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.

Keywords: Citrobacter rodentium; CpxRA; bacterial gene expression; intestinal infection; two component systems; virulence.

Figure 1

Survival of susceptible mice after infection with Cpx TCS mutant strains of Citrobacter rodentium. (A) TCSs are used by bacteria in order to sense environmental stress. The membrane bound histidine kinase (CpxA) is activated by an external signal and propagates a cascade of phosphorylation leading to a transcriptional response by the cytoplasmic response regulator (CpxR). The Cpx TCS has a reported upstream outer membrane sensor, NlpE, and a periplasmic inhibitor, CpxP. (B) Female C3H/HeJ mice were infected by oral gavage with 2–3 × 10⁸ colony forming units of wild-type C. rodentium, or a TCS mutant strain. Survival was monitored for 30 days post infection. The log-rank (Mantel-Cox) method was used to determine statistical significance. (PG - peptidoglycan), (**P < 0.01, n = 5).

Figure 2

Bacterial burden in female C3H/HeJ mice after infection with Cpx TCS mutant strains of Citrobacter rodentium. Female C3H/HeJ mice were infected as described previously, and fecal bacterial burden was assessed at day 3 (A), 6 (B), and 9 (C) post infection by plating on MacConkey agar and counting Colony Forming Units (CFU). At day 9 post infection, due to significant illness manifestation, in the absence of fecal matter, colon was homogenized and plated on MacConkey agar. A Mann-Whitney test was used to determine significance between each mutant strain and wild-type (**p < 0.01; *p < 0.05). (LOD = Limit of detection; day 3, day 6: n = 5; day 9: n = 3–5; black bar denotes the median).

Figure 3

In vivo localization of wild-type and Cpx TCS mutant strains of Citrobacter rodentium in the intestine of C3H/HeJ mice. Localization of C. rodentium wild-type and Cpx TCS mutant strains in distal colon samples of day 9 infected C3H/HeJ mice. Sections stained with DAPI (blue) and anti-Citrobacter LPS (green). White boxes on 10X images denote the area of the 20X higher magnification image. Representative images shown from n = 3–4 biological replicates. Scale bars 100 μm.

Figure 4

In vitro adherence of wild-type and Cpx TCS mutant strains of Citrobacter rodentium on HeLa cells. HeLa cells were infected with wild-type C. rodentium or a Cpx TCS mutant strain for 8 hrs. The samples were fixed and stained with DAPI and anti-Citrobacter LPS. Samples were imaged on a Zeiss Axiovert 200M microscope. Total number of bacteria per HeLa cell were counted. The 3 biological replicates for each strain are color-coded. Each replicate consists of 10 fields of view. A non-parametric one-way ANOVA was used to determine statistical significance (****P < 0.0001). Bars denote the mean.

Figure 5

General gene expression profile of all mutant strains relative to wild-type. Strains were grown in DMEM and RNA was extracted from three biological replicates. The expression profiles of mutant strains were compared to that of the wild-type C. rodentium. (A) Venn diagram of the downregulated genes in each deletion strain. (B) Venn diagram of the upregulated genes in each deletion strain. (C) Genes differentially expressed in ΔcpxRA were clustered with Hierarchical clustering, using Pearson Uncentered correlation. Upregulated genes are shown in red, and downregulated genes are in green. Genes displayed are differentially expressed in the ΔcpxRA C. rodentium strain. An unpaired t-test was used to determine statistical significance (p < 0.05).