Elucidating the role of the Cpx envelope stress response in the colonization and virulence of Citrobacter rodentium
Creators: Gilliland, Ashley Rebecca
Publisher: name: University of Alberta Library
Year: 2022

<p>The murine attaching and effacing (A/E) pathogen, Citrobacter rodentium, is used as an infection model in vivo for the A/E pathogens enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). All three A/E pathogens harbor the Locus of Enterocyte Effacement (LEE) which encodes a Type III Secretion System (T3SS) and other virulence factors that are required for the adherence to intestinal epithelial cells, formation of pedestals and injection of effector proteins. During colonization, these pathogens face a myriad of challenges associated with the gastrointestinal tract including acidic pH, bile, oxygen gradients, mucus, and microbiome-mediated colonization resistance. To ameliorate the environmental stressors encountered, pathogens like C. rodentium, utilize two-component systems (TCS) to sense and appropriately respond to changes in the surrounding environment by moderating gene expression. The Cpx envelope stress response (ESR), consisting of the sensor histidine kinase CpxA and the response regulator CpxR, has previously been shown to be required for C. rodentium colonization and virulence in vivo. The purpose of this thesis was to investigate the observed attenuation by analyzing genes up and downregulated in the presence of the Cpx ESR to determine which, if any, were required for pathogenesis. Using transcriptomic and proteomic datasets from previous research as well as luminescent reporter assays to confirm Cpx-dependent upregulation, the genes yebE, ygiB, bssR, and htpX were chosen for further study. Here we showed that the virulence-inducing condition high-glucose Dulbecco’s modified Eagle medium (HG-DMEM) strongly activated the Cpx ESR and further induced the expression of our four genes of interest. After gene deletion by allelic exchange, it was determined that only the ΔcpxRA mutant had reduced colonization and was attenuated in vivo in C57Bl/6J and C3H/HeJ mice, while the ΔyebE, ΔygiB, ΔbssR, and ΔhtpX mutants remained virulent. To further investigate the colonization defect seen in the ΔcpxRA mutant, we conducted growth experiments in buffered simulated colonic fluid (SCF). Interestingly, we were able to replicate the observed colonization abilities of our mutants seen in vivo as only the ΔcpxRA mutant experienced a growth defect in SCF. In addition, SCF highlighted an extreme sensitivity to sub-inhibitory levels of oxidative stress as well as various growth defects in our mutants. Niche differentiation and pathogen expansion via aerobic respiration is important for C. rodentium infection and our data suggests that the Cpx ESR is necessary in the colonic environment to mediate relevant stressors. Following these findings, we turned focus to investigate genes downregulated by the Cpx ESR. The downregulated genes espV, mpc, kfcC, pspA and pspF, identified from the transcriptomic and proteomic datasets, were confirmed to have reduced expression in the presence of the Cpx ESR in HG-DMEM. espV and mpc encode a non-LEE encoded T3SS and LEE regulator respectively, and the kfc operon encodes a putative fimbria. This data supports the notion that the Cpx ESR downregulates virulence factors and could implicate that controlled virulence gene expression contributes to cell viability in vivo. Finally, after bioinformatic analyses indicated an interaction between yebE and pspACE, all genes which undergo regulation impacted by the Cpx ESR, we generated various mutant strains and luminescent reporters to experimentally determine possible interactions. Here we found the presence of YebE influences the Phage Shock Protein (Psp) response, measured by pspA-lux expression, and the absence of an intact Psp response induces yebE expression. Overall, these data contribute to the overall knowledge surrounding C. rodentium colonization and virulence as well as proposes novel interactions between the inner membrane stress responses, CpxRA and Psp, as well as the inner membrane protein YebE.</p>