The cell biology of Leishmania is investigated as a model of both intra- and extracellular parasitism. Emphasis is placed on characterizing the biochemical and physiological functions of its surface membrane (SM) and secreted components toward defining their roles in parasite survival and development. Leishmania promastigotes possess a number of SM transport systems for the active accumulation of certain essential amino acids and nucleosides. In order to identify these, a series of unique, radiolabeled, substrate specific, affinity-binding reagents were devised, synthesized and used to covalently label the SM of living L. donovani promastigotes in situ. Such labeling was specifically and competitively inhibited (>90+%) in the presence of excess unlabeled substrate. Cumulatively, two different, externally oriented, SM transporters are identified in these studies. The first, specific for methionine was composed of 2 polypeptides with apparent molecular weights of about 54 and 56 kDa and the second was specific for adenosine transport and consisted of a single polypeptide chain of about 33 kDa. Studies are in progress to further characterize these essential proteins. Additional chemical properties of the SM acid phosphatase were delineated. The secretory acid phosphatase (SACP) was shown to possess the unique [PO4-6Gal(beta),4Man], repetitive epitope in common with the SM lipophophoglycan (LPG). Both SAcP and LPG were terminally co-processed in and transported through the Golgi compartment. Restriction and genetic analyses of wild type and LPG- mutants are being used to identify genes involved in LPG expression. A 420 bp DNA coding fragment for SAcP was cloned and used to identify and isolate a full length copy of this gene. Methods are devised for the continuous in vitro cultivation of "amastigote-like" forms of L. donovani at 37 degrees C. The availability of such forms should facilitate studies of parasite gene regulated differentiation and development. The current results are of relevance toward the development of improved diagnostic, chemotherapeutic and immunoprophylactic agents.
NIH ExPorter
NIH ExPorter