The cellular transformation of the Leishmania protozoan parasite from the promastigote to the amastigote stages takes place in the phagolysosomal compartment of vertebrate host macrophage cells. This cytodifferentiation is a prerequisite for parasite survival and for the establishment of the infection in the mammalian host. The differential screening of a L. donovani amastigote cDNA library using stage-specific cDNA probes allowed the identification of the A2 genes, which are only expressed by the parasite as an amastigote. A2 specific transcripts are developmentally expressed in Leishmania in response to a combination of pH and temperature shifts, conditions associated with the transfer from the insect vector to the phagolysosomal compartment in macrophages. The coding genes are clustered on a 850 kb chromosome and arranged in tandem arrays with copies of another gene, the A2rel gene. The developmental expression is mediated post-transcriptionally and involves elements located in the 3$ prime$ untranslated region; accurate processing by trans splicing is essential for the A2 mRNAs developmental accumulation in cells transferred into phagolysosomal conditions. The A2 protein product is composed mostly of highly repeated domains, carries a functional signal peptide at its amino-terminal end, and shares sequence homology with other developmentally expressed proteins in unrelated parasites of humans. Using A2 locus sequences, vectors and transfection systems were designed and developed to differentially express reporter or selectable markets specifically in the amastigote stage.

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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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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.

No description available.