BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ENZYMES SECRETED BY LEISHMANIA

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The basic cell biology, biochemistry and molecular biology of the human pathogen, Leishmania, are investigated as a model of parasitism. Our focus centers on characterizing both the basic biochemical functions and gene structure of several unique parasite secretory enzymes with an aim toward defining their essential roles in parasite survival, growth and transmission. In that regard, L. donovani (Ld) promastigotes (Pro) constitutively release two different isoforms (110 and 130 kDa) of secretory acid phosphatase (SAcP) in vitro. We isolated and characterized the genes (SAcP-1 and SAcP-2) which encode each of these isoenzymes. Their in vitro-transcription/translation products were specifically immunoprecipitated with antibodies against the purified, native SAcPs. SAcP-1 and SAcP-2 are single copy genes which are transcribed in both Ld Pro and amastigotes. The locus of these two tandemly linked genes is a single about 1.25 Mb-sized Ld chromosome which is also present in other Leishmania indicating its structural conservation. Moreover, visceral leishmaniasis patients were shown to possess specific antibodies against the Ld SAcPs indicating its presence in human disease. In other studies, an Ld chitinase gene (CHI) was isolated and characterized. Transcripts of CHI were identified from Ld Pros and anti-CHI peptide sera reacted on Western blots with a single about 52 kDa Ld Pro protein. The locus of CHI was mapped to a single about 450kb sized chromosome in Ld and other leishmanias indicating its structural conservation across species boundaries. In collaboration with Dr. Bates, the Ld CHI probe was used to identify the homologous gene in Leishmania mexicana, an organism which produces human cutaneous disease. Finally, we identified a new about 38 kDa secreted nuclease in Ld Pros. Based on its activity and constitutive secretion, we assume that it must play an essential role in parasite acquisition of nucleo-bases via hydrolysis of host-derived nucleic acids.