The Virulence Effect of CpxRA in Citrobacter rodentium Is Independent of the Auxiliary Proteins NlpE and CpxP

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.

The CpxR regulates type VI secretion system 2 expression and facilitates the interbacterial competition activity and virulence of avian pathogenic Escherichia coli

“…Collectively, these results suggested that CpxR promotes the expression of APEC T6SS2. This result is consistent with the regulatory role in Citrobacter rodentium [36]. Additional, the decreased expression of the T6SS2 hcp2B operon, including the interbacterial effector encoding the gene xmtU , might be responsible for the decreased invasion, diminished survival and impaired competition of the mutant strain ΔcpxR.…”

“…It has been shown that the Cpx TCS plays roles in regulating the T6SS in Citrobacter rodentium [36]. Hence, we sought to determine whether the decreased interbacterial activity of the mutant strain ΔcpxR was due to changes in T6SS2 expression.…”

“…The E. coli CpxR regulon contains hundreds of genes, according to genome-wide screens for the putative CpxR binding sequence [56, 57]. In addition to regulating protein folding and degradation, CpxR also plays roles in regulating T6SS expression in Citrobacter rodentium [36]. T6SSs are a common strategy used by many pathogens to mediate successful infections in hosts [7–14, 16].…”

“…The Cpx TCS affects bacterial virulence through regulating the expression of virulence genes involved in envelope stress relief, biofilm formation, adherence, motility and secretion systems (type III, type IV and type VI) [26–36]. The activation of Cpx TCS in Shigella sonnei , Legionella pneumophila , Xenorhabdus nematophila and Yersinia pestis results in increased expression of virulence genes and contributes to pathogenesis [26, 30, 32, 33, 37, 38].…”

“…The activation of Cpx TCS in Shigella sonnei , Legionella pneumophila , Xenorhabdus nematophila and Yersinia pestis results in increased expression of virulence genes and contributes to pathogenesis [26, 30, 32, 33, 37, 38]. The Cpx TCS also regulates T6SS expression, and affects the virulence of Citrobacter rodentium [31, 36]. However, constitutive activation of the Cpx TCS results in downregulation of virulence genes and attenuated virulence of Haemophilus ducreyi [39, 40].…”