RNAi screen to identify host factors involved in Chlamydia pathogenesis

说明(申请人提供)：衣原体是一种革兰氏阴性细菌病原体，可感染多种宿主并导致各种疾病，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被称为包涵体的膜结合的间隔内复制，该包涵体可能作为免疫监视的保护罩，也充当与宿主细胞交换因子的“过滤器”。尽管衣原体作为一种人类病原体非常重要，但人们对感染过程中涉及的细菌和宿主因素知之甚少。这种知识的匮乏主要是由于衣原体不是一种遗传上易驯化的生物，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的发病机制，我们在模式生物黑腹果蝇中开发了一种遗传学方法。我们已经证明，果蝇SL2细胞构成了研究衣原体感染的一个可行的模型。我们证明，与哺乳动物细胞中的情况类似，豚鼠的感染型(EB)，沙眼和生殖器感染的豚鼠模型，进入果蝇SL2细胞，分化为复制型(RB)，在膜结合的间隔内复制，并从RBS分化回EBS。使用这个模型系统，我们已经进行了全基因组RNAi筛选，并识别了450个宿主，当耗尽时，它们增加或减少了豚鼠隐孢子虫的细胞内生长。我们现在建议：(1)确认在初级筛查中确定的候选对象，(2)确定与衣原体感染相关的宿主因子，(3)在哺乳动物系统中验证选定的衣原体特异性候选对象。总之，我们的遗传学方法应该有助于识别衣原体致病中涉及的未被发现的宿主因素。我们的长期目标是确定已确定的候选者的功能，以便更好地了解衣原体与其宿主之间的复杂相互作用，并潜在地确定治疗的新靶点。

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.