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

Nichols, Katie (2016). Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression MPhil Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland. doi:10.14264/uql.2016.388

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Author Nichols, Katie
Thesis Title Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression
Formatted title
Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
DOI 10.14264/uql.2016.388
Publication date 2016-07-01
Thesis type MPhil Thesis
Supervisor Mark Schembri
Total pages 210
Language eng
Subjects 0605 Microbiology
0604 Genetics
Formatted 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 vat-vatX 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 infected by vat-containing UPEC strains, demonstrating Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated cytotoxin secreted by UPEC during infection.

This project also investigated the role of periplasmic chaperones in the translocation of UPEC-associated type Va and type Vc AT proteins. In the absence of the periplasmic chaperone Skp, expression of the AT proteins Vat, antigen 43 (Ag43) and the trimeric AT E. coli immunoglobulin binding protein D (EibD) was reduced. Ag43 and EibD-mediated biofilm formation in the K-12 strain MG1655 (harbouring plasmids encoding Ag43 or EibD, respectively) was significantly reduced in a skp mutant. Impaired Ag43-mediated auto-aggregation and decreased intracellular concentrations of monomeric EibD were also observed in an MG1655skp mutant. Finally, secretion of Vat in a CFT073skp mutant was also reduced. Overall, this work demonstrated an important role for Skp in the translocation and secretion of AT proteins.
Keyword Vat
Autotransporter
Periplasmic chaperones
Ag43
EibD
Translocation and assembly module

Document type: Thesis
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Created: Mon, 20 Jun 2016, 03:25:08 EST by Katie Nichols on behalf of Learning and Research Services (UQ Library)