Two-component system that controls bacterial membrane synthesis

BACKGROUND OF THE INVENTION

[0003]

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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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

[0010] The present invention provides a mutant Neisseria bacterium having excessive outer membrane. The bacterium may be Neisseria gonorrhoeae or Neisseria meningitidis.

[0011] 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.

[0012] 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.

[0013] 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.

DESCRIPTION OF THE DRAWINGS

[0014]
FIG. 1 shows a diagram of the bmr locus of N. gonorrhoeae (GC), N. meningitidis (NM) and N. cinerea (NC).

[0015]
FIG. 2 shows the alignment of the amino acid sequences of the BmrR open reading frames (ORF) from N. gonorrhoeae (GC) (SEQ ID NO:2, SEQ ID NO:3), N. meningitidis (NM) (SEQ ID NO:1; SEQ ID NO:10; SEQ ID NO:11) and N. cinerea (NC) (SEQ ID NO:4); and the alignment of the amino acid sequences of the BmrS ORFs from GC (SEQ ID NO:6), NM (SEQ ID NO:5), and NC (SEQ ID NO:7)

[0016]
FIG. 3 shows scanning electron micrographs of wild type and mutant N. gonorrhoeae.

[0017]
FIG. 4 shows scanning electromicrographs of wild type and mutant N. meningitidis.

DETAILED DESCRIPTION OF THE INVENTION

[0018]

Neisseria gonorrhoeae
and N. meningitidis are strictly human pathogens. Current knowledge of the pathogenesis of these organisms suggests that these bacteria encounter different environments within the host during infection. However, little is known of how Neisseria sense and respond to these different environments or what systems contribute to virulence. Many bacteria respond to changes in their environment through changes in the expression level of particular genes. Two-component regulatory systems are now recognized as significant mediators of signaling in bacteria, relaying environmental signals that produce changes in gene expression patterns. During signal transduction by a typical two-component regulator, a membrane sensor protein detects a specific environmental stimulus presumably either through a direct interaction with a ligand or through conformational changes induced by changes in environmental conditions. Such a regulatory system might be useful as a diagnostic marker of virulence in Neisseria.

[0019] However, information regarding signaling within the gonococcus is limited. Previously a two-component regulator-like system was reported in pilus gene expression, although the environmental cue to which the system responds is unknown. Since membrane blebbing has been found to be associated with virulence, it was thought that a quantitative assay of the amount of membrane would be indicative of increased virulence. In order to further investigate a possible two-component regulator-mediated signaling pathway in N. gonorrhoeae, the polymerase chain reaction (PCR) with degenerate oligonucleotide primers was used to amplify a region from the DNA-binding component of a novel N. gonorrhoeae two-component regulator.

[0020] This regulatory system is known to be present in other Neisseria species and to have a role in outer-membrane blebbing. The term “blebbing” is used to mean the bubble- or blister-like extrusion of membrane from the surface of the bacterial cell. The term “high-blebbing” is used to describe bacteria showing such extensive outer membrane formation. “Blebs” are easily separated from the outer membrane by mild physical manipulation, such as differential centrifugation.

[0021] Outer membrane bleb formation by Neisseria meningitidis and N. gonorrhoeae has long been recognized as an important factor in Neisseria infection, particularly that due to N. meningitidis. Numerous membrane blebs were found at the tissue surface during ultrastructural analysis of cervical gonorrhoea (Evans et al. J. Infect. Dis. (1977) 136:248-255) and a meningococcus recovered from the plasma of an infected patient contained multiple, long membranous protrusions typical of blebbing (Brandtzaes et al. J. Infec. Dis. (1989) 155:195-204). Lipooligosaccharide (LOS), which is a component of the blebs shed by the gonococcus and meningococcus, has major physiological effects during sepsis and meningitis caused by N. meningitidis and the plasma LOS levels are closely correlated to prognosis. Furthermore, compartmentalization of LOS production correlates with the clinical presentation in meningococcal infection. LOS levels in patients defined as having septicaemia showed high levels in plasma (median 3500 ng/ml) and low levels in cerebral spinal fluid, while with patients with meningitis, LOS was detectable in the plasma of three out of 19 patients and in the CSF in 18 of 19 patients, with median levels of 2500 ng/ml.

[0022] From these observations, it is possible that blebbing is both the indicium and the cause of pathogenicity in Neisseria. Agents that control or inhibit blebbing may be useful in therapy against Neisseria infections. However, the standard laboratory cultures of Neisseria typically show little or no blebbing after repeated laboratory passage and are therefore not useful in screening for agents that control or inhibit blebbing. Identification of the degree of blebbing of newly isolated strains from clinical samples may be an early indication of the virulence of the pathogen and whether immediate, aggressive therapy is necessary.

[0023] Factors affecting the blebbing process are poorly understood. Strains of meningococcal serogroup A, B and C release membrane blebs in the log phase of growth but not in the lag phase, suggesting some form of regulation. The identification of a putative regulatory system, the mutation of which produces clear differences in the blebbing process is potentially of great interest to improvement of therapeutic strategies for treatment of Neisseria infections in which blebbing is a serious complication.

[0024] During the course of investigation, mutation of the genes controlling blebbing was successful, and after insertion into a strain, the resultant high-blebbing bacterium is a “super blebber” that shows extensive membrane blebbing in all culture conditions. Unlike virulent, high-blebbing clinical isolates of Neisseria, these super blebber mutants, termed bmrRS, are stable and do not revert to non-blebbing strains on repeated culture. For this reason, they are particularly useful as controls to estimate the virulence of clinical samples, for the production of neisserial membranes for use as a vaccine and for screening of antibiotics targeted at virulent Neisseria.

[0025] Outer membrane proteins and LOS are known to be useful as vaccines. (See, e.g., U.S. Pat. No. 5,902,586 issued May 11, 1999 to Jennings et al.) Because the super blebbers produce two to four times as much outer membrane as do wild type strains, they are a particularly useful source of neisseria vaccine material. Thus, a novel two-component regulatory system has been identified in N. gonorrhoeae, N. meningitidis and N. cinerea. DNA sequence analysis suggested that the system may be non-functional in laboratory strains of Neisseria. However, experimental mutation of the system in each of the three species resulted in increased membrane production in the mutant compared to the wild type. This correlated with an increase in outer membrane blebbing. These data suggest that outer membrane blebbing, which is regarded as an important contributor to the pathogenesis of neisserial diseases, is regulated as part of a coordinated response to environmental cues.

[0026] The references identified in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques, and/or compositions employed herein.

[0027] The following examples are included to demonstrate the preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents, such as DNA constructs, that are both chemically and biologically equivalent may be substituted for the agents described herein while the same or equivalent results would be achieved. It will also be apparent that the techniques are not limited by the order in which the steps are carried out. All such substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit and scope of the invention as defined by the appended claims.

[0028] The present invention provides a target for control of the neisserial membrane synthesis. The present invention also provides a quantitative assay of neisserial membrane. The present invention also provides a neisserial bacterium with increased amounts of membrane, more specifically, blebs of outer membrane, which bacterium is useful as a production cell for isolation of vaccine related membrane proteins and glycolipids. The present inventions also provides a standard with which to compare clinical isolates of Neisseria spp. in order to estimate the pathogenicity of such isolates. The present invention also provides a screening assay for drugs that are specifically effective against virulent strains of Neisseria.

[0029] The invention is illustrated by the following examples.

EXAMPLE 1

Construction of the Super Blebber

[0030] A. Bacterial Strains and Plasmids

[0031] Wild type Neisseria strains used were N. gonorrhoeae 1291, N. meningitidis NMB and N. cinerea 601. Neisseria were grown on solid GC Base medium (Difco Laboratories, Detroit) supplemented with amino acids and vitamins. For growth of kantamycin resistant Neisseria, BHI agar was used (Difco) supplemented with 2.5% FCS. Liquid Neisseria cultures were grown in proteose peptone broth (proteose peptone No. 3, Difco) 15 grams per liter; soluble starch (Difco) 1 gram per liter, dibasic potassium phosphate 4 grams per liter; monobasic potassium phosphate 1 gram per liter; sodium chloride 15 grams per liter or in Morse's defined medium.

[0032]

Escherichia coli
strain XL1-Blue (Stratagene, LaJolla, Calif.) was used to maintain plasmids and was grown in liquid culture using LB broth or on solid medium using LB agar. Antibiotics were used at the following concentrations: ampicillin 100 μg/ml; kanamycin 50 μg/ml (E. coli) or 25 μg/ml (Neisseria), erythromycin 150 μg/ml (E. coli) or 1 μg/ml (Neisseria).

[0033] pBluescript (Stratagene), pUC18 (Pharmacia, Piscataway, N.J.) and pCRII.I (Invitrogen, Carlsbad, Calif.) were used as cloning vectors. PUC4K (Pharmacia) was used as a source of kanamycin cassette. Erythromycin resistance cassette was derived from pKErmC′ plasmids (Zhou, D. G. and M. A. Apicella (1996) Gene 171:133-134.)

[0034] Plasmid DNA was prepared using Qiagen plasmid DNA preparation (Qiagen Inc., Chatsworth, Calif.). Chromosomal DNA was prepared from plate grown bacteria which were scraped from the plate into 100 μl of phosphate buffered saline (PBS). Cells were then pelleted by spinning for one minute at top speed in a microfuge before resuspension in 250 μl of TNE (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA.) 25 l of 10% SDS were added followed by 25 μl of Proteinase K (25 mg/ml). The reaction was incubated overnight at 55° C. and then extracted twice with phenol and then twice with phenol/chloroform. DNA was precipitated by addition of 10 μl 3M sodium acetate pH 5.4 and 1 ml 100% ethanol, lifted from the tube on the end of a pipette tip and washed by repeated transfer to tubes containing 500 μl 70% ethanol. Washed DNA samples were air dried before resuspension in 100 μl TE RNase.

[0035] DNA sequencing was performed at the University of Iowa DNA sequencing facility using dye terminator sequencing chemistry with AmpliTaq DNA polymerase, FS enzyme (PE Applied Biosystems, Foster City, Calif.). The reactions were run on and analyzed using an Applied Biosystems Model 373A stretch fluorescent automated sequencer.

[0036] PCR was performed using Boehringer Mannheim (Philadelphia) reagents according to the protocols provided. PCRDOP was performed using degenerate oligonucleotides as described by Wren et al (FEMS Microbial Letters (1992) 99:287-291). PCR products were directly cloned using the TA cloning system (Invitrogen) according to the manufacturer's instructions.

[0037] B. Results of PCR Amplification and Cloning in N. gonorrhoeae

[0038] PCR using the degenerate oligonucleotides described by Wren produced a product of the expected size (322 bp) from a N. gonorrhoeae genomic DNA template. The product was cloned and numerous clones were analyzed through DNA sequencing. This revealed that the PCR product contained many different DNA fragments that were clearly not from two-component regulator genes, which is in contrast to the report in which the same primer sequences amplified a region of two-component systems from numerous other bacteria without this apparent non-specific effect.

[0039] A single clone of 322 bp was obtained which through analysis of its deduced amino-acid sequence clearly contained the 5′ region of the DNA-binding component of a two-component regulatory system. This clone was not identical to the pilAB system of N. gonorrhoeae.

[0040] Using the 322 bp PCR product as a probe, several genomic libraries were screened, but no positive signals were observed, although a Southern blot of N. gonorrhoeae genomic DNA identified several restriction fragments to which the probe stringently hybridized. Attempts to clone these fragments, all of which were 4 kbp or greater in size, failed. These fragments, which might have contained the entire two-component system sought, could not be cloned, suggesting that the system was toxic to E. coli when carried at high copy number. However, two Sau3AI fragments which were identified by the Southern analysis were successfully cloned and found to contain open reading frames (ORFs) that encoded for the majority of a two-component regulator, based on their deduced amino acid sequence. Successive cloning of other small genomic fragments, containing small parts of the two-component regulator, followed by reconstruction, produced a sequence contig of 3.97 kbp. that contained the two ORFs of the putative two-component regulator and partial ORFs upstream and downstream of the system. We have named this two-component system bmr (for bacterial membrane regulator, see below). The upstream ORF was homologous to a hypothetical membrane protein of Bacillus subtilis (Genbank submission number P42308) and the downstream ORF was homologous to the cytoplasmic axial filament protein CafA, of E. coli (FIG. 1.) N. gonorrhoeae bmrRS has been deposited with the American Type Culture Collection (P.O. Box 1549, Manassas, Va., 20108) under the accession number PTA-801.

[0041] Comparison of the deduced amino acid sequence of the putative two-component regulator to the amino acid sequences of the Genbank database revealed homology to numerous other two-component regulators. The highest degree of homology was to the BasRS system of E. coli and Salmonella typhimurium and the putative Bas homologue in Haemophilus influenzae. The Bas system of E. coli and S. typhimurium is involved in regulation of lipopolysaccharide (LPS) substitution by phosphate and aminoarabinose, that in the case of S. typhimurium is implicated in LPS changes during the course of infection. This degree of homology is frequently found in bacteria. Biochemical capabilities of the bacteria are modified and extended by the duplication and subsequent mutation of genes, resulting in gene products with considerable degrees of homology, but having different functions and characteristics, as is seen in the bas and bmr gene products.

[0042] An interesting feature of N. gonorrhoeae bmr was the presence of a stop-codon at the fourteenth codon position of the DNA-binding component that was predicted to terminate translation of this ORF at that Point (FIG. 2.)

[0043] C. Cloning of bmr From Other Neisseria

[0044]

N. meningitidis
is closely related to N. gonorrhoeae and N. cinerea is a non-pathogenic member of the Neisseria genus. Southern blots of genomic DNA from these two bacteria, using the N. gonorrhoeae 322 bp PCR product as a probe, identified stringently hybridizing restriction fragments in each of these organisms. Using a strategy similar to that described above, the bmr locus was cloned from N. meningitidis and N. cinerea. The predicted amino acid sequences of the three loci are highly homologous, strongly suggesting that the equivalent locus from the three species had been cloned (FIG. 2). However, in N. meningitidis, a T to G substitution altered the premature TAA stop-codon of N. gonorrhoeae to a GAA glutamate codon. In N. cinerea, the codon was also for glutamate, but read GAG. However, the N. meningitidis locus contains a 591 bp deletion compared to both N. gonorrhoeae and N. cinerea that results in deletion of the 3′ 486 bp of the putative DNA-binding component-encoding ORF, the intergenic region and the 5′ 69 bp of the putative sensor-encoding ORF (FIG. 1.) In addition, other nucleotide differences in the N. meningitidis locus immediately preceding the deletion cause frame shifts of the BmrR ORF (FIG. 2.) Thus, N. meningitidis is not predicted to encode a BmrR protein. Although the 5′ 23 codons of the sensor-encoding ORF are deleted, an alternative initiation codon may allow translation of a near-full length sensor protein in N. meningitidis. The bmr locus was amplified by PCR from numerous N. meningitidis strains, including several from serotypes A, C and W 135. The primers used (5′ CCTGTCCGTGTGTTGCAATC 3′ (SEQ ID NO:8) and 5′ CCGTTCCCGTTATCCTCCAC 3′) (SEQ ID NO:9)) amplify the region starting 166 bp upstream of the initiating codon of the deleted BmrR ORF and ending 200 bp upstream of the termination codon of the putative BmrS ORF. All of these PCR reactions produced a band of the same size as that from strain NMB (i.e., approximately 600 bp smaller than the fragment amplified from N. gonorrhoeae) (data not shown) suggesting that the deletion in bmr is common to many, if not all, N. meningitidis strains. N. meningitidis bmrRS has been deposited with the American Type Culture Collection (P.O. Box 1549, Manassas, Va., 20108) under accession number PTA-800.

[0045] An N. gonorrhoeae bmrS insertion mutant was constructed by insertion of an erthromycin resistance cassette at the site indicated in FIG. 1. An insertion mutant of N. meningitidis was constructed by insertion of a kanamycin resistance cassette within the sensor coding region (FIG. 1.) The locus was mutated in N. cinerea by deletion of an internal BsrGI fragment and insertion of a kanamycin resistance cassette in its place. This mutation deletes the 3′ 418 bp of bmrR and the 5′ 565 bp of bmrS (FIG. 1).

[0046]
FIG. 2 shows the alignment of the amino acid sequences of the BmrR and BmrS ORFs from the three Neisseria spp. The GC BmrR ORF contains a stop codon (*) at the fourteenth codon. The NM BmrR sequence contains a small deletion compared to the GC and NC sequences after the 34th codon, resulting in a frameshift (denoted by the amino acid sequence written on the line above). The amino acid sequences in frame with the N-terminal NM BmrR ORF is closed by the presence of a stop codon (*). Following the frameshift, a small insertion results in the presence of the amino acids FVPLA (SEQ ID NO:12) in the NM sequence that is absent from both GC and NC. Following this is a large deletion that removes the remainder of the BmrR ORF and the N-terminal 23 codons of the BmrS ORF. Outside of this region, the three sequences are highly homologous.

EXAMPLE 2

Characteristics of the Super Blebbers

[0047] A. Chemical Analysis of the Outer Membrane of the Super Blebbers

[0048] The effect of the bmr mutation on several parameters was investigated by comparison of the N. gonorrhoeae wild-type and the mutant. Overnight cultures (10 ml) were killed by the addition of 0.5% phenol for two hours and outer membrane enriched samples were isolated as described by Zollinger et al., International Symposium, Hanasaari, Espoo, Finland (1991). Samples were analyzed by SDS-PAGE on a 6-15% gradient gel. LOS was isolated by phenol/chloroform/petroleum ether extraction as described by Galanos et al. Eur. J. Biochem.(1969) 9:45-249 and analyzed as described by Lee et al. J. Biol. Chem. (1995) 270:271151-9. The growth rate and outer membrane protein profile of the mutant were unaltered from the wild type.

[0049] Because of the homology of the Neisseria locus to the previously characterized bas locus of E. coli and S. typhimurium and the involvement of this system in LPS modulation in those bacteria, LOS was isolated from wild type and mutant and analyzed by mass spectrometry. No difference was found, showing that despite the homology, the system functioned differently in Neisseria and those other bacteria.

[0050] B. Morphology of the Super Blebbers

[0051] For scanning electron microscopy (SEM), samples of proteose peptone broth cultures were taken directly without centrifugation or washing. Processing included treatment with 1% osmium tetroxide prior to dehydration through a graded ethanol series, with a final clearance in hexamethyldisilane (HMDS from Polysciences, Inc. Warrington, Pa.). After coating with gold-palladium, the specimens were viewed on an S-4000 Hitachi scanning electron microscope at 5kV accelerating voltage.

[0052] For transmission electron microscopy, the samples were dehydrated in a graded ethanol series prior to embedment in LR White resin (Ted Pella, Inc., Redding, Calif.) and sectioned to approximately 85 nm thickness using an ultra-microtome. Samples were counter-stained with 5% uranyl acetate for viewing with an H-7000 Hitachi transmission electron microscope at 75kV accelerating voltage.

[0053] SEM analysis revealed that the wild type and mutant bacteria differed in two ways. Wild type N. gonorrhoeae were observed to be distinct diplococci with a smooth appearance. (FIGS. 3A and 3B) However, the mutant produced large numbers of blebs. (FIGS. 3C and 3D) Shedding of outer-membrane particles (blebbing) is a characteristic of Neisseria. In these analyses the mutant bacteria were blebbing to a much higher degree than the wild type. In addition, many mutant diplococci were joined to others by membranous connections (FIGS. 3C and 3E). When viewed by TEM, the connections did not contain electron dense material of the cytoplasm, suggesting that they consist of hollow membrane tubes joining pairs of diplococci. (FIG. 3F). These connections were not observed with the wild type.

[0054] The morphological changes seen in the N. meningitidis bmr mutant are shown in FIGS 4A and 4B. As observed with the mutant N. gonorrhoeae, these mutants were also joined by membranous connections. The bmr mutant of N. cinerea showed similar changes in morphology.

[0055] C. Phospholipid Analysis

[0056] The electron micrographs suggested that the mutants had alterations in their membranes that resulted in an increase in membrane material. To test this hypothesis, wild type and mutant N. gonorrhoeae were grown in the presence of 14C-labeled acetate to label phospholipid and allow quantitation of membrane produced by the bacteria.

[0057] Bacteria were grown in 5 ml of Morse's defined medium supplemented with 4 μCi/ml 14C-labeled acetate. N. meningitidis was grown for 5 hours and N. gonorrhoeae was grown for 20 hours at 37° C. in a shaking incubator to achieve the appropriate bacterial growth. For N. gonorrhoeae, the blebs were separated from the bacteria by underlaying the cultures with 2.5 ml of 10% sucrose in balanced salt buffer in centrifuge tubes and centrifuging in a SW-40 Ti rotor (Beckman, Palo Alto, Calif.) at 10,000 rpm at 4° C. for 30 minutes. The supernatant above the sucrose cushion was transferred to fresh centrifuge tubes. The sucrose cushion was removed for scintillation counting. The supernatant was centrifuged in a SW-40 Ti rotor at 25,000 rpm (100,000×g) for 75 minutes at 4° C. to pellet blebs. The supernatant from this spin was removed for scintillation counting. The centrifuge tube was partially dried using cotton swabs.

[0058] Labeled phospholipids were isolated by extraction with 100 μl of chloroform and 200 μl of methanol added to the sample and vortexed for 2 minutes. A further 100 μl of chloroform was added followed by 30 seconds of vortexing. 100 μl of deionized water was added followed by 30 seconds of vortexing. The mixture was centrifuged at 13,000 rpm in a microfuge for ten minutes at room temperature. The upper layer was removed for scintillation counting. The lower layer was removed from below the interface pellet and centrifuged before scintillation counting.

[0059] Blebs were separated from whole cells and the phospholipids in both samples extracted. The counts present in both bleb and cell-associated phospholipids are presented in Table I.

1TABLE I14C-acetate Incorporation, Counts per MinuteN. gonorrhoeae strain 1291, wild type and bmr mutantExperiment 1Experiment 2Experiment 3Wild TypeTotal CPM7.4 × 1075.7 × 1077.1 × 107CPM in cell-5.7 × 106 4 × 1064.8 × 106associatedphospholipidsCPM in bleb-2.5 × 1048.1 × 104 5 × 104associatedphospholipidsMutantTotal CPM6.8 × 1076.7 × 1076.8 × 107CPM in cell-7.3 × 1064.8 × 1065.6 × 106associatedphospholipidsCPM in bleb-5.2 × 1043.1 × 1052.1 × 105associatedphospholipidsRatio of mutant to1.31.21.2wild type cell-associated CPMRatio of mutant to2.13.84.2wild type bleb-associated CPM

[0060] In each of these three experiments, N. gonorrhoeae 1291 bmr incorporated increased amounts of label compared to wild type. Both cell-associated and bleb-associated label was increased in the mutants. The increase in cell-associated counts was slight, which agrees with microscopy observations in which the mutant and wild type bacteria did not appear different except for the presence of connections between mutant diplococci and increased level of blebbing in the mutant. As would be expected from the morphology, mutant bleb-associated counts were between two- and four-fold higher than wild type.

[0061] Labeling experiments were also conducted using N. meningitidis. For unknown reasons, it proved difficult to obtain consistent bleb-associated counts with these bacteria. The micrographs suggested that the membrane alteration observed with these mutants did not involve the type of blebbing seen in the gonococcus, but instead involved cell-associated membrane changes. However, total membrane was increased as was seen in the N. gonorrhoeae experiments. Table II shows a comparison of total outer membrane counts from both wild type and mutant bacteria. Increased incorporation of counts in the mutants was observed.

2TABLE II14C-acetate Incorporation, Counts per MinuteWild type and bmr mutant of N. meningitidis NMBExperiment 1Experiment 2Wild-TypeTotal CPM4.0 × 1086.0 × 107Total phospholipids1.0 × 1075.4 × 106MutantTotal CPM4.0 × 1086.0 × 107Total phospholipids1.9 × 1079.7 × 106Ratio of mutant to wild-type CPM1.91.8

[0062] When larger quantities of membrane are desired, the culturing and purification may be easily scaled up.

EXAMPLE 3

Use of the Neisseria bmr Mutants to Estimate Virulence of Bacteria Recovered from Clinical Samples

[0063] Swabs from suspected Neisseria infections are cultured for meningococcus or gonococcus by the methods disclosed in Example 1. A laboratory strain of Neisseria and a super blebber are grown in the same manner as controls. Overnight cultures are examined for blebbing in the SEM as described in Example 2B.

[0064] Phospholipids are isolated as described in Example 1. Phospholipids are quantitated as described by the incorporation of radionuclide or by HPLC analysis of total phospholipids. A level of phospholipid close to that of the super blebber indicates infection with a virulent neisserial pathogen, indicating that therapy should be immediate and aggressive.

[0065] As seen in Table III, a quantitative estimate on a scale of 1 (no blebbing) to 3 (many blebs and filamentous connections between cells) and the phospholipid content that correlates with blebbing are made to determine virulence of the clinical sample.

3TABLE IIIPhospholipidBlebbingCPM*DiagnosisN. gonorrhoeae strain16 × 1041291Super blebber32 × 105Hypothetical Sample #116 × 104Not virulent,therapy notneededHypothetical Sample #221 × 105Moderatevirulence, therapyindicatedHypothetical Sample #33>1.8 × 105 Highly virulent,immediate andaggressivetherapy indicated*Phospholipid content is expressed as counts per minute 14C-acetate incorporation into phospholipid fraction.

EXAMPLE 4

Use of Super Blebbers to Screen Biocidal or Biostatic Agents

[0066] A ninety-six well culture is set up with N. gonorrhoeae Strain 1291 and the N. gonorrhoeae super blebber, and the N. meningitidis strain NMB and the N. meningitidis super blebber. Dilutions of the potentially selective antibiotics are added to respective wells. An antibiotic is considered selective if it inhibits the mutant organisms that show excessive blebbing as indicated by no growth in the super blebber chambers at lower concentrations than those that inhibit growth in the strains 1291 and/or NMB.

[0067] An compound may affect a specific property of the bacterium without outright killing of the organism. Such a compound may be particularly useful in that it does not disturb the normal microflora of a patient while inhibiting the specific property that renders a pathogen virulent. It would be particularly useful to identify a compound that targets the Neisseria blebbing process. A ninety-six well culture is set up with a recently isolated, high-blebbing Neisseria that shows excessive outer membrane formation as indicated by blebbing. Dilutions of the potentially selective antibiotics are added to respective wells. An antibiotic is considered selective if it inhibits the blebbing at lower concentrations that those that inhibit growth. Such an antibiotic may be considered to have converted the high-blebbing, virulent bacterium to a non-virulent form.

[0068] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the previous disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be readily appreciated by those of skill in the art that many variations and modifications may be applied to the compositions and methods and used in the steps or in the sequence of steps of the methods described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents, such as vectors, primers and bacterial strains, that are both chemically and physiologically equivalent may be substituted for the agents described herein while the same or similar results would be achieved. All such similar variations and modifications apparent to those of skill in the art are deemed to be within the scope and concept of the invention defined by the appended claims.

[0069] All cited publications, patents, and patent applications are incorporated by reference herein, as though individually incorporated by reference.

	Number	Date	Country
Parent	09439226	Nov 1999	US
Child	10288986	Nov 2002	US

Two-component system that controls bacterial membrane synthesis

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