Studies with AggR: The master regulator of virulence in enteroaggregative Escherichia coli

Christie, Gabrielle Sophie ORCID: 0000-0002-2703-0416 (2023). Studies with AggR: The master regulator of virulence in enteroaggregative Escherichia coli. University of Birmingham. Ph.D.

Preview

Christie2023PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (22MB) | Preview

Abstract

Escherichia coli exists as part of the human flora in the gastrointestinal tract. Some strains cause intestinal and extra-intestinal infections, and these bacteria are amongst the most prevalent of Gram-negative pathogens. There are many pathotypes of diarrhoeagenic E. coli, including enteroaggregative E. coli (EAEC). EAEC is a significant cause of diarrhoeal disease in low-, middle- and high-income countries. Aggregative adherence fimbriae (AAFs) mediate EAEC adherence to epithelial cells. A ‘typical’ EAEC strain contains the master virulence regulator, AggR, which is a transcription factor, encoded on a virulence plasmid (pAA), that has over 40 virulence genes in its regulon. Previous work demonstrated that AggR acts as a monomer as a Class II activator of transcription at its dependent promoters. The aim of this project was to investigate complex AggR-regulated promoters, focussing on a bi-directional promoter, and to examine variations in the organisation of dependent promoters between strains. A further objective was to define the key determinants for AggR activity with an aim of understanding how its activity is regulated.
The yicS-nlpA bi-directional promoter is known to be present on the chromosome in more than ten E. coli strains, though it has been identified in at least 30 more strains. The YicS protein is thought to be involved in biofilm formation in avian pathogenic E. coli (APEC), but YicS is not required for biofilm formation in EAEC 042. An investigation of the regulation of the yicS and nlpA promoters, from E. coli K-12, EAEC strain 042 and EAEC strain H92/3, by AggR identified the key determinants that permit AggR-dependent activation of the yicS promoter in EAEC 042, weaker activation in EAEC H92/3, and no activation in E. coli K-12. In contrast, in all these strains, the nlpA promoter is weakly repressed by AggR.
In my study, the genome of the ‘atypical’ EAEC strain H149/5, which lacks a virulence plasmid, was sequenced and several AggR-activated promoters were identified. I identified the aap and caf1M genes, which demonstrates that this strain carries the dispersin (Aap) surface protein and Caf1M, a homologue of AAF proteins. An AggR-like transcription factor was identified, CfaD H149/5, that is chromosomally encoded. The AraC subgroup transcription regulators, Rns and AggR, could substitute for CfaD for activation of promoters from H149/5. My results showed that CfaD H149/5 has a different activation profile to AggR.
Through mutational analysis, key determinants for AggR activity were located throughout both the N-terminal domain (NTD) and the C-terminal domain (CTD). None of the AggR mutants were strongly trans-dominant over wild-type AggR and this argues that AggR might function by pre-recruitment. An investigation of how AggR activity is regulated suggested the existence of a bi-stable virulence switch. Consistent with this, I showed that expression of AggR is not dependent on any external signal. Measurement of cell-to-cell variation of AggR activity indicates that a small proportion of cells reach a critical threshold of AggR production, and I argue that this ‘flips’ the virulence switch to the ‘on’ state. Taken together, the available experimental data suggest that the regulation of AggR activity is dependent on a feedforward loop with a bi-stable switch.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):