Elucidation of Cryptosporidium virulence mechanisms using Toxoplasma gondii

描述(申请人提供)：弓形虫和微小隐孢子虫可导致因艾滋病而免疫受损的人受到致命感染。对于艾滋病患者，基本上没有有效的治疗方法来对抗微小的弓形虫，对弓形虫的治疗选择有限。缺乏针对微小弧菌的治疗选择在很大程度上是因为微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，并拥有丰富的分子遗传学工具用于分析。我们的目标一直是对弓形虫进行功能基因组学研究，以发现毒力基因。为此，我们将签名标记突变(STM)应用于弓形虫，并筛选了一个包含6300多个插入突变体的文库。我们从这个文库中分离了39个无毒克隆，并在34个无毒突变体中鉴定了该基因的中断。对于这个提议，我们将检查在STM筛查中发现的六个突变，这些突变在微小隐孢子虫的同源序列的基因中被破坏。由于微小隐孢子虫不包含慢性包囊阶段的感染，我们希望关注在急性感染过程中有缺陷的突变株。第一个目标将确定被破坏的基因对这六个突变体的急性感染的贡献。然后，我们将选择两个突变体进行互补研究，方法是重新表达被破坏的弓形虫基因或表达来自弓形虫启动子的微小弧菌同源基因。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，并确定微小隐孢子虫的同源基因在功能上是否相似。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微小弧菌的毒力基因。这项研究可能会为弓形虫和微小弧菌发现新的药物靶点，并为潜在抑制剂的研究创造工具。与公共卫生相关的弓形虫和微小隐孢子虫对艾滋病免疫受损的人造成致命感染，目前几乎没有有效的治疗选择。缺乏对微小弧菌的治疗在很大程度上是由于微小弧菌不能在细胞培养中繁殖的事实。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微小弓形虫的毒力基因，并可能导致发现针对弓形虫和微小弓形虫的新的药物靶点。

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.