Elucidation of Cryptosporidium virulence mechanisms using Toxoplasma gondii

描述（由申请人提供）：刚地弓形虫和细小隐孢子虫在艾滋病免疫功能低下的人群中引起致命感染。对于艾滋病患者来说，基本上没有针对细小梭菌的有效治疗方法，而针对弓形虫的治疗选择也很有限。缺乏针对小弧菌的治疗方案主要是由于小弧菌不能在细胞培养中繁殖。相比之下，弓形虫很容易在细胞培养中繁殖，并且有丰富的分子遗传工具可用于其分析。我们的目标是对弓形虫进行功能基因组学研究，以发现毒力基因。为此，我们将特征标记诱变（STM）应用于弓形虫，并筛选了超过6300个插入突变体。从这个文库中，我们分离出39个无毒性克隆，并鉴定出34个无毒性突变体中被破坏的基因。为了这个提议，我们将研究在STM筛选中发现的6个突变体，这些突变体在小孢子虫中具有同源序列的基因中被破坏。因为小芽胞杆菌不包含慢性囊肿期感染，我们想把重点放在急性感染期间有缺陷的突变体上。第一个目标是确定被破坏的基因对这六种突变体急性感染的贡献。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弓形虫基因或从弓形虫启动子中表达小弓形虫同源基因。这些实验将使我们能够确认破坏基因对急性毒力的贡献，并确定小孢子虫的同源基因是否在功能上类似。这一建议将使我们能够利用弓形虫分子遗传学的力量来揭示小弓形虫的毒力基因。本研究可能为弓形虫和小弓形虫发现新的药物靶点，并为研究潜在的抑制剂创造工具。刚地弓形虫和细小隐孢子虫在艾滋病免疫功能低下者中引起致命感染，目前很少或没有有效的治疗方案。缺乏针对小孢子虫的治疗很大程度上是由于小孢子虫不能在细胞培养中繁殖。这一建议将使我们能够利用弓形虫分子遗传学的力量来发现细小弓形虫的毒力基因，并可能导致发现新的弓形虫和细小弓形虫的药物靶点。

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.