Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression

Abstract

Uropathogenic Escherichia coli (UPEC) are the primary cause for all urinary tract infections.Autotransporter (AT) proteins are virulence factors secreted by the type V secretion pathway. This project examined the prevalence, regulation and expression of the vacuolating AT toxin (Vat), and the role of periplasmic chaperone proteins in AT translocation.The vacuolating autotransporter (AT) toxin (Vat) contributes to Uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Vat is a serine protease AT of Enterobacteriaceae (SPATE) with cytotoxic activity. Here we characterised Vat and investigated its regulation in UPEC.We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed it that was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, 73 and 95) and these strains secreted the Vat protein.Further analysis of the vat genetic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator, which we termed vatX. The vatvatX genes were present in the UPEC reference strain CFT073 and RT-PCR revealed both genes are co-transcribed. Over-expression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator H-NS; thus the hns gene was mutated in CFT073 (to generate CFT073hns).Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hns compared to wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients iv Declaration by authorThis thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.