Regulation of the Edwardsiella ictaluri type III secretion system by pH and phosphate concentration through EsrA, EsrB, and EsrC

Abstract

A recently described Edwardsiella ictaluri type III secretion system (T3SS) with functional similarity to the Salmonella pathogenicity island 2 T3SS is required for replication in channel catfish head-kidney-derived macrophages (HKDM) and virulence in channel catfish. Quantitative PCR and Western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Mutagenesis studies demonstrated that expression is under the control of the EsrAB two-component regulatory system. EsrB also induces upregulation of the AraC-type regulatory protein EsrC, which enhances expression of the EscB/EseG chaperone/effector operon in concert with EsrB and induces expression of the pEI1-encoded effector, EseH. EsrC also induces expression of a putative type VI secretion system translocon protein, EvpC, which is secreted under the same low-pH conditions as the T3SS translocon proteins. The pEI2-encoded effector, EseI, was upregulated under low-pH and low-phosphate conditions but not in an EsrB- or EsrC-dependent manner. Mutations of EsrA and EsrB both resulted in loss of the ability to replicate in HKDM and full attenuation in the channel catfish host. Mutation of EsrC did not affect intracellular replication but did result in attenuation in catfish. Although EsrB is the primary transcriptional regulator for E. ictaluri genes within the T3SS pathogenicity island, EsrC regulates expression of the plasmid-carried effector eseH and appears to mediate coordinated expression of the T6SS with the T3SS.

Fig. 1.

Two-dimensional polyacrylamide gel electrophoresis analyses of extracellular protein products of wild-type Edwardsiella ictaluri (WT) and T3SS regulatory gene mutants cultured in MM19 at pH 5.5. The T3SS translocon proteins EseB, EseC, and EseD and the T6SS protein EvpC are indicated by arrows. Circles indicate areas on the gel where protein spots occur in the WT ECP but are absent in the mutant ECP. Gel experiments were repeated three times, and representative gels are shown.

Fig. 2.

Western blot detection of T3SS proteins fused to the Flag epitope in Edwardsiella ictaluri whole-cell lysates. Strains carrying the fusions were cultured in MM19 at pH 7.0, MM19 at pH 5.5, and MMP at pH 5.5. Proteins carrying the Flag epitope were labeled with anti-Flag antibody followed by HRP-conjugated goat anti-mouse antibody to detect EsrC, EseB, and EseG. Detection of EseI required mouse anti-Flag antibody followed by biotinylated goat anti-mouse antibody and HRP-conjugated streptavidin. Loading of equivalent protein amounts across lanes was verified using mouse anti-DnaK antibody, biotinylated goat anti-mouse antibody, and HRP-conjugated streptavidin.

Fig. 3.

Replication of Edwardsiella ictaluri strains carrying mutations in T3SS regulatory gene esrA (A), esrB (B), or esrC (C) in HKDM. Strains carrying complementation plasmids are labeled with pesrA, pesrB, or pesrC. Bars indicate the mean fold replication (± standard error of the mean [SEM]) at 5 and 10 h postinfection for triplicate assays. Asterisks indicate a significant difference from the WT fold increase at the same time point (P ≤ 0.05).

Fig. 4.

Cumulative mortality of channel catfish following infection by Edwardsiella ictaluri strains carrying mutations in T3SS regulatory gene esrA (A), esrB (B), or esrC (C). Strains carrying complementation plasmids are labeled with pesrA, pesrB, or pesrC. Bars indicate the mean daily cumulative percent mortality (± SEM) in triplicate challenge tanks. Mortality curves with the same letter show no significant difference in cumulative percent mortality (P > 0.05), based on analysis of arcsine-transformed data.

Fig. 5.

Proposed model for pH and phosphate regulation of the E. ictaluri T3SS.