Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC 3168, Australia.
Department of Molecular and Translational Science, Monash University, Clayton, VIC 3168, Australia.
Proc Natl Acad Sci U S A. 2022 Aug 23;119(34):e2204332119. doi: 10.1073/pnas.2204332119. Epub 2022 Aug 17.
Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.
附着和破坏（AE）肠细胞上的肠致病性（EPEC）需要 EPEC 型 III 分泌系统（T3SS）。在感染过程中，两种注入宿主细胞的 T3SS 效应子是非典型激酶 NleH1 和 NleH2。然而，在感染过程中 NleH1 和 NleH2 激酶活性的宿主靶标尚未报道。在这里，磷酸蛋白质组学鉴定出微绒毛蛋白 Eps8 中的 Ser775 是 NleH1 和 NleH2 磷酸化的真正靶标。这两种激酶都通过以前未被识别的非典型“富含脯氨酸”基序 PxxDY 与 Eps8 相互作用，该基序结合了 Eps8 的Src 同源性 3（SH3）结构域。对 Eps8 SH3 结构域与来自 NleH 的富含脯氨酸基序之一的肽结合的结构分析表明，肽的 N 端部分采用 II 型多脯氨酸螺旋，其 C 端“DY”片段与 SH3 结构域形成多个接触。NleH1 或 NleH2 对 Ser775 的磷酸化抑制了 Eps8 的捆绑活性，并促使 Eps8 在 EPEC 感染过程中从 AE 病变中分散。这一发现表明，NleH1 和 NleH2 在 EPEC 感染过程中改变了 Eps8 的细胞定位和 AE 病变的细胞骨架组成。
