U.S. patent application number 11/030569 was filed with the patent office on 2005-11-10 for autoinducer molecule.
This patent application is currently assigned to University of Iowa. Invention is credited to Eberhard, Anatol, Gray, Kendall M., Greenberg, Everett P., Iglewski, Barbara H., Passador, Luciano, Pearson, James P., Tucker, Kenneth D..
Application Number | 20050250841 11/030569 |
Document ID | / |
Family ID | 34860228 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250841 |
Kind Code |
A1 |
Pearson, James P. ; et
al. |
November 10, 2005 |
Autoinducer molecule
Abstract
Autoinducer molecules, e.g., N-(3-oxododecanoyl)homoserine
lactone, for Pseudomonas aeruginosa are described. The molecules
regulate gene expression in the bacterium. Therapeutic compositions
and therapeutic methods involving analogs and/or inhibitors of the
autoinducer molecules also are described. The molecules are useful
for treating or preventing infection by Pseudomonas aeruginosa.
Inventors: |
Pearson, James P.;
(Cambridge, MA) ; Gray, Kendall M.; (Iowa City,
IA) ; Passador, Luciano; (Rochester, NY) ;
Tucker, Kenneth D.; (Germantown, MD) ; Eberhard,
Anatol; (Brooktondale, NY) ; Iglewski, Barbara
H.; (Fairport, NY) ; Greenberg, Everett P.;
(Iowa City, IA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
University of Iowa
University of Rochester
Ithaca College
|
Family ID: |
34860228 |
Appl. No.: |
11/030569 |
Filed: |
January 5, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11030569 |
Jan 5, 2005 |
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09541873 |
Apr 3, 2000 |
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6936447 |
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09541873 |
Apr 3, 2000 |
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08456864 |
Jun 1, 1995 |
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6057288 |
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08456864 |
Jun 1, 1995 |
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08104487 |
Aug 9, 1993 |
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5591872 |
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Current U.S.
Class: |
514/424 ;
514/432; 514/445; 514/460; 514/471 |
Current CPC
Class: |
C12N 1/38 20130101; A61K
31/366 20130101; Y02A 50/30 20180101; Y02A 50/473 20180101; Y10S
435/874 20130101; A61K 31/382 20130101; A61K 31/4015 20130101; A61K
31/381 20130101; Y10S 435/875 20130101 |
Class at
Publication: |
514/424 ;
514/460; 514/471; 514/432; 514/445 |
International
Class: |
A61K 031/4015; A61K
031/366; A61K 031/382; A61K 031/381 |
Goverment Interests
[0002] This research was supported by grants and fellowships from
the Cystic Fibrosis Foundation, Office of Naval Research
(N00014-80-6570), National Institute of Allergy and Infectious
Diseases (33713), and National Science Foundation (DIR9017262).
Claims
1. (canceled)
2. An autoinducer molecule comprising a molecule of the formula:
7wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and R is
a C.sub.11-C.sub.14 acyl moiety that may be substituted or a moiety
having at least seven members containing a ring structure that may
be substituted; the molecule being able to regulate the activity of
the LasR protein of Pseudomonas aeruginosa.
3-4. (canceled)
5. The autoinducer molecule of claims 2 wherein R is a C.sub.12
acyl moiety.
6-7. (canceled)
8. The autoinducer molecule of claim 2 wherein R contains a
heterocyclic ring structure.
9. The autoinducer molecule of claim 8 wherein the heterocyclic
ring structure has five to seven ring members.
10. The autoinducer molecule of claim 9 wherein the heterocyclic
ring structure contains oxygen.
11. The autoinducer molecule of claim 2 wherein R contains a
carbocyclic ring structure.
12. The autoinducer of claim 11 wherein the carbocyclic ring
structure is a fused ring system.
13. The autoinducer molecule of claim 2 wherein the molecule is
purified from the native source.
14. The autoinducer molecule of claim 13 wherein the native source
is the culture media of Pseudomonas aeruginosa.
15. The autoinducer molecule of claim 2 wherein the molecule is
synthesized by chemical means.
16. The autoinducer molecule of claim 2 wherein the molecule is an
optically active isomer.
17. The autoinducer molecule of claim 16 wherein the isomer is the
L-isomer.
18. The autoinducer molecule of claim 16 wherein the isomer is the
D-isomer.
19-20. (canceled)
21. An analog of an autoinducer molecule comprising a molecule of
the formula 8wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or
NH; and R is a C.sub.11-C.sub.14 acyl moiety that may be
substituted or a moiety having at least seven members containing a
ring structure that may be substituted; that affects the activity
of the LasR protein.
22. The analog of claim 21 wherein the analog inhibits the
autoinducer activity of the N-(3-oxododecanoyl)homoserine
lactone.
23. The analog of claim 21 wherein the analog synergistically
enhances the autoinducer activity of N-(3-oxododecanoyl)homoserine
lactone.
24. The analog of claim 21, wherein the analog is an agonist of the
LasR protein of Pseudomonas aeruginosa.
25. The analog of claim 21, wherein the analog is an antagonist of
the LasR protein of Pseudomonas aeruginosa.
26-43. (canceled)
44. A composition comprising an autoinducer molecule of the
formula: 9wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH;
and R is a C.sub.11-C.sub.14 acyl moiety that may be substituted or
a moiety having at least seven members containing a ring structure
that may be substituted; the molecule being able to regulate the
activity of the LasR protein of Pseudomonas aeruginosa; and a
pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/541,873 filed on Nov. 19, 2001, which is a
Continued Prosecution Application of Ser. No. 09/541,873 filed on
Apr. 3, 2000, which is a Continuation Application of Ser. No.
08/456,864 filed on Mar. 17, 1999, which in turn is a Continued
Prosecution Application of Ser. No. 08/456,864 filed Jun. 1, 1995,
which in turn is a Divisional Application of Ser. No. 08/104,487
filed Aug. 9, 1993.
BACKGROUND
[0003] The Gram-negative bacterium Pseudomonas aeruginosa is an
opportunistic human pathogen that causes infections in
immunocompromised hosts, and colonizes the lungs of individuals
with cystic fibrosis (Hoiby, N. (1974) Acta Patholgica Microbiol.
Scand. Sect. B. 82, 551-558; Reynolds, H. Y., Levine, A. S., Wood,
R. E., Zierdt, C. H., Dale, D. C. and Pennington, J. L. (1975) Ann.
Intern. Med. 82, 819-832). This bacterium produces a number of
extracellular virulence factors including exotoxin A, which is
encoded by the toxA gene (Iglewski, B. H. and Kabat, D. (1975)
Proc. Natl. Acad. Sci. USA. 72, 22842288; Iglewski, B. H., Sadoff,
J. C., Bjorn, M. J., and Maxwell, E. S. (1978) Proc. Natl. Acad.
Sci. USA. 75, 3211-3215), two elastolytic proteases, encoded by the
lasA and lasB genes, and an alkaline protease encoded by the aprA
gene (Morihara, K. and Homma, J. Y. (1985) in Bacterial Enzymes and
Virulence, ed. Holder, I. A. (CRC Press, Boca Raton, Fla.) pp.
41-79; Bever, R. A. and Iglewski, B. H. (1988) J. Bacteriol. 170,
4309-4313; Kessler, E. and Saffrin, M. (1988) J. Bacteriol. 170,
5241-5247).
[0004] Autoinducer molecules are capable of regulating the gene
expression of certain microorganisms. Bycroft et al. (WO92/18614)
describe a class of autoinducer molecules which includes
N-(.beta.-ketocaproyl) L-homoserine lactone and
N-(.beta.-hydroxybutyryl) homoserine lactone. All of the
exemplified autoinducer molecules of Bycroft et al. contain
C.sub.1-C.sub.7 side chains. However, autoinducer molecules with
side chains of greater length or cyclic side chains are not
exemplified by Bycroft et al.
[0005] Bycroft et al. state that Pseudomonas aeruginosa is affected
by N-(.beta.-ketocaproyl) homoserine lactone. As recently as 1993,
researchers have believed that N-(.beta.-ketocaproyl) homoserine
lactone is the autoinducer molecule of P. aeruginosa. (Stewart, G.
S. A. B. and P. Williams (1993) ASM News, 59, 241-46)
SUMMARY OF THE INVENTION
[0006] The present invention is based, in least in part, on the
discovery that the autoinducer molecule for Pseudomonas aeruginosa
is N-(3-oxododecanoyl)homoserine lactone and not
N-(.beta.-ketocaproyl)homos- erine lactone as previously believed.
Upon the discovery of this novel autoinducer molecule, it was
realized that autoinducer molecule(s) containing a fatty moiety or
a moiety having at least seven members in the R moiety of the
formula set forth below: 1
[0007] are involved in the regulation of gene expression. In the
above formula, n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and
R is a fatty hydrocarbon or acyl moiety that may be substituted or
a moiety having at least seven members containing a ring structure
that may be substituted. The present invention further pertains to
autinducer molecules of the following formula: 2
[0008] wherein X and Y are as defined above and Z.sub.1 and Z.sub.2
are independently selected from the group consisting of hydrogen,
.dbd.O, .dbd.S, and .dbd.NH.
[0009] The present invention also pertains to analogs of the
autoinducer molecule that affect the activity of the LasR protein,
e.g., inhibit the autoinducer activity or synergistically enhance
the autoinducer activity. The present invention even further
includes inhibitors of the autoinducer activity of
N-(3-oxododecanoyl)homoserine lactone. The present invention also
pertains to methods of selecting such inhibitors and analogs. These
methods involve the contact of the autoinducer molecule with the
suspected inhibitor or synergist followed by the measuring of the
ability of the treated autoinducer molecule to perform its intended
function. From these steps, it is determined whether the suspected
inhibitor or synergist inhibits or enhances the ability of the
autoinducer molecule to stimulate the activity of the selected
gene.
[0010] The present invention also pertains to therapeutic
compositions comprising an agent having the ability to inhibit the
activity of the LasR protein of Pseudomonas aeruginosa and/or
inhibit the autoinducer activity of N-(3-oxododecanoyl)homoserine
lactone and a pharmaceutically acceptable carrier. The agent can be
the analogs or inhibitors as described above and in further detail
below.
[0011] The present invention even further pertains to a method of
inhibiting the infectivity of Pseudomonas aeruginosa and a method
of treating an immunocompromised individual infected with
Pseudomonas aeruginosa. Both of these methods involve the
administration to an individual of a therapeutically effective
amount of the agents and/or therapeutic compositions described
above that inhibit the activity of the LasR protein and/or inhibit
the autoinducer activity of N-(3-oxododecanoyl)homoserine lactone.
An example of an immunocompromised individual in an individual
afflicted with cystic fibrosis.
[0012] The present invention further pertains to a culture medium
containing as an added compound an autoinducer molecule as
described and methods of controlling the expression of a gene in
bacteria or cells using the described autoinducer molecules and/or
analogs or inhibitors thereof.
[0013] The present invention also pertains to analogs of the
autoinducer molecule that inhibit the induction of virulence
factors by the autoinducer molecule or LasR. The virulence factors
include exotoxin A, elastolytic proteases, and an alkaline
protease.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows the dose-response curve for P. aeruginosa
autoinducer in ethyl acetate extracts of bacterial culture fluid.
Culture fluid extracts were of P. aeruginosa PA01 (.cndot.), P.
aeruginosa PA01-R1 (.largecircle.), E. coli with pLasI-1
(.box-solid.), and E. coli without pLasI-1 (.quadrature.). As
defined in Materials and Methods, a unit of autoinducer activity is
that amount required for half-maximal activation of the lasB
promoter in E. coli (pKDT17).
[0015] FIG. 2 shows the HPLC analysis of autoinducer extracts.
Extracted autoinducer was from an E. coli (pLasI-1) culture medium
(.largecircle.), and from a P. aeruginosa PA01 culture medium
(.cndot.). Methanol-gradient HPLC was as described in Materials and
Methods. Each fraction was 2 ml. The dashed line indicates the
methanol concentration. The percent of activity recovered in the
major peak for either bacterium was >75%. The white triangle
indicates where N-(3-oxohexanoyl)homoserine lactone elutes, and the
black triangle indicates where N-(3-oxo-octanoyl)homoseri- ne
lactone elutes.
[0016] FIG. 3 shows the dose-response curve for activity of
synthetic N-(3-oxododecanoyl)homoserine lactone in the P.
aeruginosa autoinducer bioassay.
[0017] FIG. 4 shows the known autoinducer structures. From the top,
N-(3-hydroxybutanoyl)homoserine lactone, the inducer of
luminescence in Vibrio harveyi; N-(3-oxohexanoyl)homoserine
lactone, the inducer of V. fischeri luminescence;
N-(3-oxooctanoyl)homoserine lactone, the inducer of conjugal
transfer genes in Agrobacterium tumefaciens; and
N-(3-oxododecanoyl)homoserine lactone, the P. aeruginosa
autoinducer of the present invention.
DETAILED DESCRIPTION
[0018] The present invention pertains to autoinducer molecules of
the formula: 3
[0019] wherein n is 2 or 3; Y is O, S, or NH; X is O, S, or NH; and
R is a fatty hydrocarbon or acyl moiety that may be substituted or
a moiety having at least seven members containing a ring structure
that may be substituted. The autoinducer molecule regulates the
activity of the LasR protein of Pseudomonas aeruginosa. In
addition, the present invention pertains to optically active
isomers of the autoinducer molecule. The autoinducer molecule can
be purified from the native source using conventional techniques or
can be derived synthetically by chemical means. Included in the
invention are optically active isomers of the claimed autoinducer
molecule as well as analogs of the claimed autoinducer
molecule.
[0020] The language "autoinducer molecule" is intended to include a
molecule involved in the regulation of gene expression, e.g., it
may increase or decrease gene expression, of a microorganism.
Typically, autoinducer molecules are produced by microorganisms,
such as bacteria, during metabolism. The autoinducer molecules then
regulate gene expression, for example, by combining with a
transcriptional activator protein.
[0021] The language "fatty hydrocarbon or acyl moiety" is intended
to include a long straight or branched chain moiety having seven or
more carbon atoms. For example, the fatty acyl moiety can be of the
following formula: 4
[0022] wherein n is 4 or more. The preferred fatty acyl moieties
include C.sub.7-C.sub.14 acyl moieties, more preferred are
C.sub.10-C.sub.14 acyl moieties, and most preferred is the C.sub.12
acyl moiety. Fatty hydrocarbon or acyl moieties include saturated
and unsaturated moieties as well as substituted moieties, for
example, by substituting a S for an O. Examples of fatty
hydrocarbon moieties include fatty alkyl, fatty alkenyl, and fatty
alkynyl moieties.
[0023] The language "ring structure" is intended to include
arrangements of atoms which form one or more rings. The ring
structures can contain heterocyclic ring(s), e.g., oxygen, sulfur,
or nitrogen containing, or can contain carbocyclic ring(s). The
ring structure further can be a fused ring system. Examples of ring
structures include 5 to 7 membered heterocyclic rings, napthyl, and
phenyl. The ring structures further can be substituted with groups
that do not effect the molecule's ability to perform its intended
function as described above.
[0024] The substituents on the "R" moiety are substituents which do
not detrimentally effect the molecule's ability to perform its
intended autoinducer function. Examples of such substituents
include hydrocarbon groups, e.g., lower alkyl, alkenyl and alkynyl
groups, keto groups and halogen containing substituents. Examples
of such substituents include butyl, propyl, methyl, butenyl,
propenyl and butynyl groups.
[0025] The language "able to regulate the activity" is intended to
include the activation of or an act to increase the operation of
another molecule, e.g., the LasR protein.
[0026] The language "LasR protein of Pseudomonas aeruginosa" is
intended to include the transcriptional activator proteins of the
bacteria Pseudomonas aeruginosa. LasR proteins of P. aeruginosa
include proteins encoded by the lasR gene of P. aeruginosa. The
LasR proteins are global regulators of genes involved in the
virulence of P. aeruginosa.
[0027] The language "isomer" is intended to include molecules
having the same molecular formula as the autoinducer molecule but
possessing different chemical and physical properties due to a
different arrangement of the atoms in the molecule. Isomers include
both optical isomers and structural isomers.
[0028] The language "optically active" is intended to include
molecules that have the ability to rotate a plane of polarized
light. An optically active isomer includes the L-isomer and the
D-isomer of the claimed autoinducer molecule. The L-isomer of
N-(3-oxododecanoyl)homoserine lactone is the active form. The
D-isomer shows a small amount of activity and can inhibit the
ability of the L-isomer to activate the LasR protein by attaching
to the autoinducer binding domain of the LasR protein.
[0029] The language "purified from the native source" is intended
to include an autoinducer molecule of the above formula that has
been manufactured by an organism. "Purified from the native source"
includes isolating the autoinducer molecule from the culture media
or cytoplasm of bacteria such as Pseudomonas aeruginosa using
conventional techniques.
[0030] The language "synthesized by chemical means" is intended to
include autoinducer molecules of the claimed formula that have been
made artificially outside of an organism. The invention includes
the claimed autoinducer made by a scientist in a laboratory from
chemical precursors using standard chemical synthesis techniques.
For example, the claimed autoinducer molecules can be synthesized
using the protocol of Eberhard et al. (Eberhard, A., Burlingame, A.
L., Eberhard, C., Kenyon, G. L., Nealson, K. H. and Oppenheimer,
N.J. (1981) Biochemistry 20, 2444-2449) from commercially available
precursors. The starting materials can be modified to produce the
desired end product.
[0031] The present invention further pertains to autinducer
molecules of the following formula: 5
[0032] wherein X and Y are as defined above and Z.sub.1 and Z.sub.2
are independently selected from the group consisting of hydrogen,
O, S, and NH.
[0033] The preferred autoinducer molecule of the present invention
is of the formula: 6
[0034] This autoinducer molecule is a novel chemical compound which
is at least part of the present invention. The new chemical
compound has utility as an autoinducer and also may have other
utilities. As an autoinducer, it can regulate the activity of the
transcriptional protein of Pseudomonas aeruginosa, LasR. The
chemical name of the autoinducer molecule is
N-(3-oxododecanoyl)homoserine lactone. The autoinducer molecule can
be purified from the native source or can be derived synthetically
by chemical means. Included in the invention are optically active
isomers of the claimed autoinducer molecule as well as analogs of
the claimed autoinducer molecule.
[0035] The language "analog" is intended to include molecules that
are structurally similar but not identical to the claimed
autoinducer molecule N-(3-oxododecanoyl)homoserine lactone. For
example, the length of the fatty acyl moiety can be varied
producing an analog or one of the keto groups can be removed from
the fatty acyl moiety. Analogs include autoinducer molecules that
are structurally similar to the claimed autoinducer molecule but
can inhibit rather than stimulate the activity of the LasR protein
or analogs which act synergistically to enhance the ability of the
claimed autoinducer to increase the activity of the LasR protein.
One of ordinary skill in the art would be able to select analogs
which are useful within the present invention using the selection
methods described below.
[0036] The present invention also pertains to methods of selecting
inhibitors or synergists of the autoinducer molecule,
N-(3-oxododecanoyl)homoserine lactone. The method comprises
contacting the autoinducer molecule with a suspected inhibitor or
synergist, measuring the ability of the treated autoinducer
molecule to stimulate the activity of a selected gene then
determining whether the suspected inhibitor or synergist represses
or enhances the activity of the autoinducer molecule. Actual
inhibitors and synergists of the autoinducer molecule are then
selected. For example, a suspected inhibitor can be mixed with
N-(3-oxododecanoyl)homoserine lactone and the mixture then combined
with E. coli MG4 which produces .beta.-galactosidase in the
presence of N-(3-oxododecanoyl)homoserine lactone. The amount of
.beta.-galactosidase can then be compared to a standard to
determine if the suspected inhibitor represses the ability of
N-(3-oxododecanoyl)homos- erine lactone to stimulate the production
of .beta.-galactosidase in E. coli MG4.
[0037] The language "inhibitors of the autoinducer molecule of P.
aeruginosa" is intended to include molecules that interfere with
the ability of the autoinducer molecule to stimulate the activity
of the LasR protein of P. aeruginosa. Inhibitors include molecules
that degrade or bind to N-(3-oxododecanoyl)homoserine lactone. The
inhibitors can compete with the autoinducer molecule not allowing
it to perform its intended function.
[0038] The language "synergist of the autoinducer molecule of P.
aeruginosa" is intended to include molecules that enhance the
ability of the autoinducer molecule to stimulate the LasR protein.
Synergists include molecules that bind to either
N-(3-oxododecanoyl)homoserine lactone or the LasR protein.
[0039] The present invention also pertains to methods of selecting
inhibitory and synergistic analogs of the claimed autoinducer. The
method comprises mixing a known amount of the autoinducer molecule
with a known amount of the suspected inhibitory or synergistic
analog, measuring the ability of the treated autoinducer molecule
to stimulate the activity of a selected gene then determining
whether the suspected inhibitory or synergistic analog represses or
enhances the activity of the autoinducer molecule. Actual
inhibitory or synergistic analogs of the autoinducer molecule are
then selected.
[0040] The present invention further pertains to methods of
inhibiting the infectivity of P. aeruginosa, methods for treating
an immunocompromised host infected by P. aeruginosa, e.g., a person
afflicted with cystic fibrosis, as well as therapeutic
compositions. The methods comprise administering to an individual a
therapeutically effective amount of an agent that is capable of
inhibiting the activity of the LasR protein.
[0041] The language "inhibiting the infectivity of P. aeruginosa"
is intended to include methods of affecting the ability of P.
aeruginosa to initially infect or further infect an organism. This
includes using agents that prevent the LasR protein from activating
the transcription of extracellular virulence factors such as
exotoxin A and elastolytic proteases by P. aeruginosa.
[0042] The language "agent" is intended to include molecules that
inhibit the ability of the LasR protein to activate transcription
of extracellular virulence factors. Agents include inhibitors of
N-(3-oxododecanoyl)homoserine lactone. Agents also include analogs
of N-(3-oxododecanoyl)homoserine lactone that can directly inhibit
the LasR protein of P. aeruginosa or can compete with
N-(3-oxododecanoyl)homoserin- e lactone. Inhibitory agents can be
selected using the method described above.
[0043] The language "administering a therapeutically effective
amount" is intended to include methods of giving or applying an
agent to an organism which allow the agent to perform its intended
therapeutic function. The therapeutically effective amounts of the
agent will vary according to factors such as the degree of
infection in the individual, the age, sex, and weight of the
individual, and the ability of the agent to inhibit the activity of
the LasR protein of P. aeruginosa in the individual. Dosage regima
can be adjusted to provide the optimum therapeutic response. For
example, several divided doses can be administered daily or the
dose can be proportionally reduced as indicated by the exigencies
of the therapeutic situation. Administering also includes
contacting the agent with the LasR protein outside of an organism
such as with a culture of bacteria.
[0044] The agent can be administered in a convenient manner such as
by injection (subcutaneous, intravenous, etc.), oral
administration, inhalation, transdermal application, or rectal
administration. Depending on the route of administration, the agent
can be coated with a material to protect the agent from the action
of enzymes, acids and other natural conditions which may inactivate
the agent.
[0045] The agent can also be administered parenterally or
intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
may contain a preservative to prevent the growth of
microorganisms.
[0046] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. In all cases, the
composition must be sterile and must be fluid to the extent that
easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0047] Sterile injectable solutions can be prepared by
incorporating the agent in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the agent into a sterile
vehicle which contains a basic dispersion medium and the required
other ingredients from those enumerated above.
[0048] The agent can be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The agent and other
ingredients can also be enclosed in a hard or soft shell gelatin
capsule, compressed into tablets, or incorporated directly into the
individual's diet. For oral therapeutic administration, the agent
can be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. Such compositions and
preparations should contain at least 1% by weight of active
compound. The percentage of the compositions and preparations can,
of course, be varied and can conveniently be between about 5 to
about 80% of the weight of the unit. The amount of agent in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
[0049] The tablets, troches, pills, capsules and the like can also
contain the following: a binder such as gum gragacanth, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin or a
flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring. When the dosage unit form is a capsule, it can contain,
in addition to materials of the above type, a liquid carrier.
Various other materials can be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules can be coated with shellac, sugar or both. A
syrup or elixir can contain the agent, sucrose as a sweetening
agent, methyl and propylparabens as preservatives, a dye and
flavoring such as cherry or orange flavor. Of course, any material
used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic in the amounts employed. In
addition, the agent can be incorporated into sustained-release
preparations and formulations.
[0050] The language "pharmaceutically acceptable carrier" is
intended to include solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
agent, use thereof in the therapeutic compositions and methods of
treatment is contemplated. Supplementary active compounds can also
be incorporated into the compositions.
[0051] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
individual to be treated; each unit containing a predetermined
quantity of agent is calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the novel dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the agent and the particular therapeutic effect
to be achieve, and (b) the limitations inherent in the art of
compounding such an agent for the treatment of P. aeruginosa
infection in individuals.
[0052] The principal agent is compounded for convenient and
effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in an acceptable dosage unit.
In the case of compositions containing supplementary active
ingredients, the dosages are determined by reference to the usual
dose and manner of administration of the said ingredients.
[0053] The language "an immunocompromised host" is intended to
include an organism that has an immune system that is incapable of
reacting to pathogens. The host can be immunocompromised due to a
genetic disorder, disease or drugs that inhibit immune response. An
immunocompromised host includes an individual afflicted with cystic
fibrosis or who is taking corticosteroids or immunosuppressive
agents.
[0054] The language "infected with Pseudomonas aeruginosa" is
intended to include an organism that is found to have the bacteria,
Pseudomonas aeruginosa, present in its body in an infective form.
For example, Pseudomonas aeruginosa often infects the lungs of
cystic fibrosis patients. Even a small number of Pseudomonas
aeruginosa found in an organism can constitute infection with
Pseudomonas aeruginosa.
[0055] The present invention further pertains to a culture medium
containing as an added compound N-(3-oxododecanoyl)homoserine
lactone at a concentration effective to stimulate or promote
cellular metabolism, growth or recovery. For example, the culture
medium could be used to support growth of Pseudomonas
aeruginosa.
[0056] The language "culture medium" is intended to include a
substance on which or in which cells grow. The autoinducer molecule
can be included in commercially available cell culture media.
Culture media include broths, agar, and gelatin.
[0057] The present invention also pertains to a method of
regulating the expression of a gene. The method comprises inserting
a gene into bacteria chosen for enhancement of gene expression by
an agent capable of stimulating the activity of the LasR protein
and incubating the bacteria with an agent capable of stimulating
the activity of the LasR protein. The method further can include
the steps of allowing the gene expression to reach a desired level
and then incubating the bacteria with an agent capable of
inhibiting the activity of the LasR protein.
[0058] The present invention also pertains to a method for
detecting the presence or absence of Pseudomonas aeruginosa in a
sample. The method includes the steps of obtaining a sample fluid
suspected of containing Pseudomonas aeruginosa and detecting the
presence or absence of the autoinducer molecule as an indication of
the presence or absence of Pseudomonas aeruginosa in the sample.
The presence or absence of the autoinducer molecule can be detected
using the bioassay described below.
[0059] The present invention also pertains to analogs of the
autoinducer molecule that inhibit the induction of virulence
factors by the autoinducer molecule or LasR. The virulence factors
include exotoxin A, elastolytic proteases, and an alkaline
protease.
[0060] The invention is further illustrated by the following
non-limiting examples. The contents of all of the references,
published patent applications, and issued patents cited throughout
this application are expressly incorporated by reference.
EXAMPLES
Materials and Methods
[0061] Bacterial Strains, Plasmids and Culture Conditions
[0062] The E. coli strains used were TBl (Gibco-Bethesda Research
Laboratories Life Technologies (1984) Focus 6, 4), MG4 (Railing,
G., Bodrug, S. and Linn, T. (1985) Mol. Gen. Genet. 201, 379-386),
and VJS533 (Stewart, V. J. and Paroles, J. V., Jr. (1988) J.
Bacteriol. 170, 1589-1597). The P. aeruginosa strains used were
PA01, which contains functional lasR and lasI genes, and PAO-RI,
which is a lasR-lasI-mutant derived from PAO1 (Gambello, M. J. and
Iglewski, B. H. (1991) J. Bacteriol. 173, 3000-3009). The plasmids
used were pLasI-1, a lasI expression vector (Passador, L., Cook, J.
M., Gambello, M. J., Rust, L. and Iglewski, B. H. (1993) Science
260, 1127-1130), pKDT17, which contains a lasB::lacZ reporter of
lasB promoter activity and lasR under control of the lac promoter,
and pHV200I.sup.-, which contains the V. fischeri luminescence gene
cluster with an inactive luxI. These plasmids are all ColEl
replicons containing an ampicillin-resistance marker. Construction
of pKDT17 involved cloning into SmaI-digested pUCP18 (Schweizer, H.
P. (1991) Gene 97, 109-112) an 800-bp lasR fragment from pMJG1.7
(Gambello, M. J. and Iglewski, B. H. (1991) J. Bacteriol. 173,
3000-3009), which extended from the EcoRV site 59-bp upstream of
the lasR transcriptional start to the AluI site 22 bp beyond the
lasR translational stop codon to construct pKDT11. In this plasmid,
lasR is under control of the lac promoter. An intermediate
construct containing only two PvuII sites was made by subcloning
the 800-bp fragment in pUC18 to form pKDT13. This intermediate
construct was digested with PvuII and the plac-lasR fragment was
cloned in TthIII-digested pTS400 (Brumlik, M. J. and Storey, D. G.
(1992) Molec. Microbiol. 6, 337-344). The resulting plasmid was
called pKDT17. The plasmid, pHV200.sup.- was derived from the lux
regulon-containing pHV200 (Gray, K. M. and Greenberg, E. P. (1992)
J. Bacteriol. 174, 4384-4390) by introducing a frameshift mutation
in luxI. This was accomplished by digestion of pHV200 with BglII,
filling in the single-stranded overhangs with taq polymerase and
treating with T4 DNA ligase.
[0063] For production of PAI, cultures of P. aeruginosa PAO1 or E.
coli TB1 containing pLasI-1 were grown to the late-logarithmic
phase in A medium (Maniatis, T., Fritsch, E. F., and Sambrook, J.
(1992) Molecular Cloning: A Laboratory Manual, ed. Nolan C. (Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), 2nd Ed)
supplemented with 0.4% glucose, 0.05% yeast extract, and 1 mM
MgSO.sub.4 with shaking at 37.degree. C., unless otherwise
specified. For subsequent use in autoinducer bioassays, E. coli MG4
containing pKDT17 was grown in supplemented A medium at 30.degree.
C. with shaking, and E. coli VJS533 containing pHV200I.sup.- was
grown in L broth (Silhavy, T. J., Berman, M. L. and Enquist, L. W.
(1984) Experiments with Gene Fusions (Cold Spring Harbor Lab., Cold
Spring Harbor, N.Y.), P. 217) at 30.degree. C. with shaking. For
plasmid screening and maintenance, ampicillin (100 .mu.g/ml) was
included in E. coli cultures, and carbenicillin (200 ug/ml) was
included in cultures of P. aeruginosa.
[0064] Plasmid Purification and Transformation Procedure
[0065] Plasmids were purified and manipulated (Maniatis, T.,
Fritsch, E. F., and Sambrook, J. (1992) Molecular Cloning: A
Laboratory Manual, ed. Nolan C. (Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.), 2nd Ed) as described elsewhere. The
transformation procedure used was described by Hanahan (Hanahan, D.
(1983) J. Mol. Biol. 166, 557-580).
[0066] Autoinducer Bioassays
[0067] The quantitative assay that was developed for PAI was based
on a previous report that E. coli MG4 containing a plasmid with
lasR and a lasB::lacZ transcriptional fusion showed a 25-fold
induction of .beta.-galactosidase, when grown in a medium in which
a recombinant E. coli containing lasI had been grown previously as
compared to a medium in which E. coli without lasI had been grown.
It has been found that this PAI activity could be extracted and
concentrated in ethyl acetate as described for
N-(3-oxohexanoyl)homoserine lactone, the V. fischeri autoinducer or
VAI (Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L.,
Nealson, K. H. and Oppenheimer, N.J. (1981) Biochemistry 20,
2444-2449; Nealson, K. H. (1977) Arch. Microbiol. 112, 73-79). For
the bioassay, overnight cultures of E. coli containing the
lasB-promoter reporter, pKDT 17 were diluted in supplemented A
medium to an optical density of 0.1 at 660 nm, and stored on ice.
Each bioassay consisted of 2 ml of the cell suspension plus the
test sample. After 5.5 h at 30.degree. C. with shaking,
.beta.-galactosidase activity was measured by the CHCl.sub.3-sodium
dodecyl sulfate method described by Miller (Miller, J. A. (1976)
Experiments in Molecular Genetics (Cold Spring Harbor Lab., Cold
Spring Harbor, N.Y.), pp. 352-355). As described in Example 1,
there was a linear dose response to autoinducer in this bioassay.
Without addition of autoinducer, .beta.-galactosidase activities
were 25.+-.10 Miller units, and with saturating amounts of
autoinducer, .alpha.-galactosidase activities were 1300.+-.200
Miller units. A unit of P. aeruginosa autoinducer activity is
defined as that amount required to achieve 1/2-saturation in the
bioassay.
[0068] The quantitative assay for VAI was based on those described
elsewhere (Kaplan, H. B. and Greenberg, E. P. (1985) J. Bacteriol.
163, 1210-1214; Bainton, N. J., Bycroft, B. W., Chhabra, S. R.,
Stead, P., Gledhill, L., Hill, P. J., Rees, C. E. D., Winson, M.
K., Salmond, G. P. C., Stewart, G. S. A. B. and Williams, P.
(1992)Gene 116, 87-91; Nealson, K H. (1977) Arch. Microbiol. 112,
73-79). E. coli VJS533 containing pHV200I.sup.- was used to test
for V. fischeri autoinducer activity. The plasmid, pHV200I.sup.-
contains all of the V. fischeri genes necessary for autoinducible
luminescence in E. coli, however, the gene encoding autoinducer
synthase, luxI, is inactivated such that E. coli containing
pHV200I.sup.- is not luminescent without addition of VAI. Overnight
cultures of E. coli containing pHV200I.sup.- were diluted to an
optical density of 0.01 at 660 nm in an assay medium consisting of
0.05% tryptone, 0.03% glycerol, 100 mM NaCl, 50 mM MgSO.sub.4, and
10 mM potassium phosphate, pH 7. Each bioassay consisted of 1 ml of
the cell suspension plus the test sample. After 3 hours at room
temperature, luminescence was measured by using a Beckman LS 1800
Scintillation Counter that was set for single photon counting.
Synthetic VAI (Kaplan, N. B., Eberhard, A., Widrig, C. and
Greenberg, E. P. (1985) J. Radiolabelled Cmpds. and Pharmaceut. 22,
387-395) was used to construct a standard curve. A unit of activity
is defined as that amount required to achieve a half-maximal
response [equivalent to approximately 25 nM
N-(3-oxohexanoyl)homoserine lactone].
[0069] Purification of the P. aeruginosa Autoinducer Produced by E.
coli Containing pLasI-1
[0070] The procedure for PAI purification was based on that
described previously for purification of VAI (Eberhard, A.,
Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H. and
Oppenheimer, N. J. (1981) Biochemistry 20, 2444-2449). Cells and
culture fluid were separated by centrifugation (10,000.times.g for
10 min. at 4.degree. C.). The culture fluid was then passed through
a 0.2 .mu.m pore-size filter, and the filtered material was
extracted twice with equal volumes of ethyl acetate plus 0.1
ml/liter glacial acetic acid. The combined extracts were pooled,
water was removed with magnesium sulfate, and the ethyl acetate was
removed by rotary evaporation at 40-45.degree. C. The residue was
dissolved in 6 ml of ethyl acetate. The ethyl acetate was removed
by rotary evaporation and the residue was then extracted with 5 ml
of ethanol. The ethanol solution was was dried by rotary
evaporation and the residue was dissolved in ethyl acetate. The
ethyl acetate was removed and the residue was extracted in 5 ml of
ethanol. This ethanol extract was dried, and dissolved in ethyl
acetate. Finally, the sample was dried and dissolved in 0.2 ml of
methanol. This sample was further purified by High Performance
Liquid Chromatography (HPLC) with a C18 reverse phase column
(0.46.times.25 cm). The P. aeruginosa autoinducer activity was
first eluted as a sharp peak at 73 to 78% methanol in a linear 20
to 100% gradient of methanol and water. Fractions constituting this
peak were pooled, dried by rotary evaporation and the residue
dissolved in ethyl acetate plus acetic acid. The ethyl acetate was
removed, the residue was dissolved in 0.1 ml of methanol and this
solution was subjected to further purification by HPLC, eluting
isocratically with 65% methanol in water. Fractions containing
autoinducer activity were dried, dissolved in ethyl acetate plus
acetic acid and stored at -20.degree. C.
[0071] Chemical Synthesis of P. aeruginosa Autoinducer
[0072] Synthesis of PAI, N-(3-oxododecanoyl)-L-homoserine lactone
was similar to that described by Eberhard et al (Eberhard, A.,
Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H. and
Oppenheimer, N. J. (1981) Biochemistry 20, 2444-2449) for synthesis
of the V. fischeri autoinducer, N-(3-oxohexanoyl)homoserine
lactone. The major difference was that ethyl 3-oxododecanoate was
used instead of ethyl 3-oxohexanoate. The ethyl 3-oxododecanoate
was prepared from decanoyl chloride and the dilithio dianion of
monoethyl hydrogen malonate as described previously (Wierenga, W.
and Skulnick, H. K. (1979) J. Org. Chem. 44, 310-311). The ethylene
glycol ketal of ethyl 3-oxododecanoate was prepared as described
for ethyl 3-oxohexanoate (Eberhard, A., Burlingame, A. L.,
Eberhard, C., Kenyon, G. L., Nealson, K. H. and Oppenheimer, N. J.
(1981) Biochemistry 20, 2444-2449) except that Dowex-50 sulfonic
acid cation exchange resin was used in place of p-toluene sulfonic
acid as described by Goswami et al (Goswami, A., Beale, J. M., Jr.,
Chapman, R. L., Miller, D. W. and Rosazza, J. P. (1987) J. Natural
Prod 50, 49-54). The sodium salt was prepared as described
(Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L.,
Nealson, K. H. and Oppenheimer, N. J. (1981) Biochemistry 20,
2444-2449). The sodium 3-oxododecanoate was incubated with
equimolar amounts of L-homoserine lactone HCl (Sigma Chemical Co.,
St. Louis, Mo.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(Aldrich, Milwaukee, Wis.), and the resulting ethylene glycol ketal
of N-(3-oxododecanoyl)-L-h- omoserine lactone was deprotected by
acid treatment (Eberhard, A., Burlingame, A. L., Eberhard, C.,
Kenyon, G. L., Nealson, K. H. and Oppenheimer, N. J. (1981)
Biochemistry 20, 2444-2449) to yield
N-(3-oxododecanoyl)-L-homoserine lactone. This compound was
purified by preparative HPLC using a 20 to 100% methanol gradient
as described above. A sharp peak of autoinducer activity eluted at
73 and 78% methanol, exactly where the major peak of natural
autoinducer was found to elute. The fractions containing activity
were taken to dryness by rotary evaporation, dissolved in ethyl
acetate, and this solution was stored at -20.degree. C. prior to
further analysis.
[0073] Spectra
[0074] Proton NMR was performed at the University of Iowa College
of Medicine NMR Facility on a Varian Unity 500 MHz instrument.
Infrared spectroscopy was performed on a Nicolet 205 FTIR. Chemical
ionization mass spectrometry was performed at the University of
Iowa College of Medicine Mass Spectrometry Facility on a Nermag
RIO-10C instrument with a desorption chemical ionization probe. The
reagent gas was ammonia. High-resolution fast atom bombardment was
performed at the University of Nebraska--Midwest Center for Mass
Spectrometry.
Example 1
Extraction and Purification of PAI from Culture Media
[0075] Ethyl acetate extracts of E. coli (pLasI-1) culture medium
or P. aeruginosa PAO1 culture medium contained PAI activity in an
amount equivalent to that in the culture medium prior to
extraction. Bioassays on extracts of media from cultures grown to
an equivalent optical density of 0.3 in supplemented A medium
indicated the P. aeruginosa culture and the E. coli (pLasI-1)
culture produced roughly equivalent amounts of PAI (FIG. 1).
[0076] Because it has been reported that P. aeruginosa produces the
V. fischeri autoinducer, N-(3-oxohexanoyl)homoserine lactone
(Bainton, N. J., Bycroft, B. W., Chhabra, S. R., Stead, P.,
Gledhill, L., Hill, P. J., Rees, C. E. D., Winson, M. K., Salmond,
G. P. C., Stewart, G. S. A. B. and Williams, P. (1992) Gene 116,
87-91), this compound was tested for activity as an inducer for the
lasB promoter. Also, a homolog of the VAI was tested,
N-(3-oxooctanoyl)homoserine lactone, which has recently been
reported to serve as the autoinducer in conjugal transfer gene
activation in the Gram-negative bacterium, Agrobacterium
tumefaciens, a plant pathogen (Zhang, L., Murphy, P. J., Kerr, A.
and Tate, M. (1993) Nature 362,446-448). VAI had no detectable
activity when tested in the P. aeruginosa autoinducer bioassay at
concentrations as high as 1 uM. This compound gives a maximal
response at about 50 nM in the V. fischeri autoinducer bioassay.
The A. tumefaciens autoinducer (AAI), however, did show
considerable activity (Table 1). This suggested that PAI was
N-(3-oxooctanoyl)homoserine lactone or a compound related to
it.
1TABLE 1 Influence of P. aeruginosa , V. fischeri, and A.
tumeficians Autoinducers on lasB Promoter Activity in E. coli MG4
(pKDT17) .beta.-galactosidase Autoinducer added (Miller
units).sup.1 None 26 +/- 2 50 nM N-(3-oxohexanoyl)homoserine
lactone (VAI) 33 +/- 3 500 nM N-(3-oxohexanoyl)homoserine lactone
(VAI) 34 +/- 1 50 nM N-(3-oxooctanoyl)homoserine lactone (AAI) 56
+/- 5 500 nM N-(3-oxooctanoyl)homoserine lactone (AAI) 733 +/- 50
1.0 unit P. aeruginosa autoinducer.sup.2 735 +/- 110 3.0 units P.
aeruginosa autoinducer.sup.2 1470 +/- 90 .sup.1P. aeruginosa
autoinducer assays were performed as described in Materials and
Methods. .beta.-galactosidase activity is a measure of lasB::lacZ
promoter activity. Numbers are the average of four experiments .+-.
the ranges. .sup.2The P. aeruginosa autoinducer was an ethyl
acetate extract of the culture medium in which E. coli (pLasI-1)
was grown.
[0077] When PAI from E. coli containing pLasI-1 or P. aeruginosa
PAO1 was subjected to HPLC, a single major peak of activity was
observed (FIG. 2). In the case of PAI produced by E. coli (pLasI-1)
there was a small peak of activity that eluted just after the major
peak, and with P. aeruginosa there was a small peak of activity
that eluted just prior to the major peak.
N-(3-oxooctanoyl)homoserine eluted well ahead of the major peak of
PAI (FIG. 2). This showed that PAI is not
N-(3-oxooctanoyl)homoserine lactone. The V. fischeri autoinducer,
N-(3-oxohexanoyl)homoserine lactone elutes ahead of
N-(3-oxooctanoyl) homoserine lactone. The chromatographic behavior
of the PAI, together with the finding that AAI shows activity in
the bioassay for P. aeruginosa autoinducer suggests that PAI is an
N-acyl-homoserine lactone with a hydrophobic side-chain, which is
longer than that of AAI.
[0078] Because it has been reported that P. aeruginosa produces the
V. fischeri autoinducer (Bainton, N. J., Bycroft, B. W., Chhabra,
S. R., Stead, P., Gledhill, L., Hill, P. J., Rees, C. E. D.,
Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B. and
Williams, P. (1992) Gene 116, 87-91), fractions were tested from
HPLC for induction of luminescence by using the V. fischeri
autoinducer bioassay. In fact, there was a peak of activity that
eluted at the same location as synthetic
N-(3-oxohexanoyl)homoserine lactone. There was also a peak that
eluted at the same location as synthetic
N-(3-oxooctanoyl)homoserine lactone, which shows some activity with
V. fischeri (Eberhard, A., Widrig, C., MacBath, P. and Schineller
(1986) Arch. Microbiol. 146, 35-40), and, as has been shown, is an
inducer of the P. aeruginosa lasB (Table 1). However, only low
levels of these compounds were present in extracts of either P.
aeruginosa or E. coli (pLasI-1) culture media. There was no
significant VAI activity in the PAI peaks shown in FIG. 2.
Apparently, this P. aeruginosa autoinducer cannot cross-react with
the V. fischeri LuxR protein to activate the luminescence
genes.
Example 2
Analysis of Purified P. aeruginosa Autoinducer
[0079] The level of PAI activity in extracts of P. aeruginosa PAO1
medium was similar to the level in extracts of E. coli containing
pLasI-1 (FIG. 1). The autoinducer produced by E. coli containing
pLasI-1 was purified because P. aeruginosa produces a great variety
of extracellular compounds (Nicas, T. I. and Iglewski, B. H. (1985)
Can. J. Microbiol. 31, 387-392) that could complicate purification
of the autoinducer. Using the purification procedure described in
the Materials and Methods, approximately 300 .mu.g of PAI was
obtained from 3 L of culture fluid.
[0080] Analysis of the purified PAI by proton NMR in D.sub.2O at
25.degree. C. showed a spectrum that was remarkably similar to that
for VAI (Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G.
L., Nealson, K. H. and Oppenheimer, N. J. (1981) Biochemistry 20,
2444-2449) except that the integrations of the methyl triplet at
0.85 ppm, and the CH.sub.2 multiplet at 1.28 ppm indicated the
purified PAI had a longer alkyl chain than does VAI. Based on the
similarity between the proton NMR spectrum of PAI and VAI, and the
ratio of the methyl triplet and the methylene multiplet, it seemed
likely that the purified compound was N-(3-oxododecanoyl)homoserine
lactone. Chemical ionization mass spectrometry showed a strong
quasimolecular (M+H).sup.+ ion with an m/z of 298. This is
consistent with the conclusion from the proton NMR analysis that
the compound was N-(3-oxododecanoyl)homoserine lactone. The
chemical composition was confirmed by high-resolution fast atom
bombardment, which more precisely established the mass of the
purified compound. The m/z of the (M+H).sup.+ was 298.2018. This
corresponded to a chemical composition of C.sub.16H.sub.27NO.sub.4.
This is the composition of N-(3-oxododecanoyl)homoserine
lactone.
Example 3
Analysis of Synthetic N-(3-oxododecanoyl)homoserine lactone
[0081] As a confirmation of the conclusion that the purified PAI
was N-(3-oxododecanoyl)homoserine lactone, this compound was
synthesized. The synthetic compound had chromatographic and
spectral properties indistinguishable from those of the material
purified from culture medium and was biologically active.
[0082] The dose response in a P. aeruginosa autoinducer bioassay
indicates the half-maximal response occurs at 3 to 5 nM
N-(3-oxododecanoyl) L-homoserine lactone (FIG. 3). This is in the
range found for the V. fischeri (Kaplan, H. B. and Greenberg, E. P.
(1985) J. Bacteriol. 163, 1210-1214) and A. tumefaciens (Goswami,
A., Beale, J. M., Jr., Chapman, R. L., Miller, D. W. and Rosazza,
J. P. (1987) J. Natural Prod. 50, 49-54) autoinducer systems where
half saturations occur at about 50 nM and 5 nM, respectively.
DISCUSSION
[0083] Based on the evidence presented in the above examples, the
autoinducer, which serves in conjunction with the LasR protein to
activate a number of P. aeruginosa virulence genes, is
N-(3-oxododecanoyl)homoserine lactone. This autoinducer has a
longer acyl side chain than related autoinducers from other
bacteria (FIG. 4). It was reported elsewhere that P. aeruginosa
produces the V. fischeri autoinducer, N-(3-oxohexanoyl)homoserine
lactone (Bainton, N. J., Bycroft, B. W., Chhabra, S. R., Stead, P.,
Gledhill, L., Hill, P. J., Rees, C. E. D., Winson, M. K., Salmond,
G. P. C., Stewart, G. S. A. B. and Williams, P. (1992) Gene 116,
87-91), and it was suggested that VAI was the inducer required
together with LasR for activation of specific P. aeruginosa
virulence genes (Jones, S., Yu, B., Bainton, N. J., Birdsall, M.,
Bycroft, B. W., Chhabra, S. R., Cox, A. J. R., Golby, P., Reeves,
P. J., Stephens, S., Winson, M. K., Salmond, G. P. C., Stewart G.
S. A. B. and Williams, P. (1993) EMBO J. 12, 2477-2482). The
analysis performed in the above Examples confirmed that a compound,
which serves to induce the V. fischeri lux genes is produced by the
lasI gene product, and this compound has the behavior of V.
fischeri autoinducer in HPLC. However, relatively low levels of
this compound were synthesized by lasI-containing P. aeruginosa or
E. coli (the concentration of this compound was 0.5% of the PAI
concentration in extracts of P. aeruginosa PAO1), and neither this
compound nor authentic VAI (at concentrations as high as 500 nM)
showed P. aeruginosa autoinducer activity. In fact, the lasI gene
product appears to catalyze the synthesis of a number of related
compounds by E. coli or P. aeruginosa, including compounds that
behaved as did N-(3-oxohexanoyl)homoserine lactone and
N-(3-oxooctanoyl)homoserine lactone when subjected to
methanol-water gradient HPLC. However,
N-(3-oxododecanoyl)homoserine lactone is the most abundantly
produced of these related compounds. Based on the data in FIGS. 1
and 3, approximately 120 ng/ml of this autoinducer was present in a
culture of P. aeruginosa grown in supplemented A medium as
described. This is equivalent to 400 nM PAI, and is in about
40-fold excess of the concentrations required with LasR to fully
activate the lasB promoter. In contrast, it is estimated that
approximately 0.5 ng/ml of VAI was present in the culture of P.
aeruginosa.
[0084] AAI shows activity not only as the A. tumefaciens (Zhang,
L., Murphy, P. J., Kerr, A. and Tate, M. (1993) Nature 362,
446-448) autoinducer, but also shows activity together with the
LuxR protein in activation of V. fischeri luminescence genes
(Eberhard, A., Widrig, C., MacBath, P. and Schineller (1986) Arch.
Microbiol. 146, 35-40), and together with the LasR protein in
activation of the P. aeruginosa lasB (Table 1). The concentration
of AAI required for activity as the P. aeruginosa autoinducer was
higher than the concentration of PAI required for activity, and the
maximal response was lower (Table 1, FIG. 3). VAI,
N-(3-oxohexanoyl)homoserine lactone, did not show any detectable
activity as a P. aeruginosa autoinducer (Table 1). Apparently, the
LasR protein shows specificity with respect to the N-acyl side
chain length. This also has been shown for the V. fischeri LuxR
protein (Eberhard, A., Widrig, C., MacBath, P. and Schineller
(1986) Arch. Microbiol. 146, 35-40) and the A. tumefaciens TraR
(Zhang, L., Murphy, P. J., Kerr, A. and Tate, M. (1993) Nature 362,
446-448).
[0085] It has recently become apparent that regulatory circuits
homologous to the LuxR-LuxI regulatory circuit in V. fischeri and
the LasR-LasI circuit in P. aeruginosa are common to a number of
diverse Gram-negative bacteria. At this time, four different
autoinducer structures (including PAI as described here) are known
(FIG. 4). The luminescence genes in Vibrio harveyi are controlled
by N-(3-hydroxybutanoyl)homoserine lactone or
N-(.beta.-hydroxybutyryl)homoserine lactone (Cao, J.-G. and
Merghen, E. A. (1989) J. Biol. Chem. 264, 21670-21676; Cao, J.-G.
and Meighen, E. A. (1993) J. Bacteriol. 175, 3856-3862) but
homologs of luxI and luxR in this organism have not been
identified. A number of bacteria have been reported to produce
N-(3-oxohexanoyl)homoserine lactone, VAI (Bainton, N. J., Bycroft,
B. W., Chhabra, S. R., Stead, P., Gledhill, L., Hill, P. J., Rees,
C. E. D., Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B.
and Williams, P. (1992) Gene 116, 87-91). In one of these bacteria
at least, Erwinia carotovora, the luxI and luxR homologs expI and
expR have been identified (Pirhonen, M., Flego, D., Heikinheimo, R.
and Palva, E. T. (1993) EMBO J. 12, 2467-2476). The expI gene
directs the synthesis of an autoinducer that is required together
with the expR product for induction of extracellular protease in a
fashion reminiscent of PAI control of extracellular protease
induction in P. aeruginosa. Conjugal transfer genes in A.
tumifaciens are controlled by AAI (Zhang, L., Murphy, P. J., Kerr,
A. and Tate, M. (1993) Nature 362, 446-448) together with a
transcriptional activator encoded by traR (Piper, K. R., von
Bodman, S. B. and Farrand, S. K. (1993) Nature 362,448-450). The
gene or genes required for AAI synthesis have not yet been
described.
EQUIVALENTS
[0086] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific embodiments described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the following claims.
* * * * *