Comparisons between Colony Phase Variation of Neisseria gonorrhoeae FA1090 and Pilus, Pilin, and S-Pilin Expression

The gonococcal pilus is a primary virulence factor, providing the initial attachment of the bacterial cell to human mucosal tissues. Pilin, the major subunit of the pilus, can carry a wide spectrum of primary amino acid sequences which are generated by the action of a complex antigenic variation system. Changes in the pilin amino acid sequence can produce different pilus-dependent colony morphotypes, which have been previously shown to reflect phase variation of pili on the bacterial cell surface. In this study, we further examined the relationships between changes in pilus-dependent colony morphology, pilin sequence, pilus expression, and pilus function in Neisseria gonorrhoeae FA1090. A group of FA1090 colony variants expressed different pilin sequences and demonstrated different levels of pilin, S-pilin, and pilus expression. The analysis of these colony variants shows that they do not represent two distinct phases of pilus expression, but that changes in pilin protein sequence produce a spectrum of S-pilin production, pilus expression, and pilus aggregation levels. These different levels of pilus expression and aggregation influence not only colony morphology but also DNA transformation efficiency and epithelial cell adherence.     

Evidence for functionally distinct pili expressed by Neisseria meningitidis.

In order to investigate possible functional consequences of phase and antigenic variation of meningococci, the attachment of 15 strains of Neisseria meningitidis to human erythrocytes was studied by a nitrocellulose hemadsorption assay. This assay allows the study of individual meningococcal colonies with respect to erythrocyte attachment. Of the 15 strains studied, 7 demonstrated binding of human erythrocytes (HA+). Among these seven strains, the percentage of colonies that were HA+ ranged from 0.2 to 97%. Meningococcal colonies that did not produce pilin (the major structural subunit of pili) did not demonstrate erythrocyte binding (HA-). The HA+ colony phenotype was correlated with assembly of pilin into pili and expression of pili on the meningococcal surface. However, only some piliated colonies bound human erythrocytes. This could not be explained by differences between piliated HA+ and HA- colonies in the amount of pilin produced or by differences in number of pili expressed per diplococcus. Pili of five of the meningococcal strains with HA+ colonies were antigenically related to gonococcal pili (class I meningococcal pili), but HA+ colonies were also seen in two meningococcal strains expressing class II meningococcal pili. Changes from HA+ to HA- and from HA- to HA+, in the presence of continuing pilin production and pilus assembly, occurred at frequencies of up to 10(-2)/CFU per generation. Such frequencies resemble those of phase and antigenic variation described previously for Neisseria species pilin. These studies indicate that phase variation influences the ability of meningococci to attach to human cells and suggest that meningococci may express functionally different pili.     

Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria.

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.     

Loss of pili and decreased attachment to human cells by Neisseria meningitidis and Neisseria gonorrhoeae exposed to subinhibitory concentrations of antibiotics.

Recent evidence has suggested that surface structures of pathogenic bacteria, which are important in attachment to human mucosal surfaces, may be absent on bacteria grown in the presence of subinhibitory concentrations of antibiotics. We studied the effect of tetracycline and penicillin on meningococcal and gonococcal pili. Subinhibitory concentrations of tetracycline and penicillin were found to markedly reduce the number of pili per meningococcus or gonococcus and the percentage of meningococci or gonococci with pili, as determined by negative-staining electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations suggested that tetracycline decreased expression of pili by inhibiting synthesis of pilin subunits. In contrast, pilin subunit synthesis was unaltered by penicillin, suggesting a defect in assembly of pilin subunits or in anchoring of assembled pili. The decrease in the number of pili that occurred with subinhibitory concentrations of both tetracycline and penicillin was accompanied by a marked decrease in the ability of the organisms to attach to human cells. Gonococci or meningococci removed from the influence of subinhibitory concentrations of the antibiotics regained piliation, and attachment returned to levels near those of controls. The expression of meningococcal and gonococcal pili may be affected by factors that influence synthesis of pilin subunits or factors that interfere with the assembly and anchoring of pili in the outer membrane.     

Distribution of specific DNA sequences among pathogenic and commensal Neisseria species.

Several traits, including pili and the outer membrane proteins P.II and H.8, have been associated with pathogenic Neisseria species. We examined several Neisseria species for DNA sequence homology to cloned pilin, P.II, and H.8 genes. Strains of Neisseria gonorrhoeae and N. meningitidis showed hybridization to all of these genes. Commensal strains showed little hybridization to any of these genes. Strains of N. lactamica and N. cinerea showed intermediate patterns of hybridization. Generally, organisms that expressed a given trait showed DNA homology to the corresponding cloned gene. However, we observed pili on some commensal strains that did not show hybridization to the cloned gonococcal pilin gene.     

Characterization of the CsfC and CsfD proteins involved in the biogenesis of CS5 pili from enterotoxigenic Escherichia coli

The region required for biosynthesis of CS5 pili consists of six csf genes, with csfA encoding the major subunit. In this study, we describe the characterization of two of the genes constituting the region, csfC and csfD, but also identify the true morphology of the CS5 pilus by high resolution electron microscopy. CsfD was shown to be essential in the initiation of CS5 pilus biogenesis, did not possess any chaperone-like activity for the major subunit, and was an integral minor component of the pilus structure. Studies on CsfD translocation across the outer membrane in Escherichia coli K-12 using a csfA mutant also showed that CsfD is likely to be the first pilin subunit assembled. A specific in-frame deletion in the csfC gene resulted in the complete absence of cell surface CS5 pili and prevented the translocation of CsfA and CsfD pilins across the outer membrane. Specific cell localization studies showed an accumulation of CsfC in the outer membranes of E. coli K-12, while complementation experiments with homologous outer membrane assembly genes from CS1 and CFA/I pili systems were unable to restore assembly of CS5 pili. The CS5 pilus was shown to be a 2 nm flexible fibrillar structure, which adopted a predominantly open helical conformation under the electron microscope.     

Expression of type IV pili by Moraxella catarrhalis is essential for natural competence and is affected by iron limitation

Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.     

Identification of hifD and hifE in the pilus gene cluster of Haemophilus influenzae type b strain Eagan.

Haemophilus influenzae produces surface structures called pili that promote adherence to human cells. Three genes encoding the major pilus structural component (pilin), chaperone, and usher proteins (designated hifA, -B, and -C, respectively) have been identified previously. In this study, transposon mutagenesis and DNA sequence analysis identified two open reading frames (ORFs) downstream of, and in the same orientation as, hifC. These genes have been designated hifD and hifE. Both genes have predicted C-terminal amino acid homology to HifA, and mutations in either gene resulted in the loss of morphologic and functional pili, indicating that hifD and hifE encode pilus structural components and are required for pilus expression. Another ORF, identified immediately downstream of hifE, has a predicted amino acid sequence that is 70% identical to an aminopeptidase of Escherichia coli called PepN, and a mutation within this ORF did not alter pilus expression. These data indicate that the pepN homolog is not required for pilus biogenesis and that one end of the pilus gene cluster has been defined.     

Conservation of Neisseria gonorrhoeae pilus expression regulatory genes pilA and pilB in the genus Neisseria.

The pili of Neisseria gonorrhoeae mediate bacterial adhesion to the host-susceptible tissues. We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression. Besides this regulatory function, pilA participates in an essential function for bacterial viability. Here we show that pilA and pilB homologs are also present in a variety of other members of the Neisseriaceae family of bacteria in contrast to the gonococcal pilin gene which hybridizes only to the pathogenic Neisseria species.     

Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated.     

Purification and characterization of a pilus of a Vibrio cholerae strain: a possible colonization factor.

A new flexible type of pilus was purified from Vibrio cholerae non-O1, non-0139 strain NAGV14 and characterized. The molecular mass of the pilin was estimated to be 20 kDa, and the antigenicity differed from that of known pili such as toxin-coregulated pili, mannose-sensitive hemagglutinating pili, V10 pili, and Al-1841 pili. The NAGV14 pilus was regarded as a colonization factor because the purified pili adhered to rabbit intestine and adhesion was inhibited by treating the organisms with the Fab fraction of an antipilus antibody. An intestinal receptor blockade using purified pili failed to inhibit adhesion of the organisms. The NAGV14 pili adhered to the surface of live V. cholerae. An antigen cross-reacting with the NAGV14 pili was widely and specifically distributed among V. cholerae strains irrespective of serotype and biotype. The amino acid sequence of the pilin was homologous with that of MshA. The NAGV14 pili did not agglutinate human and rabbit erythrocytes.     

Detection of pilus subunits (pilins) and filaments by using anti-P pilin antisera.

P pilus filaments are important in binding to globoside through an adhesin located at the tip of the pilus. There is considerable antigenic variation among P pili, and the immunologic response is usually serotype specific. We purified denatured pilin subunits and used them as immunogens to prepare more broadly cross-reactive antisera. Although antifilament antisera (AFA) detected predominantly the homologous strain, antisubunit antisera (ASA) prepared from two different strains detected P pili in 16 of 16 and 14 of 16 P-piliated strains by Western blotting (immunoblotting). The binding of ASA to the homologous pilus filament was inhibited by only 3 of 17 strains. ASA agglutinated only two of nine heterologous strains and immunoprecipitated pili from one of three heterologous strains. By immunoelectron microscopy ASA was seen to bind to pilus filaments but not as strongly as AFA. Antiserum raised to the denatured pilin subunit was not substantially more reactive with pilus filaments derived from heterologous strains than was AFA. ASA was, however, a very useful probe for detecting most P pilins.     

Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.     

Serial isolates of Pseudomonas aeruginosa from a cystic fibrosis patient have identical pilin sequences.

Five isolates of Pseudomonas aeruginosa (CD2, CD3, CD4, CD5, and CD10) from a patient with cystic fibrosis were examined with regard to several genotypic and phenotypic characteristics to determine whether the patient was colonized with one or several distinct strains. Isolates CD2, CD3, and CD4 were obtained from a single sputum sample, and CD5 and CD10 were obtained 1 and 2 years later, respectively. On the basis of colonial morphology, serotyping, and antibiograms, the five isolates appeared to be different strains. However, Southern blot analysis with a 1.2-kilobase DNA probe containing the P. aeruginosa PAK pilin gene indicated that all five strains were identical at that genetic locus. The pilin genes of the five isolates were cloned and sequenced at the nucleotide level and found to be identical. Southern blot analysis with a probe from a separate region of the P. aeruginosa chromosome, a 741-base-pair PstI-NruI DNA fragment adjacent to the exotoxin A gene, also revealed genetic identity among these five clinical isolates. On this basis, it was concluded that this patient was colonized with a single strain of P. aeruginosa and that the strain had remained genetically stable over a period of 2 years. The predicted pilin sequence of the CD isolates was almost identical to that of strain PA103 (97% homology) and serologically related to PAO pilin, with which it shared 80% homology. No immunological cross-reactivity was detected between the CD and PAK pilins, which shared the least homology (62%) among the four pilins considered in this study. Although all five CD isolates contained identical pilin genes, three had acquired mutations which prevented normal expression of the pilus system. CD3 was a putative regulatory mutant which was unable to produce normal amounts of pilin, and CD4 and CD10 were putative assembly mutants which produced normal amounts of pilin but were unable to assemble the pilin subunit into intact pili.     

Soluble Pilin of Neisseria gonorrhoeae Interacts with Human Target Cells and Tissue

Pili of Neisseria gonorrhoeae are phase-variable surface structures that mediate adherence to host target cells. Each pilus is composed of thousands of major pilus subunits, pilins, pilus-associated protein PilC, and possibly other components. Piliated and nonpiliated gonococcal clones may secrete a soluble smaller pilin (S-pilin) that is cleaved after amino acid 39 of the mature pilin protein. Here, purified S-pilin was found to migrate as a 61- to 64-kDa double band on nondenaturing gels, suggesting the formation of tetrameric S-pilin proteins with two isomeric forms. In situ studies of binding to formalin-fixed tissue sections demonstrated the binding of S-pilin to human tissue but not to tissue from mouse or rat organs, showing the presence of a human-specific receptor-binding domain within the pilin polypeptide. Pretreatment of the target tissues with proteinase K decreased gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect gonococcal binding. In overlay assays, purified S-pilin bound to a band with a migration pattern and size similar to those of CD46, a cellular pilus receptor. Further, binding of N. gonorrhoeae to target cells and tissues could be blocked by both CD46 antibodies and purified S-pilin. These data argue that S-pilin interacts with a protein domain(s) of the CD46 receptor on human cells.     

Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.     

Sortase A utilizes an ancillary protein anchor for efficient cell wall anchoring of pili in Streptococcus agalactiae

Pili are putative virulence factors and promising vaccine candidates in Streptococcus agalactiae (group B Streptococcus [GBS]) infection, a leading cause of neonatal sepsis and meningitis. The genes necessary for pilus synthesis and assembly are clustered in pilus islands (PI). Each gene encodes three structural subunits (a backbone and two ancillary proteins) bearing a C-terminal LPXTG motif and two subfamily C sortases (SrtC) involved in covalent polymerization of the subunits. GBS strains also possess the conserved “housekeeping” sortase A (SrtA), but its role in pilus assembly is unclear. To address this issue, pilus expression and cell wall anchoring were analyzed in srtA deletion mutants. Loss of SrtA did not affect pilus polymerization. However, pilus expression on the cell surface was reduced, and pili accumulated in the culture supernatant. Furthermore, cell-associated pili could be readily released by detergent treatment, indicating that SrtA is involved in covalent anchoring of pili to the cell wall. When each of the genes comprising PI-2a was systematically deleted, only the absence of ancillary subunit GBS150 or the SrtC required for incorporation of GBS150 into pili mimicked the srtA mutant phenotype. Thus, from these data a model for GBS pilus assembly can be proposed in which PI sortases are responsible for polymerization of the pilus structure, while SrtA is required to covalently attach it to the cell wall, utilizing ancillary pilus subunit GBS150 as the anchor protein.     

A Foreign Protein Incorporated on the Tip of T3 Pili in Lactococcus lactis Elicits Systemic and Mucosal Immunity

The use of Lactococcus lactis to deliver a chosen antigen to the mucosal surface has been shown to elicit an immune response in mice and is a possible method of vaccination in humans. The recent discovery on Gram-positive bacteria of pili that are covalently attached to the bacterial surface and the elucidation of the residues linking the major and minor subunits of such pili suggests that the presentation of an antigen on the tip of pili external to the surface of L. lactis might constitute a successful vaccine strategy. As a proof of principle, we have fused a foreign protein (the Escherichia coli maltose-binding protein) to the C-terminal region of the native tip protein (Cpa) of the T3 pilus derived from Streptococcus pyogenes and expressed this fusion protein (MBP*) in L. lactis. We find that MBP* is incorporated into pili in this foreign host, as shown by Western blot analyses of cell wall proteins and by immunogold electron microscopy. Furthermore, since the MBP* on these pili retains its native biological activity, it appears to retain its native structure. Mucosal immunization of mice with this L. lactis strain expressing pilus-linked MBP* results in production of both a systemic and a mucosal response (IgG and IgA antibodies) against the MBP antigen. We suggest that this type of mucosal vaccine delivery system, which we term UPTOP (for unhindered presentation on tips of pili), may provide an inexpensive and stable alternative to current mechanisms of immunization for many serious human pathogens.Pili of Gram-positive bacteria are filamentous structures that extend outward from the bacterial surface and are covalently anchored to the bacterial cell wall. They are believed to be the primary means of attachment to the appropriate environmental receptor for the organism, which, for pathogens, is within the human host. The backbone of the pilus in Gram-positive bacteria is composed of multiple covalently linked identical subunits (major pilin), to which one or more minor pilin subunits are covalently attached. Pilin proteins are synthesized with an N-terminal Sec signal, which is cleaved during transit through the cytoplasmic membrane, and a C-terminal cell wall sorting signal (CWSS), which contains an LPXTG (or similar) amino acid motif, followed by a hydrophobic region and a positively charged C terminus. Pilus assembly is catalyzed by a pilus-specific sortase family transpeptidase, which cleaves the CWSS motif between the threonine (T) and glycine (G) residues and forms a covalent bond between this T and a conserved lysine (K) residue of another major pilin subunit. As this process repeats, the pilus is polymerized until it is covalently linked to the cell wall by either the “housekeeping” sortase, which is responsible for anchoring most surface proteins of Gram-positive bacteria to the cell wall, or the pilus-specific sortase (for reviews, see references 21, 35, and 38).We have been investigating assembly of T3 pili of Streptococcus pyogenes, an important human pathogen. In this organism, the T3 pilus locus (19) encodes the major pilin (T3) and the minor pilins Cpa and OrfB, the pilus-specific transpeptidase SrtC2, and SipA2, which is required for pilus polymerization by SrtC2 (44). Our investigations into the biogenesis of T3 pili have identified the residues of T3 and Cpa required for (i) polymerization of T3 and (ii) incorporation of Cpa into the pilus structure. We have demonstrated that lysine residue 173 (K173) (29) and the CWSS (QVPTG) of the T3 major pilin subunit (2, 29) are required for polymerization of T3. This indicates that individual T3 subunits are polymerized into the pilus structure by covalent bonds between K173 of T3 and the threonine of the CWSS (T315) of the adjacent T3 subunit. We have also demonstrated that K173 of T3, along with the CWSS (VPPTG) of Cpa, are required for incorporation of the minor pilin, Cpa, into the pilus (29). Thus, the K173 residue of T3 is required for T3-T3 linkage and is also required for covalent linkage of Cpa to the T3 pilus, demonstrating that Cpa is located at the tip of T3 pili, a conclusion supported by immunogold electron microscopy (EM) (29).Identification of the residues required for attachment of Cpa, the tip protein, to the T3 pilus suggested to us that genetic engineering could be used to produce a Gram-positive bacterial strain in which a foreign protein would be covalently linked by the bacterium to the pilus tip in place of Cpa. In the present study, we used the Escherichia coli maltose-binding protein (MBP) as a model protein to test this idea. We identified amino acid residues of the primary structure of Cpa that are sufficient for incorporation of a foreign protein into T3 pili in vivo by SrtC2. We propose that this approach constitutes a novel technology for presentation of foreign polypeptides external to the bacterial envelope, which we call UPTOP (for unhindered presentation of polypeptides on tips of pili). We suggest that any Gram-positive bacterium can be used as the host for UPTOP. We also propose that UPTOP can be used to present vaccine antigens to the immune system. As proof of this principle, we constructed a strain of the probiotic bacterium Lactococcus lactis engineered to produce T3 pili with the model protein MBP covalently linked at the pilus tips. We show in this study that mucosal administration to mice of this vaccine strain generates both an IgG and an IgA response to the model protein.     

The Meningococcal Minor Pilin PilX Is Responsible for Type IV Pilus Conformational Changes Associated with Signaling to Endothelial Cells

Neisseria meningitidis crosses the blood-brain barrier (BBB) following the activation of the β2-adrenergic receptor by the type IV pili (TFP). Two components of the type IV pili recruit the β2-adrenergic receptor, the major pilin PilE and the minor pilin PilV. Here, we report that a strain deleted of PilX, one of the three minor pilins, is defective in endothelial cell signaling. The signaling role of PilX was abolished when pili were not retractable. Purified PilX was unable to recruit the β2-adrenergic receptor, thus suggesting that PilX was playing an indirect role in endothelial cell signaling. Considering the recent finding that type IV pili can transition into a new conformation (N. Biais, D. L. Higashi, J. Brujic, M. So, and M. P. Sheetz, Proc. Natl. Acad. Sci. U. S. A. 107:11358-11363, 2010), we hypothesized that PilX was responsible for a structural modification of the fiber and allowed hidden epitopes to be exposed. To confirm this hypothesis, we showed that a monoclonal antibody which recognizes a linear epitope of PilE bound fibers only when bacteria adhered to endothelial cells. On the other hand, this effect was not observed in PilX-deleted pili. A deletion of a region of PilX exposed on the surface of the fiber had phenotypical consequences identical to those of a PilX deletion. These data support a model in which surface-exposed motifs of PilX use forces generated by pilus retraction to promote conformational changes required for TFP-mediated signaling.     

Structural and serological relatedness of Haemophilus influenzae type b pili.

The structural and serological relatedness of the pilus proteins of several isolates of Haemophilus influenzae type b cultured from patients with invasive disease and from different anatomic sites within the same patient was examined. Epithelial cell-adherent variants of 25 nonadherent parent isolates were obtained by selection for organisms that adhered to human erythrocytes. Outer membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of an additional 24- to 24.5-kilodalton protein among all adherent variants but absent from all nonadherent parent isolates. Polyclonal rabbit antiserum against the intact native pilus protein of H. influenzae M43 cross-reacted with 20 of 25 adherent H. influenzae in both immunodot and slide-agglutination assays. No differences in reactivity among isolates cultured from more than one anatomic site in the same patient were noted. Anti-M43 pilus antiserum had bactericidal activity against both the homologous strain and a heterologous strain that demonstrated serologic identity in the immunodot and slide agglutination assays. The adherence of these strains to human epithelial cells in vitro was inhibited by Fab fragments purified from the antipilus antiserum. These data indicate that a remarkable degree of homogeneity in pilin subunit size exists among the pili of H. influenzae type b and that major antigenic determinants are shared among most of these pili. Also, antibodies directed against H. influenzae pilus proteins may be able to contribute to host defenses through serum bactericidal activity and by blocking the adherence of this bacterium to host epithelial cells.