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

Abstract

Systemic infections caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide and are also potentially threatening to human health. Pathogens must be able to precisely modulate gene expression to facilitate their survival and the successful infection. The Cpx two-component signal transduction system (TCS) regulates surface structure assembly and virulence factors implicated in Gram-negative bacterial pathogenesis. However, the roles of the Cpx TCS in bacterial fitness and pathogenesis during APEC infection are not completely understood. Here, we show that the Cpx TCS response regulator CpxR is critical to the survival and virulence of APEC. Inactivation of cpxR leads to significant defects in the interbacterial competition activity, invasion and survival of APEC in vitro and in vivo. Moreover, activation of CpxR positive regulates the expression of the APEC type VI secretion system 2 (T6SS2). Further investigations revealed that phosphorylated CpxR directly bound to the T6SS2 hcp2B promoter region. Taken together, our results demonstrated that CpxR contributes to the pathogensis of APEC at least through directly regulating the expression and function of T6SS2. This study broadens understanding of the regulatory effect of Cpx TCS, thus elucidating the mechanisms through which Cpx TCS involved in bacterial virulence.

Figure 1

CpxR is essential for efficient colonization and virulence of APEC. A Determination of bacterial virulence. Seven-day-old ducks were infected with APEC strains, and the mortality was monitored until 7 days post-infection. Negative controls were injected with PBS. B–E Bacterial colonization, survival and competition in ducks. Seven-day-old ducks were infected with APEC strains in noncompetitive (B–D) and competitive (E) assays. Ducks were sacrificed at 24 h post-infection, and bacteria were recovered from the lungs, livers and spleens. The competitive indices were calculated and shown for the competitive assays (E). Nonparametric Mann–Whitney U-test was carried out to determine statistical significance (*P < 0.05).

Figure 2

CpxR contributes to the invasion of APEC to DF-1 cells. The ability of APEC strains to adhere to (A) and invade into (B) DF-1 cells was compared. The mutant strain ΔcpxR showed significantly higher DF-1 cells invasion than the wild-type and complemented strains. Results are shown as relative adhesion and invasion capacity compared with those of the wild-type strain. Error bars indicate standard deviations. Statistical significance analysis was performed by using one-way ANOVA (***P < 0.001).

Figure 3

CpxR facilitates interbacterial competition of APEC in vitro. The indicated APEC donor and recipient strains were co-incubated at a ratio of 5:1, as shown by the dashed line. After incubation for 6 h at 30 °C, the donor and recipient strains were recovered. The competition outcomes between donor and recipient strains were calculated and are shown. A, B The interbacterial competition activities of wild-type, mutant and complemented strains were compared. C The interbacterial competition activity was also measured in the wild-type and mutant strains with NlpE overexpression. Statistical significance was assessed by using one-way ANOVA. (*P < 0.05; **P < 0.01).

Figure 4

CpxR regulates the expression of T6SS2 genes in APEC. A The transcription levels of T6SS2 core genes were analyzed by qRT-PCR. B The levels of Hcp2B in APEC strains were confirmed with Western blotting. C qRT-PCR analysis for the transcriptional levels of the T6SS2 genes hcp2B, vgrG and xmtU in the absence or presence of NlpE in the wild-type and mutant strains. D The levels of Hcp2B in APEC strains with or without NlpE overexpression were determined by Western blotting. The qRT-PCR data are shown as relative expression ratios compared with that of the wild-type strain. For Western blotting, anti-DnaK antibody was used as a control. The expression of the Hcp2B protein was determined by quantifying the grayscale in Image J software. Two-way ANOVA was carried out to determine statistical significance (*P < 0.05; ***P < 0.001).

Figure 5

CpxR directly binds the T6SS2 hcp2B promoter region of APEC. A The sequence and schematic representation of the hcp2B promoter region. The bold line shows the sequence used in the lacZ fusion and EMSA analyses. The putative CpxR binding site is indicated with boxes. The putative −35 and −10 elements of the promoter are indicated with underlining. B EMSA for the binding of CpxR-P protein to the hcp2B promoter region. The hcp2B promoter DNA fragment with or without the CpxR binding site was amplified and biotin-labeled, and the biotin-labeled probe was mixed with increasing amounts of CpxR-P protein. For the specific competitive EMSA, CpxR-P protein was incubated with both the biotin-labeled and the unlabeled DNA probes. The biotin-labeled DNA was detected with a chemiluminescent substrate. The concentrations of CpxR-P protein and probes are shown below the figure.

Figure 6

Schematic diagram of CpxR-mediated regulation of T6SS2 expression in APEC. According to previous studies, many environmental stresses and surface adhesion sensed by the NlpE protein can activate the Cpx response [21, 22]. After activation, the CpxA protein phosphorylates the CpxR protein. The phosphorylation of CpxR enhances its binding to the T6SS2 hcp2B promoter and consequently upregulates transcription of the hcp2B operon. The upregulation of T6SS2 contributes to increased antibacterial competition, invasion and survival, which are required for the virulence of APEC.