Enteropathogenic Escherichia coli (EPEC) utilizes the type 3 secretion system (T3SS) to translocate effector proteins into host cells that hijack cell signaling pathways to promote infection, colonization, and survival. Prior studies have shown that phosphorylation of Y152 and Y153 on the multi-effector T3SS chaperone CesT influences effector secretion and host colonization dynamics. However, additional phosphosites, the kinase(s) responsible for CesT phosphorylation, and the downstream effect(s) remain poorly characterized. Herein, we used a combination of biochemical and functional assays to show CesT tyrosine phosphorylation levels do not change in the absence or presence of the T3SS effector kinases NleH1 and/or NleH2. Instead, we demonstrate that when NleH1 and NleH2 are in complex with CesT, they function as serine kinases that target the S146 and/or S147 phosphosites, consistent with their role as Ser/Thr kinases on host nuclear factor-kappa B signaling pathways. We show that the CesT S146E, S147A, and S147E variants have an impaired ability to interact with CsrA using pull-down assays, and an EPEC ΔnleH1 ΔnleH2 strain displays increased NleA translation and host translocation compared to wild-type EPEC, supporting the role of CesT S146 and S147 phosphorylation in regulating CsrA repression of nleA mRNA. Our data support a model where the phosphorylation of CesT at S146 and/or S147 influences NleA translation levels by impairing CesT-CsrA complex formation, which in turn modulates the levels of NleA translation and translocation into host cells. This study further highlights the complex post-translational regulatory functioning of CesT in orchestrating T3SS effector secretion hierarchy during infection.
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