title: Characterization of the contributions a detoxified Stx phage prophage makes towards the fitness and virulence of its bacterial host.
Creators: name: Cai, Yueyi
Publisher: name: University of Liverpool Repository
Year: 2024

description: Shiga toxin (Stx) producing Escherichia coli are called STEC. Stx-phages can horizontally transfer Shiga toxin-encoding genes (stx) between bacteria. The production of Stx by lysogens in the digestive tract can cause bloody diarrhoea with intestinal cramping, diarrhoea, haemorrhagic colitis, and sometimes leads to HUS (haemolytic uraemic syndrome), with each disease being progressively worse and to some extent governed by the amount of Stx produced during the resulting STEC infection. In addition, most Stx-phages are lambdoid phages, of which λ phage is the archetype. In general, the size of most Stx-phage genomes is ~50% larger than the size of the λ phage genome. However, there is very little research focusing on the function of this additional DNA.
One of the well-studied Stx-phges is Φ24B. Previous work showed that the expression of two genes of unknown function from Φ24B, vb_24B_13c and res, are only expressed whilst the prophage is integrated stably in the host cell. In addition, unpublished work performed by Dr. Veses-Garcia showed that vb_24B_13c was under the control of same promoter that drives CI expression, pRM, and the genes for CI, vb_24B_13c and res are likely to be co-transcribed as a single transcript and have been shown to contribute to the motility of E. coli lysogen at 37°C.
Therefore, this project was aimed to investigate evidence, to prove that prophage gene vb_24B_13c, can alter the motility of lysogens at the transcriptional level. Multiple approaches were included in this project, such as motility assays, NanoString nCounter, RNA-seq (from previous data), and bioinformatic analyses. The production of genetic constructs, and the development of assays (both biochemical and molecular biological) were used to ascertain the function of vb_24B_13c. It was found that vb_24b_13c significantly inhibited the growth of both MC1061 and MC1061Φ24B::Cat at 37°C, and at 30°C, it independently increased the motility of naïve E. coli MC1061.
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It was also found that temperature is a key factor in changing the lysogen expression profile.
At the same time, this project further studied the effects of the well-known phage- encoded regulators CI and CII co-expression of CIII on their host. By overexpressing these phage regulators in both naïve and lysogenic cells, their functions were determined, while excluding the effects from phage genes involved in self-induced lysis. It was found that at 30°C, overexpressed CI can enhance motility in both naïve cells and lysogens. At both 37°C and 30°C, CI can promote the TCA cycle by upregulating TCA cycle genes, while CII and CIII can inhibit the expression of genes related to maltose transport and metabolism, the TCA cycle, and glycolysis. In addition, this project further studied the temperature-determined phage-bacteria interaction, finding that temperature can affect lysogen flagella assembly and motility, as well as their metabolic pathways.
Overall, this experiment demonstrated that prophage DNA, which constitutes only about 1.1% of the E. coli genome, is powerful enough to reprogram the host's expression profile and behavior.