Elucidation of Cryptosporidium virulence mechanisms using Toxoplasma gondii

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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.