Lai Tzung-Huei, Kumagai Yumi, Hyodo Mamoru, Hayakawa Yoshihiro, Rikihisa Yasuko
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093, USA.
J Bacteriol. 2009 Feb;191(3):693-700. doi: 10.1128/JB.01218-08. Epub 2008 Oct 31.
Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of A. phagocytophilum and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both pleC and pleD were expressed in an HGA patient. During A. phagocytophilum development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other A. phagocytophilum recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of A. phagocytophilum lysate with c-di-[(32)P]GMP detected an approximately 47-kDa endogenous protein, presumably c-di-GMP downstream receptor. A new hydrophobic c-di-GMP derivative, 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited A. phagocytophilum infection in HL-60 cells. Our results suggest that the two-component PleC-PleD system is a diguanylate cyclase and that a c-di-GMP-receptor complex regulates A. phagocytophilum intracellular infection.
嗜吞噬细胞无形体是人类粒细胞无形体病（HGA）的病原体，其基因预测编码三种传感激酶，其中一种注释为PleC，还有三种反应调节因子，其中一种是PleD。在本研究之前，PleC和PleD在嗜吞噬细胞无形体的 obligatory 细胞内寄生中的作用及其生化活性尚不清楚。本研究通过证明pleC和pleD在一名HGA患者中均有表达，阐明了这些因素的相关性。在嗜吞噬细胞无形体在人早幼粒细胞HL-60细胞中发育期间，PleC和PleD在指数生长期同步上调，并在细胞外释放之前下调。重组PleC激酶结构域（rPleCHKD）具有组氨酸激酶活性；当磷酸化保守位点被丙氨酸取代时未观察到活性。重组PleD（rPleD）以磷酸化的rPleCHKD作为磷酰供体具有自身激酶活性，但与其他两种重组组氨酸激酶无此活性。rPleCHKD不能作为突变型rPleD（保守天冬氨酸，磷酸化位点，被丙氨酸取代）或其他两种嗜吞噬细胞无形体重组反应调节因子的磷酰供体。rPleD在体外具有二鸟苷酸环化酶活性，可从GTP生成环（c）二鸟苷酸。嗜吞噬细胞无形体裂解物与c-二-[(32)P]鸟苷酸的紫外线交联检测到一种约47 kDa的内源性蛋白，推测为c-二鸟苷酸下游受体。一种新的疏水性c-二鸟苷酸衍生物，2'-O-二（叔丁基二甲基甲硅烷基）-c-二鸟苷酸，抑制嗜吞噬细胞无形体在HL-60细胞中的感染。我们的结果表明，双组分PleC-PleD系统是一种二鸟苷酸环化酶，并且c-二鸟苷酸-受体复合物调节嗜吞噬细胞无形体的细胞内感染。
