Regulation of virulence determinants in pathogenic \(\textit{Escherichia coli}\)

Alhammadi, Munirah (2023). Regulation of virulence determinants in pathogenic \(\textit{Escherichia coli}\). University of Birmingham. Ph.D.

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Abstract

Enteroaggregative and uropathogenic \(\textit{Escherichia coli}\) are both pathogenic strains that likely evolved from commensal \(\textit{E. coli}\) by acquisition of virulence genes. These pathogens, EAEC and UPEC, can cause problems in the intestines how pathogens, initiate their infection, it is important to consider how they behave in nutritionally challenging conditions, such as low glucose levels in the environments and how they co-exist with other pathogens and microbiota. Many bacteria can respond to environmental changes using small effector molecules that activate the regulator proteins that are needed to cope with the circumstances. One of the best studied transcription regulators in \(\textit{E. coli}\) is cyclic AMP receptor protein (CRP) which can regulate more than 100 genes/operon in \(\textit{E. coli}\) , hence, comes the name a global regulator.

The regulation of virulence in EAEC has been intensively studied, and AggR is known to be the master regulator for the expression of most chromosomally-encoded and plasmid-encoded of virulence genes. However, for some genes that both analysis of promoter sequences, and experimental data, show that they are not involved in the AggR regulon. My project focuses on the regulation of such EAEC virulence determinants, during starvation condition. One of these is Pic (Protein involved in colonisation), encoded by chromosomal \(\textit{pic}\) gene, which encodes an important enzyme, needed in the early stage of infection. The architecture of \(\textit{pic}\) promoter was compared between EAEC and UPEC using different methodologies, using site- direct mutagenesis assays, the 5'rapid amplification of cDNA ends (5'RACE) method, and promoter activity measurements. My results show that \(\textit{pic}\) is a CRP-dependent promoter with an unusual promoter architecture. In EAEC strain 042: it is the first example of an ‘ambiguous’ CRP-activiated promoter.

My unexpected results from studying \(\textit{pic}\) regulation leads me to ask if there are more virulence genes involves in the EAEC CRP regulon. Hence, my second objective was to use chromatin immunoprecipitation with high- throughput sequencing (ChIP-seq) with the wild type EAEC 042 to locate the full complement of DNA site for CRP. My results showed more than 300 CRP targets in the EAEC 042 genome. Comparison of my data with the situation in commensal \(\textit{E. coli}\) K-12 strains revealed 31 targets that were specific genes for EAEC 042, and I focussed further work on these. The results of these new CRP targets were validated using \(\textit{in vivo}\) and \(\textit{in vitro}\) experiments. Results showed involvement of CRP at some of these virulence genes and the bladder, of the host. To understand targets, whereas, at other, CRP appeared to play a role in chromosomal organisation. One outcome from the CRP ChIP-seq data was discovery of a very strong DNA site for CRP in the regulatory region upstream of the genes encoding polysialic acid transport proteins, KpsMII, located in EAEC 042 chromosome. In my investigations, I used \(\textit{in vivo}\) and \(\textit{in vitro}\) assays to demonstrate the involvement of CRP and the possibility of indirect regulation by CRP of \(\textit{kpsMII}\) expression. My data argue that bacterial pathogens use global transcription regulators to overcome challenging conditions like starvation hence and assure the production of virulence factors in the early stages of infection.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):