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.     

Genes for CS2 pili of enterotoxigenic Escherichia coli and their interchangeability with those for CS1 pili.

We have cloned and sequenced the DNA needed for production of CS2 pili in Escherichia coli K-12. The four open reading frames, cotB, cotA, cotC, and cotD, show homology with the genes needed for production of CS1 and CFA/I pili, which are also found on enterotoxigenic E. coli associated with human diarrheal disease. We also report that CotA plus CotB interact with the CS1 gene products CooC and CooD to form pili that can be visualized by electron microscopy and, conversely, that the CS1 gene products CooA and CooB interact with CotC and CotD to form pili.     

Genetic analysis of the transfer region of the IncN plasmid N3

Using lambda::Tn5 insertion mutagenesis and screening for conjugation, the boundaries of the IncN plasmid N3 transfer region were determined. Sensitivity to phage IKe infection was used to monitor that part of the N3 transfer region which harbours genes for pilus synthesis and assembly. We cloned this region, creating plasmid pBG21. Escherichia coli cells transformed with pBG21 became sensitive to phage IKe and produced pili, as shown by electron microscopy. Various plasmid constructions containing parts of the pilus-encoding region were used for expression in a minicell system and for expression in an in vitro translation system, thus characterizing for the first time some of the gene products of domain I (Winans and Walker, 1985a) of the transfer region.     

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.     

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.     

Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate.

Isolates of Escherichia coli from human urinary tract infections frequently express adherence properties found less often among normal intestinal isolates. These properties include adherence to human uroepithelial cells and primary monkey kidney cells, as well as D-mannose-resistant hemagglutination of human erythrocytes, and they are mediated by a pilus type different from type 1. The genes encoding this pilus type (pyelonephritis-associated pili, pap) and those encoding type 1 pili have been cloned from a urinary tract infection isolate of E. coli and transferred to an E. coli K-12 derivative. The recombinant plasmids were found to express functional pili and to endow the new host with all of the adherence properties of the urinary tract infection isolate. Both pilus types were found to be genetically distinct, and unlike the adherence genes from bovine, porcine, and human diarrheal isolates, both were found to be chromosomally encoded.     

Type 1 pili are not necessary for colonization of the streptomycin-treated mouse large intestine by type 1-piliated Escherichia coli F-18 and E. coli K-12.

Escherichia coli F-18, an excellent colonizer of the streptomycin-treated mouse large intestine, produces type 1 pili. E. coli F-18 FimA-, type 1 pilus negative, and E. coli F-18 FimH-, type 1 pilus positive but adhesin negative, were constructed by bacteriophage P1 transduction of defective fimA and fimH genes from the E. coli K-12 strains ORN151 and ORN133, respectively, into E. coli F-18. Adhesion of E. coli F-18 to an immobilized mannose-bovine serum albumin glycoconjugate was about sixfold greater than that of either E. coli F-18 FimA- or E. coli F-18 FimH-, and adhesion of E. coli F-18 to immobilized cecal epithelial cell brush border membranes was between two- and threefold greater than that of E. coli F-18 FimA- or E. coli F-18 FimH-. When either E. coli F-18 FimA- or E. coli FimH- was fed to streptomycin-treated mice together with E. coli F-18, the pilus-negative and adhesin-negative strains colonized as well as their type 1-piliated parent. Essentially the same result was observed when the type 1-piliated E. coli K-12 strain ORN152 was fed to streptomycin-treated mice together with a nearly isogenic K-12 FimA- strain, ORN151. Furthermore, when streptomycin-treated mice were fed E. coli F-18 FimA- or E. coli F-18 FimH- together with E. coli F-18 Col-, which also makes type 1 pili but is a poor colonizer relative to E. coli F-18 because it grows poorly in mucus in the presence of E. coli F-18, the F-18 FimA- and F-18 FimH- strains colonized well (10(6) to 10(7) CFU/g of feces), whereas the number of E. coli F-18 Col- in feces decreased rapidly to 10(2) CFU/g of feces. These data show that in streptomycin-treated mice, the inability to produce functional type 1 pili has no effect on the ability of E. coli F-18 and E. coli K-12 to colonize the large intestine.     

Identification and characterization of the locus for diffuse adherence, which encodes a novel afimbrial adhesin found in atypical enteropathogenic Escherichia coli

The O26 serogroup of enteropathogenic Escherichia coli (EPEC) is one of the serogroups most frequently implicated in infant diarrhea and is also common among enterohemorrhagic E. coli (EHEC) strains. The most common O26 strains belong to EPEC/EHEC serotype O26:H11 and are generally Shiga toxin (Stx) positive. Stx-negative E. coli strains that are negative for the EPEC EAF plasmid and bundle-forming pilus (Bfp) are classified as atypical EPEC. Here, we report a novel adhesin present in an stx-negative bfpA-negative atypical EPEC O26:H11 strain isolated from an infant with diarrhea. A cloned 15-kb genomic region from this strain, designated the locus for diffuse adherence (lda), confers diffuse adherence on HEp-2 cells when expressed in E. coli K-12. Sequence analysis of lda revealed a G+C content of 46.8% and 15 open reading frames sharing homology with the E. coli K88 fae and CS31A clp fimbrial operons. The lda region is part of a putative 26-kb genomic island inserted into the proP gene of the E. coli chromosome. Hybridization studies have demonstrated the prevalence of the minor structural subunit gene, ldaH, across E. coli serogroups O5, O26, O111, and O145. A second plasmid-encoded factor that contributed to the Hep-2 adherence of this strain was also identified but was not characterized. Null mutations that abolish adherence to HEp-2 cells can be restored by plasmid complementation. Antiserum raised against the major structural subunit, LdaG, recognizes a 25-kDa protein from crude heat-extracted protein preparations and inhibits the adherence of the E. coli DH5alpha lda(+) clone to HEp-2 cells. Electron microscopy revealed a nonfimbrial structure surrounding the bacterial cell.     

Genetics of digalactoside-binding adhesin from a uropathogenic Escherichia coli strain.

The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin. A cosmid clone from this strain has been isolated that, when harbored in E. coli K-12, expressed Pap pili and this adhesin (R. Hull et al., Infect. Immun. 33:933-938, 1981). By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E. coli K-12 strain P678-54. The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon. Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum. This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA.     

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.     

Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli.

One of the chromosomal segments associated with the virulence of Shigella flexneri and transferred to Escherichia coli K-12 by conjugation has been shown to code for the production of aerobactin and for the synthesis of an iron-regulated 76,000-dalton (76K) outer membrane protein. Analysis of various E. coli K-12-S. flexneri transconjugants showed that the genes involved with the synthesis of aerobactin and with the production of the 76K protein were linked to the mtl region of the S. flexneri chromosome. S. flexneri itself synthesized a 76K protein in its outer membrane under iron restriction as well as traces of 81K and 74K proteins. An examination of four enteroinvasive strains of E. coli showed that each produced aerobactin and a 76K outer membrane protein during iron-restricted growth. The profile of the iron-regulated proteins expressed by the enteroinvasive strains of E. coli was virtually identical to that expressed by the laboratory-constructed E. coli K-12-S. flexneri hybrids under the same growth conditions.     

Regulation of expression of Escherichia coli pilus K99.

An immunoassay demonstrated that the assembled K99 pilus on the surface of Escherichia coli grown in minimal medium appeared during the logarithmic phase of growth, but the synthesis of K99 subunits, as measured by nonequilibrium two-dimensional gel electrophoresis, occurred throughout the life cycle of the cell. Contrary to other reports, the addition of glucose to the growth medium did not affect K99 pilus assembly or subunit synthesis, although in a K99+ adenyl cyclase (cya) mutant, subunit synthesis was reduced. There was no reduction in the amount of assembled K99 on the cell surface of the cya mutant compared with the wild-type parent. The addition of L-alanine to minimal medium repressed K99 synthesis. However, if L-threonine or L-isoleucine was also included in the growth medium, the effect of L-alanine was reduced. Chloramphenicol caused a complete inhibition of K99 subunit synthesis, but assembly proceeded normally. Growth at 18 degrees C inhibited both subunit synthesis and pilus assembly. Approximately 92% of all cellular K99 was associated with the outer membrane, and 4% was associated with the inner membrane. No K99 was detected in the cytoplasm.     

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.     

Immunoglobulin-mediated agglutination of and biofilm formation by Escherichia coli K-12 require the type 1 pilus fiber

The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to Escherichia coli is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the fimA gene. A minor component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We found that the aggregation of E. coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM alpha-CH(3)Man. The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in which E. coli was agglutinated by SIgA, the binding of SIgA to E. coli was not increased by the presence of the pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin.     

Cloning and expression in Escherichia coli of LKP pilus genes from a nontypeable Haemophilus influenzae strain.

Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C. C. To, S. C. M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C. M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C. C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity.     

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.     

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.     

Purification and characterization of Serratia marcescens US5 pili.

The pili of Serratia marcescens US5 isolated from a patient with urinary tract infection were purified and characterized. During the aeration culture, the pili were detached from the bacteria and were precipitated by the addition of ammonium sulfate. The purification of the pili was carried out by ion-exchange chromatography and gel filtration on Sepharose 4B. In electron microscopy, the purified pilus showed a filament of 3 nm in diameter and 0.3 micron in average length. The molecular weight of the protein subunit of the purified pili was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two protein bands appeared. One has a molecular size of 19,000 daltons, and the other has a molecular size of 39,000 daltons. The isoelectric point was 3.7. The content of hydrophobic amino acids in purified pili subunits was 42% of the total amino acid content. Further purification of pili by isopycnic centrifugation failed to remove the large protein band. No identical protein bands to pili proteins were detected in the electrophoresis pattern of the outer membrane proteins extracted from S. marcescens US5 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These two proteins might be a dimer of a small molecule. A survey of clinically isolated strains of S. marcescens revealed that more than 60% of the strains had this type of pili. These results suggest that these pili are widely distributed among strains of S. marcescens.     

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.