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.     

PilC of pathogenic Neisseria is associated with the bacterial cell surface

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.     

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.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well-defined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.     

Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock-out mutants

The phase-variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full-length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P⁺) variants of N. gonorrhoeae MS11 led initially to a non-piliated (P⁻) phenotype; however, spontaneous P⁺ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P⁺ mutants; this protein exhibited no immunological cross-reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

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.     

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.     

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.     

Dual Role for Pilus in Adherence to Epithelial Cells and Biofilm Formation in Streptococcus agalactiae

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation.     

Role of the Eikenella corrodens pilA locus in pilus function and phase variation

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.     

Structural Characterization of Novel Pseudomonas aeruginosa Type IV Pilins

Pseudomonas aeruginosa type IV pili, composed of PilA subunits, are used for attachment and twitching motility on surfaces. P. aeruginosa strains express one of five phylogenetically distinct PilA proteins, four of which are associated with accessory proteins that are involved either in pilin posttranslational modification or in modulation of pilus retraction dynamics. Full understanding of pilin diversity is crucial for the development of a broadly protective pilus-based vaccine. Here, we report the 1.6-Å X-ray crystal structure of an N-terminally truncated form of the novel PilA from strain Pa110594 (group V), which represents the first non-group II pilin structure solved. Although it maintains the typical T4a pilin fold, with a long N-terminal α-helix and four-stranded antiparallel β-sheet connected to the C-terminus by a disulfide-bonded loop, the presence of an extra helix in the αβ-loop and a disulfide-bonded loop with helical character gives the structure T4b pilin characteristics. Despite the presence of T4b features, the structure of PilA from strain Pa110594 is most similar to the Neisseria gonorrhoeae pilin and is also predicted to assemble into a fiber similar to the GC pilus, based on our comparative pilus modeling. Interactions between surface-exposed areas of the pilin are suggested to contribute to pilus fiber stability. The non-synonymous sequence changes between group III and V pilins are clustered in the same surface-exposed areas, possibly having an effect on accessory protein interactions. However, based on our high-confidence model of group III PilA_PA14, compensatory changes allow for maintenance of a similar shape.     

Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins

Pathogenic Neisseria express type IV pili (tfp), which have been shown to play a central role in the interactions of bacteria with their environment. The regulation of piliation thus constitutes a central element in bacterial life cycle. The PilC proteins are outer membrane-associated proteins that have a key role in tfp biogenesis since PilC-null mutants appear defective for fibre expression. Moreover, tfp are also subjected to retraction, which is under the control of the PilT nucleotide-binding protein. In this work, we bring evidence that fibre retraction involves the translocation of pilin subunits to the cytoplasmic membrane. Furthermore, by engineering meningococcal strains that harbour inducible pilC genes, and with the use of meningococcus-cell interaction as a model for the sequential observation of fibre expression and retraction, we show that the PilC proteins regulate PilT-mediated fibre retraction.