Two-component system that controls bacterial membrane synthesis

Details Two-component system that controls bacterial membrane synthesis Two-component system that controls bacterial membrane synthesis

1999-11-12

2003-02-11

Tate, Christopher R. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S006120, C435S007800, C435S029000, C435S032000, C435S243000, C435S871000

Reexamination Certificate

active

06518037

ABSTRACT:

BACKGROUND OF THE INVENTION
Neisseria gonorrhoeae
(“gonococcus”) and
N. meningitidis
(“meningococcus”) are Gram-negative diplococci that are strictly human pathogens.
N. gonorrhoeae
causes primarily urethritis in males and pelvic inflammatory disease in females.
N. meningitidis
is the causative agent of middle ear infections and meningitis.
As is commonly found in other bacterial pathogens, strains of Neisseria sp. vary greatly in pathogenicity. Treatment and outcome of neisserial infections are dictated by pathogenicity. For example,
N. meningitidis
is frequently found in the throat of normal humans, where it can reside as a commensal without causing symptoms. However, virulent strains of
N. meningitidis
may cause a fulminating meningitis, resulting in brain damage or death before treatment can control the disease. Diagnostic methods to date have not been useful in distinguishing the mild, commensal
Neisseria meningitidis
from the virulent strains.
The standard method of diagnosing a bacterial disease is culturing, followed by identification by immunoreactivity, morphology, and biochemical reactions. In the case of neisserial pathogens, because of its fastidious growth requirements, the organism has often lost viability and will no longer grow in culture once isolated from the patient. U.S. Pat. No. 4,446,230 discloses a test method and bacterial strain for the laboratory diagnosis of gonorrhea. This strain can be maintained in a laboratory and will become transformed by exogenous neisserial DNA, even from a non-viable clinical sample. The transformation corrects an induced specific growth requirement, thereby permitting the strain to grow. However, this test does not distinguish virulent from non-virulent strains.
In vivo models of
N. gonorrhoeae
infection using male human volunteers have been done to elucidate the factors that contribute to bacterial virulence. However, such studies are costly and limited in scope. Recent tissue culture models of gonococcal infection have begun to define the interactions between the bacterium and the host. In brief, it is evident that the bacteria attach and invade human urethral epithelial cells, a niche that probably represents the primary site of infection during the course of urethritis. Gonococci reside and replicate within vacuoles within these cells. Infected cells can rupture or be shed into the urethral lumen, releasing gonococci to invade neighboring epithelial cells or to be excreted in the urine. Several gonococcal components, including pili, Opa proteins and lipooligosaccharide (LOS) have been implicated in the ability of gonococcus to attach to and invade host cells, and implicated in the pathogenesis of gonococcal infection.
Our current knowledge clearly suggests that the gonococcus exists within different environments during the course of infection, such as extra- and intra-cellular and vacuolar locations. It is also likely that the site of infection of
N. gonorrhoeae,
the genital tract, differs significantly between males and females. How
N. gonorrhoeae
adapts to these different conditions is not well understood. It has been observed that a gonococcus thought to be pathogenic quickly converts to a less virulent type under the conditions of culture that are used to identify the bacterium and to test for antibiotic sensitivity.
Current treatment of neisserial infections is with broad spectrum antibiotics. However, treatment with broad spectrum antibiotics leads to the disturbance of the natural microflora, leaving the patient susceptible to infections with such opportunistic pathogens as
Candida albicans
and Gardnerella. An antibiotic targeted specifically at pathogenic, rather than commensal Neisseria, would avoid this complication. It is currently difficult to test virulent clinical isolates of Neisseria to determine each strain's sensitivity to targeted-specificity antibiotics because of the difficulty of maintaining virulence during growth of the strains in culture. Therefore, such an antimicrobial sensitivity test as is disclosed in U.S. Pat. No. 5,789,173 may not be useful in determining neisserial sensitivity to antibiotics.
If the factors that convert a commensal, mildly or non-pathogenic
Neisseria meningitidis
or
N. gonorrhoeae
into a pathogenic, invasive bacterium were known, it would be possible to use the identification of such factors as an aid for the diagnosis and therapy of neisserial disease. There is a need to determine these so that they can be used as a target in screening bacteriostatic or bacteriocidal drugs that are selectively effective against virulent Neisseria.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a mutant Neisseria bacterium having excessive outer membrane. The bacterium may be
Neisseria gonorrhoeae
or
Neisseria meningitidis.
The present invention also provides a method of identifying a pathogenic bacterium comprising culturing under identical conditions 1) a mutant Neisseria bacterium having excessive outer membrane, 2) a wild type Neisseria and 3) a clinical sample taken from a patient suspected of having a neisserial infection; examining the extent of blebbing in each cultured bacterium; and comparing the extent of blebbing of the clinical sample to that to the bacterium having excessive outer membrane and to that of the wild type Neisseria so as to determine the pathogenicity of the clinical sample.
The present invention further provides a method of identifying an inhibitor compound of virulent Neisseria providing a culture containing a bacterium having a mutation which results in blebbing of the outer membrane and the test inhibitor compound, and providing a culture containing a wild type bacterium having no blebbing and the test inhibitor; and comparing the growth of the mutant bacterium to that of the wild type in order to identify an inhibitor compound that inhibits the growth of the mutant to a greater degree than the inhibitor compound inhibits the growth of the wild type.
The present invention further provides individual, isolated proteins coded for or otherwise under the control of the two-component system, the presence of which quickly and easily determined and is indicative of virulence.


REFERENCES:
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