Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection
Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection
复制标题
DOI：
10.1111/j.1462-5822.2006.00871.x
复制
发表时间：
2007-05-01
影响因子：
3.4
通讯作者：
Kennedy, Elizabeth L.
中科院分区：
文献类型：
--
作者：
IJdo, Jacob W.;Carlson, Adam C.;Kennedy, Elizabeth L.
嗜吞噬细胞无形体是一种细胞内病原体，感染并存活于嗜中性粒细胞中。助理嗜吞噬细胞菌基因组编码一个4型分泌系统（T4 SS），该系统可通过毒力因子的易位促进细胞内存活，但迄今为止，尚未鉴定出此类因子。由于几种细胞内生物的T4 SS易位蛋白通过宿主细胞激酶进行酪氨酸磷酸化，我们研究了A.嗜吞噬细胞蛋白在感染过程中。在A.在嗜吞噬细胞菌与HL-60细胞或PMN接触时，190 kDa的细菌蛋白AnkA被越来越多的酪氨酸磷酸化。A.嗜吞噬细胞噬菌体结合宿主细胞而不进入足以进行AnkA酪氨酸磷酸化。体外Src激酶测定表明，纯化的AnkA在大肠杆菌中表达的磷酸化的酪氨酸位于C-末端部分的AnkA。类似地，在COS-7细胞中表达的AnkA在C-末端通过Src进行酪氨酸磷酸化。磷酸化酪氨酸位于EPIYA基序中，其显示与SH 2结构域结合的共有序列。免疫沉淀研究表明，AnkA结合宿主细胞磷酸酶SHP-1在感染早期。EPIYA基序的磷酸化和SH 2结构域的存在是AnkA-SHP-1相互作用所必需的。我们的结论是，AnkA是一个易位的毒力因子，是酪氨酸磷酸化的宿主细胞激酶易位到宿主细胞感染过程中的早期。A.嗜吞噬细胞菌可以通过SHP-1募集来操纵宿主细胞。
Anaplasma phagocytophilum is an intracellular pathogen that infects and survives in neutrophilic granulocytes. The A. phagocytophilum genome encodes a type four secretion system (T4SS) that may facilitate intracellular survival by translocation of virulence factors, but to date, no such factors have been identified. Because T4SS-translocated proteins of several intracellular organisms undergo tyrosine phosphorylation by host cell kinases, we investigated tyrosine phosphorylation of A. phagocytophilum proteins during infection. Within minutes after incubation of A. phagocytophilum with HL-60 cells or PMN, a 190 kDa bacterial protein, AnkA, was increasingly tyrosine-phosphorylated. A. phagocytophilum binding to host cells without entry was sufficient for AnkA tyrosine phosphorylation. An in vitro Src kinase assay demonstrated that purified AnkA expressed in Escherichia coli was phosphorylated at tyrosines located at the C-terminal portion of AnkA. Similarly, AnkA expressed in COS-7 cells underwent tyrosine phosphorylation by Src at the C-terminus. The phosphorylated tyrosines were located in EPIYA motifs that display the consensus sequence for binding to SH2 domains. Immunoprecipitation studies demonstrated AnkA binding to the host cell phosphatase SHP-1 during early infection. Phosphorylation of the EPIYA motifs and the presence of the SH2 domains were necessary for AnkA-SHP-1 interaction. We conclude that AnkA is a translocated virulence factor that is tyrosine-phosphorylated by host cell kinases upon translocation into the host cell early during infection. A. phagocytophilum may manipulate the host cell through SHP-1 recruitment.