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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI018945-04
Application #
3128330
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1982-05-01
Project End
1990-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Saha, A K; Crans, D C; Pope, M T et al. (1991) Inhibition of human seminal fluid and Leishmania donovani phosphatases by molybdate heteropolyanions. J Biol Chem 266:3511-7
Saha, A K; Mukhopadhyay, N K; Dowling, J N et al. (1990) Characterization of a phosphomonoesterase from Brucella abortus. Infect Immun 58:1153-8
Butcher, B A; Shome, K; Estes, L W et al. (1990) Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization. Exp Parasitol 71:49-59
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Mukhopadhyay, N K; Shome, K; Saha, A K et al. (1989) Heparin binds to Leishmania donovani promastigotes and inhibits protein phosphorylation. Biochem J 264:517-25
Saha, A K; Dowling, J N; Mukhopadhyay, N K et al. (1989) Legionella micdadei protein kinase catalyzes phosphorylation of tubulin and phosphatidylinositol. J Bacteriol 171:5103-10
Mukhopadhyay, N K; Saha, A K; Smith, W et al. (1989) Inhibition of neutrophil and natural killer cell function by human seminal fluid acid phosphatase. Clin Chim Acta 182:31-40
Glew, R H; Saha, A K; Das, S et al. (1988) Biochemistry of the Leishmania species. Microbiol Rev 52:412-32
Mukhopadhyay, N K; Saha, A K; Lovelace, J K et al. (1988) Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania. J Protozool 35:601-7

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