U.S. patent application number 11/679815 was filed with the patent office on 2007-09-20 for novel aconitase.
Invention is credited to Lee A. Bulla, Mehmet Candas.
Application Number | 20070219269 11/679815 |
Document ID | / |
Family ID | 33490468 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070219269 |
Kind Code |
A1 |
Candas; Mehmet ; et
al. |
September 20, 2007 |
NOVEL ACONITASE
Abstract
A bacterial protein which converts 2-methyl citrate to 2-methyl
isocitrate is a previously unknown target for antibacterial agents.
The protein of this activity is associated with mucoid bacteria and
inhibitors of production or activity of this protein in combination
with propionic acid mitigate the virulence of these bacteria.
Inventors: |
Candas; Mehmet; (Dallas,
TX) ; Bulla; Lee A.; (Tioga, TX) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE
SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
33490468 |
Appl. No.: |
11/679815 |
Filed: |
February 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10441919 |
May 19, 2003 |
7183100 |
|
|
11679815 |
Feb 27, 2007 |
|
|
|
60459885 |
Apr 1, 2003 |
|
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|
Current U.S.
Class: |
514/557 ;
435/243; 435/245; 435/4; 435/69.1; 530/350; 536/23.2 |
Current CPC
Class: |
C12N 9/88 20130101; C12Y
402/01003 20130101 |
Class at
Publication: |
514/557 ;
435/243; 435/245; 435/004; 435/069.1; 530/350; 536/023.2 |
International
Class: |
A61K 31/19 20060101
A61K031/19; C07H 21/00 20060101 C07H021/00; C07K 14/00 20060101
C07K014/00; C12N 1/00 20060101 C12N001/00; C12N 1/36 20060101
C12N001/36; C12P 21/02 20060101 C12P021/02; C12Q 1/00 20060101
C12Q001/00 |
Claims
1. A method to mitigate the virulence of mucoid bacteria, which
method comprises contacting said bacteria with propionic acid or a
compound which generates propionic acid metabolically in
combination with an agent which inhibits aconitase C (acnC)
activity or production of acnC protein.
2. The method of claim 1, wherein said acnC activity is provided by
a protein comprising an amino acid sequence at least 95% homologous
to the amino acid sequence of acnC as set forth in FIG. 2.
3. A method to identify a compound that, in combination with
propionic acid, inhibits the virulence of mucoid bacteria, which
method comprises measuring the activity of acnC in the presence and
absence of a candidate compound; comparing the activity in the
presence and absence of said candidate compound, wherein a decrease
in activity in the presence of said compound, as compared to its
absence, identifies said compound as able to mitigate virulence of
mucoid bacteria in the presence of propionic acid.
4. The method of claim 3, wherein said acnC activity is provided by
a protein comprising the amino acid sequence at least 95%
homologous to the amino acid sequence of acnC as set forth in FIG.
2.
5. The method of claim 3, wherein said assessing is by measuring
the decrease in concentration of 2-methyl citrate or the increase
in concentration of 2-methyl isocitrate.
6. The method of claim 1, wherein the production of acnC is
inhibited by administering to said bacteria a nucleotide sequence
which is the complement of mRNA encoding said acnC or an expression
system for said mRNA.
7. The method of claim 1, wherein the production of acnC is
inhibited by modifying said bacteria to contain a nucleotide
sequence which forms a triple helix with at least a portion of the
acnC gene.
8. The method of claim 1, wherein the activity of said aconitase C
is inhibited by modifying the bacteria to contain an antibody
immunoreactive with said aconitase C protein or a fragment of said
antibody which retains this activity.
9. The method of claim 8, wherein said providing is accomplished by
modifying said bacteria to contain an expression system for said
antibody or fragment.
10. An isolated protein which has at least 95% sequence homology to
the amino acid sequence of aconitase C shown in FIG. 2 over its
entire length and which exhibits enzymic activity to convert
2-methyl citrate to 2-methyl isocitrate.
11. The protein of claim 10, which has the amino acid sequence set
forth in FIG. 2 or a fragment of said sequence which exhibits said
activity.
12. A nucleotide sequence which encodes the protein of claim
10.
13. A nucleotide sequence which encodes the protein of claim
11.
14. The nucleotide sequence of claim 13, which is the nucleotide
sequence encoding acnC set forth in FIG. 2.
15. A nucleic acid which comprises an expression system for the
protein of claim 10, which expression system comprises a nucleotide
sequence encoding said protein operably linked to control sequences
for its expression.
16. The nucleic acid of claim 15, wherein said control sequences
are effective in prokaryotes.
17. A recombinant host cell modified to contain the nucleic acid of
claim 16.
18. A method to produce a protein having aconitase C activity,
which method comprises culturing the cells of claim 17, whereby the
expression of said nucleotide sequence is effected.
19. A method to treat a subject for bacterial infection which
method comprises administering to said subject a compound which
generates propionic acid metabolically in combination with an agent
that inhibits aconitase C (acnC) activity or production of acnC
protein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of copending U.S. Ser. No.
10/441,919 filed 19 May 2003, which claims priority to U.S. Patent
Application Ser. No. (USSN) 60/459,885, filed Apr. 1, 2003. The
contents of this application is incorporated herein by
reference.
TECHNICAL FIELD
[0002] The invention relates to methods to control the growth and
virulence of mucoid bacteria and to regulate their production of
exopolysaccharide biofilms. The invention also is directed to
methods to screen for useful antibiotics. Such screening methods
employ a novel aconitase whose properties have heretofore been
unknown.
BACKGROUND ART
[0003] Several species of bacteria are able to secrete
exopolysaccharides or alginates that are essential for virulence as
the exudate provides a mechanism for adherence and colonization.
One particularly important example of such bacteria is Pseudomonas
aeruginosa, a common soil bacterium which inhabits individuals
generally, but is particularly destructive in subjects with cystic
fibrosis. Cystic fibrosis is an autosomal recessive genetic
disorder linked to dysfunctional CFTR chloride channels on cell
surfaces. It is characterized by production of thick mucus which
prevents clearance of bacteria, resulting in chronic infection and
inflammation. Because P. aeruginosa produces biofilm in the lungs
and digestive systems of these subjects, and the subjects are
unable to clear this biofilm, P. aeruginosa infection is a major
cause of death among such individuals.
[0004] Many bacteria exhibit mucoid (resembling mucus) phenotype as
a response to their growth environment. The mucoidy is generated by
bacterial production of extracellular polysaccharides
(exopolysaccharide or EPS). Various EPS molecules that include
frucose, rugose and glucose residues have been characterized.
Examples of bacteria producing mucoid phenotype include alginate
producing Pseudomonas and Azotobacter species (i.e., P. aeruginosa,
Azotobacter vinelandii), rugose producing Vibrio species (i.e.,
Vibrio cholerae), xanthan producing Xanthomonas species (i.e.,
Xanthomonas campestris), gellan producing Sphingomonas species
(i.e., S. paucimobilis), curdlan-type EPS producing Cellulomonas,
Alcaligenes and Agrobacterium species (i.e., Cellulomonas
flavigena, Alcalifenes faecalis) and Shewanella, Bordetella and
Streptococcus species producing various uncharacterized EPS, among
others. In fact, under unfavorable growth conditions, many bacteria
can switch to a mucoid phenotype to resist the environmental stress
and adapt to unfavored conditions. Biofilms represent a typical
structured adaptation environment in which many bacteria co-exist
and secrete extracellular polysaccharides which aid them to stick
to surfaces for growth and colonization, provide a protective
barrier around them and adapt to their environment in a microbial
community. The exopolysaccharides produced by mucoid Pseudomonas
that occupy the lungs of cystic fibrosis patients are generally
alginates which are O-glycosyl linked D-mannuronate and
L-guluronate residues.
[0005] It is understood that, in order to produce these biofilms,
the essential components required must be made available by the
metabolic system of the bacterium. The present invention provides
means to disrupt this ability by disabling an essential step in
this metabolic sequence.
Disclosure of the Invention
[0006] The present inventors have discovered that mucoid bacteria
contain an aconitase, designated herein that encoded by acnC which
catalyzes the conversion of 2-methyl citrate to 2-methyl
isocitrate. This aconitase, which has an activity different from
aconitases previously known, is required for the clearance of
propionate; propionic acid is a known toxic agent for mucoid
bacteria as described in PCT publication WO 01/30997, the
disclosure of which is incorporated herein by reference.
Accordingly, disruption of the activity or production of acnC
protein along with the administration of propionic acid, or of
compounds which generate propionic acid, has a deleterious effect
on mucoid producing bacteria. This effect resides, in large part,
in inhibiting the production of the biofilm and thus inhibiting the
ability of the bacterium to survive in its environment.
[0007] Thus, in one aspect, the invention is directed to a method
to mitigate the virulence of a mucoid bacterial culture or
infection, which method comprises contacting the bacteria contained
in said culture or infection with an effective amount of propionic
acid or a substance which generates propionic acid in combination
with effecting inhibition of the production or activity of acnC
protein.
[0008] In another aspect, the invention is directed to a method to
screen for compounds that enhance the toxicity of propionic acid to
exopolysaccharide-producing bacteria, which method comprises
assessing the ability of candidate compounds to inhibit the
activity of acnC protein. This method comprises determining the
conversion of 2-methyl citrate to 2-methyl isocitrate in the
presence of acnC protein and testing this in the presence and
absence of a candidate compound. Compounds whose presence reduces
the level of 2-methyl isocitrate produced, or which are otherwise
shown to inhibit acnC protein, are identified as useful in
enhancing the toxicity of propionic acid or its precursors.
[0009] In a third aspect, the invention is directed to a
composition of matter which comprises an isolated form of acnC
protein, compositions which comprise recombinant materials for its
production and methods for producing acnC protein using the
recombinant materials. The invention also is directed to antisense
or triplex forming nucleic acid molecules and other inhibitors for
the production of acnC protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a diagram showing the catabolism of propionate in
Pseudomonas aeruginosa.
[0011] FIGS. 2A-2D show the nucleotide and deduced amino acid
sequence of aconitase C from P. aeruginosa strain 01 (PA01) as well
as homologous enzymes from other bacteria, and position of the
encoding gene. In FIG. 2A, the nucleotide sequence and deduced
amino acid sequence of the acnC protein is shown. FIG. 2B shows the
amino acid sequence. In FIG. 2C, homologs for the acnC protein
having at least 85% similarity were retrieved through BLAST
searches and alignment of these sequence using DNASTAR. FIG. 2D
shows a comparison of the location of the gene in P. aeruginosa and
the corresponding genetic positions in S. typhimurium and E.
coli.
[0012] FIG. 3 shows a graph of the effect of 0.5% propionate added
to TSBD medium containing 50 mM glutamate on wildtype PA01 as
compared to PA01 with an acnC gene disruption.
[0013] FIG. 4 shows the effect of the addition of propionate to
clinical mucoid isolates.
MODES OF CARRYING OUT THE INVENTION
[0014] While many currently used antibiotics and drugs target the
ability of bacteria to grow, they do not necessarily reduce the
ability of these bacteria to infect the host, to adapt, and to
produce virulence factors. The present invention, by elucidating
the function and structure of a protein product and its encoding
gene that is involved in virulence and metabolic adaptation,
provides an entirely new target for the design and development of
new anti-infectives, antibacterial compounds, and biofilm control
agents. As this is a new target for antibacterial drugs, resistance
to such drugs has not developed.
[0015] This target is exemplified herein by the isolation and
manipulation of a gene from Pseudomonas aeruginosa designated
aconitase C (acnC). Disruption of acnC completely abolishes
bacterial growth in the presence of propionic acid and results in a
significant reduction in the virulence factors associated with P.
aeruginosa, including the production of biofilms. Hence, inhibition
of the production of this protein or inhibition of its activity
will attenuate microbial virulence in the presence of propionic
acid or a material which generates it. No eukaryotic counterpart to
aconitase C is known.
[0016] It is demonstrated herein that disruption of aconitase C
activity destroys the ability of mucoid bacteria to grow in the
presence of propionic acid. Accordingly, it is clear that
inhibitors of this activity, either those which inhibit the
production of the protein or those which inhibit the activity of
the protein itself will be useful in making mucoid bacteria more
susceptible to propionic acid and to agents which generate
propionic acid.
[0017] Previously described aconitase activities catalyze the
dehydration of citric acid to cis aconitate. The presently isolated
aconitase C is capable of this activity as well, but has the
additional feature of catalyzing the conversion of 2-methyl citrate
to 2-methyl isocitrate as shown in FIG. 1. This pathway is critical
to propionate metabolism as shown. Propionic acid is converted to
propionyl CoA and condensed with oxalacetate to obtain 2-methyl
citrate. 2-Methyl citrate must be isomerized to 2-methyl isocitrate
in order to complete the metabolic fate of propionic acid. 2-Methyl
citrate is metabolized to succinate and pyruvate, components of the
citric acid pathway. Inhibition of aconitase C thus diminishes the
ability of the organism to metabolize the toxic propionic acid.
[0018] "Aconitase C" and "acnC" are used interchangeably and refer
to any nucleotide sequence encoding the protein with enzymatic
activity, the protein itself, and the gene locus which results in
the production of the protein. In order to be defined as "aconitase
C" or "acnC" the protein must exhibit the ability to convert
2-methyl citrate to 2-methyl isocitrate. This activity can readily
be verified using routine enzymatic assays. In some drawings and
text herein, the corresponding materials are also labeled "acaB."
In Pseudomonas, the acnC encoding gene is found downstream in the
propionate operon from prpC and upstream of prpD.
[0019] One assay is analogous to that employed for determining
levels of aconitase activity known in the prior art, based on the
sequence of reactions shown below: ##STR1## The enzymatic process
shown above is monitored spectrophotometrically based on
measurement of increase in OD at 340 nm with formation of NADPH
from NADP.sup.+. The assay components include citrate and
isocitrate dehydrogenase. Under appropriate conditions, the rate of
NADPH production is proportional to aconitase activity. One
aconitase unit will convert 1.0 micromol of citrate to isocitrate
per minute at 25.degree. C., pH 7.4 (Gardner and Fridovisch, J.
Chem. (1992) 267:8757-8763).
[0020] As the aconitase C of the present invention converts
2-methyl citrate to 2-methyl isocitrate, and as 2-methyl isocitrate
is also oxidized with NADP.sup.+ in the presence of isocitrate
dehydrogenase, a similar assay that couples spectrophotometric
measurement of NADPH production at 340 nm can be used to determine
levels of aconitase C of the present invention.
[0021] The foregoing, and any other appropriate assay for the
conversion of 2-methyl citrate to 2-methyl isocitrate, can be used
as a screening assay to identify compounds that will be useful in
modifying the virulence of mucoid bacteria. Compounds which inhibit
this activity will be useful in this regard. Thus, the activity of
a preparation of purified and isolated aconitase C protein,
recombinantly produced aconitase C protein, or even an impure
preparation of aconitase C protein is tested for this conversion
activity in both the presence and absence of candidate compound.
Compounds whose presence results in a decrease in this activity are
identified as useful compounds for reducing the virulence of mucoid
bacteria. As a preliminary screen, the traditional aconitase
activity assay shown above can be used as a surrogate in
identifying compounds with the desired activity. However, assays
using the aconitase C protein per se are preferred.
[0022] The screening assay using aconitase C protein per se is
facilitated by virtue of the availability of recombinant materials
for production of the required aconitase C protein. Described
herein, as illustrative, is the amino acid sequence of the
aconitase C gene derived from P. aeruginosa. Homologous proteins
from other prokaryotic organisms could also be used, and can
readily be retrieved using standard techniques with the information
contained in the P. aeruginosa gene as a guide. Thus, the protein
encoded by the gene in the P. aeruginosa strain 01 comprises 869
amino acids and the nucleotide sequence and deduced amino acid
sequence are shown in FIG. 2A. The protein is found in the
cytoplasm and is probably present in association with bacterial
membrane and/or other enzymes involved in propionate catabolism.
The aconitase activity in PA acnC::Gm mutant is decreased when
measured by a traditional aconitase assay indicating that the
catalytic site of the protein may resemble the catalytic site of
the known aconitases (acnA and acnB) involving conserved cysteine
and arginine residues folded to structure a docking region for an
iron-sulfur (4Fe-4S) cubane cluster as well as interaction with
substrates including citrate and isocitrate.
[0023] Proteins with homology to the PA01 aconitase C protein also
share the ability to convert 2-methyl citrate to 2-methyl
isocitrate. Thus, included within the scope of the invention are
proteins which exhibit at least 85%, preferably 90%, preferably
95%, and more preferably 98% homology over the entire sequence to
the sequence shown in FIG. 2A and which exhibit aconitase C
activity. "Aconitase C activity" is defined herein as the ability
to convert 2-methyl citrate to 2-methyl isocitrate. Fragments of
these sequences of shared homology which retain aconitase C
activity are also included within the scope of the invention. FIG.
2B shows homologous sequences that are known to occur in other
bacteria as retrieved through BLAST searches.
[0024] Similarly, proteins encoded by a nucleotide sequence which
hybridizes under stringent conditions to a nucleotide sequence
encoding the amino acid sequence of the PA01 aconitase C protein
are also included within the scope of the invention. The stringency
of hybridization is defined by the wash conditions subsequent to
the hybridization itself and "stringent" conditions are defined as
washing in 0.1% SSC at 65.degree. C.
[0025] Thus, proteins which are within the scope of the present
invention may be defined in terms of their ability to convert
2-methyl citrate to 2-methyl isocitrate in combination (1) with
specified homology to the acnC sequence set forth in FIG. 2A or (2)
with structural characteristics as defined by the ability of a
nucleotide sequence encoding them to hybridize to a nucleotide
sequence encoding this amino acid sequence.
[0026] While nucleic acids which encode aconitase C proteins can be
defined in terms of nucleotide sequences degenerate with that set
forth in FIG. 2A as encoding acnC, nucleic acids comprising
nucleotide sequences useful in the design of probes or PCR primers
for recovery of acnC proteins from strains of bacteria other than
PA01 and for the design of nucleic acids used to inhibit or
modulate the production of native acnC will be defined structurally
in terms of their homology to the non degenerate nucleotide
sequence set forth as encoding acnC in FIG. 2A. Thus, such
nucleotide sequences will have at least 85% homology, preferably
90% homology, preferably 95% homology, and more preferably 98%
homology to the nucleotide sequence set forth as encoding acnC in
FIG. 2A or alternatively in terms of their ability to hybridize to
this nucleotide sequence or its complement--i.e., which hybridize
under stringent conditions to these sequences.
[0027] As stated above, one approach to modulating the virulence of
mucoid bacteria comprises contacting such bacteria with a compound
which inhibits the aconitase C activity in combination with a
source of propionate. Such compounds can be identified through the
screening assay described above, or may already be known to block
aconitase C activity by virtue of their ability to bind the
aconitase C protein. Thus, antibodies or other specific binding
partners for the aconitase C proteins of the invention may be
employed. "Antibodies" include, in addition to immunoglobulins in
general, immunoreactive portions, such as the F(ab) or F(ab') or
F(ab').sub.2 fragments; antibodies may also be prepared as
single-chain forms--i.e., scFv antibodies. Various ways to
manipulate antibodies for particular purposes are also well known;
thus, included within the invention are humanized forms of
antibodies of the invention or antibodies which are modified to
correspond to the species to which they may be administered. Other
specific binding partners include aptamers--i.e., nucleic acids
which optionally have been selected through known rounds of
selection using the Selex.RTM. system, for example, for specific
binding to proteins with aconitase C activity. Such aptamers may be
"traditional" nucleic acids or modified forms thereof, such as
peptide nucleic acids. Thus, aconitase C activity may be inhibited
in a variety of ways, including direct binding of the protein by
antibodies or aptamers, and by compounds which have been shown to
inhibit the activity empirically.
[0028] In addition to use of compounds which inhibit aconitase C
activity, methods to inhibit the production of the aconitase C
protein itself may also be employed. Such known methods include use
of antisense constructs and formation of a triple helix at a
critical position in the gene. In these methods, of course, the
native, non-degenerate nucleotide sequence must be targeted. Thus,
suitable targets for triplex formation or antisense inhibition
include nucleotide sequences which encode aconitase C activity and
which have at least 85%, preferably 90% homology, preferably 95%
homology and more preferably 98% homology to the nucleotide
sequence shown to encode acnC in FIG. 2 or nucleotide sequences
which hybridize under stringent conditions as defined above to that
nucleotide sequence. Typically, oligonucleotides which operate
through a mechanism of antisense complementarity are generated in
situ. Thus, vectors containing transcriptional controls may be used
to generate antisense RNA comprising nucleotide sequences
complementary to the mRNA encoding aconitase C (the structural
characteristics of which are described above). As triplex formation
operates at the gene level, the oligonucleotides for triplex
formation are generally directly supplied.
[0029] In the method of the invention, the mucoid bacteria for
which virulence is sought to be modulated is treated both with
materials which inhibit production or activity of aconitase C and
with a source of propionate. The propionate source may be propionic
acid itself or a fatty acid with an odd number of carbon atoms in
the chain which generates propionic acid metabolically. Other
materials known to be metabolized to propionic acid, such as
ibuprofen, could also be substituted. Thus, the propionic acid
portion of the treatment is contributed by any compound which is
itself propionic acid or generates propionic acid in situ.
[0030] The propionic acid source and the means for modulating
aconitase C activity (which means include direct inhibition of
activity and inhibition of production of this protein) are supplied
either simultaneously or sequentially to the mucoid bacterium
target. If the targeted bacterium is present in an in vitro
environment, e.g., in a foodstuff or other composition to be
decontaminated, the propionic acid source and the modulator of acnC
activity can be supplied directly to this material. Alternatively,
the offending bacterium may have infected an organism, in which
case the appropriate materials are supplied to the organism per se.
Thus, for example, if the targeted bacterium has infected an
animal, such as a mammal or avian host, the mammal or avian host is
provided a propionic acid source and a modulator of acnC. Such
administration can be by any traditional means and the materials
formulated appropriately to their nature. Thus, administration may
be by injection, transmucosal, transdermal, topical, local,
systemic, oral, or in a variety of paradigms well known to
practitioners.
[0031] When the combination of a propionic acid generating compound
and inhibitor or modular of acnC is used to treat a subject, the
choice of propionic acid generating compound is made appropriate to
lack of toxicity and ability of the treated subject to metabolize
the precursor to the desired product; thus, for example, propionic
acid per se would not be used in mammalian subjects. Suitable
formulations are also provided as is known in the art for effective
routes of administration.
[0032] By "treat" bacterial infection is meant any positive change
with regard to the health of the subject related to the underlying
infection, not necessarily a complete recovery. Thus, reduction of
the mucoid production by the bacteria, amelioration of symptoms,
slowing the progression of bacterial growth, and the like, are all
within the scope of "treating."
[0033] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Isolation of the Aconitase C Gene from P. aeruginosa
[0034] The genome of P. aeruginosa wildtype strain 01, a mucoid
producing strain, designated herein PA01 was subjected to PCR to
obtain a 900 base pair aconitase C encoding fragment. The forward
primer was GTNGGNACNGAYTCNCAYACN and the backward primer was
NCKNCCYTCRAARTTNCKRTT. The amplified fragment was sequenced and the
amino acid sequence encoded was deduced. The complete nucleotide
sequence and deduced amino acid sequence are shown in FIG. 2A.
[0035] The position of the aconitase C encoding gene in P.
aeruginosa is compared to the location of the corresponding
encoding sequence in S. typhimurium and E. coli in FIG. 2C. E. coli
and S. typhimurium catabolize propionate using proteins encoded by
the prpBCDE operon (prp operon). These prp operons have been shown
to contain a set of genes--prpR, prpB, prpC, prpD and prpE. The
prpD gene in these bacteria has been proposed to encode a protein
with 2-methylcitrate dehydratase enzyme activity catalyzing the
conversion of 2-methylcitrate into 2-methylisocitrate (Horswill and
Escalante-Semerena, Biochemistry (2001) 40:4703-4713). In P.
aeruginosa, the prp operon contains a different set of genes, some
of which are homologous to the genes in the E. coli and S.
typhimurium prp operons. The P. aeruginosa prp operon contains
prpR, prpB, acnC, yraM, and prpD. Neither E. coli nor typhimurium
comprise acnC in the prp operon or elsewhere in their genomes. No
sequences homologous to acnC gene have been found in any eukaryotic
genome.
[0036] Deduction of the aconitase C protein sequence, which
contains 869 amino acids, permitted comparison using the BLAST
similarity search program to known aconitases. The protein showed
61% similarity using this program to E. coli aconitase A, 60%
similarity to P. aeruginosa aconitase A, 61% similarity to human
IRP1, 53% similarity to human IRP2, and 41% similarity to pig
mitochondrial aconitase.
EXAMPLE 2
Disruption of the Aconitase C Coding Sequence
[0037] The approximately 900 base pairs of the aconitase C insert
obtained in Example 1 was amplified by PCR from the PA01 genome
using the primers GTGGCACCGACAGCCATAC and GCGCCCGTCGAAGTTGCGGTT.
The amplified fragment was ligated into pBluescript-2 (KS+)
(Stratagene) which had been cleaved with EcoRV and treated with
tack DNA polymerase and dTTP to form intermediate plasmid pBSacnC.
An approximately 1 kb DNA that encodes gentamicin resistance
(Gm.sup.R) was isolated from pUCGm described in Schweizer, H. D.,
Biotechniques (1993) 15:831-834 by digesting with Smal. This
amplified segment was cloned into the Stul site which resides in
the acnC coding sequence in pBSacnC to generate pBSacnCGm. The
approximately 2 kb DNA fragment which contains the acnC sequence
which was disrupted with the Gm.sup.R cassette was isolated by
treating this plasmid with HindIII and PstI and filled in with
Klenow and dNTP's. This fragment was ligated to the SmaI ends of
the conjugation plasmid pEX100T (Schweizer, H. D., et al., Gene
(1995) 158:15-22) to obtain pEXacnCGm.
[0038] This plasmid was used to transform E. coli S17-1 and
recombinant cells containing the plasmid were combined with an
approximately equal amount of PA01 cells and plated on LB plates
for conjugation. After conjugation, the bacteria were plated on
Pseudomonas isolation agar plates containing Gm for selection of
residue P. aeruginosa cells. Mutants for disruption of the acnC
gene were further selected on PIA plates containing Gm and 4%
sucrose. The successful transformants are designated PA01* or
PA01-acaB:Gm.
EXAMPLE 3
Effects of acnC Gene Disruption
[0039] The effect of acnC gene disruption on various virulent
activities of PA01 were determined.
[0040] Exotoxin A production, determined by Western analysis of
stationary phase supernatants did not show any change. However,
hemolytic activity (tested by zone clearance on blood agar plates);
proteolytic activity (tested by zone clearance on milk agar
plates); elastase activity (tested by zone clearance on
2XYT-elastin plates); pyoverdin production (tested by pigment
production on F agar plates); and pyocyanin production (tested by
pigment production of P agar plates) were all diminished
substantially.
[0041] When tested for growth on various carbon sources, no
difference was observed when glucose, glutamate, citrate,
isocitrate, succinate, acetate, pyruvate, butyrate, hexonate, or
glyoxalate was used as a carbon source. However, although the
wildtype could grow well on propionate, pentanate and the
combination of glyoxalate and propionate as carbon sources, the
acnC disrupted strain did not grow under these conditions.
[0042] FIG. 3 shows illustrative results of the effect of the
inclusion of 0.05% propionate in the tryptic soy broth deferrated
(TSBD) plus 100 .mu.M iron medium with and without 50 mM glutamate.
As shown, growth is diminished in the presence of propionate in
PA01* as compared to wildtype.
[0043] The two strains were also tested for aconitase activity
using the known conversion of aconitase citric acid to cis aconitic
acid. PA01 and PA01* were grown in TSBD supplemented with 100 .mu.M
iron and the aconitase activity was determined at 6, 10 and 14
hours. PA01 exhibited 5, 15, and 19 units of activity per milligram
of protein at these time points, respectively, while PA01*
exhibited only 3, 10, and 16 units, respectively. PA01* also
exhibited slightly reduced growth under these conditions.
[0044] Thus, disruption of the acnC gene completely abolishes
growth in media where propionate is the carbon source, and
diminishes growth in propionate-containing media with other carbon
sources. Diminished aconitase activity in converting citrate to cis
aconitate is also shown.
[0045] In addition, it has been shown that chemotaxis is affected
and expression of several virulence factors is diminished.
EXAMPLE 4
Studies on Additional Strains
[0046] In addition to PA01, an additional mucosal strain, FRD1 with
gentamicin resistance was subjected to disruption of the acnC gene,
as was a mucoid clinical isolate. For comparison, aconitase A was
also similarly disrupted. All of the strains behaved similarly in
respect to their ability to grow on propanediol in the presence and
absence of propionic acid. All of the strains were able to grow in
1,2-propanediol, but were less able to grow using 1,3-propanediol
as carbon source. Addition of propionic acid to the medium
completely abolished the ability of both PA01 and FRD1 with
disrupted aconitase C genes to grow under these conditions;
disruption of the acnA gene did not result in this effect.
[0047] FRD1 is much more susceptible to propionate inhibition than
wildtype PA01. While PA01 is able to grow on 0.4% propionate,
inhibition of FRD1 growth occurs at levels of 0.1% propionate and
inhibition is complete as low as 0.2% propionate in the medium.
[0048] It has also been noted that aconitase activity (measured as
the conversion of citric acid to cis aconitate) is different in
PA01 wildtype as compared to FRD1. While this activity was slightly
higher in FRD1 after 4 hours of culture as compared to PA01, after
7 hours of culture the activity was statistically higher in PA01
cells and roughly equivalent after 14 hours.
[0049] FIG. 4 shows the effect of the addition of propionate on the
growth of various mucoid clinical isolates in comparison with a
non-mucoid strain, BH1, and a revertant strain, BAB. As shown, the
mucoid strains demonstrate substantial growth inhibition at low
concentrations of propionate.
[0050] It has also been shown that the results of propionate
treatment can be obtained using odd-numbered chain fatty acids in
the medium, as the metabolic products of these fatty acids include
propionate. This is shown in Table 1 below which shows results of
the addition of propionate, butyrate, hexanoic acid and pentanoic
acid to M9 medium on wildtype PA01*, and PA01 with two irrelevant
disruptions--acaA and icdl. As shown, both the presence of 0.4%
propionate and 0.4% pentanoate disrupt the growth of the strain
with a disrupted acnC gene, but these additions have no effect on
the other strains tested. TABLE-US-00001 TABLE 1 Dis- Disrupted
PA01* rupted PA01 wt acaA (Disrupted acnC) IcdI M9/propionate 0.4%
+ + - + M9/butyrate 0.4% +/poor +/poor +/poor +/poor M9/hexanoate
0.4% + + + + M9/pentanoate 0.4% + + - +
[0051] The effect of the addition of 0.4% propionate to LB medium
containing wildtype PA01, PA01*, and the more sensitive mucoid
strain FRD1, was also tested. The wildtype does not respond to 0.4%
propionate, but the modified strain PA01* containing a disrupted
acnC gene does show diminished growth, as does the more
propionate-sensitive strain FRD1.
EXAMPLE 5
Effect of TCA Intermediates
[0052] FRD1, as set forth in Example 4, shows enhanced sensitivity
to propionate, possibly due to reduced levels of TCA cycle
activity, since propionic acid drains the TCA cycle intermediate
oxalacetic acid.
[0053] This effect was tested by supplementing M9 minimal media
with various TCA cycle components in the presence of 0.4%
propionate. While glucose as a carbon source failed to reverse the
negative effects on growth of propionate, the cell cycle
intermediates acetate, aspartate, glutamate and malate were
successful in doing so.
EXAMPLE 6
Effect of Ibuprofen on Growth of FRD1
[0054] Ibuprofen is known to generate propionic acid when
metabolized and was thus tested for its ability to inhibit the
growth of FRD1. Other propionate-generating compounds include
certain .alpha..sub.4.beta..sub.1 integrin antagonists described by
DuPlantier, A. J., et al., Bioorg. Med. Chem. Let. (2001)
11:2593-2596. Using absorbance at 600 nm as the criterion, growth
inhibition was detected at a concentration of 0.5 mg/ml of
ibuprofen added to 2 ml LB cultures; the percent inhibition at this
level was 19%. Inhibition increased in a dose-dependent manner to
72% at 5 mg/ml, and was similar at 10 mg/ml (73%).
EXAMPLE 7
Effect of Propionate on Attachment
[0055] As mucoid production is necessary for attachment, the effect
on mucoid production of addition of propionate can be tested by
assessing the level of attachment of the cells to plastic tubes.
This was measured as the absorbance at 570 nm of plastic tubes.
Levels of as low as 0.1% propionate were able to diminish
significantly the attachment of FRD1 cells to plastic tubes; the
effect on attachment of PA01 was less dramatic.
EXAMPLE 8
Effect of Propionate on Antibiotic Susceptibility of PA01 and
FRD1
[0056] The effect of various concentrations of propionate on the
ability of antibiotics to curtail the growth of PA01 or FRD1 was
tested. Levels of 0.2% propionate appeared to have no effect on
PA01 growth in the presence of the antibiotics amikacin,
carbenicillin, ciprofloxacin, tobramycin, and tetracycline. Growth
was assured by the diameter of colonies formed in the presence of
discs containing amikacin (AN-30), 30 .mu.g, carbenicillin
(CB-100), 100 .mu.g, ciprofloxacin (CIP-5), 5 .mu.g, tobramycin
(NN-10), 10 .mu.g, or tetracycline (TE-30), 30 .mu.g. On the other
hand, 0.2% propionate appeared to diminish the effect of CB-100 and
CIP-5 on inhibiting growth of FRD1. There appears to be little, if
any, effect on the efficiency of remaining antibiotics tested.
* * * * *