Leishmania infantum possesses a complex family of histone H2A genes: structural characterization and analysis of expression.

We have studied the genomic organization and transcription of the histone H2A genes in the protozoan parasite Leishmania infantum. In the parasite genome 2 gene clusters exist, each containing 3 H2A gene copies. Sequence analyses showed the existence of significant sequence divergence among the H2A genes, mainly in their 5'- and 3'-untranslated regions (UTRs). Also, the existence of allelic alternatives has been evidenced. Based on the divergence in the 3'UTR regions, we have defined 3 classes of H2A transcripts, which are present at different levels in L. infantum promastigotes. However, transcription of the 3 classes of H2A genes occurs at similar levels, as measured by nuclear run-on assays, indicating that their abundance is regulated post-transcriptionally. Also, differences in regulation were observed among the H2A transcripts: the levels of transcripts with 3'-UTR type I and type III are affected by growth phase whereas transcripts with 3'-UTR type II, that are barely detected, remain constant. It is likely that the complexity, in both gene organization and differential expression exhibited by the L. infantum H2A genes, is imposed by the nature of the post-transcriptional mechanisms of regulation operating in this parasite.

Characterisation of a monoclonal antibody recognising specifically the HSP70 from Leishmania.

Heat-shock protein 70 (HSP70) is ubiquitously distributed along the evolutionary scale and has such an amino acid sequence conservation that it is considered the most evolutionarily conserved protein. In order to obtain immunological tools specific against Leishmania infantum HSP70, hybridomas were established that secreted monoclonal antibodies (mAbs) against recombinant L. infantum HSP70. One of them, named mAb 2B8D2, specifically reacted with the Leishmania protein and did not recognise HSP70 from the related kinetoplastid Trypanosoma cruzi. The use of synthetic peptides allowed us to determine the B-cell epitope recognised by this mAb, an epitope located in the divergent C-terminal domain of the protein. Remarkably, the mAb possesses the capacity to immunoprecipate HSP70 from promastigote extracts of L. infantum. The fact that human HSP70 is not recognised by this mAb assures the usefulness of this antibody for diagnostic purposes and studies involving Leishmania infection of macrophages.

Leishmania infantum: gene cloning of the GRP94 homologue, its expression as recombinant protein, and analysis of antigenicity.

The complete nucleotide sequence for the Leishmania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 sequence possesses both an N-terminal signal peptide and a putative endoplasmic reticulum retention signal, consisting of the C-terminal tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism in which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum GRP94 gene was expressed in Escherichia coli and the recombinant protein used to evaluate its antigenicity and immunogenicity. Eighty-four percent of sera from dogs with visceral leishmaniasis reacted with the protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by the L. infantum GRP94, we think that this protein constitutes a valuable molecule for diagnostic purposes and a potential candidate for studies of protective immunogenicity.