RNAi screen to identify host factors involved in Chlamydia pathogenesis

描述（由申请人提供）：衣原体是革兰氏阴性细菌病原体，可感染广泛的宿主并引起各种疾病，包括发展中国家可预防的失明、性传播疾病和肺炎。衣原体侵入细胞并在膜结合区室（称为内含物）内复制，所述膜结合区室潜在地充当抵抗免疫监视的保护屏障，并且还充当与宿主细胞交换因子的“过滤器”。尽管衣原体作为人类病原体的首要重要性，但对感染过程中涉及的细菌和宿主因素知之甚少。知识的缺乏主要是由于衣原体不是一种遗传上易处理的生物体，以及在哺乳动物宿主中进行遗传方法的困难。 为了进一步深入了解衣原体致病机制，我们在模式生物果蝇中开发了一种遗传方法。我们已经表明，果蝇SL2细胞构成了一个可行的模型来研究衣原体感染。我们证明，类似于哺乳动物细胞中的情况，感染型（EB）的C。豚鼠生殖道和眼部感染豚鼠模型。沙眼衣原体感染后，进入果蝇SL2细胞，分化成复制型（RB），在膜结合区室内复制，并从RB分化回EB。利用这个模型系统，我们进行了全基因组RNAi筛选，并鉴定了450个宿主，当耗尽时增加或减少C。细胞内生长。我们现在提议：（1）确认在初步筛选中鉴定的候选者，（2）鉴定特异性参与衣原体感染的宿主因子，（3）在哺乳动物系统中验证所选择的衣原体特异性候选者。总而言之，我们的遗传学方法应导致发现宿主因素参与衣原体的发病机制。我们的长期目标是表征所确定的候选人的功能，以便更好地了解衣原体与其宿主之间的复杂相互作用，并可能确定新的治疗靶点。

DESCRIPTION (provided by applicant): Chlamydia are gram-negative bacterial pathogens that infect a wide range of hosts and cause various diseases, including preventable blindness in developing countries, sexually transmitted disease and pneumonia. Chlamydia invade cells and replicate within a membrane-bound compartment, named the inclusion, that potentially serves as a protector shield against immune surveillance and also acts as a "filter" to exchange factors with the host cell. Despite the primary importance of Chlamydia as a human pathogen, little is known about the bacterial and host factors involved in the infection process. This paucity of knowledge is mainly due to the fact that Chlamydia is not a genetically tractable organism and to the difficulty of conducting genetic approaches in the mammalian host. To further get insights into the mechanisms involved in Chlamydia pathogenesis, we have developed a genetic approach in the model organism Drosophila melanogaster. We have shown that Drosophila SL2 cells constitute a viable model to study Chlamydia infection. We demonstrated that, similar to the situation in mammalian cells, infectious forms (EB) of C. caviae, the guinea pig model of genital and ocular infection of C. trachomatis, enter Drosophila SL2 cells, differentiate into the replicative form (RB), replicate within a membrane-bound compartment and differentiate back from RBs to EBs. Using this model system, we have performed a genome wide RNAi screen and identify 450 host that when depleted increased or decreased C. caviae intracellular growth. We now propose: (1) to confirm the candidates identified in the primary screen, (2) to identify the host factors specifically involved in Chlamydia infection, (3) to validate the selected Chlamydia-specific candidates in a mammalian system. Altogether, our genetic approach should lead to the identification of uncovered host factors involved in Chlamydia pathogenesis. Our long-term goal is to characterize the function of the identified candidates in order to better understand the complex interaction between Chlamydia and its host and potentially identify novel targets for therapeutic treatment.