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

Abstract

Citrobacter rodentium is a murine pathogen used to model intestinal infections caused by the human diarrheal pathogens enterohemorrhagic and enteropathogenic Escherichia coli. During infection, bacteria use two-component systems (TCSs) to detect changing environmental cues within the host, allowing for rapid adaptation by altering the expression of specific genes. In this study, 26 TCSs were identified in C. rodentium, and quantitative PCR (qPCR) analysis showed that they are all expressed during murine infection. These TCSs were individually deleted, and the in vitro and in vivo effects were analyzed to determine the functional consequences. In vitro analyses only revealed minor differences, and surprisingly, type III secretion (T3S) was only affected in the ΔarcA strain. Murine infections identified 7 mutants with either attenuated or increased virulence. In agreement with the in vitro T3S assay, the ΔarcA strain was attenuated and defective in colonization and cell adherence. The ΔrcsB strain was among the most highly attenuated strains. The decrease in virulence of this strain may be associated with changes to the cell surface, as Congo red binding was altered, and qPCR revealed that expression of the wcaA gene, which has been implicated in colanic acid production in other bacteria, was drastically downregulated. The ΔuvrY strain exhibited increased virulence compared to the wild type, which was associated with a significant increase in bacterial burden within the mesenteric lymph nodes. The systematic analysis of virulence-associated TCSs and investigation of their functions during infection may open new avenues for drug development.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection

“…A). However, in our experiments, the ΔcpxRA mutant was still able to cause lethal infection in about half of the mice, in contrast to the 100% survival previously reported (Thomassin et al , ; ). The reason for the differing results is unknown, since all C3H/HeJ mice originated from the same supplier.…”

“…Citrobacter rodentium harbours 26 two-component systems (2CSs); mutants in only six of these 2CSs are attenuated in the mouse infection model, with ΔcpxRA having the largest virulence defect (Thomassin et al, 2017). The main question we set out to answer in this study is why CpxAR is so important for host infection.…”

Here, there, and everywhere: How pathogenicEscherichia colisense and respond to gastrointestinal biogeography

“…Two‐component systems (TCSs) are crucial to the regulation of bacterial signalling, allowing for rapid responses to changing environmental cues. C. rodentium has 26 identified two‐component systems expressed in vivo , typically consisting of a membrane‐associated histidine kinase and a cytoplasmic response regulator (Thomassin et al, ). Deletion of the CpxRA, ArcAB, RcsBC, RstAB, UhpAB, and ZraRS TCSs in particular significantly attenuate virulence, with CpxRA having the largest effect on C. rodentium in vivo (Thomassin et al, ).…”

“…C. rodentium has 26 identified two‐component systems expressed in vivo , typically consisting of a membrane‐associated histidine kinase and a cytoplasmic response regulator (Thomassin et al, ). Deletion of the CpxRA, ArcAB, RcsBC, RstAB, UhpAB, and ZraRS TCSs in particular significantly attenuate virulence, with CpxRA having the largest effect on C. rodentium in vivo (Thomassin et al, ). CpxRA plays an important role in the envelope stress response of E. coli , by monitoring membrane protein folding, as reviewed by Guest and Raivio ().…”

“…To avoid or bypass changing obstacles within the gut environment, there are several tools pathogens use to tailor the expression of virulence genes. CpxRA having the largest effect on C. rodentium in vivo (Thomassin et al, 2017). CpxRA plays an important role in the envelope stress response of E. coli, by monitoring membrane protein folding, as reviewed by Guest and Raivio (2016).…”