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.     

Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili,or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH₄)₂SO₄ concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.     

The role of fibronectin in the pathogenesis of meningococcal infection

We have characterized an interaction of 20 strains of Neisseria meningitidis serogroups A, B, C, 29E, W-135 and Z with immobilized fibronectin of human plasma. The adhesion of meningococci to fibronectin was determined by the extent of piliated cells and did not depend on the meningococcal serogroup. Binding of non-piliated or weakly piliated strains (2-5% of piliated cells in the stock) was sufficiently greater than those piliated (8-10%), where the adhesion to fibronectin was not at all observed. The examination of two well-piliated strains showed that the loss of pili resulted in the increase of bacterial adhesion to fibronectin. Constants of association and dissociation of piliated and non-piliated strains to fibronectin were calculated. The role of meningococci-fibronectin interaction in the pathogenesis of meningococcal infection is discussed.     

Some Properties of the Pili of Corynebacterium renale

Some properties of the pili of the gram-positive bacteria Corynebacterium renale were described. A relationship was found between the morphological features of pili and the types of C. renale. Strains of types I and III usually possessed a small number of pili, whereas those of type II possessed numerous pili. Thick and long bundles of pili characteristic of C. renale were frequently observable in type II strains. Piliation of C. renale was stable under various cultural conditions. No ability to agglutinate red blood cells was demonstrated by piliated strains of C. renale. Pili were isolated from the cells of C. renale and studied serologically by immunodiffusion. The pili of a type II strain were serologically identical with the pili of another type II strain but not with those of the strains belonging to types I and III. The pili were serologically distinct from the cell wall. The pili were broken into short pieces by boiling, but their antigenicity was increased after boiling.     

A myxobacterial S-motility protein dances with poles

Coordinated movement of packs of S-motile Myxococcus xanthus cells relies on extrusion and retraction of pili that are located at one cell pole. At regular intervals the pili switch their polar location and cells reverse direction. Recently, the FrzS S-motility protein was observed to localize predominantly to the piliated pole. In time, FrzS was redeployed to the opposite pole and its sequestration at the new site coincided with cell reversal. The C-terminal region of FrzS, a response regulator homolog, is rich in coiled-coil motifs and is required for dynamic localization and proper motility. These results raise the possibility that proper spatial control of FrzS has an important role in the regulation of cell reversal and S-motility.     

Opc- and pilus-dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides

The interplay between four surface-expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non-sialylated (L8 immuno-type) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc-mediated invasion of host cells by piliated and non-piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc-expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc-mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine-glycine-aspartic acid (RGD) sequence. Cellular invasion in piliated Opc⁺ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin αvβ3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.     

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 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.     

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.     

Genetic and mass spectrometry analyses of the unusual type IV-like pili of the archaeon Methanococcus maripaludis

The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.     

Production of Pili (Fimbriae) by Pseudomonas fluorescens and Correlation with Attachment to Corn Roots

Pseudomonas fluorescens isolates 13525 and 2-79 were grown in Luria broth and low-nutrient medium (LNM). Pililike fibrils were very rarely produced in Luria broth but were abundantly produced in LNM. In LNM the pili were peritrichously distributed and had diameters ranging from 3 to 8 nm. Pili were purified from strain 2-79, and the pilin subunit was found to have a molecular weight of about 34,000. Strain 2-79 produced two colony types on Luria agar, nonmucoidal and mucoidal. Cells in LNM cultures of the nonmucoidal colony type were highly piliated, and cells from the mucoidal type were nearly devoid of pili. The presence of pili on nonmucoidal isolate 2-79 was quantitatively correlated with hydrophobic attachment to polystyrene, hemagglutination, and attachment to corn roots.     

Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent M_r and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent M_r values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.     

Pili mediated agglutination of Serratia marcescens in human urine

Of 51 strains of Serratia marcescens isolated from patients with urinary or respiratory tract infections, 35 agglutinated in human urine. The agglutinating strains possessed numerous pili which were morphologically distinct from common pili or type I pili. The diameter of the pili was 3 nm and the average length was 0.3 micrometer. Electron microscopic examination showed that 80% or more of the cells of the agglutinating strains and 0 to 8% of the cells of the nonagglutinating strains were piliated. When an agglutinating strain was heated at 55 C for 10 min, it lost its agglutinating capacity and concomitantly its pili. These results suggest that the agglutination might occur because of interactions between the pili and some factors in human urine. The urinary slime appears to contain these agglutinating factors.     

Type 1 pili enhance the invasion of Salmonella braenderup and Salmonella typhimurium to HeLa cells.

The relationship between type 1 pili-associated adhesion and invasion to HeLa cells by Salmonella braenderup and S. typhimurium was studied. When the clinical isolates of these strains were grown in L-broth, they showed both type 1 pili formation and mannose-sensitive adhesion to HeLa cells. On the other hand, the type 1 pili-defective mutants, which were obtained either by repeated subcultures on L-agar plates or by the transposon Tn1-insertion mutagenesis of the S. braenderup and S. typhimurium strains, concomitantly lost mannose-sensitive adhesion to HeLa cells. When the HeLa cells were incubated with Salmonella, the type 1 piliated strains invaded the HeLa cells with much higher infection rate than did the type 1 pili-defective strains. The invasion of type 1 piliated strains to HeLa cells was markedly inhibited in the presence of D-mannose. The infectivity of the strain, which lost type 1 pili but still had mannose-resistant adhesion, was slightly higher than that of the strains defective in both mannose-sensitive and mannose-resistant adhesion. These results suggested that type 1 pili have a role in enhancing the invasion of S. braenderup and S. typhimurium to HeLa cells.     

Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus , T4P are unipolarly localized and undergo pole-to-pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non-piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole-to-pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP–PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.     

Type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus

The hair‐like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo‐proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm‐promoting function in type IV pili‐producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non‐piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well‐studied type IV pili‐producing heterotrophic bacteria.     

Pili and the interaction of Aeromonas species with humanperipheral blood polymorphonuclear cells

Abstract The interaction of differentiallu piliated Aeromonas strains expressing pili of two broadly different morphologic types (short, rigid (S/R) and/or long, wavy (L/W)) with human peripheral blood mononuclear leukocytes (PMN) was investigated to determine whether host defense cells might exert a selective pressure on pili expression in vivo accounting for the different pili phenotypes of clinical and environmental strains. A majority of Aeromonas veronii biotype sobria strains from water (6/6) and faeces (8/11) readily associated with PMN (>60% PMN with adherent and/or internalised bacteria), irrespective of their degree, or predominant type, of piliation. Rigid pili of Aeromonas species did not promote interaction with PMN. However, the majority (55%) of strains which interacted well with PMN were adherent to HEp-2 cells. Interactio with PMN is unlikely to be the reason few S/R pili are seen on faecal strains, but it may be a selective pressure on L/W adhesive pili, or other OMP adhesins, resulting in the shedding of strains which have lost critical adhesins.     

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.     

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.