Identification and Characterisation of New Adhesins in Uropathogenic Escherichia coli

Luke Allsopp (2011). Identification and Characterisation of New Adhesins in Uropathogenic Escherichia coli PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

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Author Luke Allsopp
Thesis Title Identification and Characterisation of New Adhesins in Uropathogenic Escherichia coli
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2011-07
Thesis type PhD Thesis
Supervisor Prof Mark Schembri
Dr Makrina Totsika
Total pages 183
Total colour pages 21
Total black and white pages 162
Subjects 03 Chemical Sciences
Abstract/Summary Escherichia coli (E. coli) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. The biological significance of these AT adhesins and their role in UPEC pathogenesis has not been fully investigated. Detailed molecular characterisation of three of these AT proteins from UPEC CFT073 – UpaH, UpaB and UpaC – was undertaken in this study. A new AT-encoding gene, termed upaH, was identified in a 6.5 kb unannotated intergenic region in the genome of UPEC CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535 kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonisation of the bladder in single-challenge experiments. However, in competitive colonisation experiments, CFT073 significantly out-competed its upaH isogenic mutant strain in urine and the bladder. The genes encoding UpaH variants were cloned from five UPEC strains and their contribution to biofilm formation was assessed using the continuous-flow chamber model. The sequence of UpaH varies between different UPEC strains and this sequence variation is associated with differences in the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined in this study retained a conserved ability to mediate binding to several extracellular matrix (ECM) proteins. Regulation of the upaH gene was also investigated, with H-NS identified as a repressor of upaH expression in UPEC. Two conventional AIDA-I type AT proteins, UpaB and UpaC, were cloned and expressed in an E. coli K-12 recombinant strain. Expression of UpaB promoted adherence to several ECM proteins while expression of UpaC significantly increased biofilm formation. Specific antisera were generated against each protein and immunofluorescence microscopy and immunogold labelling with electron microscopy revealed that UpaB and UpaC are both located at the cell surface. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In the mouse UTI model, deletion of the upaB gene in CFT073 significantly reduced early colonisation of the bladder. These results show that UPEC CFT073 encodes the capacity to express multiple AT proteins. The work adds significant new knowledge to our understanding of AT proteins in UPEC and their contribution to adherence, biofilm formation and colonisation of the mouse urinary tract.
Keyword CFT073
Escherichia coli (E. coli)
ECM binding
Additional Notes Colour 26, 31, 35, 37, 58-62, 79, 83, 109, 111, 115, 164, 166-170, 173 A3 29, 156

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Created: Tue, 01 Nov 2011, 13:31:33 EST by Mr Luke Allsopp on behalf of Library - Information Access Service