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Abstract
Citrobacter rodentium is a murine pathogen used to model the intestinal infection caused by Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC), two diarrheal pathogens responsible for morbidity and mortality in developing and developed countries, respectively. During infection, these bacteria must sense and adapt to the gut environment of the host. In order to adapt to changing environmental cues and modulate expression of specific genes, bacteria can use two-component signal transduction systems (TCS). We have shown that the deletion of the Cpx TCS in C. rodentium leads to a marked attenuation in virulence in C3H/HeJ mice. In E. coli, the Cpx TCS is reportedly activated in response to signals from the outer-membrane lipoprotein NlpE. We therefore investigated the role of NlpE in C. rodentium virulence. We also assessed the role of the reported negative regulator of CpxRA, CpxP. We found that as opposed to the ΔcpxRA strain, neither the ΔnlpE, ΔcpxP nor the ΔnlpEΔcpxP strains were significantly attenuated, and had similar in vivo localization to wild-type C. rodentium. The in vitro adherence of the Cpx auxiliary protein mutants, ΔnlpE, ΔcpxP, ΔnlpEΔcpxP, was comparable to wild-type C. rodentium, whereas the ΔcpxRA strain showed significantly decreased adherence. To further elucidate the mechanisms behind the contrasting virulence phenotypes, we performed microarrays in order to define the regulon of the Cpx TCS. We detected 393 genes differentially regulated in the ΔcpxRA strain. The gene expression profile of the ΔnlpE strain is strikingly different than the profile of ΔcpxRA with regards to the genes activated by CpxRA. Further, there is no clear inverse correlation in the expression pattern of the ΔcpxP strain in comparison to ΔcpxRA. Taken together, these data suggest that in these conditions, CpxRA activates gene expression in a largely NlpE- and CpxP-independent manner. Compared to wildtype, 161 genes were downregulated in the ΔcpxRA strain, while being upregulated or unchanged in the Cpx auxiliary protein deletion strains. This group of genes, which we hypothesize may contribute to the loss of virulence of ΔcpxRA, includes T6SS components, ompF, the regulator for colanic acid synthesis, and several genes involved in maltose metabolism.
Highlights
Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC) are Gram-negative food-borne diarrheal pathogens, transmitted through the fecal-oral route (Mundy et al, 2005)
To assess the virulence contribution of the Cpx transduction systems (TCSs) auxiliary proteins (Figure 1A), we used sacB gene-based allelic exchange to generate knock out strains of C. rodentium, each lacking either NlpE, CpxP, or a double mutant
The mice infected with the Cpx auxiliary protein mutant strains succumbed to infection with similar kinetics as the cohort infected with wild-type C. rodentium (Figure 1B)
Summary
Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC) are Gram-negative food-borne diarrheal pathogens, transmitted through the fecal-oral route (Mundy et al, 2005). They are responsible for high morbidity and mortality in both the developed and developing world. EHEC, EPEC, and C. rodentium are members of a group of pathogens known for their ability to form attaching and effacing lesions (A/E lesions) during infection (Mundy et al, 2005). The bacteria attach to intestinal epithelial cells, efface the microvillar architecture, and form actin-rich pedestals beneath the adherent bacteria (Moon et al, 1983; Mundy et al, 2005)
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