U.S. patent application number 10/794922 was filed with the patent office on 2004-08-05 for autoinducer synthase modulating compounds and uses therefore.
This patent application is currently assigned to The Board of Trustees of the University of Illinois. Invention is credited to Cronan, John E. JR., Greenberg, E. Peter, Parsek, Matthew R., Plapp, Bryce V..
Application Number | 20040152660 10/794922 |
Document ID | / |
Family ID | 26789418 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152660 |
Kind Code |
A1 |
Cronan, John E. JR. ; et
al. |
August 5, 2004 |
Autoinducer synthase modulating compounds and uses therefore
Abstract
Provided are compositions and methods useful for modulating the
activity of autoinducer synthase catalysts. A method for
identifying modulators of the autoinducer synthesis reaction is
also provided. Such modulators are useful for controlling bacterial
growth and can be used for therapeutic treatment of bacterial
infections particularly in immunocompromised subjects. They are
also useful in treating disease states associated with autoinducer
synthesis and biofilm development.
Inventors: |
Cronan, John E. JR.;
(Urbana, IL) ; Plapp, Bryce V.; (Iowa City,
IA) ; Greenberg, E. Peter; (Iowa City, IA) ;
Parsek, Matthew R.; (Iowa City, IA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
The Board of Trustees of the
University of Illinois
Urbana
IL
|
Family ID: |
26789418 |
Appl. No.: |
10/794922 |
Filed: |
March 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10794922 |
Mar 5, 2004 |
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09227488 |
Jan 6, 1999 |
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6723321 |
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60094988 |
Jul 31, 1998 |
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Current U.S.
Class: |
514/46 ;
514/47 |
Current CPC
Class: |
C07H 19/16 20130101;
C07H 19/207 20130101; Y02A 50/30 20180101; A61K 31/7076 20130101;
Y02A 50/473 20180101; Y10S 424/832 20130101; C12Q 1/25
20130101 |
Class at
Publication: |
514/046 ;
514/047 |
International
Class: |
A61K 031/7076 |
Claims
What is claimed is:
1. A method for modulating the activity of an autoinducer synthase
molecule comprising: providing an effective amount of a compound
capable of modulating the binding of a substrate to the homoserine
lactone substrate binding site of the autoinducer synthase, thereby
modulating the activity of the autoinducer synthase molecule.
2. The method of claim 1, wherein the said autoinducer synthase
molecule is selected from the group of bacterial quorum system
autoinducer synthase molecules consisting of LuxI, AinS, LucM,
LasI, RhlI, PhzI, TraI, CarI, HsII, EsaI, EagI, YenI, SwrI, and
AhyI.
3. The method of claim 2, wherein the said autoinducer synthase is
RhlI.
4. The method of any of claims 1-3, wherein said compound inhibits
the binding of said substrate to the homoserine lactone substrate
binding site of the autoinducer synthase.
5. The method of claim 1, wherein the said compound promotes the
binding of said substrate to the homoserine lactone substrate
binding site on the autoinducer synthase.
6. The method of claim 1, wherein the said substrate is S-adenosyl
methionine.
7. The method of claim 4, wherein the said compound is a thio
derivative of a purine nucleotide.
8. The method of claim 7, wherein the said compound is an alkylated
thio derivative of a purine nucleotide.
9. The method of claim 8, wherein the said derivative is S-adenosyl
homocysteine.
10. The method of claim 8, wherein the said derivative is
S-adenosyl cysteine.
11. The method of claim 8, wherein the said derivative is
5'-methylthioadenosine.
12. The method of claim 4, wherein the said compound is
sinefungin.
13. The method of claim 1, wherein the said modulation of the
autoinducer synthase molecule activity occurs in vivo.
14. The method of claim 1, wherein the said modulation of the
autoinducer synthase molecule activity occurs in vitro.
15. A method of selecting a compound capable of modulating the
activity of the autoinducer synthase molecule comprising: providing
an effective amount of a test compound; and determining whether the
activity of the autoinducer synthase molecule is modulated; and
selecting the compound which modulates the activity of the
autoinducer synthase molecule.
16. The method of claim 15, further comprising determining the
extent of the modulation of the autoinducer synthase molecule by
the said compound.
17. The method of claim 16 wherein the extent to which the activity
of the autoinducer synthase molecule is modulated is determined by:
providing a sufficient amount of a labeled homoserine lactone
substrate; allowing the reaction to proceed to completion; and
determining the extent of conversion of the labeled homoserine
lactone substrate to a homoserine lactone product.
18. The method of claim 17, wherein, the homoserine lactone
substrate is S-adenosylmethionine.
19. A method for producing a highly active bacterial recombinant
autoinducer synthase molecule comprising: introducing DNA encoding
said bacterial autoinducer synthase molecule into a bacterial host
cell of the same species such that the autoinducer synthase
molecule is overexpressed, thereby producing the highly active
recombinant autoinducer synthase molecule.
20. The method of claim 19, wherein the said purity of the
autoinducer synthase is in the range of from about 50% to about
100% pure.
21. An autoinducer synthase molecule produced by the method of
claim 19.
22. An amino acid sequence of SEQ ID NO. 1 comprising amino acids
24-104.
23. The amino acid sequence of claim 22 comprising acids 24-73.
24. The amino acid sequence of claim 22 comprising Arginine 24,
Glutamic Acid 46, Aspartic Acid 48 and Glutamic Acid 101.
25. A method for modulating the formation of bacterial quorum
system autoinducers comprising: providing an effective amount of a
compound capable of modulating the binding of a substrate to the
homoserine lactone substrate binding site of the autoinducer
synthase, thereby modulating the formation of bacterial quorum
system autoinducers.
26. A method for modulating bacterial biofilm development
comprising: providing an effective amount of a compound capable of
modulating the binding of a substrate to the homoserine lactone
substrate binding site of an autoinducer synthase, thereby
modulating the development of bacterial biofilms.
27. A method of inhibiting the infectivity of a pathogenic bacteria
in a subject comprising administering a therapeutically effective
amount of an autoinducer synthase blocker, such that the
infectivity of a pathogenic bacteria is inhibited.
28. A method of treating an immunocompromised individual by
administering a therapeutically effective amount of an autoinducer
synthase blocker to an immunocompromised individual, such that
treatment occurs.
29. A therapeutic composition comprising an autoinducer synthase
blocker and a pharmaceutically acceptable carrier.
30. The therapeutic composition of claim 29, wherein the
autoinducer synthase blocker is a molecule which inhibits the
autoinducer synthase activity of RhlI.
31. A method of treating a subject for a disease state associated
with biofilm development comprising administering a therapeutically
effective amount of an autoinducer synthase blocker, such that
biofilm development is inhibited.
32. A method for treating a subject for a state associated with
autoinducer synthesis comprising administering an effective amount
of an autoinducer synthase blocker such that treatment occurs.
33. A purified autoinducer synthase molecule which is at least
about 50% pure and being biologically active.
34. The autoinducer synthase molecule of claim 33 which is about
95% pure.
35. The autoinducer synthase molecule of claim 33 which is
RhlI.
36. A biologically active autoinducer synthase molecule
substantially free of other contaminants.
37. The autoinducer synthase molecule of claim 33 or 36 which is
substantially free of inclusion bodies.
38. A highly soluble autoinducer synthase molecule having
biological activity.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/227,488 filed on Jan. 6, 1999, which claims priority to
U.S. Provisional Patent Application No. 60/094,988 filed on Jul.
31, 1998, the entire contents of which are hereby incorporated by
reference (including the originally filed claims).
BACKGROUND
[0002] Quorum sensing is cell density-dependent regulation of genes
that involves a freely diffusible molecule synthesized by the cell
called an autoinducer (Fuqua et al., 1996, Salmond et al., 1995,
Sitnikov et al, 1995). The paradigm system for quorum sensing is
the lux system of the luminescent marine bacterium, Vibrio
fischeri. V. fischeri exists at low cell densities in sea water and
also at very high cell densities within the light organs of various
marine organisms, such as the squid Euprymna scolopes (Pesci et al.
1997, Ruby, 1996). At high cell densities, the V. fischeri genes
encoding the enzymes required for light production are expressed.
These genes are part of the lux ICDABEG operon and are regulated by
the gene products of luxI and luxR (Baldwin et al., 1989, Eberhard
et al., 1991, Gray et al. 1992). LuxI is an autoinducer synthase
that catalyzes the formation of the V. fischeri autoinducer (VAI),
N-(3oxohexanoyl) homoserine lactone (Eberhard et al. 1981, Seed et
al. 1995). The autoinducer freely diffuses across the cell membrane
and at high cell densities, reaches a critical concentration
(Kaplan et al. 1985). At this critical concentration, VAI interacts
with LuxR, a DNA-binding transcriptional regulator. The LuxR-VAI
complex then binds to an upstream sequence of the lux operon called
the "lux box", and activates transcription (Devine et al. 1989,
Hanzelka et al. 1995, Stevens et al. 1994). Since one of the genes
of the operon is luxI, an autoregulatory loop is formed.
[0003] Many gram-negative bacteria have been shown to possess one
or more quorum sensing systems (Fuqua et al., 1996, Salmond et al.,
1995). These systems regulate a variety of physiological processes,
such as conjugal plasmid transfer in the plant pathogen
Agrobacterium tumefaciens and antibiotic production in Erwinia
stewartii. The systems typically have acylated homoserine lactone
ring autoinducers, in which the homoserine lactone ring is
conserved. The acyl side chain, however, can vary in length and
degree of substitution. Pseudomonas aeruginosa has two quorum
sensing systems, las and rhl (Brint et al. 1995, Hanzelka et al.
1996, Baldwin et al., 1989, Passador et al. 1993, Pearson et al.
1997, Pesci et al. 1997). The two systems have distinct autoinducer
synthases (lasI and rhll), transcriptional regulators (lasR and
rhlR), and autoinducers (N-(3-oxododecanoyl) homoserine lactone
(HSL) and N-butyryl HSL) (Sitnikov et al, 1995, Stevens et al.
1994). N-(3-oxododecanoyl) homoserine lactone is synthesized by
LasI along with a small amount of N-(3-oxooctanoyl) HSL and
N-(3-oxohexanoyl) HSL, while RhlI makes primarily N-butyryl HSL and
a small amount of N-hexanoyl (Pearson et al. 1994, Winson et al.
1995). The rhl and las systems are involved in regulating the
expression of a number of secreted virulence factors, biofilm
development, and the stationary phase sigma factor (RpoS) (Brint et
al. 1995, Davies et al. 1998, Latifi et al. 1996, Ochsner et al.
1995, Pesci et al. 1997). Expression of the rhl system requires a
functional las system, therefore the two systems in combination
with RpoS constitute a regulatory cascade (Pesci et al. 1997, Seed
et al. 1995).
[0004] Quorum sensing systems are essential for communication
between bacterial cells in many environments, including living
biofilms. Biofilms contain distinct microcolonies, separated by
discrete water channels. Biofilms are characterized by an extensive
matrix of acidic polysaccharides, which protect the biofilm
bacteria from biocides. In order to synthesize the polysaccharide
matrix, the bacteria communicate through the quorum sensing system.
Bacterial biofilms are ubiquitous and are seen in substrates
ranging from sewage pipes and medical implants to teeth and the
lungs of immunocompromised hosts.
SUMMARY OF THE INVENTION
[0005] The present invention is based, at least in part, on the
discovery that bacterial autoinducer synthase molecules catalyze
the synthesis of homoserine lactone autoinducers in a highly
specific interaction of particular homoserine lactone substrates.
This provided the capability to develop compositions and methods to
modulate the quorum sensing capabilities of bacterial cells by
controlling autoinducer production.
[0006] The present invention provides a method for identifying
modulators of the autoinducer synthesis reaction, which promote or
inhibit the production of homoserine lactones. Such modulators are
useful for controlling bacterial growth and can be used for
therapeutic treatment of bacterial infections particularly in
immunocompromised subjects. They are also useful in treating
disease states associated with biofilm development.
[0007] The invention pertains to methods that modulate the activity
of an autoinducer synthase molecule by providing an effective
amount of a compound capable of binding to the homoserine lactone
substrate binding site on the autoinducer synthase molecule. The
invention includes modulation of the activity of autoinducer
synthase molecules including LuxI, AinS, LucM, LasI, RhlI, PhzI,
TraI, HslI, EsaI, EagI, YenI, SwrI, and AhyI.
[0008] The present invention pertains to methods of selecting a
compound capable of modulating the activity of the autoinducer
synthase molecule by providing an effective amount of the
potentially modulating compound, and determining whether the
compound effectively modulates the activity of the autoinducer
synthase, then selecting those compounds which do modulate the
activity of the autoinducer synthase. Methods are also provided
where the extent of the modulation of the activity of the
autoinducer synthase is determined. A preferred embodiment pertains
to a method for determining the extent of modulation by the
potentially modulating compound by providing a sufficient amount of
a labeled homoserine lactone substrate, allowing the reaction to go
to completion, then determining the extent of the conversion of the
labeled homoserine lactone substrate to homoserine lactone
product.
[0009] The present invention also pertains to a method for
producing highly active recombinant autoinducer synthase molecules
by introducing DNA encoding the autoinducer synthase molecules into
a bacterial host cell of the same species. The invention also
pertains to the autoinducer synthase produced by the methods of the
invention. In preferred embodiments, the purity of the autoinducer
synthase is in the range from about 50-100%. In preferred
embodiments, the purity of the autoinducer synthase is in the range
from about 75-100%. In preferred embodiments, the purity of the
autoinducer synthase is in the range from about 85-100%. In
particularly preferred embodiments, the purity of the autoinducer
synthase is about 95%. In a preferred embodiment, the autoinducer
synthase of the RhlI quorum sensing system of P. aeruginosa is
produced by the methods of the invention. In a particularly
preferred embodiment, the RhlI autoinducer synthase has the amino
acid sequence of SEQ ID NO 1. In a preferred embodiment, the RhlI
autoinducer synthase has an active portion which includes amino
acids 24-104. In a preferred embodiment, the RhlI autoinducer
synthase has an active portion which includes amino acids 24-73. In
a preferred embodiment, the RhlI autoinducer synthase has an amino
acid sequence which includes Arginine 24, Glutamic Acid 46,
Aspartic Acid 48 and Glutamic Acid 101.
[0010] The present invention also pertains to a method for
modulating the formation of bacterial quorum system autoinducers by
providing compounds which modulate the production of bacterial
autoinducers by blocking the binding of homoserine lactone
substrates to the homoserine lactone binding site on the
autoinducer synthase molecules. In preferred embodiments, the
autoinducer synthase molecules are RhlI.
[0011] The present invention also pertains to a method of
modulating biofilm development in an immunocompromised individual
by administering a therapeutically effective amount of an
autoinducer synthase blocker. In preferred embodiments, the method
is provided wherein the immunocompromised individual is afflicted
with cystic fibrosis or HIV.
[0012] The present invention also pertains to a method of
inhibiting the infectivity of a pathogenic bacteria by
administering a therapeutically effective amount of an autoinducer
synthase blocker molecule.
[0013] The invention pertains to a method of treating a subject for
a disease state associated with biofilm development by
administering a therapeutic composition of an autoinducer synthase
blocker molecule and a pharmaceutically effective carrier. In a
preferred embodiment, the invention pertains to a method of
treating a human with cystic fibrosis or HIV.
[0014] The invention also pertains to a method of treating a
subject for a state associated with autoinducer synthesis by
administering an effective amount of an autoinducer synthase
blocker.
[0015] The invention further pertains to a purified autoinducer
synthase molecule. In preferred embodiments, the autoinducer
synthase molecule is at least about 50% pure. In particularly
preferred embodiments, the autoinducer synthase molecule is at
least about 95% pure.
[0016] The invention also pertains to a biologically active
autoinducer synthase molecule. In preferred embodiments, the
autoinducer synthase molecule is substantially free of other
contaminants. In particularly preferred embodiments, the
autoinducer synthase molecule is substantially free of inclusion
bodies.
[0017] The invention also pertains to a highly soluble autoinducer
synthase molecule having biological activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A is a representation of a high performance liquid
chromotography analysis demonstrating that butyryl homoserine
lactone is produced from S-adenosylmethionine.
[0019] FIG. 1B is a graphic representation of the performance
criteria of an assay which measures autoinducer synthase
activity.
DETAILED DESCRIPTION
[0020] The present invention pertains to a method for modulating
the activity of an autoinducer synthase molecule by providing an
effective amount of a compound capable of modulating the binding of
the substrate to the homoserine lactone binding site of the
autoinducer synthase molecule, such that the activity of the
autoinducer synthase molecule is modulated.
[0021] The language "modulating the activity" is intended to
include changes in the activity of the autoinducer synthase
molecule which affect the molecule's ability to function as a
catalyst or facilitate the synthesis of an autoinducer molecule.
The changes include both promotion and inhibition of the activity
of the autoinducer synthase molecule. The activity can be the
autoinducer synthase's ability to interact with the substrate to
form the autoinducer. All measurable change in activity is
included. Change in activity can be measured directly by
determining an increase or decrease in autoinducer synthase
activity from a previously determined standard or baseline level.
Alternatively, the change in activity can be measured indirectly by
determining an increase or decrease in the amount of product, e.g.
autoinducer, e.g. homoserine lactone autoinducer,produced or the
amount of substrate consumed from a previously determined standard
or baseline level.
[0022] The language "autoinducer synthase molecule" is intended to
include molecules, e.g. proteins, which catalyze or facilitate the
synthesis of autoinducer compounds, e.g. in the quorum sensing
system of bacteria. It is also intended to include active portions
of the autoinducer synthase protein contained in the protein or in
fragments or portions of the protein. The language "active
portions" is intended to include the portion of the autoinducer
synthase protein which contains the homoserine lactone binding
site. Table 1 contains a list of exemplary autoinducer synthase
proteins of the quorum sensing systems of various gram-negative
bacteria.
[0023] Autoinducer synthase molecules can be obtained from
naturally occurring sources, e.g., by purifying cellular extracts,
can be chemically synthesized or can be recombinantly produced.
Recombinantly produced autoinducer synthase molecules can have the
amino acid sequence of a naturally occurring form of the
autoinducer synthase protein. They can also have a similar amino
acid sequence which include mutations such as substitutions and
deletions (including truncation) of a naturally occurring form of
the protein. Autoinducer synthase molecules can also include
compounds which are structurally similar to the structures of
naturally occurring autoinducer synthase proteins.
[0024] The language "purified autoinducer synthase molecule" is
intended to include an autoinducer synthase molecule which has been
as least partially isolated from other types of molecules with
which it is typically associated in its naturally occurring
environment, e.g., cellular preparations. In preferred embodiments,
autoinducer synthase molecules are in preparations in which at
least about 50% of the molecules are autoinducer synthase
molecules. In particularly preferred embodiments, autoinducer
synthase molecules are in preparations in which at least about 95%
of the molecules are autoinducer synthase molecules.
[0025] The language "biologically active autoinducer synthase
molecule" is intended to include an autoinducer synthase molecule
which can function as a catalyst or facilitate the synthesis of an
autoinducer molecule. In preferred embodiments, the autoinducer
synthase molecule is substantially free of contaminants, e.g.,
other cellular contaminants. In particularly preferred embodiments,
the autoinducer synthase molecule is substantially free of
inclusion bodies.
[0026] The language "highly soluble" is art-recognized and is
intended to include an autoinducer synthase molecule which is
sufficiently soluble such that it functions within the methods
described herein, e.g., substantially free of inclusion bodies. In
preferred embodiments, the autoinducer synthase molecule is
biologically active.
[0027] In a related aspect, the invention further pertains to a
method for producing a highly active recombinant autoinducer
synthase molecule, e.g., bacterial. This method includes
introducing DNA encoding an autoinducer synthase molecule,; e.g.,
bacterial, into a host cell of the same species, such that a highly
active recombinant autoinducer synthase molecule is produced by the
host cells. The molecule can be overexpressed within this method.
In preferred embodiments, the host cells are grown at temperatures
in the range of about 15.degree. C.-45.degree. C. In preferred
embodiments, the host cells are grown at temperatures in the range
of about 25.degree. C.-35.degree. C. In a particularly preferred
embodiment, the cells are grown at about 30.degree. C.
[0028] The term "highly active" is intended to include an
autoinducer synthase molecule having a functional activity such
that it can catalyze an interaction which produces an autoinducer
product. Autoinducer synthase molecules can have functional
activity in the range of a V.sub.max of 2.1 mol of butyryl HSL
min.sup.-1 mol.sup.-1 of RhlI for butyryl ACP to 15.5 mol of
butyryl HSL min.sup.-1 mol.sup.-1 of RhlI for S-adenosyl
methionine. In general, such molecules are non-aggregated monomeric
proteins.
[0029] The term "overexpressed" is intended to include levels of a
protein produced in a recombinant host cell which are greater than
those found in a non-genetically altered cell.
[0030] The language "about [X] .degree.C." is intended to include a
range of temperatures from 3.degree. C. below the stated
temperature [X] .degree.C. to 30.degree. C. above the stated
temperature.
[0031] TraI, LuxI, RhlI are the homoserine lactone autoinducer
synthases of Agrobacterium tumefaceins, Vibrio fischeri, and
Pseudomonas aeruginosa, respectively. The term "RhlI" is intended
to include proteins which catalyze the synthesis of the homoserine
lactone autoinducer of the RhlI quorum sensing system of P.
aeruginosa, butyryl homoserine lactone. The invention also pertains
to the autoinducer synthase produced by the methods of the
invention. In preferred embodiments, the purity of the autoinducer
synthase is in the range from about 50-100%. In preferred
embodiments, the purity of the autoinducer synthase is in the range
from about 75-100%. In preferred embodiments, the purity of the
autoinducer synthase is in the range from about 85-100%. In
particularly preferred embodiments, the purity of the autoinducer
synthase is about 95%.
[0032] RhlI is also intended to include active portions of the
autoinducer synthase protein contained in the protein or in
fragments or portions of the protein. The amino acid sequence of
one RhlII protein is contained in SEQ ID NO.1 of this application.
One active portion of the RhlI protein of SEQ ID NO. 1 includes the
amino acid sequence 24-104. A second active portion of the RhlI
protein of SEQ ID NO. 1 includes the amino acid sequence 24-73. In
addition, the particular amino acids, Arginine 24, Glutamic Acid
46, Aspartic Acid 48, and Glutamic Acid 101 have been identified as
those located in highly conserved locations of RhlI.
[0033] The language "homoserine lactone substrate" is intended to
include compounds which can be used as the source of the homoserine
lactone moiety of the homoserine lactone autoinducer compounds, as
well as optically active isomers and chemically similar analogs.
The homoserine lactone moiety is shown below: 1
[0034] wherein R represents hydrogen in the homoserine lactone
substrate and the site of attachment of the acyl moiety of the
homoserine lactone autoinducer. The homoserine lactone substrate
can be obtained from naturally occurring proteins, by purifying
cellular extracts, or it can be chemically synthesized.
[0035] The primary homoserine lactone substrate for RhlI is
S-adenosyl methionine (SAM). The structure of S-adenosyl methionine
is shown below: 2
[0036] The homoserine lactone moiety is derived from the amino acid
side chain.
[0037] The language "homoserine lactone substrate binding site" is
intended to include a localized region on an autoinducer synthase
molecule which interacts, e.g., binds, with a homoserine lactone
substrate. This interaction catalyzes the reaction between a
homoserine lactone substrate and an acylated acyl carrier protein
which results in the formation of a homoserine lactone
autoinducer.
[0038] The language "acylated acyl carrier protein" is intended to
include both acylated acyl carrier proteins (ACPs) and other
compounds which are able to supply the acyl moiety to the
autoinducer such as acylated CoA. The term "acylated" is intended
to refer to acyl carrier proteins with an attached acyl moiety. The
attached acyl group can be butyryl, hexanoyl, octanoyl, or of the
following general structure including analogs and homologs known to
those skilled in the art: 3
[0039] wherein Y represents either hydrogen or an addition carbonyl
group.
[0040] The language "autoinducer compounds" is art-recognized and
is intended to include molecules, e.g., proteins which freely
diffuse across cell membranes and which activate transcription of
various factors which affect bacterial viability. Such compounds
can affect virulence and biofilm development. Autoinducer compounds
can be acylated homoserine lactones. They can be other compounds
similar to those listed in Table 1. Homoserine autoinducer
compounds are produced in vivo by the interaction of a homoserine
lactone substrate and an acylated acyl carrier protein in a
reaction catalyzed by an autoinducer synthase molecule. In isolated
form, autoinducer compounds can be obtained from naturally
occurring proteins by purifying cellular extracts, or they can be
chemically synthesized or recombinantly produced.
1TABLE 1 Summary of N-acyl homoserine lactone based regulatory
systems Regulatory Bacterial species Signal molecules.sup.a
Proteins.sup.b Target function(s) Vibrio fischeri
N-3-(oxohexanoyl)- LuxI/LuxR luxICDABEG, homoserine lactone luxR
(VAI-1) luminescence N-(octanoyl)-L-homoserine AinS/AinR.sup.c
luxICDABEG, ? lactone (VAI-2) Vibrio harveyi
N-.beta.-(hydroxybutyryl)- LuxM/LuxN- luxICDABEG, homoserine
lactone LuxO-LuxR.sup.d luminescence and (HAI-1)
polyhydroxybutyrate synthesis HAI-2 Lux?/LuxPQ- luxCDABEG
LuxO-LuxR.sup.d Pseudomonas N-3-(oxododecanyoyl)-L- LasI/LasR lasB,
lasA, aprA, toxA, aeruginosa homoserine lactone virulence factors
(PAI-1) N-(butyryl)-L-homoserine RhII/RhIR rhlAB, rhamnolipid
lactone synthesis, virulence (PAI-2) factors Pseudomonas
(PRAI).sup.e PhzI/PhzR phz, phenazine aeureofaciens biosynthesis
Agroacterium N-3-(oxooctanoyl)-L- TraI/TraR-TraM tra gens, traR, Ti
tumefaciens homoserine lactone plasmid conjugal (AAI) transfer
Erwinia carotovora VAI-1.sup.f ExpI/ExpR pel, pec, pep, subsp.
carotovora exoenzyme synthesis SCRI193 Erwinia carotovora
VAI-1.sup.f CarI/CarR cap, carbapenem subsp. carotovora antibiotic
synthesis SCC3193 Erwinia carotovora VAI-1.sup.f HsII/? pel, pec,
pep, subsp. carotovora exoenzyme synthesis 71 Erwinia stewartii
VAI-1.sup.f EsaI/EsaR wts genes, exopolysaccharide synthesis,
virulence factors Rhizobium N-(3R-hydroxy-7-cis- ?/RhiR rhiABC,
rhizosphere leguminosarum tetradecanoyl-L-homoserine genes and
stationary lactone, small bacteriocin, phase (RLAI) Enterobacter
VAI-1.sup.f EagI/EagR function unclear agglomerans Yersenia
VAI-1.sup.f YenI/YenR function unclear enterocolitica Serratia
liquifaciens N-butanoyl-L-homoserine SwrI/? swarming motility
lacton (SAI-1) N-hexanoyl-L-homoserine SwrI/? swarming motility
lacton (SAI-2) Aeromonas (AHAI).sup.e AhyI/AhyR function unclear
hydrophila Escherichia coli/?.sup.g ?/SdiA ftsQAZ, cell
division
[0041] The present invention further pertains to a method for
selecting compounds which modulate the activity of autoinducer
synthases. The method includes providing an effective amount of a
test compound, determining whether the activity of the autoinducer
synthase molecule is modulated and selecting those compounds which
modulate the activity of the autoinducer synthase molecule.
[0042] The language "autoinducer synthase modulating compounds" and
"autoinducer synthase modulators" is used interchangeably herein,
and is intended to include compounds which alter the interaction of
the substrate to the homoserine lactone binding site on the
autoinducer synthase molecule. The language "autoinducer synthase
blocking compounds" is intended to include compounds which inhibit
the binding of the homoserine lactone substrate to the homoserine
lactone binding site on the autoinducer synthase molecules. In
preferred embodiments, the autoinducer synthase blocking compounds
are thio derivatives of purine nucleotides, sinefungin, and other
related compounds.
[0043] Autoinducer synthase blocking compounds can also include
homologs or analogs of purine nucleotides, sinefungin, and other
related compounds. Autoinducer synthase blocking compounds can also
include mimics. The term "mimics" is intended to include compounds
which may not be structurally similar to autoinducer synthase
blocking compounds but mimic the activity of autoinducer synthase
blocking compounds.
[0044] The language "thio derivatives" is intended to include
compounds in which sulfur has been substituted for one or more of
the atoms originally contained in the compound. In preferred
compounds, oxygen atoms are replaced with sulfur atoms.
[0045] The language "thio derivatives of purine nucleotides" is
intended to include compounds formed by at least one substitution
of sulfur for oxygen in purine nucleotides, preferably at the 5'
position of the ribose sugar ring. It also includes any chemically
equivalent compounds, such as those resulting from substitutions,
deletions, or additions anywhere on the nucleotide, such as thio
substitutions at other places on the ribose ring or nucleic
acid.
[0046] The language "alkylated thio derivatives of purine
nucleotides" is intended to include "thio derivatives of purine
nucleotides" which have an aliphatic group bonded directly to the
sulfur which is also bound to the purine nucleotide. The aliphatic
group can be straight or branched, typically having between 1 and
22 carbon atoms. Aliphatic groups include alkyl groups, alkenyl
groups and alkynyl groups. In complex structures, the chains can be
branched or cross-linked. Alkyl groups include saturated
hydrocarbons having one or more carbon atoms, including
straight-chain alkyl groups and branched-chain alkyl groups. Such
hydrocarbon moieties may be substituted on one or more carbons
with, for example, a halogen, a hydroxyl, a thiol, an amino, an
alkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless the
number of carbons is otherwise specified, "lower aliphatic" as used
herein means an aliphatic group, as defined above (e.g., lower
alkyl, lower alkenyl, lower alkynyl), but having from one to six
carbon atoms. Representative of such lower aliphatic groups, e.g.,
lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl,
2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl,
tert-butyl, 3-thiopentyl, and the like. As used herein, the term
"amino" means --NH.sub.2; the term "nitro" means --NO.sub.2; the
term "halogen" designates --F, --Cl, --Br or --I; the term "thiol"
means SH.sub.2; and the term "hydroxyl" means --OH. Thus, the term
"alkylamino" as used herein means an alkyl group, as defined above,
having an amino group attached thereto. The term "alkylthio" refers
to an alkyl group, as defined above, having a sulphydryl group
attached thereto. The term "alkylcarboxyl" as used herein means an
alkyl group, as defined above, having a carboxyl group attached
thereto. The term "alkoxy" as used herein means an alkyl group, as
defined above, having an oxygen atom, attached thereto.
Representative alkoxy groups include methoxy, ethoxy, propoxy,
tert-butoxy and the like. The terms "alkenyl" and "alkynyl" refer
to unsaturated aliphatic groups analogous to alkyls, but which
contain at least one double or triple bond respectively. The term
"alkylated thio derivatives of purine nucleotides" is also intended
to include any homologs or analogs, which have similar autoinducer
synthase blocking activity. Preferred embodiments include
5'-methylthioadenosine, S-adenosyl homocysteine, and S-adenosyl
cysteine
[0047] The language "test compound" is intended to include
compounds which potentially modulate the activity of an autoinducer
synthase molecule. Test compounds may be purchased, chemically
synthesized or recombinantly produced. Test compounds can be
obtained from a library of diverse compounds based on a desired
activity, or alternatively they can be selected from a random
screening procedure. Preferably, the test compounds of the present
invention are molecules having a moiety which binds to the
homoserine binding site of the autoinducer synthase molecule. In
preferred embodiments the moiety is a purine nucleotide moiety. The
term "moiety" is intended to include synthetic and
naturally-occurring entities.
[0048] The term "labeled" is intended to include compounds which
are readily identifiable by, for example, radioactive labels (H3,
C14, O18, etc.) or fluorescence.
[0049] The language "an effective amount of a compound" is intended
to include the amount necessary or sufficient to produce a
modulating effect in the activity of the condition being treated.
In the case of an autoinducer synthase modulating compound, an
effective amount is the amount necessary to sufficiently promote or
inhibit the activity of the autoinducer synthase molecule such that
the amount of autoinducer produced is either decreased or
increased.
[0050] The invention pertains to a method for modulating the
formation of bacterial quorum sensing system autoinducers by
providing an effective amount of a compound capable of modulating
the binding of a substrate to the homoserine lactone substrate
binding site of the autoinducer synthase, such that the formation
of bacterial quorum sensing system autoinducers is modulated.
[0051] The language "bacterial quorum sensing system" is intended
to include the cell-to cell communication system of gram-negative
bacteria which enables population density control of gene
expression.
[0052] The language "gram-negative bacteria" is intended to include
those bacteria which stain pink when treated with Gram's Stain.
[0053] The invention pertains to a method for modulating bacterial
biofilm development by providing an effective amount of a compound
capable of modulating the binding of a substrate to the homoserine
lactone substrate binding site of an autoinducer synthase, such
that the development of the bacterial biofilm is modulated. The
term "biofilm" is intended to include biological films that develop
and persist at interfaces in aqueous environments. Biofilms are
composed of microorganisms embedded in an organic gelatinous
structure composed of one or more matrix polymers which are
secreted by the resident microorganisms.
[0054] The language "biofilm development" is intended to include
the formation growth, and modification of the bacterial colonies
contained with the biofilm structures as well as the synthesis and
maintenance of the exopolysaccharide matrix of the biofilm
structures.
[0055] The invention pertains to a method of inhibiting the
infectivity of a pathogenic bacteria in a subject comprising
administering a therapeutically effective amount of an autoinducer
synthase blocker, such that the infectivity of a pathogenic
bacteria is inhibited.
[0056] The language "inhibiting the infectivity" is intended to
include any mitigation of the ability of a pathogenic bacteria to
either initiate a disease state within a subject or to sustain a
disease state within a subject.
[0057] The language "pathogenic bacteria" is intended to include
any bacteria capable of initiating a disease state within a subject
or sustaining a disease state within a subject. Examples of such
bacteria include gram-negative bacteria, e.g. P. aeruginosa.
[0058] The invention also pertains to a method of treating a
subject for a state associated with autoinducer synthesis by
administering an effective amount of an autoinducer synthase
blocker, such that treatment occurs.
[0059] The language "state associated with autoinducer synthesis"
is intended to include states or conditions in which autoinducer
synthesis causes or increases symptoms which can be detrimental to
a subject. Examples of such states include those associated with
biofilm development, e.g., bacterial infections.
[0060] The invention also pertains to a method of treating an
immunocompromised subject by administering a therapeutically
effective amount of an autoinducer synthase blocking compound to
the subject such that treatment occurs.
[0061] The language "immunocompromised subject" is art-recognized
and is intended to include subjects having an immune system which
is compromised, at least in part. For example, the subject can be
immunocompromised due to a genetic disorder, disease or drugs that
inhibit the immune response. The compromise of the immune system
can be temporary or permanent. An immunocompromised subject
includes individuals who are afflicted with HIV or cystic fibrosis,
or who are taking corticosteroids or immunosuppressive agents.
[0062] The phrase "therapeutically-effective amount" as used herein
means an amount of a autoinducer synthase modulator, or composition
comprising such a compound which is effective for the autoinducer
synthase modulator to produce its intended function, e.g., the
modulation of the synthesis of autoinducers. The effective amount
can vary depending on such factors as the type of activity being
modulated, the particular type of autoinducer synthase molecule,
the size of the subject, or the severity of the state being treated
or the activity. One of ordinary skill in the art would be able to
study the aforementioned factors and make the determination
regarding the effective amount of the autoinducer synthase
modulator without undue experimentation.
[0063] The terms "subject" or "subjects" as used herein, means a
living animal, susceptible to the conditions or state described
herein. Examples include reptiles, rodents, horses, sheep, cattle,
dogs, cats, gorillas, and humans. It also includes healthy animals
and those suffering from diseases characterized by the infection of
pathogenic bacteria.
[0064] The invention pertains to methods of treating a subject for
a disease state associated with biofilm development by
administering a therapeutically effective amount of an autoinducer
synthase blocking compound, such that biofilm development is
inhibited.
[0065] The language "disease state associated with biofilm
development" is intended to include diseases characterized by the
growth of bacterial biofilms. In preferred embodiments the disease
state is a bacterial infection.
[0066] The phrase "pharmaceutically acceptable" is employed herein
to refer to those autoinducer synthase modulators, compositions
containing such compounds, and/or dosage forms which are, within
the scope of sound medical judgment, suitable for use in treating
human beings and animals without excessive toxicity, irritation,
allergic response, or other problem or complication, commensurate
with a reasonable benefit/risk ratio.
[0067] Autoinducer synthase modulators of the present invention can
exist in free form or, where appropriate, in salt form.
Pharmaceutically acceptable salts and their preparation are
well-known to those of skill in the art. The pharmaceutically
acceptable salts of such compounds include the conventional
non-toxic salts or the quaternary ammonium salts of such compounds
which are formed, for example, from inorganic or organic acids of
bases.
[0068] The compounds of the invention may form hydrates or
solvates. It is known to those of skill in the art that charged
compounds form hydrated species when lyophilized with water, or
form solvated species when concentrated in a solution with an
appropriate organic solvent.
[0069] This invention also relates to pharmaceutical compositions
comprising a therapeutically (or prophylactically) effective amount
of the autoinducer synthase modulator, and a pharmaceutically
acceptable carrier or excipient. Carriers include e.g. saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof, and are discussed in greater detail below.
The composition, if desired, can also contain minor amounts of
wetting or emulsifying agents, or pH buffering agents. The
autoinducer synthase modulator can be a liquid solution,
suspension, emulsion, tablet, pill, capsule, sustained release
formulation, or powder. The autoinducer synthase modulator can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Formulation may involve mixing, granulating and
compressing or dissolving the ingredients as appropriate to the
desired preparation.
[0070] The pharmaceutical carrier employed may be, for example,
either a solid or liquid.
[0071] Illustrative solid carriers include lactose, terra alba,
sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate,
stearic acid and the like. A solid carrier can include one or more
substances which may also act as flavoring agents, lubricants,
solubilizers, suspending agents, fillers, glidants, compression
aids, binders or tablet-disintegrating agents; it can also be an
encapsulating material. In powders, the carrier is a finely divided
solid which is in admixture with the finely divided active
ingredient. In tablets, the active ingredient is mixed with a
carrier having the necessary compression properties in suitable
proportions , and compacted in the shape and size desired. The
powders and tablets preferably contain up to 99% of the active
ingredient. Suitable solid carriers include, for example, calcium
phosphate, magnesium stearate, talc, sugars, lactose, dextrin,
starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl
cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange
resins.
[0072] Illustrative liquid carriers include syrup, peanut oil,
olive oil, water, etc. Liquid carriers are used in preparing
solutions, suspensions, emulsions, syrups, elixirs and pressurized
compositions. The active ingredient can be dissolved or suspended
in a pharmaceutically acceptable liquid carrier such as water, an
organic solvent, a mixture of both or pharmaceutically acceptable
oils or fats. The liquid carrier can contain other suitable
pharmaceutical additives such as solubilizers, emulsifiers,
buffers, preservatives, sweeteners, flavoring agents, suspending
agents, thickening agents, colors, viscosity regulators,
stabilizers or osmo-regulators. Suitable examples of liquid
carriers for oral and parenteral administration include water
(partially containing additives as above, e.g. cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g. glycols) and their derivatives, and oils (e.g. fractionated
coconut oil and arachis oil). For parenteral administration, the
carrier can also be an oily ester such as ethyl oleate and
isopropyl myristate. Sterile liquid carders are useful in sterile
liquid form compositions for parenteral administration. The liquid
carrier for pressurized compositions can be halogenated hydrocarbon
or other pharmaceutically acceptable propellant. Liquid
pharmaceutical compositions which are sterile solutions or
suspensions can be utilized by, for example, intramuscular,
intraperitoneal or subcutaneous injection. Sterile solutions can
also be administered intravenously. The autoinducer synthase
modulator can also be administered orally either in liquid or solid
composition form.
[0073] The carrier or excipient may include time delay material
well known to the art, such as glyceryl monostearate or glyceryl
distearate along or with a wax, ethylcellulose,
hydroxypropylmethylcellulose, methylmethacrylate and the like.
[0074] A wide variety of pharmaceutical forms can be employed. If a
solid carrier is used, the preparation can be tableted, placed in a
hard gelatin capsule in powder or pellet form or in the form of a
troche or lozenge. The amount of solid carrier will vary widely but
preferably will be from about 25 mg to about 1 g. If a liquid
carrier is used, the preparation will be in the form of a syrup,
emulsion, soft gelatin capsule, sterile injectable solution or
suspension in an ampule or vial or nonaqueous liquid
suspension.
[0075] To obtain a stable water soluble dosage form, a
pharmaceutically acceptable salt of a autoinducer synthase
modulator may be dissolved in an aqueous solution of an organic or
inorganic acid, such as a 0.3 M solution of succinic acid or citric
acid. Alternatively, acidic derivatives can be dissolved in
suitable basic solutions. If a soluble salt form is not available,
the compound is dissolved in a suitable cosolvent or combinations
thereof. Examples of such suitable cosolvents include, but are not
limited to, alcohol, propylene glycol, polyethylene glycol 300,
polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty
alcohols or glycerin hydroxy fatty acids esters and the like in
concentrations ranging from 0-60% of the total volume.
[0076] Various delivery systems are known and can be used to
administer the autoinducer synthase modulator, or the various
formulations thereof, including tablets, capsules, injectable
solutions, encapsulation in liposomes, microparticles,
microcapsules, etc. Methods of introduction include but are not
limited to dermal, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular
and (as is usually preferred) oral routes. The compound may be
administered by any convenient or otherwise appropriate route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. For treatment or prophylaxis of nasal, bronchial or
pulmonary conditions, preferred routes of administration are oral,
nasal or via a bronchial aerosol or nebulizer.
[0077] In certain embodiments, it may be desirable to administer
the autoinducer synthase modulator locally to an area in need of
treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application, by
injection, by means of a catheter, by means of a suppository, or by
means of a skin patch or implant, said implant being of a porous,
non-porous, or gelatinous material, including membranes, such as
sialastic membranes, or fibers.
[0078] In a specific embodiment, the autoinducer synthase modulator
is formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous administration
to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Where necessary, the composition may also include a solubilizing
agent and a local anesthetic to ease pain at the side of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients may be mixed prior to administration.
[0079] Administration to an individual of an effective amount of
the compound can also be accomplished topically by administering
the compound(s) directly to the affected area of the skin of the
individual. For this purpose, the compound is administered or
applied in a composition including a pharmacologically acceptable
topical carrier, such as a gel, an ointment, a lotion, or a cream,
which includes, without limitation, such carriers as water,
glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides,
fatty acid esters, or mineral oils.
[0080] Other topical carriers include liquid petroleum, isopropyl
palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene
monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
Other materials such as anti-oxidants, humectants, viscosity
stabilizers, and similar agents may be added as necessary.
Percutaneous penetration enhancers such as Azone may also be
included.
[0081] In addition, in certain instances, it is expected that the
compound may be disposed within devices placed upon, in, or under
the skin. Such devices include patches, implants, and injections
which release the compound into the skin, by either passive or
active release mechanisms.
[0082] Materials and methods for producing the various formulations
are known in the art and may be adapted for practicing the subject
invention.
[0083] The amount of compound which will be effective in the
treatment or prevention of a particular disorder or condition will
depend in part on the nature of the disorder or condition, and can
be determined by standard clinical techniques. In addition, in
vitro or in vivo assays may optionally be employed to help identify
optimal dosage ranges. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. The precise dosage level should be determined by the
attending physician or other health care provider and will depend
upon well known factors, including route of administration, and the
age, body weight, sex and general health of the individual; the
nature, severity and clinical stage of the disease; the use (or
not) of concomitant therapies; and the nature and extent of genetic
engineering of cells in the patient.
[0084] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infuision.
[0085] The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein mean the administration of a
autoinducer synthase modulator, drug or other material, such that
it enters the subject's system and, thus, is subject to metabolism
and other like processes, for example, subcutaneous
administration.
[0086] The invention also provides a pharmaceutical package or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceutical or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. The package also can include
instructions for using the autoinducer synthase modulator within
the methods of the invention.
[0087] This invention is further illustrated by the following
examples which in no way should be construed as being further
limiting. The contents of all references, pending patent
applications and published patent applications, cited throughout
this application (including the background section) are hereby
incorporated by reference.
EXAMPLES
Materials and Methods
[0088] Bacterial Strains, Plasmids and Growth Conditions
[0089] All cloning and basic genetic manipulations were performed
in E. coli XL1-Blue. All molecular biology enzymes were purchased
from New England Biolabs (Beverly, Mass.). The RhlI overexpression
vector, pRhlI-2, was constructed by cloning an 899 bp BamH I-Hind
III fragment (containing the rhlI gene under control of the tac
promoter and the lacUV5 Shine Dalgarno) from pRhlI-1 into BamH
I-Hind III-digested pBBR1MCS5 (Gm.sup.r). This plasmid, pBBR1MCS5,
is a broad host range vector containing the multicloning site from
pBluescript II SK.sup.+. The plasmid pRhlI-2 was then
electroporated into P. aeruginosa PAO-JP1 (PAO1 derivative;
transposon insertion in lasI; Tet.sup.r) and selected for PTSB agar
plates supplemented with Gm (25 .mu.g/ml) and Tet (50 .mu.g/ml).
The plasmid pRhlI-1A was constructed by cloning an EcoR I-Hind
III-digested 647 bp PCR product containing the rhlI gene fused to
the T7 gene 10 rbs into EcoR I-Hind III-digested pEX1.8 (Broad host
range derivative of pKK223-3, Cb.sup.r). This has an improved Shine
Dalgarno sequence and seven bases between the Shine Dalgarno and
the translational start site instead of the suboptimal 10 bases of
pKK223-3. The plasmid pECP61.5 was used in E. coli XL1-Blue for the
butyryl homoserine lactone (HSL) bioassay.
[0090] Electroporation was performed as follows a 500 .mu.l
inoculation of an overnight culture of PAO-JP1 was added to a fresh
culture of PTSB supplemented with Tet (50 .mu.g/ml). The culture
was grown at 37.degree. C. for about 5 hr to an OD.sub.600 of
approximately 0.45. This culture was spun down at 5000.times.g for
5 min and resuspended in 1/2 volume of ice-cold 300 mM sucrose. The
cells were pelleted at 4.degree. C. and resuspended in {fraction
(1/50)} volume of 300 mM sucrose. About 500 ng of plasmid DNA was
added to 100 .mu.l of cells and allowed to sit on ice for about 5
min. The cells were then electroporated (2.5 V, 25 .mu.Fd, 200
.OMEGA.), 700 .mu.l of fresh PTSB was added and the cells were
allowed to incubate at 37.degree. C. for 1 hr. The cells were then
plated out on selective media.
[0091] Purification of RhIl
[0092] Conditions were optimized for RhlI expression in PAO-JP1.
Crude cell lysates of cells grown under different conditions were
generated using sonication and run on a 12% SDS polyacrylamide gel
(SDS-PAGE). Protein concentrations were determined using Bradford
assays (Bio Rad, Hercules, Calif.). The gels were blotted onto
nitrocellulose and western analysis was conducted using RhlI
antisera. RhlI expression was found to be maximal in cells grown at
30.degree. C. and harvested at an OD.sub.600 of 0.850.
[0093] Ten liters of a PAO-JP1/pRhlI-2 was grown to an OD.sub.600
of 0.850 on PTSB broth in a 10 L Biostat B fermentor (B Braun
Biotec, Inc., St. Louis, Mo.). The cells were harvested by
centrifugation at 4.degree. C. for 10 min at 5,000.times.g. The
cells were washed once in ice cold purification buffer I (20 mM KPO
ph=7.5, 10% glycerol, 0.1 mM DL-Dithiothretol (DTT), 0.1 mM EDTA, 1
mM Phenylmethylsulfonyl fluoride (PMSF) and then centrifuged again.
The cell pellet was resuspended in 70 ml of purification buffer I
and subjected to lysis in a french pressure cell. Two passes were
made over the french pressure cell at 8,000 psi and the sample was
centrifuged at 12,000.times.g for 30 min at 4.degree. C. The
soluble fraction was centrifuged again at 74,000.times.g for 60
min. The crude extract was loaded onto a 5 ml HITRAP Q (Pharmacia,
Piscataway, N.J.) column equilibrated in purification buffer I. The
column was then washed and then subjected to a 75 ml 0-1 M NaCl
gradient in purification buffer I. The flow rate was 2 ml/min and
25, 3-ml fractions were collected. The protein from each fraction
was run on an SDS-PAGE polyacrylamide gel and the fraction
containing RhlI were detected by western analysis. Fractions
containing RhlI were pooled and buffer exchanged with 0 M NaCl
purification buffer II (same as buffer I, except pH=5.8) using a
Centriplus 10 spin column (Amicon, Beverly, Mass.). The pooled
fractions were loaded onto a HITRAP S (Pharmacia, Piscataway, N.J.)
column and the column was then washed in 0 M NaCl purification
buffer II. A 75 ml 0-1 M NaCl gradient was run at a flow rate of 2
ml/min and 25 fractions were collected. The fractions were
subjected to SDS-PAGE and western analysis. The RhlI-containing
fractions were pooled, buffer exchanged in purification buffer III
(same as buffer I, except pH=7.2) and concentrated down to 4 ml
volume. Two 2-ml fractions were loaded onto a 100 ml Superdex 75
column (Pharmacia, Piscataway, N.J.) and run at a flow rate of 0.4
ml/min in purification buffer III. RhlI eluted in two 5-ml
fractions. The RhlI containing fractions were pooled, run on an
SDS-PAGE gel, and stained with Coomassie blue and estimated to be
about 95% pure.
[0094] Determination of RhlI Substrates Using Bioassays
[0095] Purified RhiI was assayed for activity in vitro. Reactions
were conducted using purified RhlI (72 ng) in a 100 .mu.l reaction
containing 1.times. reaction buffer (20 mM Tris-Cl pH=7.8, 2 mM
DTT, 200 mM NaCl), 114 .mu.M SAM, and 40 .mu.M butyryl-ACP. The
reaction was allowed to proceed for 30 min at 37.degree. C. The
reaction was terminated by the addition of 4 .mu.l of 1 M HCl. The
reaction was then extracted twice with equal volumes of acetified
ethyl acetate. The organic phase was collected and dried down in a
13 mm culture tube with N2 gas. The material was resuspended in 500
.mu.l of A media and subjected to the RhlI bioassay as previously
described. Other substrates were tested in the assay by replacing
either SAM or butyryl-ACP. The compounds tested as possible
substrates were: butyryl-CoA (565 .mu.M), hexanoyl-ACP (43.8
.mu.M), octanoyl-ACP (44 .mu.M), butyrate (1 mM), S-adenosyl
homocysteine (650 .mu.M), S-adenosyl cysteine (400 .mu.M),
homoserine lactone (400 .mu.M), homocysteine (400 .mu.M),
homoserine (400 .mu.M), and methionine (400 .mu.M).
[0096] Radioactive Assay for in vitro RhlI Activity
[0097] To facilitate kinetic analysis of RhlI in vitro, an assay
was developed using C14-labeled SAM (Amersham, Arlington Heights,
Ill.). The labeled carbon is in the carboxyl group, and is thus
incorporated into the autoinducer product. When an aqueous solution
is extracted with acetified ethyl acetate, SAM remains in the
aqueous phase. However, acyl HSLs will partition into the organic
phase of an ethyl acetate extraction. After two extractions with
equal volumes of ethyl acetate, approximately 99% of the acyl HSL
is in the organic phase. The assay was performed in 1.5 ml
siliconized eppendorf tubes. The 100 .mu.l reactions contained:
1.times. reaction buffer buffer (20 mM Tris-Cl pH=7.8, 2 mM DTT,
200 mM NaCl), purified RhlI, and desired concentrations of acyl-ACP
and unlabeled SAM supplemented with radioactive SAM. The
concentration of radioactive SAM used regardless of the
concentration of unlabeled SAM was 4 .mu.M (1 .mu.l of 60
.mu.Ci/.mu.mole stock solution). Reactions were initiated by adding
72 ng of RhlI, allowed to proceed for 10 min at 37.degree. C. and
then terminated by addition of 4 .mu.l of 1 M HCl. The reactions
were extracted twice with equal volumes of acetified ethyl acetate
and centrifuged after each extraction for 5 min in a
microcentrifuge at 10,000.times.g. The ethyl acetate phase was then
added to 4 ml of Budget-solve (Research Products International,
Mount Prospect, Ill.) scintillation cocktail and then counted for 1
min on a Beckman LS1800 scintillation counter (Beckman Instruments,
Inc., Palo Alto, Calif.). The calculated total acyl HSL produced in
the reaction accounted for the ratio of unlabeled to labeled SAM.
This assay was used to monitor the activity of RhlI during the
different steps throughout the purification process and to
determine the kinetic characteristics of RhlI.
[0098] High Pressure Liquid Chromatography (HPLC) analysis of
reaction products were conducted by drying down ethyl acetate
extracts of in vitro reactions and solubilizing them in 250 .mu.l
of 20% methanol in water. The sample was then applied to a
Nucleosil C18 reverse phase 5 .mu.M HPLC column (Sigma, Inc., St.
Louis, Mo.) and a 20-100% methanol gradient was applied and
fractions collected as described previously. The 2-ml fractions
were collected in scintillation vials, 4 ml of Budget-solve were
added, and the fractions were counted in a scintillation counter.
Characteristic retention times of acyl HSLs were used to verify
radio-labeled reaction products. HPLC analysis was used to resolve
products of reactions containing both butyryl-ACP and hexanoyl-ACP
substrates.
[0099] Analysis of Kinetic Parameters of RhlI
[0100] To determine the kinetic constants of acyl HSL synthesis by
RhlI, radioactive in vitro assays were conducted using its
different substrates. The kinetic constants for the different
substrates were determined by plotting out initial velocity data
using Lineweaver-Burk plots. To generate the Lineweaver Burk plots,
Cleland's kinetics programs were used (G.X.H. and B.V.P.,
University of Iowa, .COPYRGT. 1993). To determine the apparent
K.sub.m of RhlI for SAM, a range of concentrations were used for
SAM (4-95 .mu.M) with butyryl-ACP remaining fixed at 58 .mu.M. The
K.sub.m of butyryl-ACP was determined by holding the SAM
concentration at 95 .mu.M and varying the butyryl-ACP (3.9-58
.mu.M). The amount of RhlI used in these reactions was 72 ng. The
K.sub.m's for hexanoyl ACP and octanoyl-ACP were determined by
holding the SAM concentration at 95 .mu.M and varying the
hexanoyl-ACP concentration (8.65-692 .mu.M) and the octanoyl-ACP
concentration (4.4-264 .mu.M). The amount of RhlI added for these
in vitro reactions were 144 ng and 1.44 .mu.g for hexanoyl-ACP and
octanoyl-ACP containing reactions, respectively. Butyryl-CoA
concentrations were varied (28-798 .mu.M) and 3.6 .mu.g of RhlI was
used to determine the K.sub.m of RhlI for butyryl-CoA.
EXAMPLE 1
Overexpression and Purification of an Autoinducer Synthase
[0101] Initially studies were conducted at temperatures ranging
from 20.degree. to 37.degree. C. to determine the optimal RhlI
overexpression conditions. Two RhlI overexpression plasmids were
tested for production of soluble RhlI. The plasmid pRhlI-2 was a
broad host range vector that has RhlI fused to the suboptimal Shine
Dalgamo of pKK223-3, whereas pRhlII-1A had the RhlI gene fused to a
the T7 early gene 10 Shine Dalgamo with optimal spacing. Initial
experiments in E. coli determined that although RhlI was expressed
at high levels from pRhlI-1A, almost all the RhlI produced was
insoluble. The RhlI expressed from pRhlI-2 was at as high of levels
as in pRhlI-1A, however only a small amount remained insoluble
(<10%) as determined by western analysis. Although expression in
P. aeruginosa was not as high as in E. coli, the solubility was
much improved. The greatest amount of soluble RhlI (.about.90% of
total produced in cells) generated was in PAO-JP1/pRhlI-2 cells
grown at 30.degree. C. At higher temperatures, RhlI was expressed
at higher level but it occurred as insoluble aggregates. This
suggested that P. aeruginosa ability to produce properly folded,
soluble protein, was sensitive to the levels of RhlI produced.
[0102] The purification process was monitored using SDS-PAGE and
western analysis, as well as using the in vitro radioactive assay
(see Material and Methods). The RhlI purification process yielded
pure (estimated to be.about.95% pure by SDS-PAGE), soluble, active
protein (see Table 2 below). Purified RhlI migrated in SDS-PAGE
gels at approximately 26 kDa, which is slightly larger than the
predicted value (.about.22 kDa). Similar observations have been
reported for other LuxI family members. RhlI eluted at about 25 kDa
on a superdex 75 size exclusion column, which would approximately
correspond to a monomer of RhlI in solution.
[0103] The overexpression of RhlI in a PAO-JP1 background yielded
3.6 mg of pure protein from 10 L of culture. Visual inspection of
SDS-PAGE gels shows significant increases in the RhlI band
intensity relative to contaminants in every purification step.
However, the specific activity of the different pooled fractions
following each step gradually increases until the final step where
it increases markedly (see Table 2 below). The calculated total
number of units increases from 1,649 in the S sepharose fraction to
7,430 in the S-75 fraction. Glycerol was added to the purified RhlI
preparation at a final concentration of 20%. The protein was then
aliquotted and stored at -70.degree. C. This protein was used in
subsequent in vitro reactions.
2TABLE 2 Purification of RhlI total total specific purification
activity protein purification activity step (units).sup.a (mg)
(x-fold) (units/mg) recovery % crude extract 18,228 840 1 21.7 100
Q sepharose 10,899 315 1.59 34.6 59.7 S sepharose 1,649 26.6 2.85
62 9.0 S 75 size 7,430 3.6 95.1 2,064 40.7 exclusion a-one unit is
defined as nanomoles of product formed per minute.
EXAMPLE 2
Examination of Autoinducer Synthase Substrates Using in vitro
Reactions Coupled with Bioassays
[0104] An initial screen for RhlI substrates was conducted by
extracting in vitro reactions with ethyl acetate and assaying dried
down extracts in the butyryl HSL bioassay. The amount of acyl HSL
synthesized was quantified by comparing activity in the rhl
bioassay to standard curves generated using either synthetic
butyryl HSL or hexanoyl HSL. The reacting using octanoyl-ACP was
tested in the Ralstonia solanacerarum bioassay, which is sensitive
to octanoyl HSL. The potential RhlI substrates that were tested are
listed in Table 3. The in vitro synthesis of acyl HSLs was
completely dependent upon RhlI. Likewise, acyl HSL synthesis did
not occur in the absence of one or both of the substrates. No
compound other than SAM served as a substrate for RhlI and as a
source of the homoserine lactone ring moiety of butyryl HSL. A very
small amount of activity was detected in the reactions containing
homocysteine. This activity is at least 150-fold less when compared
to reactions containing SAM (see Table 3 below).
[0105] There was a great deal of flexibility in the substrates that
can be used only by RhlI as a source of the acyl group. Both
butyryl-CoA and butyryl-ACP can serve as a substrate for butyryl
HSL synthesis, although butyryl-ACP appears to be the preferred
substrate. Butyrate did not have activity in vitro. Presumably the
acyl group has to be bound via a thioester linkage to the
phosphopantetheine moiety of either CoA or ACP. Hexanoyl-ACP is a
substrate for the synthesis of hexanoyl HSL by RhlI.
3TABLE 3 Compounds tested as potential RhlI substrates Activity in
butyryl Activity in radioactive Compound.sup.a HSL bioassay.sup.b
assay.sup.b butyryl-ACP 532 650 butyryl-ACP + NADPH ND 718
butyryl-CoA 38 82 butyrate -- -- B-OH butyryl-CoA ND --
hexanoyl-ACP 91 263 hexanoyl-CoA -- 4 octanoyl-ACP --.sup.c 28
octanoyl-CoA ND -- decanoyl-CoA ND -- S-adenosyl homocysteine -- ND
S-adenosyl cysteine -- ND homoserine lactone -- ND homocysteine
<2 ND homoserine -- ND methionine -- ND ND Not determined
.sup.a61 .mu.M SAM was used as a substrate in reactions testing
potential acyl group substrates. 39 .mu.M butyryl ACP was used in
reactions testing potential amino acid substrates. .sup.bthe units
of activity for the butyryl HSL bioassay and the radioactive assay
are nmoles butyryl HSL formed min.sup.-1 mg.sup.-1. .sup.cThis
reaction was tested in the R. solanacearum bioassay
EXAMPLE 3
Radioactive in vitro Assay for Activity of an Autoinducer
Synthase.
[0106] To facilitate the study of acyl HSL synthesis by RhlI, an in
vitro assay using C.sup.14-labeled SAM was developed. This assay
was initially used to monitor the synthesis of butyryl HSL using
the substrates SAM (65 .mu.M) and butyryl-ACP (39 .mu.M). The
reaction products were subjected to HPLC analysis to verify that
the radioactive component in the extracted ethyl acetate phase
corresponded to butyryl HSL. The major radioactive product
co-eluted with unlabeled butyryl HSL (FIG. 1A). This result
verified that RhlI is an autoinducer synthase requiring only
butyryl-ACP and SAM as substrates. A small peak of activity eluted
in the void volume of the column (FIG. 1A). Similar reactions
performed with hexanoyl-ACP or octanoyl-ACP produced peaks that
correspond to the retention times of cold hexanoyl-ACP or octanoyl
HSL, respectively. These reactions also had a very small peak of
activity in fraction #4. This peak is responsible for the small
amount of radioactivity present in the negative controls for the
radioactive in vitro reactions and was subtracted out of the total
counts for reactions. A reaction using butyryl-CoA as a substrate
and analyzed with HPLC demonstrated there was a single peak of
radioactivity corresponding to butyryl HSL. There was no detectable
hexanoyl HSL peak indicative of contamination of the Sigma
butyryl-CoA with hexanoyl-CoA as reported previously in other
studies.
[0107] This assay was shown to satisfy the basic criteria of
standard enzymatic assays. The reaction was linear over time and
dependent upon the concentration of the enzyme (see FIG. 1B). The
reactions remained linear for up to 90 min. using the enzyme
concentrations tested. The synthesis of hexanoyl HSL and octanoyl
HSL in this assay was also shown to be linear over time and
dependent upon the concentration of the enzyme. No product was
detectable if RhlI or either of the two substrates were omitted
(SAM or any of the acyl-CoAs or acyl-ACPs). This assay is dependent
upon radioactive SAM, therefore it is limited to testing different
acyl chain substrates.
[0108] The activities of different acylated compounds of substrates
were tested in the radioactive assay (Table 3). The most active
acyl substrates were butyryl-ACP and hexanoyl-ACP, similar to the
bioassay results. This analysis proved to be more sensitive than
the coupled in vitro reactions and bioassays. Octanoyl-ACP was
demonstrated to be a substrate of RhlI. It was not detected as a
substrate in the coupled in vitro reaction-bioassays. NADPH,
included with butyryl-ACP and SAM, was found to slightly increase
the reaction rate.
EXAMPLE 4
Kinetic Parameters of an Autoinducer Synthase Mediated Autoinducer
Synthesis.
[0109] To determine the kinetic parameters of RhlI, in vitro
radioactive assays were performed. A wide range of concentrations
for the tested compounds were assayed at a high, fixed
concentration of the other substrate. These initial velocity
studies were conducted at time points where the pseudo-first order
reactions was proceeding at a linear rate. The data was fitted to
Lineweaver-Burke plots and the kinetic constants were determined
for the following substrates: SAM, butyryl-ACP, butyryl-CoA,
hexanoyl-ACP, and octanoyl-ACP (Table 4). The K.sub.m of RhlI for
SAM was 14 .mu.M. For acyl substrates, RhlI had the lowest K.sub.m
for butyryl-ACP (6 .mu.M), although the K.sub.m for hexanoyl-ACP is
comparable (8.mu.M). The K.sub.m's for octanoyl-ACP and butyryl-CoA
were 43 .mu.M and 230 .mu.M, respectively. The maximum velocities
of the reactions (V.sub.max) with butyryl-ACP and hexanoyl-ACP are
comparable, whereas the velocities of the reactions containing
octanoyl-ACP and butyryl-CoA are at least 5-fold lower. Reactions
conducted with saturating levels (40 .mu.M) of both butyryl-ACP and
hexanoyl-ACP showed that butyryl-APC is the preferred substrate,
with butyryl HSL and hexanoyl HSL produced at a ratio of 20:1.
4TABLE 4 Kinetic parameters for different substrates Substrate
Apparent K.sub.m.sup.a Apparent V.sub.max.sup.b S-adenosyl
methionine 14 15.5 butyryl ACP 6 15.5 butyryl CoA 230 2.1 hexanoyl
ACP 8 9.6 octanoyl ACP 43 1.8 .sup.aThe unit of measurement is
.mu.M. .sup.bThe units of measurement are mol of butyryl HSL
min.sup.-1 mol.sup.-1 of RhlI.
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[0144] The entire contents of all of the aforementioned references
are expressly incorporated herein by reference.
[0145] Equivalents
[0146] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
Sequence CWU 1
1
1 1 200 PRT Pseudomonas aeruginosa 1 Met Ile Glu Phe Leu Ser Glu
Ser Leu Glu Gly Leu Ser Ala Ala Met 1 5 10 15 Ile Ala Glu Leu Gly
Arg Tyr Arg His Gln Val Phe Ile Glu Lys Leu 20 25 30 Gly Trp Asp
Val Val Ser Thr Ser Arg Val Arg Asp Gln Glu Phe Asp 35 40 45 Gln
Phe Asp His Pro Gln Thr Arg Tyr Ile Val Ala Met Gly Arg Gln 50 55
60 Gly Ile Cys Gly Cys Ala Arg Leu Leu Pro Thr Thr Asp Ala Tyr Leu
65 70 75 80 Leu Lys Glu Val Phe Ala Tyr Leu Cys Ser Glu Thr Pro Pro
Ser Asp 85 90 95 Pro Ser Val Trp Glu Leu Ser Arg Tyr Ala Ala Ser
Ala Ala Asp Asp 100 105 110 Pro Gln Leu Ala Met Lys Ile Phe Trp Ser
Ser Leu Gln Cys Ala Trp 115 120 125 Tyr Leu Gly Ala Ser Ser Val Val
Ala Val Thr Thr Thr Ala Met Glu 130 135 140 Arg Tyr Phe Val Arg Asn
Gly Val Ile Leu Gln Arg Leu Gly Pro Pro 145 150 155 160 Gln Lys Val
Lys Gly Glu Thr Leu Val Ala Ile Ser Phe Pro Ala Tyr 165 170 175 Gln
Glu Arg Gly Leu Glu Met Leu Leu Arg Tyr His Pro Glu Trp Leu 180 185
190 Gln Arg Thr Leu Ser Met Ala Val 195 200
* * * * *