Developmental Gene Expression in Leishmania donovani: Differential Cloning and Analysis of an Amastigote-Stage- Specific Gene

Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. During their life cycle, Leishmania protozoans exist as flagellated promastigotes in the sand fly vector and as nonmotile amastigotes in the mammalian hosts. The promastigote-to-amastigote transformation occurs in the phagolysosomal compartment of the macrophage cell and is a critical step for the establishment of the infection. To study this cytodifferentiation process, we differentially screened an amastigote cDNA library with life cycle stage-specific cDNA probes and isolated seven cDNAs representing amastigote-specific transcripts. Five of these were closely related (A2 series) and recognized, by Northern (RNA) blot analyses, a 3.5-kb transcript in amastigotes and in amastigote-infected macrophages. Expression of the amastigote-specific A2 gene was induced in promastigotes when they were transferred from culture medium at 26 degrees C and pH 7.4 to medium at 37 degrees C and pH 4.5, conditions which mimic the macrophage phagolysosomal environment. A2 genes are clustered in tandem arrays, and a 6-kb fragment corresponding to a unit of the cluster was cloned and partially sequenced. An open reading frame found within the A2-transcribed region potentially encoded a 22-kDa protein containing repetitive sequences. The recombinant A2 protein produced in Escherichia coli cells was specifically recognized by immune serum from a patient with visceral leishmaniasis. The A2 protein repetitive element has strong homology with an S antigen of Plasmodium falciparum, the protozoan parasite responsible for malaria. Both the A2 protein of Leishmania donovani and the S antigen of P. falciparum are stage specific and developmentally expressed in mammalian hosts.