Leishmania is a dimorphic parasitic protozoan which exists as a flagellated promastigote in the sandfly vector and as an intracellular amastigote in the phagolysosomal compartment of mammalian host macrophages. It is the amastigote form that is responsible for the pathology in susceptible vertebrate hosts. Leishmania donovani is responsible for visceral leishmaniasis, the most severe form of the leishmanial diseases. We have investigated the antibody response against an amastigote-specific protein, A2, which is developmentally expressed in L. donovani during promastigote-to-amastigote cytodifferentiation. A2 is conserved in L. donovani and L. mexicana species but not in other Leishmania species tested. We have shown that this characteristic contributes to its potential as a useful specific diagnostic antigen for visceral leishmaniasis. Developmental expression of A2 involves A2 mRNA untranslated regions (UTRs) and we have demonstrated that A2 UTRs can regulate expression of exogenous suicide genes throughout the Leishmania life cycle. We have shown that the A2 gene regulatory system has potential for the generation of developmentally attenuated L. donovani strains. Finally, we have performed a preliminary characterization of a gene, A2rel, that is tandemly associated with A2 genes in the genome. Contrary to A2 genes, the A2rel gene is well conserved in the Leishmania species. Although A2rel does not share sequence similarity with any known leishmanial genes characterized to date, it does appear to share characteristics with membrane-bound glycoproteins.

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Leishmania protozoans are the etiologic agents of the disease leishmaniasis. The parasite exists in two morphological forms: promastigote and amastigote. Promastigotes are found in the gut of the sandfly vector while amastigotes reside inside the vertebrate macrophage. Leishmania, an obligate intracellular parasite, resists toxic reactive oxygen species (ROS) from both endogenous and exogenous sources. Like other protozoa, Leishmania lacks some of the antioxidant defence enzymes such as catalase and glutathione peroxidase (Gpx) that are usually found in aerobic cells. Instead they possess the antioxidant thiol compound trypanothione, in association with specific trypanothione linked antioxidant enzymes such as peroxidoxins. The transformation from promastigote to amastigote is a crucial step for parasite infection and survival. The molecular basis for this transformation is not clearly understood. Recently it was shown that the peroxidoxin gene is present in multiple copies in Leishmania. In the present study we examined the potential of antisense RNA and double stranded RNA (dsRNA) to perform functional knockout of the peroxidoxin gene. Towards that end antisense RNA and dsRNA expressing plasmids, targeting the peroxidoxin gene, were constructed. Leishmania promastigotes were subsequently transfected with these plasmids and the levels of peroxidoxin gene expression were studied. The results from this study suggest that there is no apparent reduction in either the levels of peroxidoxin mRNA or the protein in the transfected promastigotes as compared to the non-transfected cells.

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In Leishmania glycosomes compartmentalize a variety of essential biochemical and metabolic pathways required for parasite viability. Biogenesis and maintenance of glycosomes involves a family of proteins called peroxins, however the molecular mechanisms governing the early events of this pathway have not been fully established. / A structural-functional analysis of the receptor LdPEX5 revealed the formation of a tetrameric structure stabilized by coiled-coil motifs. A biophysical approach showed that the tetrameric structure of LdPEX5 dissociates to dimers upon binding to the PTS1 ligand. However, the tetrameric LdPEX5 is more thermodynamically favorable to bind. Lastly, LdPEX14 modulates the LdPEX5-PTS1 interaction as the presence of LdPEX14 decreases the Kd of LdPEX5-PTS1 by ∼10 folds. / PTS1-loaded LdPEX5 docks onto the glycosomal membrane via the membrane-associated LdPEX14, an interaction that was characterized by molecular mapping and biophysical approaches. In higher eukaryotes this PEX5-PEX14 interaction involves conserved WXXXF/Y pentapeptide motifs found on PEX5 and a signature sequence found on PEX14. These three repeats in LdPEX5 do not appear to be crucial for interaction with LdPEX14 thus suggesting a unique molecular mechanism mediating the docking event. On the other hand, the conserved signature motif is crucial for interaction with LdPEX5. / The topology and nature of the interaction of LdPEX14 with the glycosomal membrane is not clear. In vivo expression of FLAG-LdPEX14-HA together with proteinase digestion confirmed that both N- and C-termini are cytosolic facing. Mapping analysis revealed that the first 63 N-terminal residues of LdPEX14 are critical for anchoring LdPEX14 to the glycosomal membrane. Interestingly, expression of the first 74 amino acids of LdPEX14 is toxic to the parasites. / Finally, the structure of LdPEX14 on the glycosomal membrane was addressed by molecular mapping and biophysical techniques. Partial trypsin digestion of recombinant LdPEX14 and molecular mapping suggested that the first 250 residues of LdPEX14 were involved in the formation of the complex. Biophysical approaches and cross-linking assays suggest that this complex may consist of ∼12-100 LdPEX14 subunits. Interestingly, the structure of LdPEX14 appears to be modulated by LdPEX5. / Considering all the results, these findings have established important molecular information concerning early events in the targeting and import of PTS1 proteins into the glycosome.

Dimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms.

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