AIDS Opportunistic Pathogens: Targeting the Methyl Citrate Cycle

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DESCRIPTION (provided by applicant): Candida glabrata is an important opportunistic pathogen in HIV seropositive and HIV seronegative populations. In the United States, most clinical series over the last 15 years show that C. glabrata accounts for 15-20% of mucosal and disseminated candidiasis, C. glabrata is innately resistant to triazole antifungals, and there is a need for development of new chemotherapeutic strategies for treatment of C. glabrata disseminated and mucosal infections. The methylcitrate cycle represents an excellent potential target for therapeutic intervention in C. glabrata. The methylcitrate cycle carried out detoxification of propionate and propionyl-CoA, produced endogenously by C. glabrata as a byproduct of the degradation of protein. The cycle degrades propionate to pyruvate in three steps, catalyzed by the Cit3, Pdh1 and Icl2 enzymes. We have shown that the entire methylcitrate cycle is strongly induced following phagocytosis by macrophages. We have also shown that disruption of PDH1 or ICL2 renders C. glabrata exquisitely sensitive to propionate, likely due to the accumulation of the metabolic toxin methylcitrate. We propose first to analyze how flux through the methylcitrate cycle is controlled; specifically we would like to know how, in C. glabrata, propionate is converted to propionyl-CoA, the entry point into the methylcitrate cycle. We hypothesize that this activity is due to dual function acetyl- and propionyl-CoA synthetases encoded by the ACS1 and/or ACS2 genes. We will test this hypothesis and determine if Acs activity controls flux through the pathway and potentiates propionate toxicity. We also have designed a whole cell assay to screen for compounds that inhibit Pdh1 or Icl2. We propose to screen the NCI DTP compound collection for compounds that target either of these enzymes. We will characterize initial hits in several ways. First, we will determine in vitro if the compounds inhibit Pdh1 or Icl2 activity; second we will test positive compounds against the orthologous enzymes of C. albicans or A. fumigatus to assess activity against other important fungal pathogens. This is a high risk project, and a new area of investigation for my lab; identification of compounds that inhibit this cycle will in the future allow characterization of the methylcitrate cycle during infection in both disseminated and mucosal models of infection. Active compounds may also provide a starting point for future development of human therapeutics. PUBLIC HEALTH RELEVANCE: Candida glabrata is an important cause of infection in HIV-positive and HIV-negative individuals. We are studying the methylcitrate cycle in C. glabrata as a potential target for new drugs. We propose screening a large collection of compounds available from the NIH to identify compounds that inhibit this pathway; some of these may ultimately lead to development of new treatments of Candida and other fungal infections.