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 coli(2000)Enteropathogenic 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 coli(1995)Localized 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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Single Particle Localization and a Novel Topology of the Enteropathogenic Escherichia coli Type IV Pilus Outer Membrane Secretin(2011)Type IV Pili (T4P) are filamentous surface appendages required for adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins are a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4Ps, type II secretion, type III secretion, and phage assembly. We determined the role of the lipoprotein transport pathway in targeting the BfpB secretin protein of the EPEC T4P to the OM. We demonstrated that concurrent mutation of genes encoding both the secretin and retraction ATPase can result in viable cells and find that these cells display paradoxically low levels of cell envelope stress response activity. To study the sub-cellular distribution of BfpB, we used Photo-activated Localization Microscopy to localize single BfpB molecules fused to photo-activated mCherry. Contrary to findings in other T4P systems, we find that BFP components predominantly have an uneven distribution throughout the cell envelope and are only found at one or both poles in a minority of cells. Furthermore, we have used an array of biophysical and biochemical techniques to construct a model topology of BfpB. We provide evidence that the N- and C-termini of this protein have a trans configuration with respect to the OM. We propose that BfpB and all secretins constitute a novel structural class of OM proteins.
