhjT 3695658
3695846 Hypothetical ORF, unclassified, unknown 171 331 yhjU
3695843 3697522 Hypothetical ORF, Regions similar to unclassified,
dehydro-genases, unknown nucleases etc. 172 332 EcXA036 yqjC
3246594 3246977 (P) Hypothetical ORF, unclassified, unknown 173 333
yqjD 3247015 3247320 Hypothetical ORF, unclassified, unknown 174
334 yqjE 3247323 3247727 Hypothetical ORF, unclassified, unknown
175 335 yqjK 3247717 3248016 Similartomukb from H. Inf. 176 336
yqjF 3248112 3248594 (P) Hypothetical ORF, Homologues in many
unclassified, bacteria, blocks; unknown secretion/ATP synthase/ftsz
177 337 EcXA037 ydeH 1620984 1621874 (P) Hypothetical ORF, Similar
to carboxy- unclassified, kinase, oxidase, unknown symporters 178
338 EcXA038 sieB 1416572 1417183 (P) Phage, transposon, or
Super-infection plasmid exclusion factor B- like 179 339 rajB
1417192 1417368 Hypothetical ORF, (b1354) unclassified, unknown 180
340 EcXA039 rhsD 522485 526765 (P) Hypothetical ORF, unclassified,
unknown 181 341 ybbC 526805 527173 Hypothetical ORF, unclassified,
unknown 182 342 ylbH 527173 527883 Hypothetical ORF, Rhs-like
element unclassified, unknown 183 343 ybbD 527864 528124
Hypothetical ORF, ATP synthase, unclassified, desaturase unknown
184 344 ylbI 528163 528354 Hypothetical ORF, unclassified, unknown
185 345 EcXA040 insB_6 351114 351389 (P) Phage, transposon, or
plasmid 186 346 insA 351308 3581811 Phage, transposon, or plasmid
187 347 yrhA 3580669 3581085 Hypothetical ORF, unclassified,
unknown 188 348 yhhZ 3579494 3580672 Hypothetical ORF,
unclassified, unknown 189 349 EcXA041 ymfD 1196090 1196755 (P)
Hypothetical ORF, No assigned role unclassified, unknown 190 350
ymfE 1196756 1197460 Hypothetical ORF, No assigned role
unclassified, unknown 191 351 EcXA042a-b rplY 2280537 2280821 (P)
Translation, post- Translation translational modification 192 352
EcXA043 hrsA 765207 767183 (P) Translation, post- translational
modification 193 353 ybgB 767201 769834 Carbon compound Unknown
catabolism 194 354 cydA 770678 772249 (D) Energy metabolism
Cytochrome D oxidase 195 355 cydB 772265 773404 Energy metabolism
196 356 EcXA044 purB 1189839 1191209 (D) Nucleotide Purine
biosynthesis biosynthesis and metabolism 197 357 EcXA045 csrA
2816983 2817168 (P) Regulatory function Carbon storage regulator
(mRNA decay factor) 198 358 serV 2816575 2816667 Unpredicted
Translation, post- Translation (tRNA) translational modification
199 359 EcXA046 fimB 4538525 4539127 (D) Cell structure 200 360
fimE 4539605 4540201 Cell structure Fimbrae 201 361 fimA 4540683
4541231 Cell structure Regulator of inversion 203 363 fimC 4541872
4542597 Cell structure 204 364 fimD 4542665 4545301 Cell structure
205 365 fimF 4545311 4545841 Cell structure 206 366 fimG 4545854
4546357 Cell structure 207 367 fimH 4546377 4547279 Cell structure
208 368 EcXA047 ydJP 1637054 1638684 (P) Hypothetical ORF,
unclassified, unknown 209 369 ydfQ 1637548 1638081 Hypothetical
ORF, unclassified, unknown 210 370 ydfR 1638078 1638389
Hypothetical ORF, unclassified, unknown 211 371 ydfS 1638394
1638684 Hypothetical ORF, Lysis protein unclassified, unknown 212
372 cspB 1639363 1639578 (P) Cell processes (mel. Adaptation,
protection) 213 373 EcXA048 yi52_7 2099917 2100933 (P) Phage,
transposon, or plasmid 214 374 yefJ 2100938 2101411 Putative
enzymes 215 375 yefI 2101413 2102531 Hypothetical ORF,
unclassified, unknown 216 376 yefH 2102516 2103106 Putative enzymes
217 377 yefG 2103087 2104079 Hypothetical ORF, unclassified,
unknown 218 378 rfc 2104082 2105248 Cell structure 219 379 yefE
2105248 2106351 Hypothetical ORF, UDP galacto- unclassified,
pyranase mutase unknown 220 380 EcXA049 yaiC 402927 404042 (P)
Hypothetical ORF, Unknown unclassified, unknown 221 381 EcXA050
yaiU 392239 393642 (P) Putative enzymes Putative auto- transporter
222 382 yaiV 393685 394353 Hypothetical ORF, Hypothetical outer
unclassified, membrane protein unknown 223 383 EcXA051a-b rpsQ
3445951 3446205 (D) Translation, post- translational modification
224 384 rpmC 3446205 3446396 Translation, post- translational
modification 225 385 rplP 3446396 3446806 Translation, post-
translational modification 226 386 rpsC 3446819 3447520
Translation, post- translational modification 227 387 rplV 3447538
3447870 Translation, post- translational modification 228 388 rpsS
3447885 3448163 Translation, post- translational modification 229
389 rplB 3448180 3449001 Translation, post- Translation
translational modification 230 390 rplW 3449019 3449321
Translation, post- Translation translational modification 231 391
rplD 3449318 3449923 Translation, post- translational modification
232 392 rplC 3449934 3450563 Translation, post- translational
modification 233 393 rpsJ 3450596 3450907 Translation, post-
translational modification 234 394 EcXA052 rplT 1797417 1797773 (D)
Translation, post- translational modification 235 395 rpmI 1797826
1798023 Translation, post- translational modification 236 396 infC
1798120
1798662 Translation, post- Translation translational modification
237 397 thrS 1798666 1800594 Translation, post- translational
modification 238 398 EcXA053 gor 3643929 3645281 (P) Biosynthesis
of Glutathione oxido- cofactors, prosthetic reductase groups and
carriers EcXA054 Same operon as EcXA031 239 399 EcXA055 rrlG
2724301 2727204 (D) Translation, post- Translation (rRNA)
translational modification 240 400 rrfG 2724089 2724208
Translation, post- Translation (rRNA) translational modification
241 401 gltW 2727389 2727464 Translation, post- Translation (tRNA)
translational modification 242 402 rrsG 2727636 2729178
Translation, post- Translation (rRNA) translational
modification
[0172] Several of the expression vectors contain fragments that
correspond to genes of unknown function or if the function is
known, it is not known whether the gene is essential. For example,
EcXA001, 003, 007, 008, 013, 015, 016, 017, 018, 019, 020, 021,
022, 023, 024, 025, 026, 027, 028, 029, 030, 032, 033, 034, 035,
036, 037, 038, 039, 040, 041, 047, 048, 049 and 050 are all
exogenous nucleic acid sequences that correspond to E. coli
proteins that have no known function or where the function has not
been shown to be essential or nonessential.
[0173] The present invention reports a number of novel E. coli
genes and operons that are required for proliferation. From the
list clone sequences identified here, each was identified to be a
portion of a gene in an operon required for the proliferation of E.
coli. Cloned sequences corresponding to genes already known to be
required for proliferation in E. coli include EcXA002, 004, 005,
010, 012, 014, 031, 02, 043, 045, 051, 052, 054, and 055. The
remaining identified sequences correspond to E. coli genes
previously undesignated as required for proliferation in the
art.
[0174] An interesting observation of the present invention is that
there are also several sequence fragments that correspond to E.
coli genes that are not thought to be required for E. coli
proliferation. Nevertheless, under the conditions described above,
the antisense expression of these gene fragments causes a reduction
in cell growth. This result implies that the genes corresponding to
the identified sequences are actually required for proliferation.
Molecule Nos. corresponding to these genes are EcXA006, 044, 046,
and 053.
[0175] Following identification of the sequences of interest, these
sequences were localized into operons. Since bacterial genes are
expressed in a polycistronic manner, the antisense inhibition of a
single gene in an operon might effect the expression of all the
other genes on the operon or the genes down stream from the single
gene identified. In order to determine which of the gene products
in an operon are required for proliferation, each of the genes
contained within an operon may be analyzed for their effect on
viability as described below.
3TABLE III Operon Boundaries Left Right Mole. No. Coordinate
Coordinate EcXA001 3606848 3608143 EcXA002 2702355 2703329 EcXA003
1015762 1017522 EcXA004 3467782 3472189 EcXA00S 4177574 4178503
EcXA006 2412767 2414911 EcXA007 3841591 3843357 EcXA008 3268266
3272548 EcXA009 1599514 1605313 EcXA010 1647406 1647458 EcXA011
1545425 1550015 EcXA012 3467782 3472189 EcXA013 2697683 2697943
EcXA014 3049135 3053470 HeXAC15 2465875 2468482 EcXA016 1877031
1877972 EcXA017 3065360 3066100 EcXA018 2151891 2160901 EcXA019
2185400 2190242 EcXA020 1877031 1877972 EcXA021 238746 239084
EcXA022 1703791 1710182 EcXA023 1585817 1588025 EcXA024 3231369
3232096 EcXA025 2042885 2050036 EcXA026 331001 331184 EcXA027c
2225343 2228405 EcXA028 2420669 2421559 EcXA029 4626424 4628091
EcXA030 3718309 3719678 EcXA031 3440255 3445786 EcXA032b 751452
753691 EcXA033 3192961 3198606 EcXA034 3717678 3717890 EcXA035
3694087 3697522 EcXA036 3246594 3248594 EcXA037 1620984 1621874
EcXA038 1416572 1417368 EcXA039 522485 528354 EcXA040 3580669
3580672 EcXA041 1196090 1197460 EcXA042 2280537 2280821 EcXA043
765207 773404 EcXA044 1189839 1191209 EcXA045 2816575 2817168
EcXA046 4538525 4547279 EcXA047 1637054 1639578 EcXA048 2099917
2106351 EcXA049 402927 404042 EcXA050 392239 394353 EcXA051 3445951
3450907 EcXA052 1797417 1800594 EcXA053 3643929 3645281 EcXA054
3440255 3445786 EcXA055 2724301 2729178
EXAMPLE 5
Identification of Individual Genes Within an Operon Required for
Proliferation
[0176] The following example illustrates a method for determining
which gene in an operon is required for proliferation. The clone
insert corresponding to Molecule No. EcXA004 possesses nucleic acid
sequence homology to the E. coli genes rspG and rspL. This molecule
corresponds to an operon containing two additional genes fusA and
tufA. The rpsL gene is the first gene in the operon. To determine
which gene or genes in this operon are required for proliferation,
each gene is selectively inactivated using homologous
recombination. Gene rpsL is the first gene to be inactivated.
[0177] Deletion inactivation of a chromosomal copy of a gene in E.
coli can be accomplished by integrative gene replacement. The
principle of this method (Hamilton, C. M., et al 1989. J.
Bacteriol. 171: 4617-4622) is to construct a mutant allele of the
targeted gene, introduce that allele into the chromosome using a
conditional suicide vector, and then force the removal of the
native wild type allele and vector sequences. This will replace the
native gene with a desired mutation(s) but leave promoters,
operators, etc. intact. Essentiality of a gene is determined either
by deduction from genetic analysis or by conditional expression of
a wild type copy of the targeted gene (trans complementation).
[0178] The first step is to generate a mutant rpsL allele using PCR
amplification. Two sets of PCR primers are chosen to produce a copy
of rpsL with a large central deletion to inactivate the gene. In
order to eliminate polar effects, it is desirable to construct a
mutant allele comprising an in-frame deletion of most or all of the
coding region of the rpsL gene. Each set of PCR primers is chosen
such that a region flanking the gene to be amplified is
sufficiently long to allow recombination (typically at least 500
nucleotides on each side of the deletion). The targeted deletion or
mutation will be contained within this fragment. To facilitate
cloning of the PCR product, the PCR primers may also contain
restriction endonuclease sites found in the cloning region of a
conditional knockout vector such as pKO3 (Link, et al 1997 J.
Bacteriol. 179 (20): 6228-6237). Suitable sites include NotI, SalI,
BamHI and SmaI. The rpsL gene fragments are produced using standard
PCR conditions including, but not limited to, those outlined in the
manufacturers directions for the Hot Start Taq PCR kit (Qiagen,
Inc., Valencia, Calif.). The PCR reactions will produce two
fragments that can be fused together. Alternatively, crossover PCR
can be used to generate a desired deletion in one step (Ho, S. N.,
et al 1989. Gene 77: 51-59, Horton, R. M., et al 1989. Gene 77:
61-68). The mutant allele thus produced is called a "null" allele
because it cannot produce a functional gene product.
[0179] The mutant allele obtained from PCR amplification is cloned
into the multiple cloning site of pKO3. Directional cloning of the
rpsL null allele is not necessary. The pKO3 vector has a
temperature-sensitive origin of replication derived from pSC101.
Therefore, clones are propagated at the permissive temperature of
30.degree. C. The vector also contains two selectable marker genes:
one that confers resistance to chloramphenicol and another, the
Bacillus subtilis sacB gene, that allows for counter-selection on
sucrose containing growth medium. Clones that contain vector DNA
with the null allele inserted are confirmed by restriction
endonuclease analysis and DNA sequence analysis of isolated plasmid
DNA. The plasmid containing the rpsL null allele insert is known as
a knockout plasmid.
[0180] Once the knockout plasmid has been constructed and its
sequence verified, it is transformed into a Rec.sup.+ E. coli host
cell. Transformation can be by any standard method such as
electroporation. In some fraction of the transformed cells,
plasmids will integrate into the E. coli chromosome by homologous
recombination between the rpsL null allele in the plasmid and the
rpsL gene in the chromosome. Transformant colonies in which such an
event has occurred are readily selected by growth at the
non-permissive temperature of 43.degree. C. and in the presence of
choramphenicol. At this temperature, the plasmid will not replicate
as an episome and will be lost from cells as they grow and divide.
These cells are no longer resistant to chloramphenicol and will not
grow when it is present. However, cells in which the knockout
plasmid has integrated into the E. coli chromosome remain resistant
to chloramphenicol and propagate.
[0181] Cells containing integrated knock-out plasmids are usually
the result of a single crossover event that creates a tandem repeat
of the mutant and native wild type alleles of rpsL separated by the
vector sequences. A consequence of this is that rpsL will still be
expressed in these cells. In order to determine if the gene is
essential for growth, the wild type copy must be removed. This is
accomplished by selecting for plasmid excision, a process in which
homologous recombination between the two alleles results in looping
out of the plasmid sequences. Cells that have undergone such an
excision event and have lost plasmid sequences including sacB gene
are selected for by addition of sucrose to the medium. The sacB
gene product converts sucrose to a toxic molecule. Thus counter
selection with sucrose ensures that plasmid sequences are no longer
present in the cell. Loss of plasmid sequences is further confirmed
by testing for sensitivity to chloramphenicol (loss of the
chloramphenicol resistance gene). The latter test is important
because occasionally a mutation in the sacB gene can occur
resulting in a loss of sacB function with no effect on plasmid
replication (Link, et. al., 1997 J. Bacteriol. 179 (20):
6228-6237). These artifact clones retain plasmid sequences and are
therefore still resistant to chloramphenicol.
[0182] In the process of plasmid excision, one of the two rpsL
alleles is lost from the chromosome along with the plasmid DNA. In
general, it is equally likely that the null allele or the wild type
allele will be lost. Therefore, if the rpsL gene is not essential,
half of the clones obtained in this experiment will have the wild
type allele on the chromosome and half will have the null allele.
However, if the rpsL gene is essential, cells containing the null
allele will not be obtained as a single copy of the null allele
would be lethal.
[0183] To determine the essentiality of rpsL, a statistically
significant number of the resulting clones, at least 20, are
analyzed by PCR amplification of the rpsL gene. Since the null
allele is missing a significant portion of the rpsL gene, its PCR
product is significantly shorter than that of the wild type gene
and the two are readily distinguished by gel electrophoretic
analysis. The PCR products may also be subjected to sequence
determination for further confirmation by methods well known to
those in the art.
[0184] The above experiment is generally adequate for determining
the essentiality of a gene such as rpsL. However, it may be
necessary or desirable to more directly confirm the essentiality of
the gene. There are several methods by which this can be
accomplished. In general, these involve three steps: 1)
construction of an episome containing a wild type allele, 2)
isolation of clones containing a single chromosomal copy of the
mutant null allele as described above but in the presence of the
episomal wild type allele, and then 3) determining if the cells
survive when the expression of the episomal allele is shut off. In
this case, the trans copy of wild type rpsL is made by PCR cloning
of the entire coding region of rpsL and inserting it in the sense
orientation downstream of an inducible promoter such as the E. coli
lac promoter. Transcription of this allele of rpsL will be induced
in the presence of IPTG which inactivates the lac repressor. Under
IPTG induction rpsL protein will be expressed as long as the
recombinant gene also possesses a ribosomal binding site, also
known as a "Shine-Dalgarno Sequence". The trans copy of rpsL is
cloned on a plasmid that is compatible with pSC101. Compatible
vectors include p15A, pBR322, and the pUC plasmids, among others.
Replication of the compatible plasmid will not be
temperature-sensitive. The entire process of integrating the null
allele of rpsL and subsequent plasmid excision is carried out in
the presence of IPTG to ensure the expression of functional rpsL
protein is maintained throughout. After the null rpsL allele is
confirmed as integrated on the chromosome in place of the wild type
rpsL allele, then IPTG is withdrawn and expression of functional
rpsL protein shut off. If the rpsL gene is essential, cells will
cease to proliferate under these conditions. However, if the rpsL
gene is not essential, cells will continue to proliferate under
these conditions. In this experiment, essentiality is determined by
conditional expression of a wild type copy of the gene rather than
inability to obtain the intended chromosomal disruption.
[0185] An advantage of this method over some other gene disruption
techniques is that the targeted gene can be deleted or mutated
without the introduction of large segments of foreign DNA.
Therefore, polar effects on downstream genes are eliminated or
minimized. There are methods described to introduce inducible
promoters upstream of potential essential bacterial genes. However
in such cases, polarity from multiple transcription start points
can be a problem. One way of preventing this is to insert a gene
disruption cassette that contains strong transcriptional
terminators upstream of the integrated inducible promoter (Zhang,
Y, and Cronan, J. E. 1996 J. Bacteriol. 178 (12): 3614-3620). The
described techniques will all be familiar to one of ordinary skill
in the art.
[0186] Following the analysis of the rpsL gene, the other genes of
the operon are investigated to determine if they are required for
proliferation.
EXAMPLE 6
Expression of the Proteins Encoded by Genes Identified as Required
for E. coli Proliferation
[0187] The following is provided as one exemplary method to express
the proliferation-required proteins encoded by the identified
sequences described above. First, the initiation and termination
codons for the gene are identified. If desired, methods for
improving translation or expression of the protein are well known
in the art. For example, if the nucleic acid encoding the
polypeptide to be expressed lacks a methionine codon to serve as
the initiation site, a strong Shine-Delgarno sequence, or a stop
codon, these sequences can be added. Similarly, if the identified
nucleic acid sequence lacks a transcription termination signal,
this sequence can be added to the construct by, for example,
splicing out such a sequence from an appropriate donor sequence. In
addition, the coding sequence may be operably linked to a strong
promoter or an inducible promoter if desired. The identified
nucleic acid sequence or portion thereof encoding the polypeptide
to be expressed is obtained by PCR from the bacterial expression
vector or genome using oligonucleotide primers complementary to the
identified nucleic acid sequence or portion thereof and containing
restriction endonuclease sequences for NcoI incorporated into the
5' primer and BglII at the 5' end of the corresponding 3'-primer,
taking care to ensure that the identified nucleic acid sequence is
positioned in frame with the termination signal. The purified
fragment obtained from the resulting PCR reaction is digested with
NcoI and BglII, purified and ligated to an expression vector.
[0188] The ligated product is transformed into DH5.alpha. or some
other E. coli strain suitable for the over expression of potential
proteins. Transformation protocols are well known in the art. For
example, transformation protocols are described in: Current
Protocols in Molecular Biology, Vol. 1, Unit 1.8, (Ausubel, et al.,
Eds.) John Wiley & Sons, Inc. (1997). Positive transformants
are selected after growing the transformed cells on plates
containing 50-100 .mu.g/ml Ampicillin (Sigma, St. Louis, Mo.). In
one embodiment, the expressed protein is held in the cytoplasm of
the host organism. In an alternate embodiment, the expressed
protein is released into the culture medium. In still another
alternative, the expressed protein can be sequestered in the
periplasmic space and liberated therefrom using any one of a number
of cell lysis techniques known in the art. For example, the osmotic
shock cell lysis method described in Chapter 16 of Current
Protocols in Molecular Biology, Vol. 2, (Ausubel, et al., Eds.)
John Wiley & Sons, Inc. (1997). Each of these procedures can be
used to express a proliferation-required protein.
[0189] Expressed proteins, whether in the culture medium or
liberated from the periplasmic space or the cytoplasm, are then
purified or enriched from the supernatant using conventional
techniques such as ammonium sulfate precipitation, standard
chromatography, immunoprecipitation, immunochromatography, size
exclusion chromatography, ion exchange chromatography, and HPLC.
Alternatively, the secreted protein can be in a sufficiently
enriched or pure state in the supernatant or growth media of the
host to permit it to be used for its intended purpose without
further enrichment. The purity of the protein product obtained can
be assessed using techniques such as Coomassie or silver staining
or using antibodies against the control protein. Coomassie and
silver staining techniques are familiar to those skilled in the
art.
[0190] Antibodies capable of specifically recognizing the protein
of interest can be generated using synthetic peptides using methods
well known in the art. See, Antibodies: A Laboratory Manual,
(Harlow and Lane, Eds.) Cold Spring Harbor Laboratory (1988). For
example, 15-mer peptides having a sequence encoded by the
appropriate identified gene sequence of interest or portion thereof
can be chemically synthesized. The synthetic peptides are injected
into mice to generate antibodies to the polypeptide encoded by the
identified nucleic acid sequence of interest or portion thereof.
Alternatively, samples of the protein expressed from the expression
vectors discussed above can be purified and subjected to amino acid
sequencing analysis to confirm the identity of the recombinantly
expressed protein and subsequently used to raise antibodies. An
Example describing in detail the generation of monoclonal and
polyclonal antibodies appears in Example 7.
[0191] The protein encoded by the identified nucleic acid sequence
of interest or portion thereof can be purified using standard
immunochromatography techniques. In such procedures, a solution
containing the secreted protein, such as the culture medium or a
cell extract, is applied to a column having antibodies against the
secreted protein attached to the chromatography matrix. The
secreted protein is allowed to bind the immunochromatography
column. Thereafter, the column is washed to remove non-specifically
bound proteins. The specifically bound secreted protein is then
released from the column and recovered using standard techniques.
These procedures are well known in the art.
[0192] In an alternative protein purification scheme, the
identified nucleic acid sequence of interest or portion thereof can
be incorporated into expression vectors designed for use in
purification schemes employing chimeric polypeptides. In such
strategies the coding sequence of the identified nucleic acid
sequence of interest or portion thereof is inserted in-frame with
the gene encoding the other half of the chimera. The other half of
the chimera can be maltose binding protein (MBP) or a nickel
binding polypeptide encoding sequence. A chromatography matrix
having antibody to MBP or nickel attached thereto is then used to
purify the chimeric protein. Protease cleavage sites can be
engineered between the MBP gene or the nickel binding polypeptide
and the identified expected gene of interest, or portion thereof.
Thus, the two polypeptides of the chimera can be separated from one
another by protease digestion.
[0193] One useful expression vector for generating maltose binding
protein fusion proteins is pMAL (New England Biolabs), which
encodes the malE gene. In the pMal protein fusion system, the
cloned gene is inserted into a pMal vector downstream from the malE
gene. This results in the expression of an MBP-fusion protein. The
fusion protein is purified by affinity chromatography. These
techniques as described are well known to those skilled in the art
of molecular biology.
EXAMPLE 7
Production of an Antibody to an Isolated E. coli Protein
[0194] Substantially pure protein or polypeptide is isolated from
the transformed cells as described in Example 6. The concentration
of protein in the final preparation is adjusted, for example, by
concentration on a 10,000 molecular weight cut off AMICON filter
device (Millipore, Bedford, Mass.), to the level of a few
micrograms/ml. Monoclonal or polyclonal antibody to the protein can
then be prepared as follows:
[0195] Monoclonal Antibody Production by Hybridoma Fusion
[0196] Monoclonal antibody to epitopes of any of the peptides
identified and isolated as described can be prepared from murine
hybridomas according to the classical method of Kohler, G. and
Milstein, C., Nature 256:495 (1975) or any of the well-known
derivative methods thereof. Briefly, a mouse is repetitively
inoculated with a few micrograms of the selected protein or
peptides derived therefrom over a period of a few weeks. The mouse
is then sacrificed, and the antibody producing cells of the spleen
isolated. The spleen cells are fused by means of polyethylene
glycol with mouse myeloma cells, and the excess unfused cells
destroyed by growth of the system on selective media comprising
aminopterin (HAT media). The successfully fused cells are diluted
and aliquots of the dilution placed in wells of a microtiter plate
where growth of the culture is continued. Antibody-producing clones
are identified by detection of antibody in the supernatant fluid of
the wells by immunoassay procedures, such as ELISA, as described by
Engvall, E., "Enzyme immunoassay ELISA and EMIT," Meth. Enzymol.
70:419 (1980), and derivative methods thereof. Selected positive
clones can be expanded and their monoclonal antibody product
harvested for use. Detailed procedures for monoclonal antibody
production are described in Davis, L. et al. Basic Methods in
Molecular Biology Elsevier, New York. Section 21-2.
[0197] Polyclonal Antibody Production by Immunization
[0198] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein or a peptide can be prepared by
immunizing suitable animals with the expressed protein or peptides
derived therefrom described above, which can be unmodified or
modified to enhance immunogenicity. Effective polyclonal antibody
production is affected by many factors related both to the antigen
and the host species. For example, small molecules tend to be less
immunogenic than larger molecules and can require the use of
carriers and adjuvant. Also, host animals vary in response to site
of inoculations and dose, with both inadequate or excessive doses
of antigen resulting in low titer antisera. Small doses (ng level)
of antigen administered at multiple intradermal sites appears to be
most reliable. An effective immunization protocol for rabbits can
be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab.
33:988-991 (1971).
[0199] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of
Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher, D., Chap. 42 in: Manual of
Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc.
For Microbiol., Washington, D.C. (1980).
[0200] Antibody preparations prepared according to either protocol
are useful in quantitative immunoassays which determine
concentrations of antigen-bearing substances in biological samples;
they are also used semi-quantitatively or qualitatively to identify
the presence of antigen in a biological sample. The antibodies can
also be used in therapeutic compositions for killing bacterial
cells expressing the protein.
EXAMPLE 8
Screening Chemical Libraries
[0201] A. Protein-Based Assays
[0202] Having isolated and expressed bacterial proteins shown to be
required for bacterial proliferation, the present invention further
contemplates the use of these expressed proteins in assays to
screen libraries of compounds for potential drug candidates. The
generation of chemical libraries is well known in the art. For
example combinatorial chemistry can be used to generate a library
of compounds to be screened in the assays described herein. A
combinatorial chemical library is a collection of diverse chemical
compounds generated by either chemical synthesis or biological
synthesis by combining a number of chemical "building blocks"
reagents. For example, a linear combinatorial chemical library such
as a polypeptide library is formed by combining amino acids in
every possible combination to yield peptides of a given length.
Millions of chemical compounds theoretically can be synthesized
through such combinatorial mixings of chemical building blocks. For
example, one commentator observed that the systematic,
combinatorial mixing of 100 interchangeable chemical building
blocks results in the theoretical synthesis of 100 million
tetrameric compounds or 10 billion pentameric compounds. (Gallop et
al., "Applications of Combinatorial Technologies to Drug Discovery,
Background and Peptide Combinatorial Libraries," Journal of
Medicinal Chemistry, Vol. 37, No. 9, 1233-1250 (1994). Other
chemical libraries known to those in the art may also be used,
including natural product libraries.
[0203] Once generated, combinatorial libraries can be screened for
compounds that possess desirable biological properties. For
example, compounds which may be useful as drugs or to develop drugs
would likely have the ability to bind to the target protein
identified, expressed and purified as discussed above. Further, if
the identified target protein is an enzyme, candidate compounds
would likely interfere with the enzymatic properties of the target
protein. Any enzyme can be a target protein. For example, the
enzymatic function of a target protein can be to serve as a
protease, nuclease, phosphatase, dehydrogenase, transporter
protein, transcriptional enzyme, and any other type of enzyme known
or unknown. Thus, the present invention contemplates using the
protein products described above to screen combinatorial chemical
libraries.
[0204] Those in the art will appreciate that a number of techniques
exist for characterizing target proteins in order to identify
molecules useful for the discovery and development of therapeutics.
For example, some techniques involve the generation and use of
small peptides to probe and analyze target proteins both
biochemically and genetically in order to identify and develop drug
leads. Such techniques include the methods described in PCT
publications No. WO9935494, WO9819162, WO9954728, the disclosures
of which are incorporated herein by reference in their
entireties.
[0205] In another example, the target protein is a serine protease
and the substrate of the enzyme is known. The present example is
directed towards the analysis of libraries of compounds to identify
compounds that function as inhibitors of the target enzyme. First,
a library of small molecules is generated using methods of
combinatorial library formation well known in the art. U.S. Pat.
Nos. 5,463,564 and 5,574,656, to Agrafiotis, et al., entitled
"System and Method of Automatically Generating Chemical Compound
with Desired Properties," are two such teachings. Then the library
compounds are screened to identify library compounds that possess
desired structural and functional properties. U.S. Pat. No.
5,684,711 also discusses a method for screening libraries.
[0206] To illustrate the screening process, the combined target and
chemical compounds of the library are exposed to and permitted to
interact with the purified enzyme. A labeled substrate is added to
the incubation. The label on the substrate is such that a
detectable signal is emitted from metabolized substrate molecules.
The emission of this signal permits one to measure the effect of
the combinatorial library compounds on the enzymatic activity of
target enzymes. The characteristics of each library compound is
encoded so that compounds demonstrating activity against the enzyme
can be analyzed and features common to the various compounds
identified can be isolated and combined into future iterations of
libraries.
[0207] Once a library of compounds is screened, subsequent
libraries are generated using those chemical building blocks that
possess the features shown in the first round of screen to have
activity against the target enzyme. Using this method, subsequent
iterations of candidate compounds will possess more and more of
those structural and functional features required to inhibit the
function of the target enzyme, until a group of enzyme inhibitors
with high specificity for the enzyme can be found. These compounds
can then be further tested for their safety and efficacy as
antibiotics for use in mammals.
[0208] It will be readily appreciated that this particular
screening methodology is exemplary only. Other methods are well
known to those skilled in the art. For example, a wide variety of
screening techniques are known for a large number of
naturally-occurring targets when the biochemical function of the
target protein is known.
[0209] B. Cell Based Assays
[0210] Current cell-based assays used to identify or to
characterize compounds for drug discovery and development
frequently depend on detecting the ability of a test compound to
inhibit the activity of a target molecule located within a cell or
located on the surface of a cell. Most often such target molecules
are proteins such as enzymes, receptors and the like. However,
target molecules may also include other molecules such as DNAs,
lipids, carbohydrates and RNAs including messenger RNAs, ribosomal
RNAs, tRNAs and the like. A number of highly sensitive cell-based
assay methods are available to those of skill in the art to detect
binding and interaction of test compounds with specific target
molecules. However, these methods are generally not highly
effective when the test compound binds to or otherwise interacts
with its target molecule with moderate or low affinity. In
addition, the target molecule may not be readily accessible to a
test compound in solution, such as when the target molecule is
located inside the cell or within a cellular compartment such as
the periplasm of a bacterial cell. Thus, current cell-based assay
methods are limited in that they are not effective in identifying
or characterizing compounds that interact with their targets with
moderate to low affinity or compounds that interact with targets
that are not readily accessible.
[0211] Cell-based assay methods of the present invention have
substantial advantages over current cell-based assays practiced in
the art. These advantages derive from the use of sensitized cells
in which the level or activity of a proliferation-required gene
product (the target molecule) has been specifically reduced to the
point where the presence or absence of its function becomes a
rate-determining step for cellular proliferation. Bacterial,
fungal, plant, or animal cells can all be used with the present
method. Such sensitized cells become much more sensitive to
compounds that are active against the affected target molecule.
Thus, cell-based assays of the present invention are capable of
detecting compounds exhibiting low or moderate potency against the
target molecule of interest because such compounds are
substantially more potent on sensitized cells than on
non-sensitized cells. The affect may be such that a test compound
may be two to several times more potent, at least 10 times more
potent or even at least 100 times more potent when tested on the
sensitized cells as compared to the non-sensitized cells.
[0212] Due in part to the increased appearance of antibiotic
resistance in pathogenic microorganisms and to the significant
side-effects associated with some currently used antibiotics, novel
antibiotics acting at new targets are highly sought after in the
art. Yet, another limitation in the current art related to
cell-based assays is the problem of identifying hits against the
same kinds of target molecules in the same limited set of
biological pathways over and over again. This may occur when
compounds acting at such new targets are discarded, ignored or fail
to be detected because compounds acting at the "old" targets are
encountered more frequently and are more potent than compounds
acting at the new targets. As a result, the majority of antibiotics
in use currently interact with a relatively small number of target
molecules within an even more limited set of biological
pathways.
[0213] The use of sensitized cells of the current invention
provides a solution to the above problem in two ways. First,
desired compounds acting at a target of interest, whether a new
target or a previously known but poorly exploited target, can now
be detected above the "noise" of compounds acting at the "old"
targets due to the specific and substantial increase in potency of
such desired compounds when tested on the sensitized cells of the
current invention. Second, the methods used to sensitize cells to
compounds acting at a target of interest may also sensitize these
cells to compounds acting at other target molecules within the same
biological pathway. For example, expression of an antisense
molecule to a gene encoding a ribosomal protein is expected to
sensitize the cell to compounds acting at that ribosomal protein
and may also sensitize the cells to compounds acting at any of the
ribosomal components (proteins or rRNA) or even to compounds acting
at any target which is part of the protein synthesis pathway. Thus
an important advantage of the present invention is the ability to
reveal new targets and pathways that were previously not readily
accessible to drug discovery methods.
[0214] Sensitized cells of the present invention are prepared by
reducing the activity or level of a target molecule. The target
molecule may be a gene product, such as an RNA or polypeptide
produced from the proliferation-required nucleic acids described
herein. Alternatively, the target may be a gene product such as an
RNA or polypeptide which is produced form a sequence within the
same operon as the proliferation-required nucleic acids described
herein. In addition, the target may be an RNA or polypeptide in the
same biological pathway as the proliferation-required nucleic acids
described herein. Such biological pathways include, but are not
limited to, enzymatic, biochemical and metabolic pathways as well
as pathways involved in the production of cellular structures such
the cell wall.
[0215] Current methods employed in the arts of medicinal and
combinatorial chemistries are able to make use of
structure-activity relationship information derived from testing
compounds in various biological assays including direct binding
assays and cell-based assays. Occasionally compounds are directly
identified in such assays that are sufficiently potent to be
developed as drugs. More often, initial hit compounds exhibit
moderate or low potency. Once a hit compound is identified with low
or moderate potency, directed libraries of compounds are
synthesized and tested in order to identify more potent leads.
Generally these directed libraries are combinatorial chemical
libraries consisting of compounds with structures related to the
hit compound but containing systematic variations including
additions, subtractions and substitutions of various structural
features. When tested for activity against the target molecule,
structural features are identified that either alone or in
combination with other features enhance or reduce activity. This
information is used to design subsequent directed libraries
containing compounds with enhanced activity against the target
molecule. After one or several iterations of this process,
compounds with substantially increased activity against the target
molecule are identified and may be further developed as drugs. This
process is facilitated by use of the sensitized cells of the
present invention since compounds acting at the selected targets
exhibit increased potency in such cell-based assays, thus; more
compounds can now be characterized providing more useful
information than would be obtained otherwise.
[0216] Thus, it is now possible using cell-based assays of the
present invention to identify or characterize compounds that
previously would not have been readily identified or characterized
including compounds that act at targets that previously were not
readily exploited using cell-based assays. The process of evolving
potent drug leads from initial hit compounds is also substantially
improved by the cell-based assays of the present invention because,
for the same number of test compounds, more structure-function
relationship information is likely to be revealed.
[0217] The method of sensitizing a cell entails selecting a
suitable gene or operon. A suitable gene or operon is one whose
expression is required for the proliferation of the cell to be
sensitized. The next step is to introduce into the cells to be
sensitized, an antisense RNA capable of hybridizing to the suitable
gene or operon or to the RNA encoded by the suitable gene or
operon. Introduction of the antisense RNA can be in the form of an
expression vector in which antisense RNA is produced under the
control of an inducible promoter. The amount of antisense RNA
produced is limited by varying the inducer concentration to which
the cell is exposed and thereby varying the activity of the
promoter driving transcription of the antisense RNA. Thus, cells
are sensitized by exposing them to an inducer concentration that
results in a sub-lethal level of antisense RNA expression.
[0218] In one embodiment of the cell-based assays, the identified
exogenous E. coli nucleotide sequences of the present invention are
used to inhibit the production of a proliferation-required protein.
Expression vectors producing antisense RNA against identified genes
required for proliferation are used to limit the concentration of a
proliferation-required protein without severly inhibiting growth.
To achieve that goal, a growth inhibition dose curve of inducer is
calculated by plotting various doses of inducer against the
corresponding growth inhibition caused by the antisense expression.
From this curve, various percentages of antisense induced growth
inhibition, from 1 to 100% can be determined. If the promoter
contained in the expression vector contains a lac operator the
transcription is regulated by lac repressor and expression from the
promoer is inducible with IPTG. For example, the highest
concentration of the inducer IPTG that does not reduce the growth
rate (0% growth inhibition) can be predicted from the curve.
Cellular proliferation can be monitored by growth medium turbidity
via OD measurements. In another example, the concentration of
inducer that reduces growth by 25% can be predicted from the curve.
In still another example, a concentration of inducer that reduces
growth by 50% can be calculated. Additional parameters such as
colony forming units (cfu) can be used to measure cellular
viability.
[0219] Cells to be assayed are exposed to the above-determined
concentrations of inducer. The presence of the inducer at this
sub-lethal concentration reduces the amount of the proliferation
required gene product to the lowest amount in the cell that will
support growth. Cells grown in the presence of this concentration
of inducer are therefore specifically more sensitive to inhibitors
of the proliferation-required protein or RNA of interest or to
inhibitors of proteins or RNAs in the same biological pathway as
the proliferation-required protein or RNA of interest but not to
inhibitors of unrelated proteins or RNAs.
[0220] Cells pretreated with sub-inhibitory concentrations of
inducer and thus containing a reduced amount of
proliferation-required target gene product are then used to screen
for compounds that reduce cell growth. The sub-lethal concentration
of inducer may be any concentration consistent with the intended
use of the assay to identify candidate compounds to which the cells
are more sensitive. For example, the sub-lethal concentration of
the inducer may be such that growth inhibition is at least about
5%, at least about 8%, at least about 10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60% at least about 75%, or more. Cells which are
pre-sensitized using the preceding method are more sensitive to
inhibitors of the target protein because these cells contain less
target protein to inhibit than wild-type cells.
[0221] In another embodiment of the cell based assays of the
present invention, the level or activity of a proliferation
required gene product is reduced using a temperature sensitive . .
. mutation in the proliferation-required sequence and an antisense
nucleic acid against the proliferation-required sequence. Growing
the cells at an intermediate temperature between the permissive and
restrictive temperatures of the temperature sensitive mutant where
the mutation is in a proliferation-required gene produces cells
with reduced activity of the proliferation-required gene product.
The antisense RNA directed against the proliferation-required
sequence further reduces the activity of the proliferation required
gene product. Drugs that may not have been found using either the
temperature sensitive mutation or the antisense nucleic acid alone
may be identified by determining whether cells in which expression
of the antisense nucleic acid has been induced and which are grown
at a temperature between the permissive temperature and the
restrictive temperature are substantially more sensitive to a test
compound than cells in which expression of the antisense nucleic
acid has not been induced and which are grown at a permissive
temperature. Also drugs found previously from either the antisense
nucleic acid alone or the temperature sensitive mutation alone may
have a different sensitivity profile when used in cells combining
the two approaches, and that sensitivity profile may indicate a
more specific action of the drug in inhibiting one or more
activities of the gene product.
[0222] Temperature sensitive mutations may be located at different
sites within the gene and correspond to different domains of the
protein. For example, the dnaB gene of Escherichia coli encodes the
replication fork DNA helicase. DnaB has several domains, including
domains for oligomerization, ATP hydrolysis, DNA binding,
interaction with primase, interaction with DnaC, and interaction
with DnaA [(Biswas, E. E. and Biswas, S. B. 1999. Mechanism and
DnaB helicase of Escherichia coli: structural domains involved in
ATP hydrolysis, DNA binding, and oligomerization. Biochem.
38:10919-10928; Hiasa, H. and Marians, K. J. 1999. Initiation of
bidirectional replication at the chromosomal origin is directed by
the interaction between helicase and primase. J. Biol. Chem.
274:27244-27248; San Martin, C., Radermacher, M., Wolpensinger, B.,
Engel, A., Miles, C. S., Dixon, N. E., and Carazo, J. M. 1998.
Three-dimensional reconstructions from cryoelectron microscopy
images reveal an intimate complex between helicase DnaB and its
loading partner DnaC. Structure 6:501-9; Sutton, M. D., Carr, K.
M., Vicente, M., and Kaguni, J. M. 1998. Escherichia coli DnaA
protein. The N-terminal domain and loading of DnaB helicase at the
E. coli chromosomal. J. Biol. Chem. 273:34255-62.), the disclosures
of which are incorporated herein by reference in their entireties].
Temperature sensitive mutations in different domains of DnaB confer
different phenotypes at the restrictive temperature, which include
either an abrupt stop or slow stop in DNA replication with or
without DNA breakdown (Wechsler, J. A. and Gross, J. D. 1971.
Escherichia coli mutants temperature-sensitive for DNA synthesis.
Mol. Gen. Genetics 113:273-284, the disclosure of which is
incorporated herein by reference in its entirety) and termination
of growth or cell death. Combining the use of temperature sensitive
mutations in the dnaB gene that cause cell death at the restrictive
temperature with an antisense to the dnaB gene could lead to the
discovery of very specific and effective inhibitors of one or a
subset of activities exhibited by DnaB.
[0223] When screening for antimicrobial agents against a gene
product required for proliferation, growth inhibition of cells
containing a limiting amount of that proliferation-required gene
product can be assayed. Growth inhibition can be measured by
directly comparing the amount of growth, measured by the optical
density of the growth medium, between an experimental sample and a
control sample. Alternative methods for assaying cell proliferation
include measuring green fluorescent protein (GFP) reporter
construct emissions, various enzymatic activity assays, and other
methods well known in the art.
[0224] It will be appreciated that the above method may be
performed in solid phase, liquid phase or a combination of the two.
For example, cells grown on nutrient agar containing the inducer of
the antisense construct may be exposed to compounds spotted onto
the agar surface. A compound's effect may be judged from the
diameter of the resulting killing zone, the area around the
compound application point in which cells do not grow. Multiple
compounds may be transferred to agar plates and simultaneously
tested using automated and semi-automated equipment including but
not restricted to multi-channel pipettes (for example the Beckman
Multimek) and multi-channel spotters (for example the Genomic
Solutions Flexys). In this way multiple plates and thousands to
millions of compounds may be tested per day.
[0225] The compounds may also be tested entirely in liquid phase
using microtiter plates as described below. Liquid phase screening
may be performed in microtiter plates containing 96, 384, 1536 or
more wells per microtiter plate to screen multiple plates and
thousands to millions of compounds per day. Automated and
semi-automated equipment may be used for addition of reagents (for
example cells and compounds) and determination of cell density.
EXAMPLE 9
[0226] The effectiveness of the above cell based assay was
validated using constructs expressing antisense RNA to E. coli
genes rplL, rplJ, and rplW encoding ribosomal proteins L7/L12, L10
and L23 respectively. These proteins are part of the protein
synthesis apparatus of the cell and as such are required for
proliferation. These constructs were used to test the effect of
antisense expression on cell sensitivity to antibiotics known to
bind to the ribosome and thereby inhibit protein synthesis.
Constructs expressing antisense RNA to several other genes (elaD,
visC, yohH, and aptE/B), the products of which are not involved in
protein synthesis were used for comparison.
[0227] First expression vectors containing antisense constructs to
either rplW or to elaD were introduced into separate E. coli cell
populations. Vector introduction is a technique well known to those
of ordinary skill in the art. The expression vectors of this
example contain IPTG inducible promoters that drive the expression
of the antisense RNA in the presence of the inducer. However, those
skilled in the art will appreciate that other inducible promoters
may also be used. Suitable expression vectors are also well known
in the art. The E. coli antisense clones encoding ribosomal
proteins L7/L12, L10 and L23 were used to test the effect of
antisense expression on cell sensitivity to the antibiotics known
to bind to these proteins. First, expression vectors containing
antisense to either the genes encoding L7/L12 and L10 or L23 were
introduced into separate E. coli cell populations.
[0228] The cell populations were exposed to a range of IPTG
concentrations in liquid medium to obtain the growth inhibitory
dose curve for each clone (FIG. 1). First, seed cultures were grown
to a particular turbidity that is measured by the optical density
(OD) of the growth solution. The OD of the solution is directly
related to the number of bacterial cells contained therein.
Subsequently, sixteen 200 ul liquid medium cultures were grown in a
96 well microtiter plate at 37 C with a range of IPTG
concentrations in duplicate two-fold serial dilutions from 1600 uM
to 12.5 uM (final concentration). Additionally, control cells were
grown in duplicate without IPTG. These cultures were started from
equal amounts of cells derived from the same initial seed culture
of a clone of interest. The cells were grown for up to 15 hours and
the extent of growth was determined by measuring the optical
density of the cultures at 600 nm. When the control culture reached
mid-log phase the percent growth of the control for each of the
IPTG containing cultures was plotted against the log concentrations
of IPTG to produce a growth inhibitory dose response curve for the
IPTG. The concentration of IPTG that inhibits cell growth to 50%
(IC.sub.50) as compared to the 0 mM IPTG control (0% growth
inhibition) was then calculated from the curve. Under these
conditions, an amount of antisense RNA was produced that reduced
the expression levels of rplW and elaD to a degree such that growth
was inhibited by 50%.
[0229] Alternative methods of measuring growth are also
contemplated. Examples of these methods include measurements of
proteins, the expression of which is engineered into the cells
being tested and can readily be measured. Examples of such proteins
include green fluorescent protein (GFP) and various enzymes.
[0230] Cells were pretreated with the selected concentration of
IPTG and then used to test the sensitivity of cell populations to
tetracycline, erythromycin and other protein synthesis inhibitors.
An example of a tetracycline dose response curve is shown in FIGS.
2A and 2B for the rplW and elaD genes, respectively. Cells were
grown to log phase and then diluted into media alone or media
containing IPTG at concentrations which give 20% and 50% growth
inhibition as determined by IPTG dose response curves. After 2.5
hours, the cells were diluted to a final OD600 of 0.002 into 96
well plates containing (1) .+-. IPTG at the same concentrations
used for the 2.5 hour pre-incubation; and (2) serial two-fold
dilutions of tetracycline such that the final concentrations of
tetracycline range from 1 .mu.g/ml to 15.6 ng/ml and 0 .mu.g/ml.
The 96 well plates were incubated at 37.degree. C. and the OD600
was read by a plate reader every 5 minutes for up to 15 hours. For
each IPTG concentration and the no IPTG control, tetracycline dose
response curves were determined when the control (absence of
tetracycline) reached 0.1 OD600. To compare tetracycline
sensitivity with and without IPTG, tetracycline IC50s were
determined from the dose response curves (FIGS. 2A-B). Cells with
reduced levels of L23 (rplW) showed increased sensitivity to
tetracycline (FIG. 2A) as compared to cells with reduced levels of
elaD (FIG. 2B). FIG. 3 shows a summary bar chart in which the
ratios of tetracycline IC50s determined in the presence of IPTG
which gives 50% growth inhibition versus tetracycline IC50s
determined without IPTG (fold increase in tetracycline sensitivity)
were plotted. Cells with reduced levels of either L7/L12 (genes
rplL, rplJ) or L23 (rplW) showed increased sensitivity to
tetracycline (FIG. 3). Cells expressing antisense to genes not
known to be involved in protein synthesis (atpB/E, visC, elaD,
yohH) did not show the same increased sensitivity to tetracycline,
validating the specificity of this assay (FIG. 3).
[0231] In addition to the above, it has been observed in initial
experiments that clones expressing antisense RNA to genes involved
in protein synthesis (including genes encoding ribosomal proteins
L7/L12 & L10, L7/L12 alone, L22, and L18, as well as genes
encoding rRNA and Elongation Factor G) have increased sensitivity
to the macrolide, erythromycin, whereas clones expressing antisense
to the non-protein synthesis genes elaD, atpB/E and visC do not.
Furthermore, the clone expressing antisense to rplL and rplJ does
not show increased sensitivity to nalidixic acid and ofloxacin,
antibiotics which do not inhibit protein synthesis.
[0232] The results with the ribosomal protein genes rplL, rplJ, and
rplW as well as the initial results using various other antisense
clones and antibiotics show that limiting the concentration of an
antibiotic target makes cells more sensitive to the antimicrobial
agents that specifically interact with that protein. The results
also show that these cells are sensitized to antimicrobial agents
that inhibit the overall function in which the protein target is
involved but are not sensitized to antimicrobial agents that
inhibit other functions.
[0233] The cell based assay described above may also be used to
identify the biological pathway in which a proliferation-required
nucleic acid or its gene product lies. In such methods, cells
expressing a sub-lethal level of antisense to a target
proliferation-required nucleic acid and control cells in which
expression of the antisense has not been induced are contacted with
a panel of antibiotics known to act in various pathways. If the
antibiotic acts in the pathway in which the target
proliferation-required nucleic acid or its gene product lies, cells
in which expression of the antisense has been induced will be more
sensitive to the antibiotic than cells in which expression of the
antisense has not been induced.
[0234] As a control, the results of the assay may be confirmed by
contacting a panel of cells expressing antisense nucleic acids to
many different proliferation-required genes including the target
proliferation-required gene. If the antibiotic is acting
specifically, heightened sensitivity to the antibiotic will be
observed only in the cells expressing antisense to a target
proliferation-required gene (or cells expressing antisense to other
proliferation-required genes in the same pathway as the target
proliferation-required gene) but will not be observed generally in
all cells expressing antisense to proliferation-required genes.
[0235] Similarly, the above method may be used to determine the
pathway on which a test antibiotic acts. A panel of cells, each of
which expresses antisense to a proliferation-required nucleic acid
in a known pathway, is contacted with a compound for which it is
desired to determine the pathway on which it acts. The sensitivity
of the panel of cells to the test compound is determined in cells
in which expression of the antisense has been induced and in
control cells in which expression of the antisense has not been
induced. If the test antibiotic acts on the pathway on which an
antisense nucleic acid acts, cells in which expression of the
antisense has been induced will be more sensitive to the antibiotic
than cells in which expression of the antisense has not been
induced. In addition, control cells in which expression of
antisense to proliferation-required genes in other pathways has
been induced will not exhibit heightened sensitivity to the
antibiotic. In this way, the pathway on which the test antibiotic
acts may be determined.
[0236] The Example below provides one method for performing such
assays.
EXAMPLE 10
Identification of the Pathway in which a Proliferation-Required
Gene Lies or the Pathway on which an Antibiotic Acts
[0237] A. Preparation of Bacterial Stocks for Assay
[0238] To provide a consistent source of cells to screen, frozen
stocks of host bacteria containing the desired antisense construct
are prepared using standard microbiological techniques. For
example, a single clone of the organism can be isolated by
streaking out a sample of the original stock onto an agar plate
containing nutrients for cell growth and an antibiotic for which
the antisense construct contains a gene which confers resistance.
After overnight growth an isolated colony is picked from the plate
with a sterile needle and transferred to an appropriate liquid
growth media containing the antibiotic required for maintenance of
the plasmid. The cells are incubated at 30.degree. C. to 37.degree.
C. with vigorous shaking for 4 to 6 hours to yield a culture in
exponential growth. Sterile glycerol is added to 15% (volume to
volume) and 100 .mu.L to 500 .mu.L aliquots are distributed into
sterile cryotubes, snap frozen in liquid nitrogen, and stored at
-80.degree. C. for future assays.
[0239] B. Growth of Bacteria for Use in the Assay
[0240] A day prior to an assay, a stock vial is removed from the
freezer, rapidly thawed (37.degree. C. water bath) and a loop of
culture is streaked out on an agar plate containing nutrients for
cell growth and an antibiotic to which the antisense construct
confers resistance. After overnight growth at 37.degree. C., ten
randomly chosen, isolated colonies are transferred from the plate
(sterile inoculum loop) to a sterile tube containing 5 mL of LB
medium containing the antibiotic to which the antisense vector
confers resistance. After vigorous mixing to form a homogeneous
cell suspension, the optical density of the suspension is measured
at 600 nm (OD600) and if necessary an aliquot of the suspension is
diluted into a second tube of 5 mL, sterile, LB medium plus
antibiotic to achieve an OD600.ltoreq.0.02 absorbance units. The
culture is then incubated at 37.degree. C. for 1-2 hrs with shaking
until the OD600 reaches OD 0.2-0.3. At this point the cells are
ready to be used in the assay.
[0241] C. Selection of Media to be Used in Assay
[0242] Two fold dilution series of the inducer are generated in
culture media containing the appropriate antibiotic for maintenance
of the antisense construct. Several media are tested side by side
and three to four wells are used to evaluate the effects of the
inducer at each concentration in each media. For example, M9
minimal media, LB broth, TBD broth and Muller-Hinton media may be
tested with the inducer IPTG at the following concentrations, 50
.mu.M, 100 .mu.M, 200 .mu.M, 400 .mu.M, 600 .mu.M, 800 .mu.M and
1000 .mu.M. Equal volumes of test media-inducer and cells are added
to the wells of a 384 well microtiter plate and mixed. The cells
are prepared as described above and diluted 1:100 in the
appropriate media containing the test antibiotic immediately prior
to addition to the microtiter plate wells. For a control, cells are
also added to several wells of each media that do not contain
inducer, for example 0 .mu.M IPTG. Cell growth is monitored
continuously by incubation at 37.degree. C. in a microtiter plate
reader monitoring the OD600 of the wells over an 18-hour period.
The percent inhibition of growth produced by each concentration of
inducer is calculated by comparing the rates of logarithmic growth
against that exhibited by cells growing in media without inducer.
The medium yielding greatest sensitivity to inducer is selected for
use in the assays described below.
[0243] D. Measurement of Test Antibiotic Sensitivity in the Absence
of Antisense Construct Induction
[0244] Two-fold dilution series of antibiotics of known mechanism
of action are generated in the culture media selected for further
assay development that has been supplemented with the antibiotic
used to maintain the construct. A panel of test antibiotics known
to act on different pathways is tested side by side with three to
four wells being used to evaluate the effect of a test antibiotic
on cell growth at each concentration. Equal volumes of test
antibiotic and cells are added to the wells of a 384 well
microtiter plate and mixed. Cells are prepared as described above
using the media selected for assay development supplemented with
the antibiotic required to maintain the antisense construct and are
diluted 1:100 in identical media immediately prior to addition to
the microtiter plate wells. For a control, cells are also added to
several wells that contain the solvent used to dissolve the
antibiotics but no antibiotic. Cell growth is monitored
continuously by incubation at 37.degree. C. in a microtiter plate
reader monitoring the OD600 of the wells over an 18-hour period.
The percent inhibition of growth produced by each concentration of
antibiotic is calculated by comparing the rates of logarithmic
growth against that exhibited by cells growing in media without
antibiotic. A plot of percent inhibition against log[antibiotic
concentration] allows extrapolation of an IC.sub.50 value for each
antibiotic.
[0245] E. Measurement of Test Antibiotic Sensitivity in the
Presence of Antisense Construct Inducer
[0246] The culture media selected for use in the assay is
supplemented with inducer at concentrations shown to inhibit cell
growth by 50 and 80% as described above and the antibiotic used to
maintain the construct. Two fold dilution series of the panel of
test antibiotics used above are generated in each of these media.
Several antibiotics are tested side by side with three to four
wells being used to evaluate the effects of an antibiotic on cell
growth at each concentration, in each media. Equal volumes of test
antibiotic and cells are added to the wells of a 384 well
microtiter plate and mixed. Cells are prepared as described above
using the media selected for use in the assay supplemented with the
antibiotic required to maintain the antisense construct. The cells
are diluted 1:100 into two 50 mL aliquots of identical media
containing concentrations of inducer that have been shown to
inhibit cell growth by 50% and 80% respectively and incubated at
37.degree. C. with shaking for 2.5 hours. Immediately prior to
addition to the microtiter plate wells, the cultures are adjusted
to an appropriate OD.sub.600 (typically 0.002) by dilution into
warm (37.degree. C.) sterile media supplemented with identical
concentrations of the inducer and antibiotic used to maintain the
antisense construct. For a control, cells are also added to several
wells that contain solvent used to dissolve test antibiotics but
which contain no antibiotic. Cell growth is monitored continuously
by incubation at 37.degree. C. in a microtiter plate reader
monitoring the OD600 of the wells over an 18-hour period. The
percent inhibition of growth produced by each concentration of
antibiotic is calculated by comparing the rates of logarithmic
growth against that exhibited by cells growing in media without
antibiotic. A plot of percent inhibition against log[antibiotic
concentration] allows extrapolation of an IC.sub.50 value for each
antibiotic.
[0247] F. Determining the Specificity of the Test Antibiotics
[0248] A comparison of the IC.sub.50s generated by antibiotics of
known mechanism of action under antisense induced and non-induced
conditions allows the pathway in which a proliferation-required
nucleic acid lies to be identified. If cells expressing an
antisense nucleic acid against a proliferation-required gene are
selectively sensitive to an antibiotic acting via a particular
pathway, then the gene against which the antisense acts is involved
in the pathway in which the antibiotic acts.
[0249] G. Identification of Pathway in Which a Test Antibiotic
Acts
[0250] As discussed above, the cell based assay may also be used to
determine the pathway against which a test antibiotic acts. In such
an analysis, the pathways against which each member of a panel of
antisense nucleic acids acts are identified as described above. A
panel of cells, each containing an inducible antisense vector
against a gene in a known proliferation-required pathway, is
contacted with a test antibiotic for which it is desired to
determine the pathway on which it acts under inducing an
non-inducing conditions. If heightened sensitivity is observed in
induced cells expressing antisense against a gene in a particular
pathway but not in induced cells expressing antisense against genes
in other pathways, then the test antibiotic acts against the
pathway for which heightened sensitivity was observed.
[0251] One skilled in the art will appreciate that further
optimization of the assay conditions, such as the concentration of
inducer used to induce antisense expression and/or the growth
conditions used for the assay (for example incubation temperature
and media components) may further increase the selectivity and/or
magnitude of the antibiotic sensitization exhibited.
[0252] The following example confirms the effectiveness of the
methods described above.
EXAMPLE 11
Identification of the Pathway in Which a Proliferation-Required
Gene Lies
[0253] Antibiotics of various chemical classes and modes of action
were purchased from Sigma Chemicals (St. Louis, Mo.). Stock
solutions were prepared by dissolving each antibiotic in an
appropriate aqueous solution based on information provided by the
manufacturer. The final working solution of each antibiotic
contained no more than 0.2% (w/v) of any organic solvent. To
determine their potency against a bacterial strain engineered for
expression of an antisense against a proliferation-required 50S
ribosomal protein, each antibiotic was serially diluted two or
three fold in growth medium supplemented with the appropriate
antibiotic for maintenance of the anti-sense construct. At least
ten dilutions were prepared for each antibiotic. 25 .mu.L aliquots
of each dilution were transferred to discrete wells of a 384-well
microplate (the assay plate) using a multi-channel pipette.
Quadruplicate wells were used for each dilution of an antibiotic
under each treatment condition (plus and minus inducer). Each assay
plate contained twenty wells for cell growth controls (growth media
replacing antibiotic), ten wells for each treatment (plus and minus
inducer, in this example IPTG). Assay plates were usually divided
into the two treatments: half the plate containing induced cells
and an appropriate concentrations of inducer (in this example IPTG)
to maintain the state of induction, the other half containing
non-induced cells in the absence of IPTG.
[0254] Cells for the assay were prepared as follows. Bacterial
cells containing a construct, from which expression of antisense
nucleic acid against rplL and rplJ, which encode
proliferation-required 50S ribosomal subunit proteins, is inducible
in the presence of IPTG, were grown into exponential growth
(OD.sub.600 0.2 to 0.3) and then diluted 1:100 into fresh media
containing either 400 .mu.M or 0 .mu.M inducer (IPTG). These
cultures were incubated at 37.degree. C. for 2.5 hr. After a 2.5 hr
incubation, induced and non-induced cells were respectively diluted
into an assay medium at a final OD.sub.600 value of 0.0004. The
medium contained an appropriate concentration of the antibiotic for
the maintenance of the anti-sense construct. In addition, the
medium used to dilute induced cells was supplemented with 800 .mu.M
IPTG so that addition to the assay plate would result in a final
IPTG concentration of 400 .mu.M. Induced and non-induced cell
suspensions were dispensed (25 .mu.l/well) into the appropriate
wells of the assay plate as discussed previously. The plate was
then loaded into a plate reader, incubated at constant temperature,
and cell growth was monitored in each well by the measurement of
light scattering at 595 nm. Growth was monitored every 5 minutes
until the cell culture attained a stationary growth phase. For each
concentration of antibiotic, a percentage inhibition of growth was
calculated at the time point corresponding to mid-exponential
growth for the associated control wells (no antibiotic, plus or
minus IPTG). For each antibiotic and condition (plus or minus
IPTG), a plot of percent inhibition versus log of antibiotic
concentration was generated and the IC50 determined. A comparison
of the IC.sub.50 for each antibiotic in the presence and absence of
IPTG revealed whether induction of the antisense construct
sensitized the cell to the mechanism of action exhibited by the
antibiotic. Cells which exhibited a significant (standard
statistical analysis) numerical decrease in the IC.sub.50 value in
the presence of inducer were considered to have an increased
sensitivity to the test antibiotic.
[0255] The results are provided in the table below, which lists the
classes and names of the antibiotics used in the analysis, the
targets of the antibiotics, the IC50 in the absence of IPTG, the
IC50 in the presence of IPTG, the concentration units for the
IC50s, the fold increase in IC50 in the presence of IPTG, and
whether increased sensitivity was observed in the presence of
IPTG.
4TABLE IV Effect of Expression of Antisense RNA to rplL and rplJ on
Antibiotic Sensitivity Conc. Fold Increase Sensitivity ANTIBIOTIC
CLASS/Names TARGET IC50 (-IPTG) IC50 (+IPTG) Unit in Sensitivity
Increased? PROTEIN SYNTHESIS INHIBITOR ANTIBIOTICS AMINOGLYCOSIDES
Gentamicin 30S ribosome function 2715 19.19 ng/ml 141 Yes
Streptomycin 30S ribosome function 11280 161 ng/ml 70 Yes
Spectinomycin 305 ribosome function 18050 <156 ng/ml Yes
Tobramycin 30S ribosome function 3594 70.58 ng/ml 51 Yes MACROLIDES
Erythromycin 50S ribosome function 7467 187 ng/ml 40 Yes AROMATIC
POYKETIDES Tetracycline 305 ribosome function 199.7 1.83 ng/ml 109
Yes Minocycline 30S ribosome function 668.4 3.897 ng/ml 172 Yes
Doxycycline 30S ribosome function 413.1 27.81 ng/ml 15 Yes OTHER
PROTEIN SYNTHESIS INHIBITORS Fusidic acid Elongation Factor G
function 59990 641 ng/ml 94 Yes Chloramphenicol 30S ribosome
function 465.4 1.516 ng/ml 307 Yes Lincomycin 50S ribosome function
47150 324.2 ng/ml 145 Yes OTHER ANTIBIOTIC MECHANISMS B-LACTAMS
Cefoxitin Cell wall biosynthesis 2782 2484 ng/ml 1 No Cefotaxime
Cell wall biosynthesis 24.3 24.16 ng/ml 1 No DNA SYNTHESIS
INHIBITORS Nalidixic acid DNA Gyrase activity 6973 6025 ng/ml 1 No
Ofloxacin DNA Gyrase activity 49.61 45.89 ng/ml 1 No OTHER
Bacitracin Cell membrane function 4077 4677 ng/ml 1 No Trimethoprim
Dihydrofolate 128.9 181.97 ng/ml 1 No Reductase activity Vancomycin
Cell wall biosynthesis 145400 72550 ng/ml 2 No
[0256] The above results demonstrate that induction of an antisense
RNA to genes encoding 50S ribosomal subunit proteins results in a
selective and highly significant sensitization of cells to
antibiotics that inhibit ribosomal function and protein synthesis.
The above results further demonstrate that induction of an
antisense construct to an essential gene sensitizes an organism to
compounds that interfere with that gene products' biological role.
This sensitization is restricted to compounds that interfere with
pathways associated with the targeted gene and it's product.
[0257] Assays utilizing antisense constructs to essential genes can
be used to identify compounds that specifically interfere with the
activity of multiple targets in a pathway. Such constructs can be
used to simultaneously screen a sample against multiple targets in
one pathway in one reaction (Combinatorial HTS).
[0258] Furthermore, as discussed above, panels of antisense
construct containing cells may be used to characterize the point of
intervention of any compound affecting an essential biological
pathway including antibiotics with no known mechanism of
action.
[0259] Another embodiment of the present invention is a method for
determining the pathway against which a test antibiotic compound is
active in which the activity of target proteins or nucleic acids
involved in proliferation-required pathways is reduced by
contacting cells with a sublethal concentration of a known
antibiotic which acts against the target protein or nucleic acid.
In one embodiment, the target protein or nucleic acid is a target
protein or nucleic acid corresponding to a proliferation-required
nucleic acid identified using the methods described above. The
method is similar to those described above for determining which
pathway a test antibiotic acts against except that rather than
reducing the activity or level of a proliferation-required gene
product using a sublethal level of antisense to a
proliferation-required nucleic acid, the activity or level of the
proliferation-required gene product is reduced using sublethal
level of a known antibiotic which acts against the proliferation
required gene product.
[0260] Interactions between drugs which affect the same biological
pathway has been described in the literature. For example,
Mecillinam (Amdinocillin) binds to and inactivates the penicillin
binding protein 2 (PBP2, product of the mrdA in E. coli). This
antibiotic inteacts with other antibiotics that inhibit PBP2 as
well as antibiotics that inhibit other penicillin binding proteins
such as PBP3 [(Gutmann, L., Vincent, S., Billot-Klein, D., Acar, J.
F., Mrena, E., and Williamson, R. (1986) Involvement of
penicillin-binding protein 2 with other penicillin-binding proteins
in lysis of Escherichia coli by some beta-lactam antibiotics alone
and in synergistic lytic effect of amdinocillin (mecillinam).
Antimicrobial Agents & Chemotherapy, 30:906-912), the
disclosure of which is incorporated herein by reference in its
entirety]. Interactions between drugs could, therefore, involve two
drugs that inhibit the same target protein or nucleic acid or
inhibit different proteins or nucleic acids in the same pathway
[(Fukuoka, T., Domon, H., Kakuta, M., Ishii, C., Hirasawa, A.,
Utsui, Y., Ohya, S., and Yasuda, H. (1997) Combination effect
between panipenem and vancomycin on highly methicillin-resistant
Staphylococcus aureus. Japan. J. Antibio. 50:411-419; Smith, C. E.,
Foleno, B. E., Barrett, J. F., and Frosc, M. B. (1997) Assessment
of the synergistic interactions of levofloxacin and ampicillin
against Enterococcus faecium by the checkerboard agar dilution and
time-kill methods. Diagnos. Microbiol. Infect. Disease 27:85-92;
den Hollander, J. G., Horrevorts, A. M., van Goor, M. L., Verbrugh,
H. A., and Mouton, J. W. (1997) Synergism between tobramycin and
ceftazidime against a resistant Pseudomonas aeruginosa strain,
tested in an in vitro pharmacokinetic model. Antimicrobial Agents
& Chemotherapy. 41:95-110), the disclosure of all of which are
incorporated herein by reference in their entireties].
[0261] Two drugs may interact even though they inhibit different
targets. For example, the proton pump inhibitor, Omeprazole, and
the antibiotic, Amoxycillin, two synergistic compounds acting
together, can cure Helicobacter pylori infection [(Gabryelewicz,
A., Laszewicz, W., Dzieniszewski, J., Ciok, J., Marlicz, K.,
Bielecki, D., Popiela, T., Legutko, J., Knapik, Z., Poniewierka, E.
(1997) Multicenter evaluation of dual-therapy (omeprazol and
amoxycillin) for Helicobacter pylori-associated duodenal and
gastric ulcer (two years of the observation). J. Physiol.
Pharmacol. 48 Suppl 4:93-105), the disclosure of which is
incorporated herein by reference in its entirety].
[0262] The growth inhibition from the sublethal concentration of
the known antibiotic may be at least about 5%, at least about 8%,
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, or at
least about 75%, or more.
[0263] Alternatively, the sublethal concentration of the known
antibiotic may be determined by measuring the activity of the
target proliferation-required gene product rather than by measuring
growth inhibition.
[0264] Cells are contacted with a combination of each member of a
panel of known antibiotics at a sublethal level and varying
concentrations of the test antibiotic. As a control, the cells are
contacted with varying concentrations of the test antibiotic alone.
The IC.sub.50 of the test antibiotic in the presence and absence of
the known antibiotic is determined. If the IC50s in the presence
and absence of the known drug are substantially similar, then the
test drug and the known drug act on different pathways. If the
IC.sub.50s are substantially different, then the test drug and the
known drug act on the same pathway.
[0265] Another embodiment of the present invention is a method for
identifying a candidate compound for use as an antibiotic in which
the activity of target proteins or nucleic acids involved in
proliferation-required pathways is reduced by contacting cells with
a sublethal concentration of a known antibiotic which acts against
the target protein or nucleic acid. In one embodiment, the target
protein or nucleic acid is a target protein or nucleic acid
corresponding to a proliferation-required nucleic acid identified
using the methods described above. The method is similar to those
described above for identifying candidate compounds for use as
antibiotics except that rather than reducing the activity or level
of a proliferation-required gene product using a sublethal level of
antisense to a proliferation-required nucleic acid, the activity or
level of the proliferation-required gene product is reduced using a
sublethal level of a known antibiotic which acts against the
proliferation required gene product.
[0266] The growth inhibition from the sublethal concentration of
the known antibiotic may be at least about 5%, at least about 8%,
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, or at
least about 75%, or more.
[0267] Alternatively, the sublethal concentration of the known
antibiotic may be determined by measuring the activity of the
target proliferation-required gene product rather than by measuring
growth inhibition.
[0268] In order to characterize test compounds of interest, cells
are contacted with a panel of known antibiotics at a sublethal
level and one or more concentrations of the test compound. As a
control, the cells are contacted with the same concentrations of
the test compound alone. The IC.sub.50 of the test compound in the
presence and absence of the known antibiotic is determined. If the
IC.sub.50 of the test compound is substantially different in the
presence and absence of the known drug then the test compound is a
good candidate for use as an antibiotic. As discussed above, once a
candidate compound is identified using the above methods its
structure may be optimized using standard techniques such as
combinatorial chemistry.
[0269] Representative known antibiotics which may be used in each
of the above methods are provided in the table below. However, it
will be appreciated that other antibiotics may also be used.
5 RESISTANT ANTIBIOTIC INHIBITS/TARGET MUTANTS Inhibitors of
Transcription Rifamycin, 1959 Rifampicin Inhibits initiation of
transcription/.beta.-subunit rpoB, crp, cyaA Rifabutin Rifaximin
RNA polymerase, rpoB Streptolydigin Accelerates transcription chain
rpoB termination/.beta.-subunit RNA polymerase Streptovaricin an
acyclic ansamycin, inhibits RNA rpoB polymerase Actinomycin D+EDTA
Intercalates between 2 successive G-C pldA pairs, rpoB, inhibits
RNA synthesis Inhibitors of Nucleic Acid Metabolism Quinolones,
1962 Nalidixic a subunit gyrase and/or topoisomerase IV, acid
Oxolinic acid gyrA gyrAorB, icd, sloB Fluoroquinolones a subunit
gyrase, gyrA and/or gyrA Ciprofloxacin, 1983 topoisomerase IV
(probable target in Staph) norA (efflux in Staph) Norfloxacin hipQ
Coumerins Novobiocin Inhibits ATPase activity of .beta.-subunit
gyrase, gyrB gyrB, cysB, cysE, nov, ompA Coumermycin Inhibits
ATPase activity of .beta.-subunit gyrB, hisW gyrase, gyrB Albicidin
DNA synthesis tsx (nucleoside channel) Metronidazole Causes
single-strand breaks in DNA nar Inhibitors of Metabolic Pathways
Sulfonamides, 1932 blocks synthesis of dihydrofolate,dihydro- folP,
gpt, pabA, pabB, Sulfanilamide pteroate synthesis,folP pabC
Trimethoprim, 1962 Inhibits dihydrofolate reductase,folA folA, thyA
Showdomycin Nucleoside analogue capable of alkylating nupC, pnp
sulfhydryl groups, inhibitor of thymidylate synthetase
Thiolactomycin type II fatty acid synthase inhibitor emrB fadB,
emrB due to gene dosage Psicofuramine Adenosine glycoside
antibiotic, target is guaA,B GMP synthetase Triclosan Inhibits
fatty acid synthesis fabl (envM) Diazoborines Isoniazid,
heterocyclic, contains boron, inhibit fatty fabl (envM) Ethionamide
acid synthesis, enoyl-ACP reductase,fabl Inhibitors of Translation
Phenylpropanoids Binds to ribosomal peptidyl transfer center
Chloramphenicol, 1947 preventing peptide translocation/binds to
rrn, cmlA, marA, ompF, S6, L3, L6, L14, L16, L25, L26, L27, but
ompR preferentially to L16 Tetracyclines, 1948, type II Binding to
30S ribosomal subunit, "A" site clnA (cmr), mar, ompF polyketides
on 30S subunit, blocks peptide elongation, Minocycline strongest
binding to S7 Doxycycline Macrolides (type I polyketides) Binding
to 50 S ribosomal subunit, 23S Erythromycin, 1950 rRNA, blocks
peptide translocation, L15, Carbomycin, Spiramycin L4, L12 rrn,
rplC, rplD, rpIV, etc mac Aminoglycosides Streptomycin,
Irreversible binding to 30S ribosomal 1944 subunit, prevents
translation or causes rpsL, strC,M ubiF Neomycin mistranslation of
mRNA/165 rRNA atpA-E, ecfB, hemAC,D,E,G, topA, Spectinomycin
rpsC,D,E, rrn, spcB atpA-atpE, cpxA, ecfB, Kanamycin hemA,B,L, topA
ksgA,B,C,D, rplB,K, Kasugamycin rpsLNJVLR rplF, ubiF Gentamicin,
1963 cpxA Amikacin rpsL Paromycin Lincosamides Binding to 50 S
ribosomal subunit, blocks Lincomycin, 1955 peptide translocation
linB, rpIN,O, rpsG Clindamycin Streptogramins Virginiamycin, 2
components, Streptogramins A&B, bind 1955 Pristinamycin to the
50S ribosomal subunit blocking Synercid: quinupristin peptide
translocation and peptide bond /dalfopristin formation Fusidanes
Inhibition of elongation factor G (EF-G) fusA Fusidic Acid prevents
peptide translocation Kirromycin (Mocimycin) Inhibition of
elongation factor TU (EF-Tu), tufA,B prevents peptide bond
formation Pulvomycin Binds to and inhibits EF-TU Thiopeptin
Sulfur-containing antibiotic, inhibits protein rplE synthesis,EF-G
Tiamuiin Inhibits protein synthesis rplC, rplD Negamycin Inhibits
termination process of protein prfB synthesis Oxazolidinones
Linezolid 23S rRNA Isoniazid pdx Nitrofurantoin Inhibits protein
synthesis, nitroreductases nfnA,B convert nitrofurantoin to highly
reactive electrophilic intermediates which attack bacterial
ribosomal proteins non- specifically Pseudomonic Acids Mupirocin
Inhibition of isoleucyl tRNA synthetase- ileS (Bactroban) used for
Staph, topical cream, nasal spray Indolmycin Inhibits
tryptophanyl-tRNA synthetase trpS Viomycin rrmA (235 rRNA
methyltransferase; mutant has slow growth rate, slow chain
elongation rate, and viomycin resistance) Thiopeptides Binds to
L11-23S RNA complex Thiostrepton Inhibits GTP hydrolysis by EF-G
Micrococcin Stimulates GTP hydrolysis by EF-G Inhibitors of Cell
Walls/Membranes .beta.-lactams Inhibition of one or more cell wall
Penicillin, 1929 Ampicillin transpeptidases, endopeptidases, and
Methicillin, 1960 glycosidases (PBPs), of the 12 PBPs only 2 amp C,
ampD, ampE, are essential: mrdA (PBP2) andftsl (pbpB, envZ, galU,
hipA, PBP3) hipQ, ompC, ompF, ompR, ptsl, rfa, tolD, tolE
Cephalosporins, 1962 tonB Binds to and inactivates PBP2 (mrdA)
alaS, argS, crp, cyaA, Mecillinam (amdinocillin) Inactivates PBP3
(fisl) envB, mrdA,B, Aztreonam (Furazlocillin) mreB, C,D Bacilysin,
Tetaine Dipeptide, inhib glucosamine synthase dppA Glycopeptides
Vancomycin, 1955 Inhib G+cell wall syn, binds to terminal
D-ala-D-ala of pentapeptide, Polypeptides Bacitracin Prevents
dephosphorylation and regeneration of lipid carrier rfa Cyclic
lipopeptide Daptomycin, Disrupts multiple aspects of membrane 1980
function, including peptidoglycan synthesis, lipoteichoic acid
synthesis, and the bacterial membrane potential Cyclic polypeptides
Polymixin, Surfactant action disrupts cell membrane pmrA 1939
lipids, binds lipid A mioety of LPS Fosfomycin, 1969 Analogue of
P-enolpyruvate, inhibits 1st murA, crp, cyaA gipT, step in
peptidoglycan synthesis - UDP-N- hipA, ptsl, uhpT acetylglucosamine
enolpyruvyl transferase, murA. Also acts as Immunosuppressant
Cycloserine Prevents formation of D-ala dimer, hipA, cycA inhibits
D-ala ligase, ddlA,B Alafosfalin phosphonodipeptide, cell wall
synthesis pepA, tpp inhibitor, potentiator of f3-lactams Inhibitors
of Protein Processing/Transport Globomycin Inhibits signal
peptidase II (cleaves lpp, dnaE prolipoproteins subsequent to lipid
modification, IspA
EXAMPLE 12
Transfer of Exogenous Nucleic Acid Sequences to Other Bacterial
Species Using the E. coli Expression Vectors or Expression Vectors
Functional in Bacterial Species Other Than E. coli
[0270] The above methods were validated using antisense nucleic
acids which inhibit the growth of E. coli which were identified
using methods similar to those described above. Expression vectors
which inhibited growth of E. coli upon induction of antisense RNA
expression with IPTG were transformed directly into Enterobacter
cloacae, Klebsiella pneumonia or Salmonella typhimurium. The
transformed cells were then assayed for growth inhibition according
to the method of Example 1. After growth in liquid culture, cells
were plated at various serial dilutions and a score determined by
calculating the log difference in growth for INDUCED vs. UNINDUCED
antisense RNA expression as determined by the maximum 10 fold
dilution at which a colony was observed. The results of these
experiments are listed below in Table VI. If there was no effect of
antisense RNA expression in an organism, the clone is minus in
Table VI. In contrast, a positive in Table VI means that at least
10 fold more cells were required to observe a colony on the induced
plate than on the non-induced plate under the conditions used and
in that organism.
[0271] Sixteen of the construts were found to inhibit growth in all
the organisms tested upon induction of antisense RNA expression
with IPTG. Those skilled in the art will appreciate that a negative
result in a heterologous organism does not mean that that organism
is missing that gene nor does it mean that the gene is unessential.
However, a positive result means that the heterologous organism
contains a homologous gene which is required for proliferation of
that organism. The homologous gene may be obtained using the
methods described herein. Those cells that are inhibited by
antisense may be used in cell based assays as described herein for
the identification and characterization of compounds in order to
develop antibiotics effective in these organisms. Those skilled in
the art will appreciate that an antisense molecule which works in
the organism from which it was obtained will not always work in a
heterologous organism.
6TABLE VI Sensitivity of Other Microorganisms to Antisense Nucleic
Acids That Inhibit Proliferation in E. coli Mol. No. S. typhimurium
E. cloacae K. pneumoniae EcXA001 + + - EcXA004 - - - EcXA005 + + +
EcXA006 - - - EcXA007 - + - EcXA008 + - + EcXA010 + + + EcXA011 - +
- EcXA012 - + - EcXA013 + + + EcXA014 + + - EcXA015 - + + EcXA016 +
+ + EcXA017 + + + EcXA018 + + + EcXA019 + + + EcXA020 + + + EcXA021
+ + + EcXA023 + + + EcXA024 + - + EcXA025 - - - EcXA026 + + -
EcXA027 + + + EcXA028 + - - EcXA029 - - - EcXA030 + + + EcXA031 + -
- EcXA032 + - - EcXA033 + + + EcXA034 + + + EcXA035 - - - EcXA036 +
- + EcXA037 - + - EcXA038 + + - EcXA039 + EcXA041 + + + EcXA042 - +
+ EcXA044 - - - EcXA045 - + - EcXA046 - - - EcXA047 + + - EcXA048 -
- - EcXA049 + - - EcXA050 - - - EcXA051 + - - EcXA052 + - - EcXA053
+ + + EcXA054 - - + EcXA055 + - -
EXAMPLE 13
Use of Identified Exogenous Nucleic Acid Sequences as Probes
[0272] The identified sequence of the present invention can be used
as probes to obtain the sequence of additional genes of interest
from a second organism. For example, probes to potential bacterial
target proteins may be hybridized to nucleic acids from other
organisms including other bacteria and higher organisms, to
identify homologous sequences. Such hybridization might indicate
that the protein encoded by the gene to which the probe corresponds
is found in humans and therefore not necessarily a good drug
target. Alternatively, the gene can be conserved only in bacteria
and therefore would be a good drug target for a broad spectrum
antibiotic or antimicrobial.
[0273] Probes derived from the identified nucleic acid sequences of
interest or portions thereof can be labeled with detectable labels
familiar to those skilled in the art, including radioisotopes and
non-radioactive labels, to provide a detectable probe. The
detectable probe can be single stranded or double stranded and can
be made using techniques known in the art, including in vitro
transcription, nick translation, or kinase reactions. A nucleic
acid sample containing a sequence capable of hybridizing to the
labeled probe is contacted with the labeled probe. If the nucleic
acid in the sample is double stranded, it can be denatured prior to
contacting the probe. In some applications, the nucleic acid sample
can be immobilized on a surface such as a nitrocellulose or nylon
membrane. The nucleic acid sample can comprise nucleic acids
obtained from a variety of sources, including genomic DNA, cDNA
libraries, RNA, or tissue samples.
[0274] Procedures used to detect the presence of nucleic acids
capable of hybridizing to the detectable probe include well known
techniques such as Southern blotting, Northern blotting, dot
blotting, colony hybridization, and plaque hybridization. In some
applications, the nucleic acid capable of hybridizing to the
labeled probe can be cloned into vectors such as expression
vectors, sequencing vectors, or in vitro transcription vectors to
facilitate the characterization and expression of the hybridizing
nucleic acids in the sample. For example, such techniques can be
used to isolate, purify and clone sequences from a genomic library,
made from a variety of bacterial species, which are capable of
hybridizing to probes made from the sequences identified in
Examples 5 and 6.
EXAMPLE 14
Preparation of PCR Primers and Amplification of DNA
[0275] The identified E. coli genes corresponding directly to or
located within the operon of nucleic acid sequences required for
proliferation or portions thereof can be used to prepare PCR
primers for a variety of applications, including the identification
or isolation of homologous sequences from other species, for
example S. typhimurium, E. cloacae, and Klebsiella pneumoniae,
which contain part or all of the homologous genes. Because
homologous genes are related but not identical in sequence, those
skilled in the art will often employ degenerate sequence PCR
primers. Such degenerate sequence primers are designed based on
conserved sequence regions, either known or suspected, such as
conserved coding regions. The successful production of a PCR
product using degenerate probes generated from the sequences
identified herein would indicate the presence of a homologous gene
sequence in the species being screened. The PCR primers are at
least 10 bases, and preferably at least 20 bases in length. More
preferably, the PCR primers are at least 20-30 bases in length. In
some embodiments, the PCR primers can be more than 30 bases in
length. It is preferred that the primer pairs have approximately
the same G/C ratio, so that melting temperatures are approximately
the same. A variety of PCR techniques are familiar to those skilled
in the art. For a review of PCR technology, see Molecular Cloning
to Genetic Engineering White, B. A. Ed. in Methods in Molecular
Biology 67: Humana Press, Totowa 1997. When the entire coding
sequence of the target gene is known, the 5' and 3' regions of the
target gene can be used as the sequence source for PCR probe
generation. In each of these PCR procedures, PCR primers on either
side of the nucleic acid sequences to be amplified are added to a
suitably prepared nucleic acid sample along with dNTPs and a
thermostable polymerase such as Taq polymerase, Pfu polymerase, or
Vent polymerase. The nucleic acid in the sample is denatured and
the PCR primers are specifically hybridized to complementary
nucleic acid sequences in the sample. The hybridized primers are
extended. Thereafter, another cycle of denaturation, hybridization,
and extension is initiated. The cycles are repeated multiple times
to produce an amplified fragment containing the nucleic acid
sequence between the primer sites.
EXAMPLE 15
Inverse PCR
[0276] The technique of inverse polymerase chain reaction can be
used to extend the known nucleic acid sequence identified in
Examples 5 and 6. The inverse PCR reaction is described generally
by Ochman et al., in Ch. 10 of PCR Technology: Principles and
Applications for DNA Amplification, (Henry A. Erlich, Ed.) W. H.
Freeman and Co. (1992). Traditional PCR requires two primers that
are used to prime the synthesis of complementary strands of DNA. In
inverse PCR, only a core sequence need be known.
[0277] Using the sequences identified as relevant from the
techniques taught in Examples 5 and 6 and applied to other species
of bacteria, a subset of exogenous nucleic sequences are identified
that correspond to genes or operons that are required for bacterial
proliferation. In species for which a genome sequence is not known,
the technique of inverse PCR provides a method for obtaining the
gene in order to determine the sequence or to place the probe
sequences in full context to the target sequence to which the
identified exogenous nucleic acid sequence binds.
[0278] To practice this technique, the genome of the target
organism is digested with an appropriate restriction enzyme so as
to create fragments of nucleic acid that contain the identified
sequence as well as unknown sequences that flank the identified
sequence. These fragments are then circularized and become the
template for the PCR reaction. PCR primers are designed in
accordance with the teachings of Example 15 and directed to the
ends of the identified sequence are synthesized. The primers direct
nucleic acid synthesis away from the known sequence and toward the
unknown sequence contained within the circularized template. After
the PCR reaction is complete, the resulting PCR products can be
sequenced so as to extend the sequence of the identified gene past
the core sequence of the identified exogenous nucleic acid sequence
identified. In this manner, the full sequence of each novel gene
can be identified. Additionally the sequences of adjacent coding
and noncoding regions can be identified.
EXAMPLE 16
Identification of Genes Required for Staphylococcus aureus
Proliferation
[0279] Genes required for proliferation in Staphylococcus aureus
are identified according to the methods described above.
EXAMPLE 17
Identification of Genes Required for Neisseria gonorrhoeae
Proliferation
[0280] Genes required for proliferation in Neisseria gonorrhoeae
are identified according to the methods described above.
EXAMPLE 18
Identification of Genes Required for Pseudomonas aeruginosa
Proliferation
[0281] Genes required for proliferation in Pseudomonas aeruginosa
are identified according to the methods described above.
EXAMPLE 19
Identification of Genes Required for Enterococcus faecalis
Proliferation
[0282] Genes required for proliferation in Enterococcus faecalis
are identified according to the methods described above.
EXAMPLE 20
Identification of Genes Required for Haemophilus influenzae
Proliferation
[0283] Genes required for proliferation in Haemophilus influenzae
are identified according to the methods described above.
EXAMPLE 21
Identification of Genes Required for Salmonella typhimurium
Proliferation
[0284] Genes required for proliferation in Salmonella typhimurium
are identified according to the methods described above.
EXAMPLE 22
Identification of Genes Required for Helicobacter pylori
Proliferation
[0285] Genes required for proliferation in Helicobacter pylori are
identified according to the methods described above.
EXAMPLE 23
Identification of Genes Required for Mycoplasma pneumoniae
Proliferation
[0286] Genes required for proliferation in Mycoplasma pneumoniae
are identified according to the methods described above.
EXAMPLE 24
Identification of Genes Required for Plasmodium ovale
Proliferation
[0287] Genes required for proliferation in Plasmodium ovale are
identified according to the methods described above.
EXAMPLE 25
Identification of Genes Required for Saccharomyces cerevisiae
Proliferation
[0288] Genes required for proliferation in Saccharomyces cerevisiae
are identified according to the methods described above.
EXAMPLE 26
Identification of Genes Required for Entamoeba histolytica
Proliferation
[0289] Genes required for proliferation in Entamoeba histolytica
are identified according to the methods described above.
EXAMPLE 27
Identification of Genes Required for Candida albicans
Proliferation
[0290] Genes required for proliferation in Candida albicans are
identified according to the methods described above.
EXAMPLE 28
Identification of Genes Required for Klebsiella pneumoniae
Proliferation
[0291] Genes required for proliferation in Klebsiella pneumoniae
are identified according to the methods described above.
EXAMPLE 29
Identification of Genes Required for Salmonella typhi
Proliferation
[0292] Genes required for proliferation in Salmonella typhi are
identified according to the methods described above.
EXAMPLE 30
Identification of Genes Required for Salmonella paratyphi
Proliferation
[0293] Genes required for proliferation in Salmonella paratyphi are
identified according to the methods described above.
EXAMPLE 31
Identification of Genes Required for Salmonella cholerasuis
Proliferation
[0294] Genes required for proliferation in Salmonella cholerasuis
are identified according to the methods described above.
EXAMPLE 32
Identification of Genes Required for Staphylococcus epidermis
Proliferation
[0295] Genes required for proliferation in Staphylococcus epidermis
are identified according to the methods described above.
EXAMPLE 33
Identification of Genes Required for Mycobacterium tuberculosis
Proliferation
[0296] Genes required for proliferation in Mycobacterium
tuberculosis are identified according to the methods described
above.
EXAMPLE 34
Identification of Genes Required for Mycobacterium leprae
Proliferation
[0297] Genes required for proliferation in Mycobacterium leprae are
identified according to the methods described above.
EXAMPLE 35
Identification of Genes Required for Treponema pallidum
Proliferation
[0298] Genes required for proliferation in Treponema pallidum are
identified according to the methods described above.
EXAMPLE 36
Identification of Genes Required for Bacillus anthracis
Proliferation
[0299] Genes required for proliferation in Bacillus anthracis are
identified according to the methods described above.
EXAMPLE 37
Identification of Genes Required for Yersinia pestis
Proliferation
[0300] Genes required for proliferation in Yersinia pestis are
identified according to the methods described above.
EXAMPLE 38
Identification of Genes Required for Clostridium botulinum
Proliferation
[0301] Genes required for proliferation in Clostridium botulinum
are identified according to the methods described above.
EXAMPLE 39
Identification of Genes Required for Campylobacter jejuni
Proliferation
[0302] Genes required for proliferation in Campylobacter jejuni are
identified according to the methods described above.
EXAMPLE 40
Identification of Genes Required for Chlamydia trachomatis
Proliferation
[0303] Genes required for proliferation in Chlamydia trachomatis
are identified according to the methods described above.
[0304] Use of Isolated Exogenous Nucleic Acid Fragments as
Antisense Antibiotics
[0305] In addition to using the identified sequences to enable
screening of molecule libraries to identify compounds useful to
identify antibiotics, the sequences themselves can be used as
therapeutic agents. Specifically, the identified exogenous
sequences in an antisense orientation can be provided to an
individual to inhibit the translation of a bacterial target
gene.
[0306] Generation of Antisense Therapeutics from Identified
Exogenous Sequences
[0307] The sequences of the present invention can be used as
antisense therapeutics for the treatment of bacterial infections or
simply for inhibition of bacterial growth in vitro or in vivo. The
therapy exploits the biological process in cells where genes are
transcribed into messenger RNA (mRNA) that is then translated into
proteins. Antisense RNA technology contemplates the use of
antisense oligonucleotides directed against a target gene that will
bind to its target and decrease or inhibit the translation of the
target mRNA. In one embodiment, antisense oligonucleotides can be
used to treat and control a bacterial infection of a cell culture
containing a population of desired cells contaminated with
bacteria. In another embodiment, the antisense oligonucleotides can
be used to treat an organism with a bacterial infection.
[0308] Antisense oligonucleotides can be synthesized from any of
the sequences of the present invention using methods well known in
the art. In a preferred embodiment, antisense oligonucleotides are
synthesized using artificial means. Uhlmann & Peymann, Chemical
Rev. 90:543-584 (1990) review antisense oligonucleotide technology
in detail. Modified or unmodified antisense oligonucleotides can be
used as therapeutic agents. Modified antisense oligonucleotides are
preferred since it is well known that antisense oligonucleotides
are extremely unstable. Modification of the phosphate backbones of
the antisense oligonucleotides can be achieved by substituting the
internucleotide phosphate residues with methylphosphonates,
phosphorothioates, phosphoramidates, and phosphate esters.
Nonphosphate internucleotide analogs such as siloxane bridges,
carbonate brides, thioester bridges, as well as many others known
in the art. The preparation of certain antisense oligonucleotides
with modified internucleotide linkages is described in U.S. Pat.
No. 5,142,047, hereby incorporated by reference.
[0309] Modifications to the nucleoside units of the antisense
oligonucleotides are also contemplated. These modifications can
increase the half-life and increase cellular rates of uptake for
the oligonucleotides in vivo. For example, .alpha.-anomeric
nucleotide units and modified bases such as
1,2-dideoxy-d-ribofuranose, 1,2-dideoxy-1-phenylribofuranose, and
N.sup.4, N.sup.4-ethano-5-methyl-cy- tosine are contemplated for
use in the present invention.
[0310] An additional form of modified antisense molecules is found
in peptide nucleic acids. Peptide nucleic acids (PNA) have been
developed to hybridize to single and double stranded nucleic acids.
PNA are nucleic acid analogs in which the entire
deoxyribose-phosphate backbone has been exchanged with a chemically
completely different, but structurally homologous, polyamide
(peptide) backbone containing 2-aminoethyl glycine units. Unlike
DNA, which is highly negatively charged, the PNA backbone is
neutral. Therefore, there is much less repulsive energy between
complementary strands in a PNA-DNA hybrid than in the comparable
DNA-DNA hybrid, and consequently they are much more stable. PNA can
hybridize to DNA in either a Watson/Crick or Hoogsteen fashion
(Demidov et al., Proc. Natl. Acad. Sci. U.S.A. 92:2637-2641, 1995;
Egholm, Nature 365:566-568, 1993; Nielsen et al., Science
254:1497-1500, 1991; Dueholm et al., New J. Chem. 21:19-31,
1997).
[0311] Molecules called PNA "clamps" have been synthesized which
have two identical PNA sequences joined by a flexible hairpin
linker containing three 8-amino-3,6-dioxaoctanoic acid units. When
a PNA clamp is mixed with a complementary homopurine or
homopyrimidine DNA target sequence, a PNA-DNA-PNA triplex hybrid
can form which has been shown to be extremely stable (Bentin et
al., Biochemistry 35:8863-8869, 1996; Egholm et al., Nucleic Acids
Res. 23:217-222, 1995; Griffith et al., J. Am. Chem. Soc.
117:831-832, 1995).
[0312] The sequence-specific and high affinity duplex and triplex
binding of PNA have been extensively described (Nielsen et al.,
Science 254:1497-1500, 1991; Egholm et al., J. Am. Chem. Soc.
114:9677-9678, 1992; Egholm et al., Nature 365:566-568, 1993;
Almarsson et al., Proc. Natl. Acad. Sci. U.S.A. 90:9542-9546, 1993;
Demidov et al., Proc. Natl. Acad. Sci. U.S.A. 92:2637-2641, 1995).
They have also been shown to be resistant to nuclease and protease
digestion (Demidov et al., Biochem. Pharm. 48:1010-1313, 1994). PNA
has been used to inhibit gene expression (Hanvey et al., Science
258:1481-1485, 1992; Nielsen et al., Nucl. Acids. Res., 21:197-200,
1993; Nielsen et al., Gene 149:139-145, 1994; Good & Nielsen,
Science, 95: 2073-2076, 1998; all of which are hereby incorporated
by reference), to block restriction enzyme activity (Nielsen et
al., supra., 1993), to act as an artificial transcription promoter
(Mollegaard, Proc. Natl. Acad. Sci. U.S.A. 91:3892-3895, 1994) and
as a pseudo restriction endonuclease (Demidov et al., Nucl. Acids.
Res. 21:2103-2107, 1993). Recently, PNA has also been shown to have
antiviral and antitumoral activity mediated through an antisense
mechanism (Norton, Nature Biotechnol., 14:615-619, 1996; Hirschman
et al., J. Investig. Med. 44:347-351, 1996). PNAs have been linked
to various peptides in order to promote PNA entry into cells (Basu
et al., Bioconj. Chem. 8:481-488, 1997; Pardridge et al., Proc.
Natl. Acad. Sci. U.S.A. 92:5592-5596, 1995).
[0313] The antisense oligonucleotides contemplated by the present
invention can be administered by direct application of
oligonucleotides to a target using standard techniques well known
in the art. The antisense oligonucleotides can be generated within
the target using a plasmid, or a phage. Alternatively, the
antisense nucleic acid may be expressed from a sequence in the
chromosome of the target cell. It is further contemplated that
contemplated that the antisense oligonucleotide contemplated are
incorporated in a ribozyme sequence to enable the antisense to
specifically bind and cleave its target mRNA. For technical
applications of ribozyme and antisense oligonucleotides see Rossi
et al., Pharmacol. Ther. 50(2):245-254, (1991), which is hereby
incorporated by reference. The present invention also contemplates
using a retron to introduce an antisense oligonucleotide to a cell.
Retron technology is exemplified by U.S. Pat. No. 5,405,775, which
is hereby incorporated by reference. Antisense oligonucleotides can
also be delivered using liposomes or by electroporation techniques
which are well known in the art.
[0314] The antisense nucleic acids of the present invention can
also be used to design antibiotic compounds comprising nucleic
acids which function by intracellular triple helix formation.
Triple helix oligonucleotides are used to inhibit transcription
from a genome. The sequences identified as required for
proliferation in the present invention, or portions thereof, can be
used as templates to inhibit microorganism gene expression in
individuals infected with such organisms. Traditionally, homopurine
sequences were considered the most useful for triple helix
strategies. However, homopyrimidine sequences can also inhibit gene
expression. Such homopyrimidine oligonucleotides bind to the major
groove at homopurine:homopyrimidine sequences. Thus, both types of
sequences based on the sequences of the present invention that are
required for proliferation are contemplated for use as antibiotic
compound templates.
[0315] The antisense oligonucleotides of this example employ the
identified sequences of the present invention to induce bacterial
cell death or at least bacterial stasis by inhibiting target gene
translation. Antisense oligonucleotides containing from about 8 to
40 bases of the sequences of the present invention have sufficient
complementary to form a duplex with the target sequence under
physiological conditions.
[0316] To kill bacterial cells or inhibit their growth, the
antisense oligonucleotides are applied to the bacteria or to the
target cells under conditions that facilitate their uptake. These
conditions include sufficient incubation times of cells and
oligonucleotides so that the antisense oligonucleotides are taken
up by the cells. In one embodiment, an incubation period of 7-10
days is sufficient to kill bacteria in a sample. An optimum
concentration of antisense oligonucleotides is selected for
use.
[0317] The concentration of antisense oligonucleotides to be used
can vary depending on the type of bacteria sought to be controlled,
the nature of the antisense oligonucleotide to be used, and the
relative toxicity of the antisense oligonucleotide to the desired
cells in the treated culture. Antisense oligonucleotides can be
introduced to cell samples at a number of different concentrations
preferably between 1.times.10.sup.-10M to 1.times.10.sup.-4M. Once
the minimum concentration that can adequately control gene
expression is identified, the optimized dose is translated into a
dosage suitable for use in vivo. For example, an inhibiting
concentration in culture of 1.times.10.sup.-7 translates into a
dose of approximately 0.6 mg/kg body weight. Levels of
oligonucleotide approaching 100 mg/kg body weight or higher may be
possible after testing the toxicity of the oligonucleotide in
laboratory animals. It is additionally contemplated that cells from
the subject are removed, treated with the antisense
oligonucleotide, and reintroduced into the subject. This range is
merely illustrative and one of skill in the art are able to
determine the optimal concentration to be used in a given case.
[0318] After the bacterial cells have been killed or controlled in
a desired culture, the desired cell population may be used for
other purposes.
EXAMPLE 41
[0319] The following example demonstrates the ability of an E. coli
antisense oligonucleotide to act as a bactericidal or
bacteriostatic agent to treat a contaminated cell culture system.
The application of the antisense oligonucleotides of the present
invention are thought to inhibit the translation of bacterial gene
products required for proliferation.
[0320] The antisense oligonucleotide of this example corresponds to
a 30 base phophorothioate modified oligodeoxynucelotide
complementary to a nucleic acid involved in proliferation, such as
Molecule Number EcXA001. A sense oligodeoxynucelotide complementary
to the antisense sequence is synthesized and used as a control. The
oligonucleotides are synthesized and purified according to the
procedures of Matsukura, et al., Gene 72:343 (1988). The test
oligonucleotides are dissolved in a small volume of autoclaved
water and added to culture medium to make a 100 micromolar stock
solution.
[0321] Human bone marrow cells are obtained from the peripheral
blood of two patients and cultured according standard procedures
well known in the art. The culture is contaminated with the K-12
strain of E. coli and incubated at 37.degree. C. overnight to
establish bacterial infection.
[0322] The control and antisense oligonucleotide containing
solutions are added to the contaminated cultures and monitored for
bacterial growth. After a 10 hour incubation of culture and
oligonucleotides, samples from the control and experimental
cultures are drawn and analyzed for the translation of the target
bacterial gene using standard microbiological techniques well known
in the art. The target E. coli gene is found to be translated in
the control culture treated with the control oligonucleotide,
however, translation of the target gene in the experimental culture
treated with the antisense oligonucleotide of the present invention
is not detected or reduced.
EXAMPLE 42
[0323] A subject suffering from an E. coli infection is treated
with the antisense oligonucleotide preparation of Example 39. The
antisense oligonucleotide is provided in a pharmaceutically
acceptable carrier at a concentration effective to inhibit the
translation of the target gene. The present subject is treated with
a concentration of antisense oligonucleotide sufficient to achieve
a blood concentration of about 100 micromolar. The patient receives
daily injections of antisense oligonucleotide to maintain this
concentration for a period of 1 week. At the end of the week a
blood sample is drawn and analyzed for the presence or absence
using standard techniques well known in the art. There is no
detectable evidence of E. coli and the treatment is terminated.
EXAMPLE 43
Preparation and Use of Triple Helix Probes
[0324] The sequences of microorganism genes required for
proliferation of the present invention are scanned to identify
10-mer to 20-mer homopyrimidine or homopurine stretches that could
be used in triple-helix based strategies for inhibiting gene
expression. Following identification of candidate homopyrimidine or
homopurine stretches, their efficiency in inhibiting gene
expression is assessed by introducing varying amounts of
oligonucleotides containing the candidate sequences into a
population of bacterial cells that normally express the target
gene. The oligonucleotides may be prepared on an oligonucleotide
synthesizer or they may be purchased commercially from a company
specializing in custom oligonucleotide synthesis, such as GENSET,
Paris, France.
[0325] The oligonucleotides can be introduced into the cells using
a variety of methods known to those skilled in the art, including
but not limited to calcium phosphate precipitation, DEAE-Dextran,
electroporation, liposome-mediated transfection or native
uptake.
[0326] Treated cells are monitored for a reduction in proliferation
using techniques such as monitoring growth levels as compared to
untreated cells using optical density measurements. The
oligonucleotides that are effective in inhibiting gene expression
in cultured cells can then be introduced in vivo using the
techniques well known in that art at a dosage level shown to be
effective.
[0327] In some embodiments, the natural (beta) anomers of the
oligonucleotide units can be replaced with alpha anomers to render
the oligonucleotide more resistant to nucleases. Further, an
intercalating agent such as ethidium bromide, or the like, can be
attached to the 3' end of the alpha oligonucleotide to stabilize
the triple helix. For information on the generation of
oligonucleotides suitable for triple helix formation see Griffin et
al. (Science 245:967-971 (1989), which is hereby incorporated by
this reference).
EXAMPLE 44
Identification of Bacterial Strains from Isolated Specimens by
PCR
[0328] Classical bacteriological methods for the detection of
various bacterial species are time consuming and costly. These
methods include growing the bacteria isolated from a subject in
specialized media, cultivation on selective agar media, followed by
a set of confirmation assays that can take from 8 to 10 days or
longer to complete. Use of the identified sequences of the present
invention provides a method to dramatically reduce the time
necessary to detect and identify specific bacterial species present
in a sample.
[0329] In one exemplary method, bacteria are grown in enriched
media and DNA samples are isolated from specimens of, for example,
blood, urine, stool, saliva or central nervous system fluid by
conventional methods. A panel of PCR primers based on identified
sequences unique to various species of microorganisms are then
utilized in accordance with Example 12 to amplify DNA of
approximately 100-200 bases in length from the specimen. A separate
PCR reaction is set up for each pair of PCR primers and after the
PCR reaction is complete, the reaction mixtures are assayed for the
presence of PCR product. The presence or absence of bacteria from
the species to which the PCR primer pairs belong is determined by
the presence or absence of a PCR product in the various test PCR
reaction tubes.
[0330] Although the PCR reaction is used to assay the isolated
sample for the presence of various bacterial species, other assays
such as the Southern blot hybridization are also contemplated.
[0331] All documents cited herein are incorporated herein by
reference in their entireties.
Sequence CWU 0
0
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References