Pilus-mediated epithelial cell death in response to infection with Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that can cause serious infections in cystic fibrosis (CF) patients. The ET12 lineage appears particularly virulent in CF; however, its pathogenesis is poorly understood and may be associated with host response. To help characterize this response, the ability of B. cenocepacia to induce cytotoxicity and apoptosis in an epithelial cell model was examined. Upon infection with B. cenocepacia strain K56-2, A549 human lung epithelial cells underwent significant cell death; propidium iodine staining and DNA fragmentation assays suggested apoptosis. Initiation of cell death was independent of the type III secretion system, biofilm formation, and secreted bacterial cytotoxins. However, the frequency of cell death was lower in cells infected with a non-piliated mutant, K56-2 cblA::Tp. Furthermore, purified cbl pili were found to directly induce cytotoxicity in A549 cells and activate caspase-9, -8, -7, and -3, the major cysteine proteinases involved in apoptosis. It appears that B. cenocepacia cbl pili, which are a distinctive feature of the ET12 lineage, act as an initiator of cytotoxicity and apoptosis. Understanding the role of cbl pili in the pathogenesis of B. cenocepacia infections offers the potential for decreasing the virulence of these potentially life-threatening organisms in CF patients.     

Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial-substratum and bacterial-bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell-cell interactions to colonization.     

Regulation and function of pilus island 1 in group B streptococcus

Group B streptococcus (GBS) pili may enhance colonization and infection by mediating bacterial adhesion to host cells, invasion across endothelial and epithelial barriers, and resistance to bacterial ingestion and killing by host phagocytes. However, it remains unclear how pilus expression is regulated and how modulation of pilus production affects GBS interactions with the human host. We investigated the regulation and function of pilus island 1 (PI-1) pili in GBS strain 2603. We found that PI-1 gene expression was controlled by the CsrRS two-component system, by Ape1, an AraC-type regulator encoded by a divergently transcribed gene immediately upstream of PI-1, and by environmental pH. The response regulator CsrR repressed expression of Ape1, which is an activator of PI-1 gene expression. In addition, CsrR repressed PI-1 gene expression directly, independent of its regulation of Ape1. In vitro assays demonstrated specific binding of both CsrR and Ape1 to chromosomal DNA sequences upstream of PI-1. Pilus gene expression was activated by acidic pH, and this effect was independent of CsrRS and Ape1. Unexpectedly, characterization of PI-1 deletion mutants revealed that PI-1 pili do not mediate adhesion of strain 2603 to A549 respiratory epithelial cells, ME180 cervical cells, or VK2 vaginal cells in vitro. PI-1 pili reduced internalization and intracellular killing of GBS by human monocyte-derived macrophages, by approximately 50%, but did not influence complement-mediated opsonophagocytic killing by human neutrophils. These findings shed new light on the complex nature of pilus regulation and function in modulating GBS interactions with the human host.     

Bovine lactoferrin interacts with cable pili of Burkholderia cenocepacia

In this study we evaluated the ability of lactoferrin, the most abundant antimicrobial protein in airway secretions, to bind the surface structures of a Burkholderia strain cystic fibrosis-isolated. Burkholderia cenocepacia is a gram-negative bacterium involved as respiratory pathogen in cystic fibrosis patient infections. This bacterium possesses filamentous structures, named cable pili that have been proposed as virulence factors because of their ability to bind to respiratory epithelia and mucin. Previously, we demonstrated that bovine lactoferrin was able to influence the efficiency of invasion of different iron-regulated morphological forms of B. cenocepacia. Bovine lactoferrin showed to efficiently inhibit invasion of alveolar epithelial cells by free-living bacteria or iron-induced aggregates or biofilm. Results of the present study demonstrate that bovine lactoferrin is also able to specifically bind to B. cenocepacia cells and show that cable pili are involved in this interaction. The attachment of bovine lactoferrin to pili led to a reduced binding of bacterial cells to mucin. Since cable pili are implicated in mediating the bacterial interactions with mucin and epithelial cells, lactoferrin binding to these structures could play an important role in neutralizing bacterial infection in cystic fibrosis patients.     

Differential interaction of bacterial species from the Burkholderia cepacia complex with human airway epithelial cells

To increase knowledge of the pathogenic potential of the Burkholderia cepacia complex (BCC), we investigated the effects of reference strains of the nine BCC species on human bronchial epithelial cells in vitro. B. multivorans exhibited the highest rates of adherence to and internalization by host cells. Two out of three clinical isolates recovered from cystic fibrosis patients confirmed the B. multivorans high adhesiveness. All four B. multivorans isolates exhibited an aggregated pattern of adherence but any of them expressed cable pili. When bacteria were centrifuged onto cell cultures to circumvent their poor adhesiveness, B. pyrrocinia exhibited the highest internalization rate, followed by B. multivorans. The percentages of apoptotic cells in cultures infected with B. cepacia, B. multivorans, B. cenocepacia (subgroups IIIA and IIIB), B. stabilis and B. vietnamiensis were significantly higher than in control non-infected cultures. All nine BCC species triggered a similar release of the inflammatory cytokine IL-8, that was not reduced by cell treatment with cytochalasin D. Hence, our data demonstrate, for the first time, that all BCC species exhibit a similar ability to induce the expression of host immune mediators whereas they differ on their ability to adhere to, invade and kill airway epithelial cells.     

Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers.

Previous studies have shown that appendage pili of Burkholderia cepacia strains isolated from patients with cystic fibrosis (CF) at The Hospital for Sick Children, Toronto, Canada, mediate adherence to mucus glycoproteins and also enhance adherence to epithelial cells. The specific pilin-associated adhesin molecule is a 22-kDa protein. In the present study we purified the major subunit pilin (17 kDa) and immunolocalized it to peritrichously arranged pili. On the basis of their novel morphological appearance as giant intertwined fibers, we refer to them as cable (Cbl) pili. Using an oligonucleotide probe corresponding to regions of the N-terminal amino acid sequence of the pilin subunit, we detected the encoding cblA gene in a chromosomal DNA library. Sequencing revealed this structural gene to be 555 bp in length, encoding a leader sequence of 19 amino acids, a cleavage site between the alanine at position 19 and the valine at position 20, and a mature pilin sequence of 165 amino acids. The calculated molecular mass is 17.3 kDa. Hydrophobic plus apolar amino acids account for 60% of the total residues. The pilin exhibits some similarities in its amino acid sequence to colonization factor antigen I and CS1 fimbriae of Escherichia coli. With the cblA gene used as a probe, hybridization assays of 59 independent isolates, including those from several geographically separated CF centers, plus environmental and clinical (non-CF) strains, gave positive results with all of the 15 CF-associated B. cepacia isolates from Toronto, plus a single strain from one other CF center (Jackson, Mississippi). The cblA gene is the first pilin subunit gene of B. cepacia to be identified.     

The PilC adhesin of the Neisseria type IV pilus-binding specificities and new insights into the nature of the host cell receptor

Type IV pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate the first contact to human mucosal epithelial cells, an interaction which is also critical for the interaction with vascular endothelial cells. The PilC proteins have been characterized as the principal pilus-associated adhesin. Here we show that PilC2 exhibits a defined cell and tissue tropism, as it binds to human epithelial and endothelial cell lines, but not to human T cells or fibroblasts. Piliated gonococci and PilC2 exhibit similar patterns of binding to human epithelial and endothelial cells, supporting the function of PilC as the key pilus adhesin. Although CD46 has previously been suggested to be a pilus receptor, several observations indicate that neisserial type IV pili and the pilus adhesin PilC2 interact with epithelial cells in a CD46 independent manner. Biochemical approaches were used to characterize the nature of host cell factors mediating binding of piliated gonococci and PilC2 protein. Our data indicate that the putative host cell receptor for gonococcal pili and the PilC2 pilus adhesin is a surface protein. Glycostructures were found to not be involved in binding. Moreover, we observed the uptake of purified PilC2 protein together with its receptor via receptor-mediated endocytosis and subsequent receptor re-exposure on the cell surface. Our data support the existence of a specific pilus receptor and provide intriguing information on the nature of the receptor.     

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.     

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.     

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.     

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.     

Pilus Gene Pool Variation and the Virulence of Corynebacterium diphtheriae Clinical Isolates during Infection of a Nematode

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.     

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.     

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.     

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.