ExsE Is a Negative Regulator for T3SS Gene Expression in Vibrio alginolyticus

Abstract

Type III secretion systems (T3SSs) contribute to microbial pathogenesis of Vibrio species, but the regulatory mechanisms are complex. We determined if the classic ExsACDE protein-protein regulatory model from Pseudomonas aeruginosa applies to Vibrio alginolyticus. Deletion mutants in V. alginolyticus demonstrated that, as expected, the T3SS is positively regulated by ExsA and ExsC and negatively regulated by ExsD and ExsE. Interestingly, deletion of exsE enhanced the ability of V. alginolyticus to induce host-cell death while cytotoxicity was inhibited by in trans complementation of this gene in a wild-type strain, a result that differs from a similar experiment with Vibrio parahaemolyticus ExsE. We further showed that ExsE is a secreted protein that does not contribute to adhesion to Fathead minnow epithelial cells. An in vitro co-immunoprecipitation assay confirmed that ExsE binds to ExsC to exert negative regulatory effect on T3SS genes. T3SS in V. alginolyticus can be activated in the absence of physical contact with host cells and a separate regulatory pathway appears to contribute to the regulation of ExsA. Consequently, like ExsE from P. aeruginosa, ExsE is a negative regulator for T3SS gene expression in V. alginolyticus. Unlike the V. parahaemolyticus orthologue, however, deletion of exsE from V. alginolyticus enhanced in vitro cytotoxicity.

Keywords: ExsACDE; ExsE; T3SS; Vibrio alginolyticus; gene expression; negative regulator.

Figure 1

ExsE exerts a negative regulatory effect on in vitro cytotoxicity. LDH assay 1.5 h post infection. Deletion of exsE in V. alginolyticus led to increased T3SS-induced cell death while intrans expression of exsE inhibited cytotoxicity in the wild-type strain. Asterisk represents statistical significance (P < 0.05). Error bars represent SEM (n = three independent replicates).

Figure 2

ExsE is a negative regulator for T3SS genes transcription in V. alginolyticus. 10 T3SS genes, including structural genes (vscY and vseE), effector genes (1687 and 1686), translocation genes (vopB and vopD) and regulatory genes (exsA, exsC, exsD and exsE), were examined using qPCR to show their transcription pattern in different V. alginolyticus strains (ZJO, ΔexsE and ΔexsE:pexsE) in TSB or contact with FHM cells. T3SS genes were transcribed after contact with host cells while transcription is limited under TSB only conditions. Deletion of exsE resulted in significantly more T3SS transcription under non-inducing conditions and transcription was inhibited by overexpression of exsE in wild-type strain even under inducing conditions. Average normalized expression data were added into individual box in the heatmap.

Figure 3

exsA can be directly activated by alternative signaling pathway. (A) The transcription of exsA was not completely inhibited by deletion of exsC or overexpression of exsD and exsE in wild-type V. alginolyticus (B) exsA has similar expression pattern in ΔexsC mutant and wild-type strain when contacted with FHM cells (lane 1, 3) suggesting other factors may also contribute to the regulation of exsA. We detected limited expression of exsA in non-inducing conditions (lane 2, 4). Endogenous dnaK served as a loading control.

Figure 4

ExsE interacts with ExsC. His-tagged ExsA, ExsC and HA-tagged ExsD, ExsE were expressed and incubated overnight at 4°C to examine protein-protein interaction. ExsA binds to ExsD (lane 3), ExsC binds to ExsD (lane 6) and ExsE binds ExsC (lane 9). Non-specific bindings were excluded in this experiment (lane 2, 5, 8, and11) and ExsE does not bind to ExsA (lane 12). + and − indicate the presence and absence of corresponding proteins, respectively.

Figure 5

ExsE is secreted in a T3SS-dependent manner under inducing conditions. FHM cells were infected with V. alginolyticus strains (ZJO, ΔexsE:pexsE and ΔvscC:pexsE) and media was collected (4 h), filtered, precipitated and probed. Recombinant ExsE was detected in the supernatant (lane 4) unless a non-function T3SS was present (lane 6).

Figure 6

Physical contact with host cells is not required to induce T3SS in V. alginolyticus. (A) Transcription of T3SS genes in V. alginolyticus was induced when cultured in cell culture media even in the absence of physical contact with eukaryotic cells. TSB and host cell contact served as negative and positive controls in this experiment. Average normalized expression data were added into individual box in the heatmap. (B) Without contacting with FHM cells, the expression of master regulator ExsA was induced to a high level (lane 3) which is comparable to host cell model (lane 4). Endogenous dnaK served as a loading control.