Compositions and methods for regulating bacterial pathogenesis

Details Compositions and methods for regulating bacterial pathogenesis Compositions and methods for regulating bacterial pathogenesis

1999-12-02

2004-04-13

Navarro, Mark (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023200, C536S023100, C536S023400, C514S04400A, C514S002600, C435S006120, C435S069100, C435S069700, C435S029000, C435S004000, C435S476000

Reexamination Certificate

active

06720415

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of bacterial diseases of humans and other mammals. In particular, the invention provides novel genes and signaling factors involved in inducing pathogenesis in certain bacteria, and methods for controlling such pathogenesis through manipulation of those factors and genes.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application to more fully describe the state of the art to which this invention pertains. The disclosure of each such publication is incorporated by reference herein.
The control of gene expression in response to cell density, or quorum sensing, was first described in the marine luminous bacteria
Vibrio fischeri
and
Vibrio harveyi
. This phenomenon has recently become recognized as a general mechanism for gene regulation in many Gram negative bacteria. Quorum sensing bacteria synthesize, release, and respond to specific acyl-homoserine lactone signaling molecules called autoinducers to control gene expression as a function of cell density. In all acyl-homoserine lactone quorum sensing systems described to date, except that of
V. harveyi
, the autoinducer synthase is encoded by a gene homologous to luxI of
V. fischeri
, and response to the autoinducer is mediated by a transcriptional activator protein encoded by a gene homologous to luxR of
V. fischeri
(Bassler and Silverman, in
Two component Signal Transduction
, Hoch et al., eds, Am. Soc. Microbiol. Washington D.C., pp 431-435, 1995). In contrast,
V. harveyi
has two independent density sensing systems (called Signaling Systems
1
and
2
), and each is composed of a sensor-autoinducer pair.
V. harveyi
Signaling System
1
is composed of Sensor 1 and autoinducer 1 (AI-
1
), and this autoinducer is N-(3-hydroxybutanoyl)-L-homoserine lactone (see Bassler et al., Mol. Microbiol. 9: 773-786, 1993).
V. harveyi
Signaling System
2
is composed of Sensor 2 and autoinducer 2 (AI-
2
) (Bassler et al., Mol. Microbiol. 13: 273-286, 1994). The structure of AI-
2
heretofore has not been determined, nor have the gene(s) involved in biosynthesis of AI-
2
been identified. Signaling System
1
is a highly specific system proposed to be used for intra-species communication and Signaling System
2
appears to be less species-selective, and is hypothesized to be for inter-species communication (Bassler et al., J. Bacteriol. 179: 4043-4045, 1997).
Reporter strains of
V. harveyi
have been constructed that are capable of producing light exclusively in response to AI-
1
or to AI-
2
(Bassler et al., 1993, supra; Bassler et al., 1994, supra).
V. harveyi
reporter strains have been used to demonstrate that a few species of bacteria produce stimulatory substances that mimic the action of AI-
2
(Bassler et al., 1997, supra).
Quorum sensing in
V. harveyi
, mediated by Signaling Systems
1
and
2
, triggers the organisms to bioluminesce at a certain cell density. These same signaling systems, particularly Signaling System
2
, are believed to trigger other physiological changes in
V. harveyi
and other bacteria possessing the same signaling system. Thus, it would be an advance in the art to identify and characterize the signaling factor autoinducer-
2
and the genes encoding the proteins required for its production. Such an advance would provide a means to identify a novel class of compounds useful for controlling mammalian enteric or pathogenic bacteria.
SUMMARY OF THE INVENTION
In accordance with the present invention, it has now been discovered that a variety of bacterial species, some of them mammalian pathogens, secrete an organic signaling molecule that stimulates the expression of luminescence in the
V. harveyi
Signaling System
2
bioassay. The molecule secreted by these organisms mimics
V. harveyi
AI-
2
in its physical and functional features. The production in bacteria of this novel signaling molecule is regulated by changes in environmental conditions associated with a shift from a free-living existence to a colonizing or pathogenic existence in a host organism. Thus, in addition to stimulating luminescence genes (specifically luxCDABE) in
V. harveyi
, the signaling molecule is expected to stimulate a variety of pathogenesis related genes in the bacterial species that produce it. A highly purified form of the signaling molecule is provided in the present invention. Also provided is a new class of bacterial genes involved in the biosynthesis of the signaling molecule.
According to one aspect, the present invention provides an isolated bacterial extracellular signaling factor comprising at least one molecule that is polar and uncharged, and having an approximate molecular weight of less than 1,000 kDa, wherein said factor interacts with LuxQ protein thereby inducing expression of a
Vibrio harveyi
operon comprising luminescence genes luxCDABE. In a preferred embodiment, the factor possesses a specific activity wherein about 0.1 to 1.0 mg of a preparation of the factor stimulates about a 1,000-fold increase in luminescense, as measured in a bioassay using a
V. harveyi
Sensor 2+ reporter strain. In a particularly preferred embodiment, the factor is purified in such a way that it possesses a specific activity wherein about 1 to 10 &mgr;g of a preparation of the factor stimulates about a 1,000-fold increase in luminescence, as measured in a bioassay using a
V. harveyi
Sensor 2+ reporter strain.
The signaling factor of the invention is produced by a variety of bacteria, including but not limited to:
Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgfdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes
and
Staphylococcus aureus.
In another aspect, the invention provides an isolated bacterial signaling factor having the formula:
In another aspect, the invention provides a method for identifying a compound that regulates the activity of a signaling factor by contacting the signaling factor with the compound, measuring the activity of the signaling factor in the presence of the compound and comparing the activity of the signaling factor obtained in the presence of the compound to the activity of the signaling factor obtained in the absence of the compound and identifying a compound that regulates the activity of the signaling factor.
In yet another aspect, the invention provides a method for detecting an autoinducer molecule in a sample by contacting the sample with a bacterial cell, or extract thereof, comprising biosynthetic pathways that produce a detectable amount of light in response to an exogenous autoinducer, the bacterial cell having at least two distinct alterations in gene loci that participate in autoinducer pathways, wherein a first alteration in a gene locus comprises an alteration that inhibits detection of a first autoinducer and wherein a second alteration in a gene locus comprises an alteration that inhibits production of a second autoinducer and measuring light produced by the bacterial cell, or extract thereof.
In another aspect, the invention provides a bacterial cell having at least two distinct alterations in gene loci that participate in autoinducer pathways, wherein a first alteration in a gene locus comprises an alteration that inhibits detection of a first autoinducer and wherein a second alteration in a gene locus comprises an alteration that inhibits production of a second autoinducer and wherein the cell is bioluminescent when contacted with an autoinducer.
In another aspect, the invention provides a method for identifying an autoinducer analog that regulates the activity of an autoinducer by contacting a bacterial cell, or extract thereof, comprising biosynthetic pathways which will produce a detectable amount of light i

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