The CpxRA Two-Component System Is Essential for Citrobacter rodentium Virulence

Abstract

dCitrobacter rodentium is a murine intestinal pathogen used as a model for the foodborne human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. During infection, these pathogens use two-component signal transduction systems to detect and adapt to changing environmental conditions. In E. coli, the CpxRA two-component signal transduction system responds to envelope stress by modulating the expression of a myriad of genes. Quantitative real-time PCR showed that cpxRA was expressed in the colon of C57BL/6J mice infected with C. rodentium. To determine whether CpxRA plays a role during C. rodentium infection, a cpxRA deletion strain was generated and found to have a colonization defect during infection. This defect was independent of an altered growth rate or a defective type III secretion system, and single-copy chromosomal complementation of cpxRA restored virulence. The C. rodentium strains were then tested in C3H/HeJ mice, a lethal intestinal infection model. Mice infected with the ⌬cpxRA strain survived infection, whereas mice infected with the wild-type or complemented strains succumbed to infection. Furthermore, we found that the cpxRA expression level was higher during early infection than at a later time point. Taken together, these data demonstrate that the CpxRA two-component signal transduction system is essential for the in vivo virulence of C. rodentium. In addition, these data suggest that fine-tuned cpxRA expression is important for infection. This is the first study that identifies a C. rodentium two-component transduction system required for pathogenesis. This study further indicates that CpxRA is an interesting target for therapeutics against enteric pathogens.

Systematic Analysis of Two-Component Systems in Citrobacter rodentium Reveals Positive and Negative Roles in Virulence

“…As expected, wild-type C. rodentium loads increased swiftly and steadily during the course of infection, reaching a peak of between 10 9 and 10 10 CFU/g of feces by day 9 postinfection. In contrast, and in agreement with our previous data, the ΔcpxRA strain displayed delayed colonization with lower levels of colonization at all time points and greater variability in bacterial burden than the wild-type strain (36). Of the other TCS deletion strains analyzed, only the ΔarcA mutant displayed significantly lower colonization levels than the wild-type strain ( Fig.…”

“…3). The already-described wild-type and ΔcpxRA strains were included in these analyses as controls (36). As expected, wild-type C. rodentium loads increased swiftly and steadily during the course of infection, reaching a peak of between 10 9 and 10 10 CFU/g of feces by day 9 postinfection.…”

“…1C, as assessed by qPCR, all unlinked HK genes were significantly expressed in vivo. Most HK sensor genes were expressed at lower levels than their respective RR genes, which is not unprecedented, since lower HK than RR transcript levels were also observed in vivo for the linked cpxRA TCS (36).…”

“…Figure 2 demonstrates that the three TCSs with the greatest influence on C. rodentium virulence are ArcAB, CpxRA, and RcsBC. We previously characterized the ΔcpxRA mutant (36). We next sought to perform a more detailed analysis of the consequences of arcA deletion on C. rodentium virulence.…”

“…This is likely due to the comparatively lower loads of C. rodentium in C57BL/6J mouse colons during early infection (35). To ensure that the expression measured was the result of the specific amplification of transcripts from C. rodentium, control qPCR experiments were performed on cDNA prepared from total RNA isolated from uninfected C3H/HeJ and C57BL/6J mice using all TCS primer pairs, except cpxR and rpoD, which we previously validated using the same criteria (36). As expected, all primer pairs were unable to amplify a product from uninfected murine samples, indicating that the signals detected are C. rodentium specific and not the result of unwanted amplification of microbiota or murine genes (data not shown).…”