| Abstract |
Cryptosporidum parvum is an opportunistic protozoan parasite frequently infecting AIDS patients and causing persistent childhood diarrhea. Culture of C. parvum in cell monolayers is far from adequate because parasite development is transient, precluding the maintenance of reference strains and limiting the testing of anti-cryptosporidial drugs. The reason for the limited development of C. parvum in vitro is unknown.
The first aim of this project is to adapt a conventional system for culturing C. parvum to a 384-well format. This miniaturized assay will enable high-throughput screening of libraries of small bioactive molecules to identify compounds, which enhance in vitro development of C. parvum . This approach differs from those attempted by others who tried to improve parasite in vitro growth by infecting different cell lines or testing culture medium supplements. The optimized 384-well system will serve to screen libraries of thousands of bioactive small molecules using high throughput methods to identify compounds, which enhance the development of C. parvum . This approach makes no assumptions on the reasons for the poor performance of current culture methods, instead relying on the power of high-throughput screening. Because the molecular target of most library compounds is known, the "hits" identified with the Cryptosporidium assay will point to host cell or parasite pathways relevant to in vitro growth. The underlying hyposthesis is that bioactive molecules can enhance infection of cell monolayers with C. parvum by up- or down-regulating host or parasite processes important for invasion, parasite growth and differentiation. This project was approved by the New England Regional Center for Excellence (NERCE) for Biodefense and Emerging Infections Diseases, giving us access to the screening facility, technical support and compound libraries at no cost. Consistent with the CFAR Developmental Grants Program, this proposal is submitted by an established investigator seeking initial funding for an innovative AIDS related project. The Specific Aims are: 1) Adapt the Cyptosporidium parvum culture method to a 384-well format. The outcome of this aim will be a miniaturized C. parvum culture method with high signal/background ratio and low variability. Infection will be quantitated by immunofluorescence and image analysis. 2) Using high throughput screening, identify small molecules which enhance the development of Cryptosporidium parvum in culture and determine the host cell or parasite pathways targeted by these compounds. Because the same compound libraries have been used in numerous screens, the NERCE database will identify metabolic pathways targeted by the hits from the Crypotsporidium assay. The identification of biological processes enhancing (or inhibiting) C. parvum infection in vitro will shed light on what may limit C. parvum in vitro growth and will generate new hypotheses to support future grant proposals.
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