Phase variation of gonococcal pili by frameshift mutation in pilC, a novel gene for pilus assembly.

Pili prepared from Neisseria gonorrhoeae contain minor amounts of a 110 kd outer membrane protein denoted PilC. The corresponding gene exists in two copies, pilC1 and pilC2, in most strains of N.gonorrhoeae. In the piliated strain MS11(P+), only one of the genes, pilC2, was expressed. Inactivation of pilC2 by a mTnCm insertion resulted in a nonpiliated phenotype, while a mTnCm insertion in pilC1 had no effect on piliation. Expression of pilC was found to be controlled at the translational level by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Nonpilated (P-), pilin expressing colony variants that did not express detectable levels of PilC were selected; all P+ backswitchers from these P-, PilC- clones were found to be PilC+. The structural gene for pilin, pilE, was sequenced and found to be identical in one P-, PilC- and P+, PilC+ pair. Most PilC- cells were completely bald whereas the PilC+ backswitcher had 10-40 pili per cell. Thus, a turn ON and turn OFF in the expression of PilC results in gonococcal pili phase variation. These results suggest that PilC is required for pilus assembly and/or translocation across the gonococcal outer membrane.     

Structural analysis of the pilE region of Neisseria gonorrhoeae P9

We have determined the nucleotide sequence of an expressed structural pilus gene (pilE) derived from Neisseria gonorrhoeae strain P9-2. Detailed analysis of nucleotide sequences upstream from pilE revealed a silent, truncated pilin gene segment that was linked to families of DNA elements (RS1 and RS3) that have previously been identified at the major silent pilin gene locus (pilS1) and at pilE of the independently isolated N. gonorrhoeae strain MS11ms. A nucleotide sequence downstream from pilE was reminiscent of the recognition sequences of several recombinases, including Tn3 tnpR product (resolvase), suggesting a possible role for site-specific events in the recombinational modulation of pilus expression.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 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.     

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.     

Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli.

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation.     

Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue

Summary
Pili of Neisseria gonorrhoeae are correlated with Increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE , or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins, in situ binding studies of N. gonorrhoeae to formalin-fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PJIC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PiiC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid-brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus-dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.     

Adherence and autoaggregation phenotypes of a Burkholderia cenocepacia cable pilus mutant

Cable pili are unique peritrichous adherence organelles expressed by certain strains of the opportunistic human pathogen Burkholderia cenocepacia. Cable pili have been proposed to facilitate binding to human epithelial cells and mucin, and may play a role in the ability of B. cenocepacia to colonise the respiratory tract of compromised hosts. In this study, a genetic approach was undertaken to assess the role of cable pili in mediating adherence as well as bacterial cell-cell interactions. The cblA gene, encoding the major pilin subunit, was insertionally inactivated, and the resulting mutant was shown to be blocked in CblA expression and in cable pilus morphogenesis. Although non-piliated, the cblA mutant was not defective in adherence to either porcine mucin or to cultured A549 human respiratory epithelial cells. Microscopic and flow cytometric analyses of B. cenocepacia cultures revealed that cable pilus expression facilitated the formation of diffuse cell networks, whereas disruption of cable pilus biogenesis enhanced autoaggregation and the formation of compact cell aggregates. Autoaggregation was observed both in culture and during B. cenocepacia infection of A549 epithelial cell monolayers. These findings indicate that cable pilus expression plays an important role in mediating B. cenocepacia cell-cell interactions, and that both cable pilus-dependent and cable pilus-independent mechanisms may contribute to B. cenocepacia adherence to cellular and acellular surfaces.     

L-pilin variants of Neisseria gonorrhoeae MS11

Phase- and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS) and the pilin expression locus (pilE). Similarly, the non-piliated L-variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilE locus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a pilE(L) locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L-variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non-revertible and non-piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pilE can be regarded as a third mechanism of pilin variation in gonococci.     

Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.     

Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA- strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PilC1 or PilC2 proteins, representing piIC phase variants generated in the absence of RecA. The A- pilC phase variants were indistinguishable from their A+ parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PilC1 nor PilC2 is obligatory for the assembly of gonococcal pili.     

Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili

Type IV pili are a major virulence factor of the obligate human pathogen Neisseria gonorrhoeae (the gonococcus; Gc). Pili facilitate bacterial adherence to epithelial cells, but their participation in later steps of epithelial infection, particularly intracellular replication and exit, is poorly understood. Using polarized T84 cells as a model for mature mucosal epithelia, pilus dynamics in piliated, Opa-expressing Gc were examined over time. T84 infection was characterized by a several-hour delay in the growth of cell-associated bacteria and by non-directional exit of Gc, the first time these phenomena have been reported. During infection, non-piliated progeny arose stochastically from piliated progenitors. Piliated and non-piliated Gc replicated and exited from T84 cell monolayers equally well, demonstrating that piliation did not influence Gc survival during epithelial infection. The frequency with which pilin variants arose from a defined piliated progenitor during T84 cell infection was found to be sufficiently high to account for the extensive pilin variation reported during human infection. However, the repertoire of variants appearing in association with T84 cells was similar to what was seen in the absence of cells, demonstrating that polarized epithelial cells can support Gc replication without selecting for a subset of pilin variants or piliation states.     

pilQ Missense mutations have diverse effects on PilQ multimer formation, piliation, and pilus function in Neisseria gonorrhoeae

Type IV pili are required for virulence in Neisseria gonorrhoeae, as they are involved in adherence to host epithelium, twitching motility, and DNA transformation. The outer membrane secretin PilQ forms a homododecameric ring through which the pilus is proposed to be secreted. pilQ null mutants are nonpiliated, and thus, all pilus-dependent functions are eliminated. Mutagenesis was performed on the middle one-third of pilQ, and mutants with colony morphologies consistent with the colony morphology of nonpiliated or underpiliated bacteria were selected. Nineteen mutants, each with a single amino acid substitution, were isolated and displayed diverse phenotypes in terms of PilQ multimer stability, pilus expression, transformation efficiency, and host cell adherence. The 19 mutants were grouped into five phenotypic classes based on functionality. Four of the five mutant classes fit the current model of pilus functionality, which proposes that a functional pilus assembly apparatus, not necessarily full-length pili, is required for transformation, while high levels of displayed pili are required for adherence. One class, despite having an underpiliated colony morphology, expressed high levels of pili yet adhered poorly, demonstrating that pilus expression is necessary but not sufficient for adherence and indicating that PilQ may be directly involved in host cell adherence. The collection of phenotypes expressed by these mutants suggests that PilQ has an active role in pilus expression and function.     

Role of chromosomal rearrangement in N. gonorrhoeae pilus phase variation

N. gonorrhoeae undergoes pilus phase and antigenic variation. During phase variation, the pilin gene is turned on and off at high frequencies. Two loci on the gonococcal chromosome from strain MS11 function as expression sites for the pilin gene (pilE1 and pilE2); many other sites apparently contain silent variant pilin sequences. We reported previously that during pilus phase variation, when cells switch from the pilus expressing state (P+) to the nonexpressing state (P-), genome rearrangement occurs. We have examined phase variation in more detail, and we report that in most P+ to P- switches a deletion of pilin gene information occurs in one or both expression sites. This deletion is due to either a simple or a multiple-step recombination event involving directly repeated sequences in the expression loci. The deletion explains the state of some P- cells, but not all. In the latter cells pilin expression is probably controlled by an undefined regulator.     

Pilin expression and processing in pilus mutants of Neisseria gonorrhoeae: critical role of Gly-1 in assembly

Spontaneous mutants of Neisseria gonorrheae failing to express pili or having diminished levels of piliation were studied with regard to pilin expression. All mutants displayed altered pilin processing detectable as the release of soluble, truncated pilin molecules (S-pilin). Of particular interest was the finding, in one mutant, that substitution of serine for glycine at position -1 of propilin, a highly conserved residue among N-metPhe and related pilins, abolished pilus expression but not S-pilin release. The degree of S-pilin processing and the levels of membrane-associated pilin varied among the different classes of mutants, suggesting that each was blocked at a distinct step of pilus biogenesis. The data support a model in which increased S-pilin processing is a result of a decreased rate of pilus polymerization.