Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium mutants and highlights a critical role for the Cpx envelope stress response in mediating stressors encountered in the gastrointestinal tract

Abstract

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. While in the host, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. To investigate genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, RNA-Seq, SILAC, and microarray data from previous research was mined and the genes yebE, ygiB, bssR, and htpX were confirmed to be strongly upregulated by the presence of CpxRA using lux reporter constructs. To determine the function of these genes in vivo, knockout mutants were tested in C57Bl/6J and C3H/HeJ mice. Although none of the mutants exhibited marked virulence phenotypes, the {Delta}cpxRA mutant had reduced colonization and attenuated virulence, as previously determined. We also found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler. In addition, we determined that the {Delta}cpxRA mutant had a growth defect in medium simulating the colon, as did several of the mutants bearing deletions in Cpx-upregulated genes. Overall, these results indicate that the {Delta}cpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.