Copy Number of Pilus Gene Clusters in Haemophilus influenzae and Variation in the hifE Pilin Gene

Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius strain F3031 contains two identical copies of a five gene cluster (hifA to hifE) encoding pili similar to well-characterized Hif fimbriae of H. influenzae type b. HifE, the putative pilus tip adhesin of F3031, shares only 40% amino acid sequence similarity with the same molecule from type b strains, whereas the other four proteins have 75 to 95% identity. To determine whether pilus cluster duplication and the hifE_F3031 allele were special features of BPF-associated bacteria, we analyzed a collection of H. influenzae strains by PCR with hifA- and hifE-specific oligonucleotides, by Southern hybridization with a hifC gene probe, and by nucleotide sequencing. The presence of two pilus clusters was limited to some H. influenzae biogroup aegyptius strains. The hifE_F3031 allele was limited to H. influenzae biogroup aegyptius. Two strains contained one copy of hifE_F3031 and one copy of a variant hifE allele. We determined the nucleotide sequences of four hifE genes from H. influenzae biogroup aegyptius and H. influenzae capsule serotypes a and c. The predicted proteins produced by these genes demonstrated only 35 to 70% identity to the three published HifE proteins from nontypeable H. influenzae, serotype b, and BPF strains. The C-terminal third of the molecules implicated in chaperone binding was the most highly conserved region. Three conserved domains in the otherwise highly variable N-terminal putative receptor-binding region of HifE were similar to conserved portions in the N terminus of Neisseria pilus adhesin PilC. We concluded that two pilus clusters and hifE_F3031 were not specific for BPF-causing H. influenzae, and we also identified portions of HifE possibly involved in binding mammalian cell receptors.     

Comparative Analysis of Haemophilus influenzae hifA (Pilin) Genes

Adherence of Haemophilus influenzae to epithelial cells plays a central role in colonization and is the first step in infection with this organism. Pili, which are large polymorphic surface proteins, have been shown to mediate the binding of H. influenzae to cells of the human respiratory tract. Earlier experiments have demonstrated that the major epitopes of H. influenzae pili are highly conformational and immunologically heterogenous; their subunit pilins are, however, immunologically homogenous. To define the extent of structural variation in pilins, which polymerize to form pili, the pilin genes (hifA) of 26 type a to f and 16 nontypeable strains of H. influenzae were amplified by PCR and subjected to restriction fragment length polymorphism (RFLP) analysis with AluI and RsaI. Six different RFLP patterns were identified. Four further RFLP patterns were identified from published hifA sequences from five nontypeable H. influenzae strains. Two patterns contained only nontypeable isolates; one of these contained H. influenzae biotype aegyptius strains F3031 and F3037. Another pattern contained predominantly H. influenzae type f strains. All other patterns were displayed by a variety of capsular and noncapsular types. Sequence analysis of selected hifA genes confirmed the 10 RFLP patterns and showed strong identity among representatives displaying the same RFLP patterns. In addition, the immunologic reactivity of pili with antipilus antisera correlated with the groupings of strains based on hifA RFLP patterns. Those strains that show greater reactivity with antiserum directed against H. influenzae type b strain M43 pili tend to fall into one RFLP pattern (pattern 3); while those strains that show equal or greater reactivity with antiserum directed against H. influenzae type b strain Eagan pili tend to fall in a different RFLP pattern (pattern 1). Sequence analysis of representative HifA pilins from typeable and nontypeable H. influenzae identified several highly conserved regions that play a role in bacterial pilus assembly and other regions with considerable amino acid heterogeneity. These regions of HifA amino acid sequence heterogeneity may explain the immunologic diversity seen in intact pili.     

Pilus-mediated adherence of Haemophilus influenzae to human respiratory mucins

Haemophilus influenzae, especially the nontypeable strains, are among the most common pathogens encountered in patients with chronic lung disease and otitis media. We and others have demonstrated that respiratory isolates of nontypeable H. influenzae bind to human mucins, but the mechanism of binding is not entirely clear. We have therefore examined the role of pili in the adherence of both type b and nontypeable H. influenzae to human respiratory mucins. We used isogenic H. influenzae strains with a mutation in the structural gene for pilin (hifA), a laboratory H. influenzae strain transformed with a type b pilus gene cluster (from strain C54), antibodies raised against H. influenzae HifA, and Escherichia coli strains carrying a cloned type b pilus gene cluster (from strain AM30) in these studies. All bacteria lacking HifA or the pilus gene cluster had decreased adherence of piliated H. influenzae to mucins, and Fab fragments of anti-HifA antibodies inhibited the adherence. E. coli strains carrying the cloned type b pilus gene cluster were six to seven times more adhesive than strains carrying the vector. The role of other putative adhesins was not examined and thus cannot be excluded, but these studies support a role for pili in the binding of H. influenzae to human respiratory mucins.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.     

Pseudomonas aeruginosa Gene Products PilT and PilU Are Required for Cytotoxicity In Vitro and Virulence in a Mouse Model of Acute Pneumonia

Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. The pilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilU mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.     

Investigation of the role of type IV Aeromonas pilus (Tap) in the pathogenesis of Aeromonas gastrointestinal infection

Although there is substantial evidence that type IV pili purified from diarrhea-associated Aeromonas species (designated Bfp for bundle-forming pilus) are intestinal colonization factors (S. M. Kirov, L. A. O'Donovan, and K. Sanderson, Infect. Immun. 67:5447-5454, 1999), nothing is known regarding the function of a second family of Aeromonas type IV pili (designated Tap for type IV Aeromonas pilus), identified following the cloning of a pilus biogenesis gene cluster tapABCD. Related pilus gene clusters are widely conserved among gram-negative bacteria, but their significance for virulence has been controversial. To investigate the role of Tap pili in Aeromonas pathogenesis, mutants of Aeromonas strains (a fish isolate of A. hydrophila and a human dysenteric isolate of A. veronii bv. sobria) were prepared by insertional inactivation of the tapA gene which encodes the type IV pilus subunit protein, TapA. Exotoxic activities were unaffected by the mutation in tapA. Inactivation of tapA had no effect on the bacterial adherence of these two isolates to HEp-2 cells. For the A. veronii bv. sobria isolate, adhesion to Henle 407 intestinal cells and to human intestinal tissue was also unaffected. There was no significant effect on the duration of colonization or incidence of diarrhea when the A. veronii bv. sobria strain was tested in the removable intestinal tie adult rabbit diarrhea model or on its ability to colonize infant mice. Evidence was obtained that demonstrated that TapA was expressed by both Aeromonas species and was present on the cell surface, although if assembled into pili this pilus type appears to be an uncommon one under standard bacterial growth conditions. Further studies into factors which may influence Tap expression are required, but the present study suggests that Tap pili may not be as significant as Bfp pili for Aeromonas intestinal colonization.     

Molecular and genetic characterization of Francisella tularensis strains of differing taxonomic status and virulence

Typing of Francisella collection strains by means of PCR on the basis of the tul4 gene coding the protein of the external membrane of 17kD and the genome region of differentiation RD1 is performed. The identification of the species and subspecies of 112 strains of Francisella tularensis was revealed. PCR on DNA targets’ loci of type IV pili genes—pilA, pilE2, pilE3, pilE4, pilE5, pilF, pilT, pilD, and pilQ—was carried out for differentiation of F. tularensis strains by virulence. The possibility was shown of distinguishing F. tularensis strains in PCR (primers A–B) on the basis of the uncovering of the gene pilA in virulent strains of three subspecies F. tularensis and F. tularensis subsp. novicida. The pilA gene was not detected in the vaccine strain 15/10 and its variants, as well as in most of avirulent F. tularensis supsp. holarctica strains. However, the fragment gene pilA was found in the attenuated strains F. tularensis subsp. tularensis and mediasiatica. No differences were found in other targets of pili genes between F. tularensis strains, with the exception of the strain F. tularensis subsp. novicida Utahl12, which did not have a fragment of the gene pilE2. The use of PCR to target the locus of the piliA gene allows differentiation of virulent F. tularensis subsp. holarctica strains from the vaccine 15/10, its variants, and avirulent strains.     

Functional Characterization of Type IV Pili Expressed on Diarrhea-Associated Isolates of Aeromonas species

Our past work has shown that long, flexible type IV pili (single or in bundles) are the predominant pili expressed on fecal isolates of diarrhea-associated species of Aeromonas (Aeromonas veronii biovar sobria and A. caviae). They represent a family of type IV pili which we have designated Bfp (for bundle-forming pili). Reports from Japan suggest that Bfp are intestinal colonization factors. This study presents compelling evidence to support this conclusion. Aeromonas bacteria and/or Bfp purified from a strain of A. veronii biovar sobria were shown to adhere to epithelial and intestinal cell lines, freshly isolated human enterocytes, and fresh and fixed human and rabbit intestinal tissues, as determined by light and electron microscopy and immunohistochemical detection. Removal of Bfp by mechanical means decreased adhesion to cell lines by up to 80%. Purified Bfp blocked adhesion of the test strain to intestinal cells in a dose-dependent manner. Adhesion was also blocked by the Fab fraction of anti-Bfp immunoglobulin G. Moreover, ultrastructural studies (ruthenium red staining and transmission and scanning electron microscopy) demonstrated for the first time that Aeromonas adhesion to human enterocytes is pilus mediated and suggested that Bfp may also promote colonization by forming bacterium-to-bacterium linkages. Bfp-positive isolates examined for type IV pilus-mediated twitching motility in agar and slide culture assays developed for Pseudomonas aeruginosa did not, however, exhibit this function.     

Twitching motility contributes to the role of pili in corneal infection caused by Pseudomonas aeruginosa

Twitching motility is a form of surface-associated bacterial movement mediated by type IV pili of Pseudomonas aeruginosa. Others have shown that pilT and pilU mutants, which are piliated but defective in twitching motility, display reduced cytotoxic capacity towards epithelial cells in vitro. Although these mutants efficiently infected lungs in vivo, they were defective in dissemination to the liver. In this study the role of twitching motility in P. aeruginosa epithelial cell invasion and corneal disease pathogenesis was explored. pilU and pilT mutants of P. aeruginosa strain PAK were compared to a nonpiliated pilA mutant and to wild-type bacteria in their ability to associate with and to invade corneal epithelial cells in vitro and to cause disease in a murine model of corneal infection. As expected, the pilA mutant demonstrated reduced association and invasion of corneal epithelial cells (P < 0.05 in both cases). The pilT mutant, but not the pilU mutant, was less invasive than wild-type PAK was (P < 0.05 versus P = 0.43), while both pilU and pilT mutants exhibited association levels similar to those of the wild type (P = 0.31 and 0.52, respectively). In vivo, all mutants were markedly attenuated in virulence and showed reduced ability to colonize the cornea at 4 and 48 h (all P values < 0.02). Thus, twitching motility contributed to the role of pili in corneal disease but was not involved in the role of pili in adherence to or invasion of corneal epithelial cells.     

Impact of Francisella tularensis pilin homologs on pilus formation and virulence

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Virulence factors for this bacterium, particularly those that facilitate host cell interaction, remain largely uncharacterized. However, genes homologous to those involved in type IV pilus structure and assembly, including six genes encoding putative major pilin subunit proteins, are present in the genome of the highly virulent Schu S4 strain. To analyze the roles of three putative pilin genes in pili structure and function we constructed individual pilE4, pilE5, and pilE6 deletion mutants in both the F. tularensis tularensis strain Schu S4 and the Live Vaccine Strain (LVS), an attenuated derivative strain of F. tularensis holarctica. Transmission electron microscopy (TEM) of Schu S4 and LVS wild-type and deletion strains confirmed that pilE4 was essential for the expression of type IV pilus-like fibers by both subspecies. By the same method, pilE5 and pilE6 were dispensable for pilus production. In vitro adherence assays with J774A.1 cells revealed that LVS pilE4, pilE5, and pilE6 deletion mutants displayed increased attachment compared to wild-type LVS. However, in the Schu S4 background, similar deletion mutants displayed adherence levels similar to wild-type. In vivo, LVS pilE5 and pilE6 deletion mutants were significantly attenuated compared to wild-type LVS by intradermal and subcutaneous murine infection, while no Schu S4 deletion mutant was significantly attenuated compared to wild-type Schu S4. While pilE4 was essential for fiber expression on both Schu S4 and LVS, neither its protein product nor the assembled fibers contributed significantly to virulence in mice. Absent a role in pilus formation, we speculate PilE5 and PilE6 are pseudopilin homologs that comprise, or are associated with, a novel type II-related secretion system in Schu S4 and LVS.     

Adhesin Expression in Matched Nasopharyngeal and Middle Ear Isolates of Nontypeable Haemophilus influenzae from Children with Acute Otitis Media

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable Haemophilus influenzae. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable H. influenzae from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five hif genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable H. influenzae associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.     

The Longus type IV pilus of enterotoxigenic Escherichia coli (ETEC) mediates bacterial self-aggregation and protection from antimicrobial agents

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. ETEC pili and non-pili adherence factors designated colonization surface antigens (CSA) are believed to be important in the pathogenesis of diarrhea. Longus, a type IV pilus identified as the CSA₂₁, is expressed in up to one-third of ETEC strains, and share similarities to the toxin-coregulated pilus of Vibrio cholerae, and the bundle-forming pilus of enteropathogenic E. coli. To identify longus phenotype and possible function, a site-directed mutation of the lngA major subunit gene in the E9034A wild type ETEC strain was constructed. Lack of longus expression from the lngA mutant was demonstrated by immunoblot analysis and electron microscopy using specific anti-LngA antibody. Formation of self-aggregates by ETEC was shown to be dependent on longus expression as the lngA mutant or wild type grown under poor longus expression conditions was unable to express this phenotype. Longus-expressing ETEC were also associated with improved survival when exposed to antibacterial factors including lysozyme and antibiotics. This suggests that longus-mediated bacterial self-aggregates protect bacteria against antimicrobial environmental agents and may promote gut colonization.     

Analysis of pilus adhesins from Haemophilus influenzae biotype IV strains

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.     

Contribution of type IV pili to the virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon (Salmo salar L.)

Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.     

Characterization of a type IV bundle-forming pilus (SFP) from a gastroenteritis-associated strain ofAeromonas veroniibiovar sobria

The colonization mechanisms of enteropathogenicAeromonasstrains are poorly characterized, but recent studies indicate that some filamentous structures are intestinal adhesins. This study describes the purification and characterization of a long, flexible pilus from a gastroenteritis-associated strain ofAeromonas veroniibiovar sobria. SDS-PAGE analysis (various conditions) of pili preparations yielded a pilin protein band of ≈21 kDa. Its N-terminal amino acid sequence was unambiguous and homologous with those of type IV pilins. Immunogold electron microscopy with rabbit antisera produced against this pilin protein (SFP) decorated single pili and rope-like bundles of pili on the bacterial surface. These were seen more frequently on strains grown at 22°C compared with 37°C and in liquid rather than on solid medium. SFP was not detected on any of 104 strains ofAeromonas(different species and sources) from our culture collection, although morphologically similar structures were seen on a number of these strains. This finding and differences among other published amino acid sequences show thatAeromonastype IV pili are antigenically diverse. Bundle-forming type IV ‘class B’ pili are important in the virulence of other enteropathogenic bacteria. The N-terminal amino acid sequence of theAeromonasSFP, however, showed closer homology to the type IV ‘class A’ pilins. Studies are in progress to investigate the role of SFP inAeromonasvirulence.     

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.     

Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.     

Characterization of pili expressed by Haemophilus ducreyi

Twelve strains of Haemophilus ducreyi isolated primarily from chancroid outbreaks in North America were examined for the presence of pili by transmission electron microscopy. We identified piliated cells in 10 of the 12 strains. Pilin extracts were prepared from the mechanically sheared cells of the 12 H. ducreyi strains as well as the stably piliated H. influenzae strain R890 and its non-piliated parent R906. Pili were present in 12 out of 12 H. ducreyi extracts and in the R890 extract but not in the R906 preparation. Pili were purified by cycles of differential pH solubilization and crystalization. In SDS-PAGE, the preparation consisted predominantly of a protein whose apparent relative molecular mass was 24000 (24 k), and an electron micrograph showed that the preparation contained pili. Three H. ducreyi strains were passed 52 times on agar plates, and extracts prepared from these strains contained pili. There was no evidence of binding of erythrocytes obtained from nine mammalian and avian species to colonies of one of the stably piliated H. ducreyi strains. We conclude that H. ducreyi expressed pili, that the relative molecular mass of the pilin monomer was 24 k, that pilus expression was not readily lost in passage and that H. ducreyi pili may not bind to an erythrocyte receptor.     

Genes Required for Assembly of Pili Associated with the Helicobacter pylori cag Type IV Secretion System

Helicobacter pylori causes numerous alterations in gastric epithelial cells through processes that are dependent on activity of the cag type IV secretion system (T4SS). Filamentous structures termed “pili” have been visualized at the interface between H. pylori and gastric epithelial cells, and previous studies suggested that pilus formation is dependent on the presence of the cag pathogenicity island (PAI). Thus far, there has been relatively little effort to identify specific genes that are required for pilus formation, and the role of pili in T4SS function is unclear. In this study, we selected 7 genes in the cag PAI that are known to be required for T4SS function and investigated whether these genes were required for pilus formation. cagT, cagX, cagV, cagM, and cag3 mutants were defective in both T4SS function and pilus formation; complemented mutants regained T4SS function and the capacity for pilus formation. cagY and cagC mutants were defective in T4SS function but retained the capacity for pilus formation. These results define a set of cag PAI genes that are required for both pilus biogenesis and T4SS function and reveal that these processes can be uncoupled in specific mutant strains.