Evolutionary Phylogeny and Molecular Characterisation of Chaperone-Usher Fimbriae from Uropathogenic Escherichia coli

Wurpel, Daniel (2013). Evolutionary Phylogeny and Molecular Characterisation of Chaperone-Usher Fimbriae from Uropathogenic Escherichia coli PhD Thesis, School of Molecular and Chemical Biosciences, The University of Queensland. doi:10.14264/uql.2014.151

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Author Wurpel, Daniel
Thesis Title Evolutionary Phylogeny and Molecular Characterisation of Chaperone-Usher Fimbriae from Uropathogenic Escherichia coli
Formatted title
Evolutionary Phylogeny and Molecular Characterisation of Chaperone-Usher Fimbriae from Uropathogenic Escherichia coli 
School, Centre or Institute School of Molecular and Chemical Biosciences
Institution The University of Queensland
DOI 10.14264/uql.2014.151
Publication date 2013
Thesis type PhD Thesis
Supervisor Mark Schembri
Makrina Totsika
Total pages 220
Language eng
Subjects 0601 Biochemistry and Cell Biology
0603 Evolutionary Biology
0605 Microbiology
Formatted abstract
Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections in humans. For the successful colonisation of the urinary tract, UPEC produce multiple surface-exposed or secreted virulence factors, including adhesins, iron uptake systems and toxins. The primary adhesins employed by UPEC are chaperone-usher (CU) fimbriae; hair-like surface organelles that mediate bacterial attachment, invasion and tissue tropism. While Type 1, P, F1C/S and AFA fimbriae are well-characterised UPEC virulence factors, several UPEC strains are able to bind to uroepithelial cells independently of these adhesins.

To investigate whether additional fimbriae are involved in uroepithelial cell adhesion, this study set out to identify and characterise fimbrial types that are either associated with UPEC isolates or related to known fimbriae involved in uropathogenesis. E. coli genomes contain a diverse array of characterised and putative CU fimbrial operons, however, the classification and annotation of individual types remains problematic. For the effective assessment of the E. coli fimbrial repertoire, this study presents a classification model for the effective typing of CU fimbriae based on usher protein phylogeny and genomic locus synteny. An iterative BLASTp usher protein query was used to identify fimbrial operons in 35 E. coli (and one Escherichia fergusonnii) genomes representing diverse pathogenic and phylogenic lineages, as well as 132 Escherichia spp. plasmids. A total of 458 CU operons were identified, which could be classified as 38 distinct fimbrial types based on the proposed evolutionary classification scheme. The majority of syntenic fimbrial operons formed monophyletic clades; exceptions were associated with mobile genetic elements. Evaluation of the distribution and prevalence of fimbrial types among E. coli pathotypes and phylogenic lineages unveiled core and group specific fimbrial types and provides insight of fimbrial ancestry and evolution within the species.

Based on the CU phylogenetic analysis, F9 fimbriae are closely related to two characterised UPEC virulence factors: type 1 and F1C/S fimbriae. Previous reports demonstrated that F9 fimbriae mediate biofilm formation and adhesion to bovine rectal cells in recombinant E. coli strains, however, the regulation and specific role of F9 fimbriae in wild-type E. coli remained to be determined. In this study, the distribution and genetic context of the ƒ9 operon was assessed among diverse E. coli lineages and pathotypes and demonstrated that ƒ9 genes are significantly more conserved in a UPEC strain collection in comparison to the well-defined E. coli reference (ECOR) collection. In the prototypic UPEC strain CFT073, the global regulator protein H-NS bound directly to the ƒ9 promoter region and was identified as a transcriptional repressor of ƒ9 gene expression at 37°C. Repression of F9 fimbriae was alleviated at 20°C, where F9 fimbriae mediated significant biofilm formation on abiotic surfaces, representing the first evidence of functional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recognise and bind to terminal Galβ1-3GlcNAc structures.

In order to investigate the production of virulence factors by wild-type UPEC isolates, a high-throughput proteomic method was developed based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles. This method was subsequently applied for a comparative proteomic analysis of fifty-four clinical UPEC strains cultured in M9 medium, resulting in the identification of 619 unique proteins, which were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Using a modified version, the surface-associated proteome of five UPEC reference strains cultured in human urine was characterised and led to the identification of 173 unique proteins. Alongside multiple characterised virulence factors, an uncharacterised F17-like fimbrial type was detected, designated uroepithelial cell binding (UCB) fimbriae in this work. UCB fimbriae are PAI-associated and unique to UPEC based on the comparative genomic analysis of CU fimbriae in E. coli. Phenotypic characterisation of a recombinant UCB E. coli strain revealed these fimbriae mediate significant biofilm formation on abiotic surfaces and attach to exfoliated human uroepithelial cells with specificity, suggesting a role in the colonisation of the human urinary tract.

This study provides a framework for the effective typing of CU fimbriae and describes for the first time a comparative proteomic analysis of the surface-associated proteome of a large set of bacterial strains using a high-throughput approach. Combined, these methods have led to the identification of a new fimbrial type involved in adhesion to human uroepithelial cells as well as several surface-associated proteins expressed by the vast majority of UPEC strains. The results provided in this study add to the overall understanding of bacterial physiology and pathogenesis, and may aid in the development of novel diagnostic, drug and vaccine applications. 
Keyword UPEC, uropathogenic E. coli

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Created: Tue, 17 Jun 2014, 23:31:15 EST by Daniel Wurpel on behalf of Scholarly Communication and Digitisation Service