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.
Charest
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Institute of Parasitology of McGill University, Ste-Anne de Bellevue, Québec, Canada.
Matlashewski
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D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis / Q000096:biosynthesis / Q000097:blood; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D015698:Genomic Library; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010963:Plasmodium falciparum / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D016053:RNA, Protozoan