Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.     

Roles of the sortases of Streptococcus pneumoniae in assembly of the RlrA pilus

Pili have been observed on the surface of several gram-positive bacteria, including Streptococcus pneumoniae. The S. pneumoniae strain TIGR4 pilus is composed of three structural subunit proteins encoded in the rlrA pathogenicity islet, RrgA, RrgB, and RrgC. RrgB comprises the pilus backbone, RrgA is observed at intervals along surface pili, while RrgC is found in a loosely defined relationship with RrgA. We investigated the incorporation of each subunit into pili and the reliance of such placement on each of the other subunits. Both accessory subunits RrgA and RrgC are present in similar quantities in pili of all sizes. However, neither protein is required for the polymerization of RrgB, suggesting a nonessential role for RrgA and RrgC in the initiation of pilus assembly. Additionally, the rlrA islet encodes three sortases, SrtC-1, SrtC-2, and SrtC-3 (formerly SrtB, SrtC, and SrtD), which are divergent in sequence from the housekeeping sortase, SrtA. We determined the contributions of these four sortases to pilus assembly and found that SrtA is dispensable for pilus assembly and localization to the cell wall. Instead, SrtC-1, SrtC-2, and SrtC-3 are responsible for pilus assembly and exhibit functional redundancy with respect to backbone assembly and cell wall localization. A level of specificity and coordination among the class C sortases was revealed by the finding that SrtC-1 and SrtC-3 are required for the incorporation of the accessory subunits and by showing a deleterious effect on pilus assembly upon alteration of the cell wall sorting signals of the accessory subunit proteins.     

Molecular architecture of Streptococcus pneumoniae TIGR4 pili

Although the pili of Gram‐positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus‐1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 μm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose‐like protrusions. These protrusions correlate to the shape of monomeric RrgB–His, which like RrgA–His and RrgC–His has an elongated, multi‐domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head‐to‐tail.     

Identification of two minor subunits in the pilus of Haemophilus influenzae.

Haemophilus influenzae type b (Hib) organisms produce pili, which mediate attachment to human cells and are multimeric structures composed of a 24-kDa subunit called pilin or HifA. Although pili from other organisms contain additional proteins accessory to pilin, no structural components other than pilin have been identified in Hib pili. Previous analysis of a Hib pilus gene cluster, however, suggested that two genes, hifD and hifE, may encode additional pilus subunits. To determine if hifD and hifE encode pilus components, the genes were overexpressed in Escherichia coli and the resulting proteins were purified and used to raise polyclonal antisera. Antisera raised against C-terminal HifD and HifE fragments reacted with H. influenzae HifD and HifE proteins, respectively, on Western immunoblots. Western immunoblot analysis of immunoprecipitated Hib pili demonstrated that HifD and HifE copurified with pili. In enzyme-linked immunosorbent assays, antisera raised against a recombinant HifE protein that contained most of the mature protein reacted more to piliated Hib than to nonpiliated Hib or to a mutant containing a hifE gene insertion. Immunoelectron microscopy confirmed that the HifE antiserum bound to pili and demonstrated that the antiserum bound predominantly to the pilus tips. These data indicate that HifD and HifE are pilus subunits. Adherence inhibition studies demonstrated that the HifE antiserum completely blocked pilus-mediated hemagglutination, suggesting that HifE mediates pilus adherence.     

Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

Pili are surface-attached, fibrous virulence factors that play key roles in the pathogenesis process of a number of bacterial agents. Streptococcus pneumoniae is a causative agent of pneumonia and meningitis, and the appearance of drug-resistance organisms has made its treatment challenging, especially in developing countries. Pneumococcus-expressed pili are composed of three structural proteins: RrgB, which forms the polymerized backbone, RrgA, the tip-associated adhesin, and RrgC, which presumably associates the pilus with the bacterial cell wall. Despite the fact that the structures of both RrgA and RrgB were known previously, structural information for RrgC was still lacking, impeding the analysis of a complete model of pilus architecture. Here, we report the structure of RrgC to 1.85 Å and reveal that it is a three-domain molecule stabilized by two intradomain isopeptide bonds. RrgC does not depend on pilus-specific sortases to become attached to the cell wall; instead, it binds the preformed pilus to the peptidoglycan by employing the catalytic activity of SrtA. A comprehensive model of the type 1 pilus from S. pneumoniae is also presented.     

Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr¹⁶⁰ in SrtB to an arginine as in SrtC (Arg¹⁶⁰) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.     

Sortase-mediated assembly and surface topology of adhesive pneumococcal pili

The rlrA genetic islet encodes an extracellular pilus in the Gram-positive pathogen Streptococcus pneumoniae. Of the three genes for structural subunits, rrgB encodes the major pilin, while rrgA and rrgC encode ancillary pilin subunits decorating the pilus shaft and tip. Deletion of all three pilus-associated sortase genes, srtB, srtC and srtD, completely prevents pilus biogenesis. Expression of srtB alone is sufficient to covalently associate RrgB subunits to one another as well as linking the RrgA adhesin and the RrgC subunit into the polymer. The active-site cysteine residue of SrtB (Cys 177) is crucial for incorporating RrgC, even when the two other sortase genes are expressed. SrtC is redundant to SrtB in permitting RrgB polymerization, and in linking RrgA to the RrgB filament, but SrtC is insufficient to incorporate RrgC. In contrast, expression of srtD alone fails to mediate RrgB polymerization, and a srtD mutant assembles heterotrimeric pilus indistinguishable from wild type. Topological studies demonstrate that pilus antigens are localized to symmetric foci at the cell surface in the presence of all three sortases. This symmetric focal presentation is abrogated in the absence of either srtB or srtD, while deletion of srtC had no effect. In addition, strains expressing srtB alone or srtC alone also displayed disrupted antigen localization, despite polymerizing subunits. Our data suggest that both SrtB and SrtC act as pilus subunit polymerases, with SrtB processing all three pilus subunit proteins, while SrtC only RrgB and RrgA. In contrast, SrtD does not act as a pilus subunit polymerase, but instead is required for wild-type focal presentation of the pilus at the cell surface.     

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.     

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility.     

Functional implications of the expression of PilC proteins in meningococci

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil^- backswitcher isolated from the hyper-adherent variant was PilC⁺ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.     

Association of RrgA and RrgC into the Streptococcus pneumoniae pilus by sortases C-2 and C-3

Pili are surface-exposed virulence factors involved in the adhesion of bacteria to host cells. The human pathogen Streptococcus pneumoniae expresses a pilus composed of three structural proteins, RrgA, RrgB, and RrgC, and requires the action of three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3, to covalently associate the Rrg pilins. Using a recombinant protein expression platform, we have previously shown the requirement of SrtC-1 in RrgB fiber formation and the association of RrgB with RrgC. To gain insights into the substrate specificities of the two other sortases, which remain controversial, we have exploited the same robust strategy by testing various combinations of pilins and sortases coexpressed in Escherichia coli. We demonstrate that SrtC-2 catalyzes the formation of both RrgA-RrgB and RrgB-RrgC complexes. The deletion and swapping of the RrgA-YPRTG and RrgB-IPQTG sorting motifs indicate that SrtC-2 preferentially recognizes RrgA and attaches it to the pilin motif lysine 183 of RrgB. Finally, SrtC-2 is also able to catalyze the multimerization of RrgA through the C-terminal D4 domains. Similar experiments have been performed with SrtC-3, which catalyzes the formation of RrgB-RrgC and RrgB-RrgA complexes. Altogether, these results provide evidence of the molecular mechanisms of association of RrgA and RrgC with the RrgB fiber shaft by SrtC-2 and SrtC-3 and lead to a revised model of the pneumococcal pilus architecture accounting for the respective contribution of each sortase.     

Kingella kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells

Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.     

The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca(2+) transients in infected epithelial cells

Purified pili and porin from Neisseria quickly mobilize calcium (Ca(2+)) stores in monocytes and epithelial cells, ultimately influencing host cell viability as well as bacterial intracellular survival. Here, we examined the Ca(2+) transients induced in human epithelial cells during infection by live, piliated N. gonorrhoeae. Porin induced an influx of Ca(2+) from the extracellular medium less than 60 s post infection. The porin-induced transient is followed by a pilus-induced release of Ca(2+) from intracellular stores. The timing of these events is similar to that observed using purified proteins. Interestingly, the porin-induced Ca(2+) flux is required for the pilus-induced transient, indicating that the pilus-induced Ca(2+) release is, itself, Ca(2+) dependent. Several lines of evidence indicate that porin is present on pili. Moreover, pilus retraction strongly influences the porin- and pilus-induced Ca(2+) fluxes. These and other results strongly suggest that the pilus and porin cooperate to modulate calcium signalling in epithelial cells, and propose a model to explain how N. gonorrhoeae triggers Ca(2+) transients in the initial stages of pilus-mediated attachment.     

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.     

Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.     

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.     

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.     

Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells

Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.