Cloning, expression, and sequence analysis of the Haemophilus influenzae type b strain M43p+ pilin gene.

By using antiserum against Haemophilus influenzae type b (Hib) strain M43p+ denatured pilin, we screened a genomic library of Hib strain M43p+ and identified a clone that expressed pilin, but not assembled pili, on its surface. Southern blot analysis revealed the presence of one structural gene, which was also present in strain M42p-, a nonpiliated variant. Five exonuclease III deletion mutants, two of which had deletions that extended into the structural gene and failed to express pilin, were used to obtain the nucleotide sequence of the structural gene. The amino acid sequence of the open reading frame agrees with 38 of 40 amino acids from the published sequence of purified Hib M43p+ pilin. The pilin gene coded for a mature protein of 193 amino acids, with a calculated molecular mass of 21,101 daltons. Comparison of the Hib M43p+ pilin amino acid sequence with those of pilins of other bacteria revealed strong conservation of amino- and carboxy-terminal regions in M43p+ and Escherichia coli F17, type 1C, and several members of the P pili family, as well as Klebsiella pneumoniae type 3 MR/K, Bordetella pertussis serotype 2, and Serratia marcescens US46 fimbriae.     

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.     

Cloning and sequence analysis of the structural pilin gene of Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius.

We have cloned and sequenced the Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (Hae) pilin gene. The sequence contained a 648-bp open reading frame encoding a mature pilin protein of 191 amino acids with a calculated mass of 20.5 kDa. There was 82% homology between the open reading frames of the BPF strain F3031 and H. influenzae type b (Hib) (strain M43) pilin genes and 71% homology at the amino acid level between the mature pilin proteins. However, areas of diversity were noted throughout the gene. A 17-bp probe corresponding to an area of diversity in the N-terminal region of the BPF-associated gene hybridized with other BPF strains but not with non-BPF Hae or Hib. In summary, the pilin protein of BPF-associated Hae is highly homologous to Hib pilin yet remains structurally distinct.     

Comparison and analysis of the nucleotide sequences of pilin genes from Haemophilus influenzae type b strains Eagan and M43.

Previous studies have demonstrated antigenic differences among the pili expressed by various strains of Haemophilus influenzae type b (Hib). In order to understand the molecular basis for these differences, the structural gene for pilin was cloned from Hib strain Eagan (p+) and the nucleotide sequence was compared to those of strains M43 (p+) and 770235 b0f+, which had been previously determined. The pilin gene of Hib strain Eagan (p+) had a 648-bp open reading frame that encoded a 20-amino-acid leader sequence followed by the 196 amino acids found in mature pilin. The translated sequence was three amino acids larger than pilins of strains M43 (p+) and 770235 b0f+ and was 78% identical and 95% homologous when conservative amino acid substitutions were considered. Differences between the amino acid sequences were not localized to any one region but rather were distributed throughout the proteins. Comparison of protein hydrophilicity profiles showed several hydrophilic regions with sequences that were conserved between strain Eagan (p+) and pilins of other Hib strains, and these regions represent potentially conserved antigenic domains. Southern blot analyses using an intragenic probe from the pilin gene of strain Eagan (p+) showed that the pilin gene was conserved among all type b and nontypeable strains of H. influenzae examined, and only a single copy was present in these strains. Homologous genes were not present in the phylogenetically related species Pasteurella multocida, Pasteurella haemolytica, and Actinobacillus pleuropneumoniae. These data indicate that the pilin gene was highly conserved among different strains of H. influenzae and that small differences in the pilin amino acid sequences account for the observed antigenic differences of assembled pili from these strains.     

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.     

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.     

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.     

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.     

Low-level pilin expression allows for substantial DNA transformation competence in Neisseria gonorrhoeae

The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.     

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.     

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.     

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.     

Paradoxical effect of pilus expression on binding of antibodies by Haemophilus influenzae.

Haemophilus influenzae type b (Hib) pili are surface proteins that are associated with the ability of Hib to attach to human epithelial cells. Like pilus expression of other bacteria, expression of Hib pili undergoes phase variation. We observed that Hib in the piliated phase (Hib p+) bound monoclonal antibodies directed against six conserved, surface-exposed, nonpilus Hib outer membrane epitopes to a greater extent than Hib in the nonpiliated phase (Hib p-). However, after extended incubation, p+ and p- cells bound these antibodies in a similar fashion. The differential in nonpilus antibody binding to p+ and p- Hib was not related to the presence of the type b capsule. In addition, Hib p+ organisms whose pilin gene was insertionally inactivated and did not produce pili and Hib in the nonpiliated phase bound the nonpilus Hib antibodies similarly. Hib p+ and p- organisms did not differ in their binding of anti-type b capsule antibody, and the binding was specific for the epitopes recognized by the antibodies. In complement-dependent bactericidal assays, the nonpilus antibodies killed Hib p+ more effectively than Hib p-. The increased binding to, and killing of, Hib p+ by a variety of nonpilus antibodies may be important for host defense against invasive Hib.     

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.     

Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae.

A Haemophilus influenzae b (Hib) membrane protein with a molecular mass of 28 kDa bound polyclonal antisera raised against a highly purified Hib fimbrial subunit. We cloned the gene encoding this protein and found that the gene was expressed in Escherichia coli. DNA sequence analysis identified an 843-bp open reading frame which predicted a 26.78-kDa protein with an amino-terminal signal sequence and a mature protein with 70% similarity to the 28-kDa lipoprotein of E. coli (F. Yu, S. Inouye, and M. Inouye, J. Biol. Chem. 261:2284, 1986). Colony blot hybridization analysis with an intergenic probe of the cloned gene demonstrated that 29 of 32 H. influenzae strains hybridize with this gene. Insertion of a chloramphenicol acetyltransferase gene into the open reading frame inactivated expression of the 28-kDa protein in E. coli. Isogenic Hib strains were derived by marker exchange mutagenesis to generate mutants which no longer expressed the 28-kDa protein as recognized with Western immunoblot analysis. There was no difference in the rate of nasopharyngeal colonization of infant rats or monkeys by the isogenic mutants which lacked the 28-kDa protein compared with colonization by the wild-type strain. In contrast, the frequency of invasion and density of bacteremia in infant rats caused by the isogenic mutants were reduced relative to those caused by the wild-type Hib strain. We conclude that this 28-kDa outer membrane protein aids transepithelial invasion of type b strains but is not essential.     

Physical linkage of the Vibrio cholerae mannose-sensitive hemagglutinin secretory and structural subunit gene loci: identification of the mshG coding sequence.

Vibrio cholerae O1 expresses a variety of cell surface factors which mediate bacterial adherence and colonization at the intestinal epithelium. The mannose-sensitive hemagglutinin (MSHA), a type IV pilus, is a potential attachment factor of the V. cholerae El Tor biotype. We describe a TnphoA mutant that is defective in its ability to hemagglutinate mouse erythrocytes. The TnphoA insertion maps to a recently identified genetic locus that encodes products that are predicted to be essential for assembly and export of the MSHA pilus. Insertional disruption at this locus in a mshA-phoA reporter strain provides evidence for a role of this locus in the latter stages of pilus assembly and/or export. These constructs have provided physical markers by which we have established close physical linkage of this secretion locus to a set of genes that includes the mshA structural gene. Sequence analysis of the intervening region between these two loci has revealed the presence of an open reading frame with homology to pilus biogenesis genes of several gram-negative bacteria. This genetic organization suggests an entire operon encoding the MSHA pilus and the components necessary for its assembly and secretion to the bacterial cell surface. The nomenclature of the MSHA structural and secretory locus has been redefined accordingly.     

Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis.

A group of high-molecular-weight surface-exposed proteins of nontypeable Haemophilus influenzae are major targets of human serum antibody (S. J. Barenkamp and F. F. Bodor, Pediatr. Infect. Dis. J. 9:333-337, 1990). To further characterize these proteins, we cloned and sequenced genes encoding two related high-molecular-weight proteins from a prototype nontypeable Haemophilus strain. The gene encoding a 120-kDa Haemophilus protein consisted of a 4.4-kbp open reading frame, and the gene encoding a 125-kDa protein consisted of a 4.6-kbp open reading frame. The first 1,259 bp of the two genes were identical. Thereafter, the sequences began to diverge, but overall they were 80% identical, and the derived amino acid sequences showed 70% identity. A protein sequence homology search demonstrated similarity between the derived amino acid sequences of both cloned genes and the derived amino acid sequence of the gene encoding filamentous hemagglutinin, a surface protein produced by the gram-negative pathogen Bordetella pertussis. Antiserum raised against a recombinant protein encoded by the 4.6-kbp open reading frame recognized both the 120- and the 125-kDa proteins in the prototype strain as well as antigenically related high-molecular-weight proteins in 75% of a collection of 125 epidemiologically unrelated nontypeable H. influenzae strains. The antiserum directed against the recombinant protein also recognized purified filamentous hemagglutinin. A murine monoclonal antibody to filamentous hemagglutinin recognized both the 120-kDa and the 125-kDa protein in the prototype strain as well as proteins identical to those recognized by the recombinant-protein antiserum in 35% of the nontypeable H. influenzae strain collection. Thus, we have identified and partially characterized a group of highly immunogenic surface-exposed proteins of nontypeable H. influenzae which are related to the filamentous hemagglutinin of B. pertussis.     

Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae

The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved. The pilA gene encodes a putative type IV pilus subunit. However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes. The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD. Taken together, these data demonstrate the effectiveness of the V. cholerae genome project for rapid identification and characterization of potential virulence factors.     

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.     

Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain.

Previous studies have suggested that the Pseudomonas aeruginosa PAK pilus adhesin moiety resides in an epithelial cell-binding domain located in the C-terminal region of the PAK pilin structural protein. Synthetic peptides Ac17red (a synthetic peptide with a sequence identical to that of PAK pilin residues 128 to 144, with the Cys-129 and Cys-142 residues being in the reduced state) and Ac17ox (a synthetic peptide with a sequence identical to that of PAK pilin residues 128 to 144, with a formed disulfide bridge between the amino acid residues Cys-129 and Cys-142), which should contain the epithelial cell-binding domain, were synthesized. Ac17red and Ac17ox both bound to buccal epithelial cells (BECs) and to ciliated tracheal epithelial cells (TECs). Ac17ox had a Km of 6.40 microM for binding to BECs, while Ac17red had a Km of 9.87 microM. Ac17red bound to the same receptor sites that purified pili did and competitively inhibited the binding of purified PAK pili to BECs. BEC glycoproteins with molecular masses of 82, 55 to 51, and 40 kilodaltons immobilized on nitrocellulose exhibited periodate-sensitive receptor activity for Ac17red; similar activity has been found for PAK pili. Ac17red, Ac17ox, and PAK pili bound to the cilia and luminal portions of the cytoplasmic membrane of human TECs, the same regions to which P. aeruginosa whole cells bind. PAK pilin has an epithelial cell-binding domain that resides in the C-terminal region of the protein.