Type-4 pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria meningitidis is considered to be a crucial early event in neisserial infections. In addition to the principal pilus subunit (pilin or PilE), both pathogens produce low quantities of a phase-variable PilC protein which is implicated in pilus biogenesis and pilus-mediated epithelial cell adherence. The identity, however, of the pilus adhesin has remained obscure. Here we describe the isolation of a PilC protein from a gonococcal overproducing strain and demonstrate its specific interaction with human epithelial cells. Our results are consistent with the cell and species tropisms of neisserial infections. Binding of PilC effectively competes with pilus-mediated, but not Opa-mediated, attachment of N. gonorrhoeae and of N. meningitidis, indicating that both pathogens interact with identical or very similar epithelial cell receptors. Immunogold electron microscopy using antisera raised against purified PilC and synthetic peptides locates PilC at the tip of gonococcal pili. PilC thus represents an essential pilus-associated adhesin, providing a rationale for selective protection against neisserial infections.
The Gram-negative bacterial pathogen Neisseria gonorrhoeae is naturally competent for transformation with species-related DNA. We show here that two phasevariable pilus-associated proteins, the major pilus subunit (pilin, or PilE) and PilC, a factor known to function in the assembly and adherence of gonococcal pili, are essential for transformation competence. The PilE and PilC proteins are necessary for the conversion of linearized plasmid DNA carrying the Neisseria-specific DNA uptake signal into a DNase-resistant form. The biogenesis of typical pilus fibers is neither essential nor sufficient for this process. DNA uptake deficiency of defined piliated pilCI,2 double mutants can be complemented by expression of a cloned pilC2 gene in trans. The PilC defect can also be restored by the addition ofpurified PilC protein, or better, pili containing PilC protein, to the mutant gonococci. Our data suggest that the two phasevariable Pil proteins act on the bacterial cell surface and cooperate in DNA recognition and/or outer membrane translocation.Neisseria gonorrhoeae, a strictly human-specific Gram-negative bacterial pathogen, belongs to a group of microorganisms that are naturally competent for DNA transformation (1). Natural transformation competence is considered to play a role in the horizontal exchange of genetic information between species, aside from transduction and conjugation (2, 3). DNA transformation appears to be particularly relevant in the case of N. gonorrhoeae, which, based on current knowledge, is not infected by any transducing phage (or phage in general) and is devoid of genetic elements capable of mobilizing chromosomal determinants. Thus, transformation is the mechanism that most probably accounts for the evolutionary signs of horizontal exchange in N. gonorrhoeae and related species (3-9).Transforming DNA is linearized during the uptake by N. gonorrhoeae (10). Rescue of linear plasmid DNA or chromosomal segments requires both a functional RecA protein and sufficient homology in the resident DNA pool; transformation is therefore limited to species-related DNA. Another restricting factor for species-related DNA is a specific nucleotide sequence required for the uptake of DNA (11,12) MATERIALS AND METHODSConstruction of Strains and Plasmids. All gonococcal strains used in this study are derived from N. gonorrhoeae MS11 (ref. 23; see Table 1). Plasmid pHEMK40 carrying the induciblepilEF3 gene (20) and pHTR93 encoding PilC (see Fig. 1) are derivatives of the conjugative N. gonorrhoeae plasmid ptetM25.2 constructed by gene replacement using the Hermes shuttle system (13,20). Gene replacements in the gonococcal genome or ptetM25.2 were carried out via transformation selected for in the presence of erythromycin (7 ,ug/ml for ermC) or chloramphenicol (Cm; 6, 12, or 20 pLg/ml for catLow, standard, or secondary catGC mutations, respectively). The conjugation of ptetM25.2 derivatives has been described (20). E. coli plasmid pES3 is plasmid pIP100 (4) with the ermC determinant inserted into the Bg...
Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coliinteracted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.
SummaryType 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis. For class I pili found in both Neisseria gonorrhoeae and N. meningitidis, PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.
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