The Leishmania spp. protozoa have an abundant surface metalloprotease called MSP (major surface protease), which in Leishmania chagasi is encoded by three distinct gene classes (MSPS, MSPL, MSPC). Although MSP has been characterized primarily in extracellular promastigotes, it also facilitates survival of intracellular amastigotes. Promastigotes express MSPS, MSPL, and two forms of MSPC RNAs, whereas amastigotes express only MSPL RNA and one MSPC transcript. We confirmed the presence of MSPC protein in both promastigotes and amastigotes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). More than 10 MSP isoforms were visualized in both amastigotes and promastigotes using two-dimensional immunoblots, but amastigote MSPs migrated at a more acidic pI. Promastigote MSPs were N-glycosylated, whereas most amastigote MSPs were not. Immuno-electron microscopy showed that two-thirds of the promastigote MSP is distributed along the cell surface. In contrast, most amastigote MSP localized at the flagellar pocket, the major site of leishmania endocytosis/exocytosis. Biochemical analyses indicated that most amastigote MSP is soluble in the cytosol, vesicles or organelles, whereas most promastigote MSP is membrane-associated and GPI anchored. Activity gels and immunoblots confirmed the presence of a novel proteolytically active amastigote MSP of higher Mr than the promastigote MSPs. Furthermore, promastigote MSP is shed extracellularly whereas MSP is not shed from axenic amastigotes. We conclude that amastigotes and promastigotes both express multiple MSP isoforms, but these MSPs differ biochemically and localize differently in the two parasite stages. We hypothesize that MSP plays different roles in the extracellular versus intracellular forms of Leishmania spp.

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Leishmania chagasi is the causative agent of visceral leishmaniasis in South America. The most abundant glycoprotein on the surface of L. chagasi promastigotes is the glycosylphosphatidylinositol (GPI) anchored protease MSP (major surface protease), also called GP63. MSP is encoded by more than 18 tandem MSP genes on a single chromosome. MSP genes are classified according to unique sequences at their 3' ends and distinct expression patterns. The five MSPS genes (MSPS1, MSPS2, etc.) express 3.0 kb RNAs in stationary phase of promastigote growth in vitro in culture. The > twelve MSPL genes express 2.7 kb RNAs in logarithmic phase of promastigote growth, and the single MSPC RNA is constitutively expressed as two RNA species (2.6 and 3.1 kb) throughout promastigote growth. The progression from logarithmic to stationary phase is accompanied by an increase in parasite virulence for a mammalian host, and a 16-fold increase in the total MSP protein associated with the cell. As such, MSP has been called a virulence factor of leishmania. Little is known about the differences between isoforms of MSP proteins encoded by the three MSP gene classes, because they have a very similar amino acid sequences. The purpose of this thesis was to study the protein expression and localization of MSPS, MSPL, and MSPC in the promastigote and amastigote stages of the L. chagasi. We took three approaches to this problem. First, we produced constructs in which the fluorescent marker GFP was flanked by putative targeting sequences of the MSPs. Second, we generated Leishmania transfectants expressing Myc-tagged fulllength MSPs and studied their localization in promastigote cells. Third, we generated antibodies to immunogenic peptides in the few regions with unique sequences that allowed us to distinguish between some of the MSP classes. One monoclonal antipeptide antibody, named C51, recognized only MSPS1 and MSPL1. Data indicated that the product of the MSPC gene runs at a higher molecular size than products of the MSPL v ABSTRACT Leishmania chagasi is the causative agent of visceral leishmaniasis in South America. The most abundant glycoprotein on the surface of L. chagasi promastigotes is the glycosylphosphatidylinositol (GPI) anchored protease MSP (major surface protease), also called GP63. MSP is encoded by more than 18 tandem MSP genes on a single chromosome. MSP genes are classified according to unique sequences at their 3' ends and distinct expression patterns. The five MSPS genes (MSPS1, MSPS2, etc.) express 3.0 kb RNAs in stationary phase of promastigote growth in vitro in culture. The > twelve MSPL genes express 2.7 kb RNAs in logarithmic phase of promastigote growth, and the single MSPC RNA is constitutively expressed as two RNA species (2.6 and 3.1 kb) throughout promastigote growth. The progression from logarithmic to stationary phase is accompanied by an increase in parasite virulence for a mammalian host, and a 16-fold increase in the total MSP protein associated with the cell. As such, MSP has been called a virulence factor of ...