Physical and genetic map of the Neisseria gonorrhoeae strain MS11-N198 chromosome

A macro-restriction map of the Neisseria gonorrhoeae chromosome was constructed using the enzymes Nhel and Spel. Combinations of one-and two-dimensional electrophoresis of completely or partially digested chromosomal DNA were performed to align the restriction fragments. The chromosome is circular, with an estimated size of 2.33Mb±35kb. A genetic map was derived from the physical map; positions of over 60 defined loci were determined by Southern hybridization.     

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.     

Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification.

We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.     

The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion

In order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue-culture line HEC-1-B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA-K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformation that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our resuits indicate that invasive-ness of GC for human epithelail cells can be determined by more than one opa gene in strain MS11 A.     

Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of All opa Genes

To better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.     

Proteoglycan receptor binding by Neisseria gonorrhoeae MS11 is determined by the HV-1 region of OpaA

The interaction of the OpaA protein of Neisseria gonorrhoeae MS11mk with heparan sulphate-containing proteoglycan receptors on Chang conjunctiva epithelial cells was examined using isolated receptor binding and cell adherence/internalization assays. OpaA deletion proteins, in which the four surface-exposed regions of the protein were deleted individually, and chimeric OpaA/B proteins, in which the surface-exposed regions of the OpaA and OpaB proteins were exchanged, were expressed in N. gonorrhoeae. The recombinant deletion proteins and the chimeric OpaA/B proteins were surface exposed in the outer membrane of N. gonorrhoeae. Isolated receptor-binding assays and Chang cell infection assays with OpaA deletion variants indicated that hypervariable region 1 was essential for the interaction of N. gonorrhoeae with the proteoglycan receptor. Expression of chimeric OpaA/B proteins confirmed the central role of hypervariable region 1 in receptor binding and demonstrated that this domain alone confers the invasive biological phenotype in a non-heparan sulphate proteoglycan-binding Opa protein. The other variable regions of OpaA enhanced receptor binding in the presence of region 1, but did not constitute binding domains on their own. The results indicate that proteoglycan receptor binding results from a hierarchical interaction between the variable domains of the OpaA protein of MS11mk.     

Biochemical comparison of pili from variants of Neisseria gonorrhoeae P9

Four different types of pili produced by variants of Neisseria gonorrhoeae P9 were isolated and characterized. The pili differed in subunit molecular weight with SDS-PAGE and in subunit isoelectric point on agarose gels. Isoelectric points of the major molecular species in Triton-agarose gels of octylglucoside solubilized pili were: delta, pI 6.5; alpha, pI 6.0; beta, pI 5.3 and gamma, pI 5.5. Amino acid analyses of pili showed close homology between different types but a reduction in the content of aspartate and serine was notable in the low molecular weight delta pili; also beta and gamma maps of tryptic/chymotryptic digests of pili with several major peptides apparently common to all four pilus types.     

Silent pilin genes of Neisseria gonorrhoeae MS11 and the occurrence of related hypervariant sequences among other gonococcal isolates

Pilin variation in Neisseria gonorrhoeae depends on a family of variant genes that undergo homologous, intragenic recombination. This work focuses on the repertoire of silent variant pilin genes in strain MS11, which contribute to the extensive variation of the expressed gene copy. A total of 17 silent copies were identified, which are, to varying degrees, truncated at their 5' coding region and grouped in seven distinct pil loci. Most silent copies belong to loci pilS1, pilS2 and pilS6, which contain six, two and three silent copies, respectively, tandemly arranged. The pilS5 and pilS7 loci each contain only a single copy. In addition, two silent copies are associated with each of the two pilE loci. By comparison with sequences present in the expressed gene of other variants of the same strain, it is suggested that each silent locus is capable of donating variant sequences into the expression locus and, thus, each silent copy can contribute to the variability of pilin expression. Often, concomitant with changes in the expressed copy, the silent copies of the pilE1 locus undergo recombinations as well. Analyses of unrelated clinical isolates of N. gonorrhoeae reveal homologies of hypervariant pilin sequences with those present in strain MS11, suggesting a limited diversity of such sequences within the gonococcal population and the existence of substantial functional constraints on the variability of pilin and pili. The data further indicate that hypervariant pilin sequences are subject to horizontal exchange and interstrain recombination.     

Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains.

The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. gonorrhoeae prepilin peptidase gene, pilDNg. A single copy of the gene is shown to be present in the chromosome. Its product promotes correct cleavage of the gonococcal prepillin in Escherichia coli cells carrying both the prepilin peptidase gene and the pilin structural gene. PilDNg also cleaves prePulG, a type IV pilin-like protein of Klebsiella oxytoca. Moreover, PilDNg complements a mutation in the gene coding for the prepilin peptidase-like protein of K. oxytoca, pulO, partially restoring PulG-PulO-dependent extracellular secretion of the enzyme pullulanase. Finally, we show that genes homologous to pilDNg are present and expressed in a variety of species in the genus Neisseria, including some commensal strains.     

Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-(γ)-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.     

Autolysis of Neisseria gonorrhoeae.

Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed.     

L form of Neisseria gonorrhoeae

Roberts, Richard B. (Walter Reed Army Institute of Research, Washington, D.C.). L form of Neisseria gonorrhoeae. J. Bacteriol. 92:1609-1614. 1966.-L forms were produced by the penicillin gradient plate technique from a recently isolated strain of Neisseria gonorrhoeae. To date, these L forms have had 30 serial passages on medium containing penicillin. Stabilized L forms developed on penicillin-free medium after 10 or more passages in the presence of penicillin. Morphological characteristics of these organisms were identical to L forms of meningococci. Medium and environmental conditions necessary for optimal growth included: Brain Heart Infusion of pH 7.2 to 7.4, 1.1 to 1.3% agar, 10 to 20% sucrose, 10 to 20% horse serum, temperature at 35 to 36 C, and increased CO(2) tension (candle jar). L forms were more resistant than the parent gonococcus to penicillin, ampicillin, methicillin, cycloserine, and cephalothin, whereas both organisms had similar sensitivities to bacitracin, vancomycin, ristocetin, novobiocin, tetracycline, and erythromycin. Revertant gonococci were produced on penicillin-free medium from L forms which had had 1, 5, and 10 serial passages. Morphology and fermentative reactions of revertant strains were identical to those of the parent gonococcus. Revertant strains produced L forms more readily than the parent organism; in fact, L forms from certain revertants did not require penicillin, but only serum and sucrose for their production and propagation on artificial medium.     

Effect of anti-pilus antisera on virulence of variants of Neisseria gonorrhoeae for cultured epithelial cells

Variants of Neisseria gonorrhoeae P9 possessing a, gamma or delta pili were shown to vary in their toxicity and virulence for human epithelial cells. Studies with antisera raised against purified pili showed that attachment and virulence were reduced to a significant degree in the presence of antisera to homologous pili. Heterologous antisera, while capable of agglutinating whole organisms, were largely ineffective in reducing attachment and cytotoxicity. Using an enzyme-linked immunosorbent assay (ELISA) system, the cross-reactivity between pili and heterologous antisera was estimated to be no more than 10-20%.