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

描述（由申请人提供）：弓形虫是一种专性细胞内病原体，是艾滋病患者和其他免疫功能低下个体的主要致病原因。目前还没有预防感染的疫苗，抗弓形虫药物的耐受性差，不能消除这种寄生虫。因此，需要新的药物靶点来改善治疗。与其他专性胞内病原体一样，弓形虫必须选择宿主细胞蛋白和途径。然而，已知弓形虫生长所需的宿主途径很少。确定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。由于弓形虫是一种真核生物，不能使用药物抑制剂来鉴定寄生虫调节的宿主细胞途径，因为这些药物可能不仅抑制宿主蛋白质，还抑制寄生虫蛋白质。RNA干扰（RNAi）不受此限制。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。我们验证了这种方法，首先证明，寄生虫的生长被抑制在细胞转染siRNA对宿主基因已知是必要的寄生虫的生长。第二，原理筛选的证明导致我们发现宿主微管在寄生虫入侵中的关键作用。为了扩展这些发现，提出了两个具体目标。在特定目标1中，我们将完成筛选我们的文库，并鉴定出显著特异性抑制弓形虫生长的siRNA。在具体目标2中，我们将确定i）在寄生虫的裂解生长周期中，宿主蛋白质何时发挥功能，ii）感染如何影响这些宿主蛋白质的表达和定位，以及ii）宿主蛋白质是否受到有毒力和无毒力弓形虫株的差异调节。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.