Identification of Host Genes Important for Growth of the AIDS Opportunistic Patho

描述(申请人提供)：弓形虫是一种专有的细胞内病原体，是艾滋病患者和其他免疫功能受损的人患病的主要原因。没有预防感染的疫苗，抗弓形虫药物耐受性差，无法消除寄生虫。因此，需要新的药物靶点来改善治疗。像其他专性细胞内病原体一样，弓形虫必须选择宿主细胞蛋白和途径。然而，几乎没有人知道弓形虫生长所需的宿主途径。识别这些宿主途径是至关重要的，因为这些途径和调节它们的寄生虫因子是新的药物靶点。由于弓形虫是真核生物，因此不能使用药物抑制剂识别受弓形虫调控的宿主细胞途径，因为这些药物不仅可能抑制宿主蛋白，还可能抑制寄生虫蛋白。RNA干扰(RNAi)不受这一限制。因此，我们将使用siRNA筛选来鉴定对寄生虫生长至关重要的宿主细胞蛋白。我们通过首先证明在细胞中针对已知对寄生虫生长所必需的宿主基因的siRNAs的感染抑制了寄生虫的生长，从而验证了这种方法。第二，原则性筛选的证据使我们发现宿主微管在寄生虫入侵中起着关键作用。为了推广这些发现，本文提出了两个具体目标。在具体目标1中，我们将完成对我们的文库的筛选，并识别出显著和特异地抑制弓形虫生长的siRNA。在特定的目标2中，我们将确定i)在寄生虫的裂解生长周期中何时发挥宿主蛋白的功能，ii)感染如何影响这些宿主蛋白的表达和定位，以及ii)宿主蛋白是否受到强毒和无毒弓形虫菌株的差异调控。总之，这些数据将确定对弓形虫生长至关重要的新宿主途径，这是开发新治疗方法的重要第一步。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular pathogen that is a major cause of disease in AIDS patients and other immune compromised individuals. There are no vaccines to prevent infections and anti-Toxoplasma drugs are poorly tolerated and cannot eliminate the parasite. Thus, new drug targets are needed to improve treatment. Like other obligate intracellular pathogens, Toxoplasma must co-opt host cell proteins and pathways. However, few host pathways needed for Toxoplasma growth are known. Identifying these host pathways is critical because these pathways and the parasite factors that regulate them are novel drug targets. Because Toxoplasma is a eukaryote, host cell pathways regulated by the parasite cannot be identified using pharmacological inhibitors because these drugs would likely inhibit not only host proteins but also parasite ones. RNA interference (RNAi) is not subject to this limitation. Thus, we will use a siRNA screen to identify host cell proteins critical for parasite growth. We validated this approach by first demonstrating that parasite growth is inhibited in cells transfected with siRNAs against a host gene known to be necessary for parasite growth. Second, a proof of principle screen led to our discovery of a critical role for host microtubules in parasite invasion. To extend these findings, two specific aims are proposed. In Specific Aim 1, we will complete screening our libraries and identify siRNAs that significantly and specifically inhibit Toxoplasma growth. In Specific Aim 2, we will determine i) when during the parasite's lytic growth cycle does a host protein function, ii) how infection influences the expression and localization of these host proteins, and ii) if a host protein is differentially regulated by virulent and avirulent Toxoplasma strains. Together, these data will identify novel host pathways critical for Toxoplasma growth, which is an important first step in developing new treatments.