Project Outline

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a major cause of diarrhoeal disease in industrialized and non-industrialized countries and has been shown to be the cause of travellers’ diarrhoea and persistent diarrhoea in children and HIV patients. EAEC strains cause disease by binding to the human gastric mucosa and establishing a thick mucoid biofilm, damaging tissue by secreting various protein toxins [1]. In spite of the widespread occurrence of EAEC strains and their impact on human health little is known about how EAEC strains control the expression of genes required to establish infection and cause disease. Furthermore, EAEC strains are becoming increasingly resistant to many clinical antibiotics, decreasing the options available to treat infections [1-4]. Thus, there is a great need to understand EAEC pathology and devise new methods to combat this important group of bacterial pathogens.

In many EAEC strains, it has been shown that the expression of important virulence determinants (e.g., the attachment adherence fimbriae (AAF) required for colonization, the anti-aggregative protein dispersin (Aap) and its dedicated secretion system) is controlled by the AggR transcription factor, the master regulator of virulence [1-3]. Recently, we have been working with EAEC strains isolated from Egyptian and Brazilian children with diarrhoea [3,4]. In these strains, we have found a new version of AggR that appears to control gene expression differently. Thus, the main aim of this project will be to determine how this new version of AggR controls virulence gene expression in these strains and determine the genes that it regulates. We have already had the genomes of many of these strains fully sequenced, and so part of this project will be to analyse their genomes, to determine each strains characteristics (e.g., antibiotic resistance gene profile, virulence determinants and plasmid replicons etc.). Once key virulence and antibiotic resistance genes have been identified, the promoters that control their expression, will be amplified by PCR and cloned into reporter gene expression plasmids. The transcription regulation of these promoters will then be examined under different environmental conditions to determine how and when the expression of these important proteins is switched on. Coupled with this, the project will also examine the binding of AggR throughout each strain’s genome, using techniques such as chromatin immunoprecipitation (ChIP-seq) and RNA-sequencing (RNA-seq) to understand where AggR binds and the genes it controls. The binding of AggR will be then confirmed using in vitro biochemical techniques, such as band shift assays (electrophoretic mobility shift assays (EMSA)). The final aim of this project will be to use synthetic biology to engineer different versions of AggR that can block specific promoters and disrupt virulence gene expression in EAEC strains. Using either bacteriophage or plasmid conjugation, these new AggR alleles will be delivered to EAEC pathogens as potential therapeutic agents to combat EAEC infection.

Methodologies

This project will provide complete training in many essential bioinformatic and molecular biology techniques, which will include, PCR, DNA cloning, site directed mutagenesis, DNA sequencing, bacterial strain manipulation, reporter gene enzyme assays, ChIP-seq, RNA-seq and various in vitro biochemical techniques (band shift assays (EMSA)/ DNAse I footprinting assays).

References

1) Harrington, S.M., Dudley, E.G. and Nataro, J.P. (2006) Pathogenesis of enteroaggregative Escherichia coli infection. FEMS Microbiol Lett. 254(1):12-8. Review.

2) Yasir, M., Icke, C., Abdelwahab, R., Haycocks, J.R., Godfrey, R.E., Sazinas, P., Pallen, M.J., Henderson, I.R., Busby S.J.W. and Browning, D.F. (2019) Organization and architecture of AggR-dependent promoters from Enteroaggregative Escherichia coli. Molecular Microbiology. 111:534-551.

3) Abdelwahab, R., Yasir, M., Godfrey, R.E., Christie, G.S., Element, S.J., Saville, F., Hassan, E.A., Ahmed, E.H., Abu-Faddan, N.H., Daef, E.A., Busby, S.J.W. and Browning, D.F. (2021) Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli from Egyptian children with diarrhoea: similarities and differences. Virulence 12:57-74

4) França, F.L.S., Wells, T.J, Browning, D.F., Nogueira, R.T., Sarges, F.S., Pereira, A. C., Cunningham, A.F., Lucheze, K., Rosa, A.C.P., Henderson, I.R. and das Graças de Luna, M. (2013) Genotypic and phenotypic characterisation of enteroaggregative Escherichia coli from children in Rio de Janeiro, Brazil. PLOS ONE 10.1371/journal.pone.0069971.

Techniques

Techniques will be chosen based on the specifics of the project as developed by the supervisor and student. Expect techniques from the following list include:

1. Bacterial whole genome sequencing and sequence analysis.
2. Molecular biology/synthetic biology: PCR, DNA manipulation, purification & cloning
3. Reporter gene assays: LacZ and GFP based systems.
4. Bacterial growth and biofilm assays.
5. Chromatin Immunoprecipitation (ChiP-seq) and RNA-seq analysis.
6. Protein expression and analysis: SDS-PAGE, cell fractionation and purification.
7. Biochemical techniques: band shift assays (EMSA)/ DNAse I footprinting assays/ in vitro transcriptions assays etc.