Identification and molecular characterization of thebfp gene cluster encoding the bundle-forming pilus of enteropathogenic Escherichia coli: A model for molecular studies of type IV pilus biogenesis
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Identification and molecular characterization of the bfp gene cluster encoding the bundle-forming pilus of enteropathogenic Escherichia coli: A model for molecular studies of type IV pilus biogenesisAbstract
Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrhea among infants in the developing world. The initial stage of EPEC pathogenesis involves the adherence of bacteria to epithelial cells in tight clusters, a pattern which has been termed localized adherence. EPEC carry 50-70 MDa plasmids, termed EPEC adherence factor (EAF) plasmids, which are sufficient to confer the localized adherence phenotype upon non-adherent E. coli strains. The EAF plasmid-encoded adherence factor has been demonstrated to be a type IV pilus which aggregates to form bundles and is thus termed the bundle-forming pilus (BFP). The gene encoding the major structural subunit of the BFP, bfpA, has been cloned from the EAF plasmid of EPEC strain E2348/69. In this dissertation, I describe the identification of the bfp gene cluster, a set of 14 essentially contiguous genes on the EAF plasmid, including bfpA and 13 downstream genes. While 10 of the proteins encoded by the genes of this cluster share sequence similarity with proteins involved in the biogenesis of other type IV pili, 4 are unique to this system. Expression of the 14 genes of the bfp gene cluster from an inducible, artificial promoter is sufficient for reconstitution of BFP biogenesis in a laboratory E. coli strain. Non-polar mutagenesis of 3 genes in the cluster, bfpU, bfpH, and bfpL, is described. Mutations in the bfpU and bfpL genes abolish BFP biogenesis, while a mutation in the bfpH gene has no detectable effect on BFP biogenesis or function. Using a monoclonal antibody raised against a BfpU-histidine fusion protein, BfpU is shown to be a periplasmic protein. This is the first evidence that a periplasmic phase of transport is involved in type IV pilus biogenesis. This well-defined system is currently being used to characterize the molecular mechanisms of type IV plus biogenesis in EPEC and should prove to be a useful model for advancing our understanding of type IV pilus biogenesis in many other important human pathogens as well.Description
University of Maryland, Baltimore. Molecular and Cellular Biology. Ph.D. 1998Keyword
Biology, MolecularBiology, Microbiology
bundle-forming pilus biogenesis
type IV pilus
Enteropathogenic Escherichia coli--pathogenicity
Fimbriae, Bacterial
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Characterization of the genes required for the expression of a type IV pilus in enteropathogenic Escherichia coliEnteropathogenic Escherichia coli (EPEC) express a type IV fimbria known as the bundle-forming pilus (BFP). This pilus is required for bacterial autoaggregation and adherence to epithelial cells in a distinctive pattern called localized adherence (LA). EPEC strains that express BFP have a large plasmid. A cluster of fourteen genes on this plasmid is sufficient to reconstitute pilus biogenesis and localized adherence in a laboratory strain of E. coli. Type IV pilus biogenesis is a poorly understood process, and the identification of all the genes required for BFP expression in EPEC makes this an attractive system to study. The first gene in the cluster, bfpA, encodes bundlin, the major structural subunit of BFP. Bundlin is expressed as a pre-protein with a characteristic type IV pilin leader sequence. We have undertaken a systematic mutagenesis of the individual genes of the bfp cluster to determine which genes are required for BFP biogenesis, LA, and autoaggregation. Here we report the construction and analysis of nonpolar mutations in seven bfp genes, bfpG, bfpB, bfpC, bfpD, bfpF, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant that is unable to express bundlin. The mutation in bfpP, the gene encoding the pre-pilin peptidase, does not affect pre-bundlin expression, but blocks pre-bundlin processing, BFP biogenesis, LA, and autoaggregation. The mutations in bfpG, bfpB, bfpC, and bfpD do not affect pre-bundlin expression or processing, but block BFP biogenesis, LA, and autoaggregation. The mutation in bfpF does not block any of these events, and in fact bfpF mutant strains adhere to epithelial cells in greater numbers than do wild-type EPEC. The mutation in bfpH has no discernable effect on BFP expression or function. We also show by sucrose density floatation gradient analysis that the association of prebundlin or bundlin with sucrose density floatation gradient fractions containing both inner and outer membrane proteins does not require any other Bfp proteins. Finally, we show that BfpC is a bitopic inner membrane protein. These results show that BfpP is the only prepilin peptidase in EPEC capable of processing prebundlin, BfpG, BfpB, BfpC, and BfpD are all required for BFP expression, BfpF is not required for BFP expression but does play a role in BFP function, and the bfpH gene is not required for any of the phenotypes examined here.
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Post-translational processing of the major structural subunit of a type IV fimbria from enteropathogenic Escherichia coliLocalized adherence (LA) to epithelial cells is a virulence-associated phenotype of enteropathogenic Escherichia coli (EPEC), a leading cause of infantile diarrhea around the world. An inducible bundle forming pilus (BFP) was proposed to be the adhesin mediating LA. The major structural subunit of BFP (bundlin) is encoded on a large EPEC plasmid by the bfpA gene, a member of the type IV fimbria gene family. Like all type IV pilins, bundlin is synthesized as a precursor which is processed at its N-terminus into the mature form after an atypical signal peptide is cleaved, and, like most fimbrial subunits of any type, bundlin has at its C-terminus two Cysteine residues which could form a disulfide bond. The gene encoding the prepilin peptidase responsible for pre-bundlin N-terminal proteolytic processing, bfpP, was cloned from the EPEC plasmid by functional complementation of a P. aeruginosa prepilin peptidase (pilD) mutant. The predicted product of bfpP is homologous to other prepilin peptidases, including TcpJ of Vibrio cholerae (30% identical amino acids), PulO of Klebsiella oxytoca (29%), and PilD of P. aeruginosa (28%). BfpP and PilD are also functionally interchangeable. Disulfide bond formation at the C-terminal domain of bundlin is catalyzed by the product of a ubiquitous E. coli gene, dsbA. Formation of this disulfide bond is required for the stability of bundlin. Mutants with either cysteine of bundlin replaced by serine fail to express detectable levels of the bundlin polypeptide. The effect of dsbA on bundlin oxidization is independent of signal peptide processing. These results clarify the early steps in biogenesis of a type IV pilus and add to our understanding of the post-translational processing of exported proteins.
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Coli surface antigen 26 acts as an adherence determinant of enterotoxigenic Escherichia coli and is cross-recognized by anti-CS20 antibodiesThe coli surface antigen 26 (CS26) of enterotoxigenic Escherichia coli (ETEC) had been described as a putative adhesive pilus based on the partial sequence of the crsH gene, detected in isolates from children with diarrhea in Egypt. However, its production and activity as adherence determinant has not been experimentally addressed. The crsH was identified as a homolog of genes encoding structural subunits of ETEC colonization factors (CFs) CS12, CS18, and CS20. These CFs, along with the recently discovered CS30, belong to the γ2 family of pili assembled by the chaperone-usher pathway (CU pili). Further, the complete CS26 locus, crsHBCDEFG, was described in an O141 ETEC strain (ETEC 100664) obtained from a diarrhea case in The Gambia, during the Global Enterics Multicenter Study. Here, we report that CS26 is a pilus of ~10 nm in diameter, with the capacity to increase the cell adherence of the non-pathogenic strain E. coli DH10B. As for other related pili, production of CS26 seems to be regulated by phase variation. Deletion of crsHBCDEFG in ETEC 100664 significantly decreased its adherence capacity, which was recovered by in trans complementation. Furthermore, CrsH was cross-recognized by polyclonal antibodies directed against the major structural subunit of CS20, CsnA, as determined by Western blotting and immunogold labeling. ETEC CS26+ strains were found to harbor the heat-labile enterotoxin only, within three different sequence types of phylogroups A and B1, the latter suggesting acquisition through independent events of horizontal transfer. Overall, our results demonstrate that CS26 is an adhesive pilus of human ETEC. In addition, cross-reactivity with anti-CsnA antibodies indicate presence of common epitopes in γ2-CFs. Copyright © 2018 Cádiz, Torres, Valdés, Vera, Gutiérrez, Levine, Montero, O'Ryan, Rasko, Stine, Vidal and Del Canto.