Leishmania donovani, the causitive agent in Kala-azar, is deficient in most acid hydrolases, but contains intense acid phosphatase activity that is localized to the surface of promastigotes grown in culture. During the previous grant period we purified this tartrate-resistant phosphatase to homogeneity, starting from a sodium cholate extract of membranes, and characterized some of its physical-chemical and kinetic properties. The enzyme exhibits protein phosphatase activity at pH 7.0 and dephosphorylates 32p-labeled macrophage membrane proteins. The phosphatase also reduces superoxide anion production by phagocytic cells and inhibits their ability to destroy bacteria. Our central hypothesis is that the cell surface acid phosphatase disarms defense mechanisms of host cells by dephosphorylating key regulatory phosphoproteins of these phagocytic cells. During the next grant period, we will continue to define the physical-chemical and kinetic properties of the tartrate-resistant acid phosphatase. We will define its composition and partially characterize its carbohydrate domains. The inhibitory effects of the polyheterometallate complexes will be investigated with the ultimate goal being to use these compounds to investigate the pathophysiological role of the leishmanial phosphatase. In terms of substrate specificity, emphasis will be placed on identifying cell surface phosphoproteins that are substrates for the enzyme. The other areas of the work plan are directed at characterizing the effects of the tartrate-resistant phosphatase on a variety of specific phagocytic cell functions; we will investigate the effect of the enzyme on: phagocytosis, the plasma membrane potential, the secretion of granule enzymes and proteins, the production of oxygen-dependent cidal compounds (e.g., O-2, H2O2, OC1-, C12) and arachidonic acid metabolites, NADPH oxidase activity, lysosomal pH and lysosomal hydrolase function. Finally, we will use antibodies against the phosphatase, as well as the phosphatase itself, to directly assess the importance of the enzyme in the killing of promastigotes by macrophages and neutrophils. The results of this study could provide insight into improved diagnostic and therapeutic strategies for leishmaniasis.
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