title: Intestinal pathogenic Escherichia coli: identification and characterisation of their virulence determinants
Creators: name: Hallam, Jennifer Caitlin
Publisher: name: University of Glasgow
Year: 2022

description: The human microbiota consists of over 100 trillion microbial cells, primarily, bacteria that reside in the gastrointestinal tract (Turnbaugh et al., 2007). The relationship between these bacteria and the host is typically symbiotic, as many microbiota-derived species play an important role in host physiology. Thus, maintaining the balance of the intestinal flora, and controlling the overgrowth of potentially pathogenic species, is important in preventing disease. Indeed, specific changes to the bacterial composition of the gut can initiate chronic inflammation of the intestine and result in the onset of gastrointestinal disorders such as inflammatory bowel disease (IBD) (Baldelli et al., 2021). The rapid advancement of sequencing methods and analytical techniques has enabled researchers to understand the microbiome and to ask the question, how do we define the microbiome and its inhabitants? There are many factors that can influence one’s microbiome, including the host genotype, lifestyle and environment (Turnbaugh et al., 2007). Therefore, it is not surprising that the gut microbiome can be up to 80-90% different between individuals, a stark contrast to the human genome, where individual humans are approximately 99.9% identical (Ursell et al., 2012). The composition of the gut microbiota is highly variable between geographical locations, particularly between humans living in markedly different socio-economic settings (Yatsunenko et al., 2012). Moreover, in certain low-income countries (LICs), many enteric pathogens are endemic throughout these regions and are frequently isolated from stool of healthy individuals, suggesting that organisms can be carried asymptomatically. Enteropathogenic E. coli (EPEC) is typically associated with diarrhoeal disease in infants, however, EPEC is frequently isolated from healthy individuals as well as from those with gastroenteritis. Indeed, it remains unknown how these ‘pathogens’ colonise individuals without any symptoms, and begs the question whether these can truly be defined as pathogens at all? Therefore, the primary aim of this thesis was to isolate and characterise the virulence determinants of asymptomatic EPEC isolates. And secondly, to identify a novel strategy for clearing these strains without compromising the composition of the host microbiota. Unfortunately, due to ceased contact with my industrial collaborator, no asymptomatic EPEC strains were received and thus these investigations did not produce any conclusive findings. In consequence, the focus of this project was redirected towards investigating virulence in a different subset of E. coli, specifically strains belonging to the B2 phylogroup which harbour the pks island, a genomic island encoding the biosynthesis genes for a genotoxin termed colibactin. In eukaryotic cells, colibactin induces DNA damage resulting in chromosomal instability and cell cycle arrest. Strikingly, E. coli strains harbouring the pks island are often overrepresented in biopsies from IBD patients and furthermore, the production of colibactin has been implicated in the promotion and development of colorectal cancer in these individuals (Arthur et al., 2012). Thus, as the composition of the human diet has been identified as a key factor in governing intestinal homeostasis, the effect of dietary amino acids on the expression of pks encoded genes was explored using RT-qPCR. Several D-amino acids exhibited the ability to inhibit expression of clbB with D-Serine exerting the strongest repressing activity in two pks+ E. coli strains CFT073 and Nissle 1917. The effect of D-Serine on the colibactin induced cellular response was also observed during infection of HeLa cells with live pks+ strains. Levels of g-H2AX (a marker of DNA double strand breaks) were significantly reduced in cells infected with D-Serine treatment. Moreover, exposure of pks+ E. coli to D-Serine during infection caused a reduction in cellular senescence that was observable at 72 hours post infection. These findings have revealed the potential of D-amino acids in reducing colibactin expression in distinct pks+ E. coli strains. Furthermore, D-Serine, and other D-amino acids are key components of our diet and therefore present opportunities for dietary supplementation that might yield important health benefits by modulating gene expression of E. coli pks+ strains present in the microbiota.