Elucidation of Cryptosporidium virulence mechanisms using Toxoplasma gondii

描述（由申请人提供）：弓形虫和隐孢子虫引起艾滋病免疫功能低下的人的致命感染。对于艾滋病患者，基本上没有针对C的有效疗法。和有限的治疗选择T.刚地。缺乏针对C的治疗选择。parvum的主要原因是C.小孢子不能在细胞培养中繁殖。与此相反，T.刚地虫在细胞培养中容易繁殖，并且具有丰富的可用于其分析的分子遗传工具。我们的目标是对T.弓形虫的毒力基因。为了这个目标，我们将特征标记诱变（STM）应用于T。gondii，并筛选了超过6300个插入突变体的文库。从这个文库中，我们分离出39个无毒克隆，我们已经确定了34个无毒突变体中的基因中断。对于这个建议，我们将检查STM筛选中发现的六个突变体，这些突变体在C.小的因为C. parvum不包含感染的慢性囊肿阶段，我们想把重点放在突变体，是在急性感染的缺陷。第一个目标是确定这六种突变体的破坏基因对急性感染的贡献。然后，我们将选择两个突变体进行互补研究，通过重新表达被破坏的T。gondii基因或C. parvum的直系同源物。弓形虫启动子这些实验将使我们能够确认被破坏的基因对急性毒力的贡献，并确定C。parvum直系同源物在功能上类似。这一提议将使我们能够利用T.弓形虫分子遗传学的研究。细小病毒毒力基因这项研究可能会发现新的药物靶点T。gondii和C. parvum，并创建用于研究潜在抑制剂的工具。公共卫生相关性弓形虫和微小隐孢子虫会导致艾滋病免疫功能低下者的致命感染，目前几乎没有有效的治疗方法。缺乏针对C. parvum的主要原因是C.小孢子不能在细胞培养中繁殖。这一提议将使我们能够利用T.弓形虫分子遗传学的研究。小锥虫毒力基因，并可能导致发现新的药物靶点，为T。gondii和C.小的

DESCRIPTION (provided by applicant): Toxoplasma gondii and Cryptosporidium parvum cause fatal infections in persons immune compromised by AIDS. For AIDS patients, there is essentially no effective therapy against C. parvum and limited therapeutic options for T. gondii. The lack of treatment options against C. parvum is largely due to the fact that C. parvum cannot be propagated in cell culture. In contrast, T. gondii is easily propagated in cell culture and has a wealth of molecular genetic tools available for its analysis. Our goals have been to perform functional genomics on T. gondii to uncover virulence genes. For this goal, we adapted signature-tagged mutagenesis (STM) to T. gondii, and screened a library of over 6300 insertion mutants. From this library we isolated 39 avirulent clones and we have identified the gene disrupted in 34 avirulent mutants. For this proposal, we will examine six mutants uncovered in the STM screen that are disrupted in genes with orthologous sequences in C. parvum. Because C. parvum does not contain a chronic cyst stage of infection, we want to focus on mutants that are defective during acute infection. The first aim will determine the contribution of the disrupted genes to acute infection for these six mutants. We will then choose two mutants for complementation studies by re-expression of the disrupted T. gondii gene or expression of the C. parvum ortholog from a T. gondii promoter. These experiments will allow us to confirm the contribution to acute virulence of the disrupted gene and to determine if the C. parvum ortholog is functionally analogous. This proposal will allow us to exploit the power of T. gondii molecular genetics to uncover C. parvum virulence genes. This research may discover new drug targets for both T. gondii and C. parvum, and create tools for the study of potential inhibitors. PUBLIC HEALTH RELEVANCE Toxoplasma gondii and Cryptosporidium parvum cause fatal infections in persons immune-compromised by AIDS, with little or no effective therapeutic options currently available. The lack of treatment against C. parvum is largely due to the fact that C. parvum cannot be propagated in cell culture. This proposal will allow us to exploit the power of T. gondii molecular genetics to uncover C. parvum virulence genes, and may result in the discovery of new drug targets for both T. gondii and C. parvum.