Bliska J B, Guan K L, Dixon J E, Falkow S
Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305.
Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1187-91. doi: 10.1073/pnas.88.4.1187.
The plasmid-encoded YopH protein is a protein-tyrosine phosphatase (PTPase; EC 3.1.3.48) that is essential for Yersinia virulence. We have investigated the molecular basis for the role of PTPase activity in Yersinia pathogenesis. Allelic recombination was employed to introduce a defined mutation into the yopH plasmid gene. Conversion of the essential Cys-403 to Ala in the catalytic domain of the protein abolished YopH PTPase activity and significantly reduced the virulence of Yersinia pseudotuberculosis in a murine infection model. 32P-labeled phosphotyrosine-containing proteins were immunoprecipitated from extracts of Y. pseudotuberculosis-infected cell monolayers and analyzed by SDS/PAGE to assess the impact of YopH on host protein phosphorylation. Major proteins of 200, 120, and 60 kDa were dephosphorylated in macrophages associated with wild-type Y. pseudotuberculosis. Selective removal of phosphate from the 120- and 60-kDa proteins was shown to be specific to the YopH PTPase activity. Phagocytosis of the bacteria was not required for this dephosphorylation activity, suggesting that YopH is functionally expressed by extracellular bacteria. These observations indicate that the essential function of YopH in Yersinia pathogenesis is host-protein dephosphorylation.
质粒编码的YopH蛋白是一种蛋白酪氨酸磷酸酶（PTPase；EC 3.1.3.48），对耶尔森菌的毒力至关重要。我们研究了PTPase活性在耶尔森菌致病机制中作用的分子基础。采用等位基因重组将一个特定突变引入yopH质粒基因。该蛋白催化结构域中关键的半胱氨酸403突变为丙氨酸后，消除了YopH的PTPase活性，并在小鼠感染模型中显著降低了假结核耶尔森菌的毒力。从感染假结核耶尔森菌的细胞单层提取物中免疫沉淀32P标记的含磷酸酪氨酸蛋白，并通过SDS/PAGE进行分析，以评估YopH对宿主蛋白磷酸化的影响。与野生型假结核耶尔森菌相关的巨噬细胞中，200 kDa、120 kDa和60 kDa的主要蛋白发生了去磷酸化。结果表明，120 kDa和60 kDa蛋白的选择性去磷酸化作用对YopH的PTPase活性具有特异性。这种去磷酸化活性不需要细菌的吞噬作用，这表明YopH由细胞外细菌功能性表达。这些观察结果表明，YopH在耶尔森菌致病机制中的基本功能是使宿主蛋白去磷酸化。
