CALCIUM AND PH HOMEOSTASIS IN PNEUMOCYSTIS CARINII

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Since the discovery of AIDS in 1981, the incidence of P. carinii pneumonia has increased dramatically. The problems of treatment of P. carinii infection in AIDS (e.g. the high rates of relapse and adverse drug reactions) have emphasized the need to develop new forms of therapy. Our research interest involves a rational approach to chemotherapeutic control of infectious agents aimed at finding new targets for drug action. We have identified that, in contrast to mammalian cells, an ATP-dependent H+-extrusion mechanism is the main mechanism that P. carinii trophozoites use for the maintenance of their intracellular pH (pHi) homeostasis. Our hypothesis is that disruption of the homeostatic mechanisms controlled by the H+-ATPase (pH homeostasis, nutrient uptake, Na+ and ca2+ extrusion) would interrupt P. carinii life cycle. Therefore, their identification could be useful for the development of new chemotherapeutic or chemoprophylactic agents against these pathogens. The specific aims of this proposal are: (1) To characterize the ATP-dependent H+-extrusion mechanism of P. carinii. These studies will involve the investigation of the Cl- requirement for H+ extrusion; the investigation of the electrogenic nature of the H+- ATPase; the study of the effect of glucose on the plasma membrane proton pump; the study of the role of the proton pump in nutrient transport; the confirmation that the product of the ATPase gene that has been cloned and sequenced from P. carinii corresponds to the plasma membrane H+-ATPase activity that we have identified; the solubilization and purification of the enzyme; and the investigation of the effect of drugs that affect the P. carinii H+-ATPase on their viability; and (2) To investigate the relation between PHI and ca2+ homeostasis in P. carinii. We will investigate the role of the P. carinii H+-ATPase in the control of Ca2+ homeostasis in these microorganisms, if changes in pHi can serve to trigger calcium signals, and if changes in pHi and in intracellular ca2+ concentration could be involved in the signaling mechanisms that mediate the attachment of P. carinii trophozoites to the host cells.