RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry.
RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry.
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DOI：
10.1371/journal.ppat.1000021
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发表时间：
2008-03-07
期刊：
影响因子：
6.7
通讯作者：
Engel, Joanne N.
中科院分区：
文献类型：
--
作者：
Elwell, Cherilyn A.;Ceesay, Alhaji;Kim, Jung Hwa;Kalman, Daniel;Engel, Joanne N.
为了阐明沙眼衣原体感染早期事件的机制，我们在果蝇S2细胞中进行了大规模的无偏RNA干扰筛选。这允许在一个简单的非冗余的，遗传上易处理的系统中识别候选宿主因子。从7，216个双链RNA（dsRNA）的文库中，我们鉴定了10226个宿主基因，包括两种酪氨酸激酶，Abelson（Abl）激酶和PDGF-和VEGF-受体相关（Pvr），其是血小板衍生生长因子受体（PDGFR）的同源物。我们进一步研究了这两种激酶在C.沙眼衣原体结合并内化到哺乳动物细胞中。这两种激酶在感染后都被磷酸化，并被募集到细菌附着的位点，但它们在感染过程中的作用是不同的。我们提供了PDGFRβ可能作为受体发挥作用的证据，因为通过RNA干扰或PDGFRβ中和抗体抑制PDGFRβ可显著降低细菌结合，而Abl激酶耗竭对结合无影响。细菌内化可以通过PDGFRβ的活化或通过Abl激酶的独立活化而发生，最终导致Rac鸟嘌呤核苷酸交换因子（GEF）、Vav 2和两种肌动蛋白成核剂WAVE 2和Corpine的磷酸化。最后，我们表明，TARP，细菌III型分泌的肌动蛋白成核剂参与进入，是一个目标的Abl激酶。总之，我们的研究结果表明，PDGFRβ和Abl激酶的功能冗余，以促进这种专性细胞内寄生虫的有效吸收。 沙眼衣原体感染是一个世界性的问题;它们是发展中国家可预防失明的主要原因，也是西方世界性传播疾病的最常见原因。结合和进入宿主细胞是这种专性细胞内寄生虫发病的关键步骤;然而关于这些过程的机制知之甚少。在这项工作中，我们描述了一个大规模的RNA干扰筛选，以确定主机的C。沙眼感染我们发现血小板衍生生长因子受体β（PDGFRβ）可以作为C.沙眼衣原体，以及C.沙眼衣原体感染导致Rac鸟嘌呤核苷酸交换因子Vav 2和几种肌动蛋白成核剂（包括WAVE 2、Corpine和TARP，一种衣原体III型分泌的效应物）的磷酸化。我们的工作提出了一个PDGFRβ和Abl激酶在C.沙眼衣原体结合，在调节这种专性细胞内寄生虫的有效摄取的细胞骨架重排中达到高潮。
To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified ∼226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRβ may function as a receptor, as inhibition of PDGFRβ by RNA interference or by PDGFRβ neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRβ or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRβ and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite. Chlamydia trachomatis infections are a worldwide problem; they are the leading cause of preventable blindness in developing nations and the most common cause of sexually transmitted disease in the Western world. Binding and entry into host cells are critical steps to the pathogenesis of this obligate intracellular parasite; however little is known regarding the mechanism of these processes. In this work, we describe a large scale RNA interference screen to identify host factors essential for early steps in C. trachomatis infection. We discover that the Platelet Derived Growth Factor Receptor β (PDGFRβ) can function as a receptor for C. trachomatis, and that activation of both PDGFRβ and Abl kinase signaling pathways by C. trachomatis leads to phosphorylation of a Rac guanine nucleotide exchange factor, Vav2, and several actin nucleators, including WAVE2, Cortactin, and TARP, a Chlamydia type III secreted effector. Our work suggests a model of redundant activation of PDGFRβ and Abl kinase upon C. trachomatis binding that culminates in cytoskeletal rearrangements that modulate efficient uptake of this obligate intracellular parasite.
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