Early signaling events involved in the entry of Rickettsia conorii into mammalian cells
Early signaling events involved in the entry of Rickettsia conorii into mammalian cells
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DOI：
10.1242/jcs.01382
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发表时间：
2004-10-01
影响因子：
4
通讯作者：
Cossart, P
中科院分区：
文献类型：
--
作者：
Martinez, JJ;Cossart, P
康氏立克次体是地中海斑疹热的病原体，在体内和体外都能附着并侵入多种细胞类型。虽然先前的研究表明，立克次体进入非吞噬细胞依赖于肌动蛋白聚合，但对立克次体-宿主细胞相互作用和肌动蛋白重排的分子细节知之甚少。我们确定了conorii将Arp2/3复合物招募到进入灶的位置，并且wasp家族成员Scar的Arp2/3结合衍生物的表达抑制了细菌进入Vero细胞，从而确定了Arp2/3是这一过程的活性成分。通过瞬时转染表达小gtpase显性阴性版本的质粒，我们发现Cdc42而非Rac1参与了conorii对Vero细胞的侵袭。使用药理学方法，我们发现这种入侵依赖于磷酸肌苷（PI） 3-激酶和蛋白酪氨酸激酶（PTK）活性，特别是src家族激酶。C-Src及其下游靶标p80 /85接触蛋白在感染过程的早期共定位于进入位点。conconi内化与其他几种宿主蛋白的酪氨酸磷酸化相关，包括局灶黏附激酶（FAK），在conconi感染的几分钟内。我们的研究结果表明，conorii R.进入非吞噬细胞依赖于Arp2/3复合物，并且涉及Cdc42， PI 3-激酶，c-Src，接触蛋白和酪氨酸磷酸化蛋白的途径相互作用调节Arp2/3激活，导致在细菌进入过程中观察到的局部肌动蛋白重排。这是第一份记录立克次体物种进入哺乳动物细胞的机制的报告。
Rickettsia conotii, the causative agent of Mediterranean spotted fever, is able to attach to and invade a variety of cell types both in vitro and in vivo. Although previous studies show that entry of R. conorii into non-phagocytic cells relies on actin polymerization, little else is known about the molecular details governing Rickettsia-host cell interactions and actin rearrangements. We determined that R. conorii recruits the Arp2/3 complex to the site of entry foci and that expression of an Arp 2/3 binding derivative of the WASP-family member, Scar, inhibited bacterial entry into Vero cells, establishing that Arp2/3 is an active component of this process. Using transient transfection with plasmids expressing dominant negative versions of small GTPases, we showed that Cdc42, but not Rac1 is involved in R. conorii invasion into Vero cells. Using pharmacological approaches, we show that this invasion is dependent on phosphoinositide (PI) 3-kinase and on protein tyrosine kinase (PTK) activities, in particular Src-family kinases. C-Src and its downstream target, p8O/85 cortactin, colocalize at entry sites early in the infection process. R. conorii internalization correlated with the tyrosine phosphorylation of several other host proteins, including focal adhesion kinase (FAK), within minutes of R. conorii infection. Our results reveal that R. conorii entry into nonphagocytic cells is dependent on the Arp2/3 complex and that the interplay of pathways involving Cdc42, PI 3-kinase, c-Src, cortactin and tyrosine-phosphorylated proteins regulates Arp2/3 activation leading to the localized actin rearrangements observed during bacterial entry. This is the first report that documents the mechanism of entry of a rickettsial species into mammalian cells.