The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosine-phosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling.
The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosine-phosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling.
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DOI：
10.1371/journal.ppat.1003954
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发表时间：
2014-02
期刊：
影响因子：
6.7
通讯作者：
Valdivia RH
中科院分区：
文献类型：
--
作者：
Chen YS;Bastidas RJ;Saka HA;Carpenter VK;Richards KL;Plano GV;Valdivia RH
沙眼衣原体（Chlamydia trachomatis）是沙眼和性传播感染的病原体，其利用III型分泌（T3 S）系统将效应蛋白递送到宿主上皮细胞中以建立复制空泡。除了磷蛋白TARP，衣原体效应，促进肌动蛋白重排，很少有因素介导的细菌进入和早期包涵体建立的特点。与许多T3 S效应器一样，TARP需要伴侣蛋白（Slc 1）有效易位到宿主细胞中。在这项研究中，我们定义了与Slc 1相关的蛋白质在侵袭性C。沙眼衣原体原体（EB）的免疫沉淀与质谱联用。我们鉴定了Ct 875，一种新的Slc 1客户蛋白和T3 S效应器，我们将其重新命名为TepP（易位早期磷蛋白）。我们提供的证据表明，T3 S效应形成大分子量的复合物与Scl 1在体外和Scl 1增强其T3 S依赖性分泌在异源耶尔森氏菌T3 S系统。我们证明，TepP是易位早期细菌进入上皮细胞，并在酪氨酸残基由宿主激酶磷酸化。然而，TepP磷酸化发生晚于TARP，这与Slc 1优先参与EB中TARP的发现一起导致我们假设这些效应物在C过程中的不同阶段易位到宿主细胞中。沙眼侵袭TepP共免疫沉淀的支架蛋白CrkI-II在感染和Crk被招募到EB在入口网站，它仍然与新生的夹杂物。重要的是，C。缺乏TepP的沙眼突变体未能招募CrkI-II到包涵体中，这为该效应子在招募宿主因子中的直接作用提供了遗传学证实。最后，感染tepP突变体的宫颈上皮细胞显示与先天性免疫反应相关的基因子集的表达改变。我们提出了一个模型，其中TepP的下游TARP招募支架蛋白在入口网站启动和放大信号级联重要的先天免疫反应的调节衣原体。 沙眼衣原体是一种专性细胞内细菌病原体，可引起一系列具有重大公共卫生意义的人类疾病。为了在其宿主内创建合适的复制生态位，衣原体通过称为III型分泌（T3 S）系统的类双链体装置将效应蛋白递送穿过哺乳动物膜。缺乏一个强大的系统，这些病原体的分子遗传操作，阻碍了在识别和表征T3 S效应的进展。在这项研究中，我们采取了一种基于质谱的方法来确定衣原体效应蛋白的基础上，他们与Slc 1，丰富的T3 S伴侣的相互作用。我们确定了一个以前未知的蛋白质，Ct 875/TepP，作为一个新的T3 S效应，并确定TepP是磷酸化易位到宿主细胞，导致招聘的主机支架蛋白Crk和推测操纵Crk依赖的信号功能。最后，我们提供了TepP在招募Crk和调节参与衣原体先天免疫反应的基因表达中的作用的遗传学确认。这项研究是衣原体中T3 S效应子功能的遗传验证的第一个例子，也是直接选择癌蛋白Crk来调节宿主细胞信号事件的细菌效应子的新例子。
Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia. Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes a range of human diseases of significant public health importance. To create a suitable replicative niche within its host, Chlamydia delivers effector proteins across mammalian membranes via a syringe-like apparatus termed a Type III secretion (T3S) system. The lack of a robust system for the molecular genetic manipulation of these pathogens has hindered progress in identifying and characterizing T3S effectors. In this study, we took a mass spectrometry-based approach to identify Chlamydia effector proteins based on their interaction with Slc1, an abundant T3S chaperone. We identified a previously uncharacterized protein, Ct875/TepP, as a new T3S effector and determined that TepP is phosphorylated upon translocation into host cells, leading to the recruitment of the host scaffolding protein Crk and presumably manipulating Crk-dependent signaling functions. Finally, we provide genetic confirmation of the role of TepP in recruiting Crk and in modulating the expression of genes involved in innate immune responses to Chlamydia. This study is the first example of genetic validation of the function of a T3S effector in Chlamydia and a new example of a bacterial effector that directly co-opts the oncoprotein Crk to modulate host cell signaling events.
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