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Abstract Background The heat stress suffered by Leishmania sp during its digenetic life-cycle is a key trigger for its stage differentiation. In Leishmania subgenera two classes of HSP70 genes differing in their 3' UTR were described. Although the presence of HSP70-I genes was previously suggested in Leishmania (Viannia) braziliensis, HSP70-II genes had been reluctant to be uncovered. Results Here, we report the existence of two types of HSP70 genes in L. braziliensis and the genomic organization of the HSP70 locus. RT-PCR experiments were used to map the untranslated regions (UTR) of both types of genes. The 3' UTR-II has a low sequence identity (55-57%) when compared with this region in other Leishmania species. In contrast, the 5' UTR, common to both types of genes, and the 3' UTR-I were found to be highly conserved among all Leishmania species (77-81%). Southern blot assays suggested that L. braziliensis HSP70 gene cluster may contain around 6 tandemly-repeated HSP70-I genes followed by one HSP70-II gene, located at chromosome 28. Northern blot analysis indicated that levels of both types of mRNAs are not affected by heat shock. Conclusions This study has led to establishing the composition and structure of the HSP70 locus of L. braziliensis, complementing the information available in the GeneDB genome database for this species. L. braziliensis HSP70 gene regulation does not seem to operate by mRNA stabilization as occurs in other Leishmania species.

Trypanosoma cruzi is a parasite characterized by a high genetic diversity and a complex biological cycle that alternates between two different hosts in which it is subjected to multiple cellular stresses such as thermal or nutritional ones. Therefore, it requires a suitable and fine gene regulation control. In trypanosomatids, gene regulation occurs mainly at the post-transcriptional level. The expression of mRNAs is attained via regulation of their processing, transport, stability, and translation, mediated by the recognition of cis elements by RNAbinding proteins (RBPs). Alba-domain proteins are a conserved DNA/RNA-binding proteins family found in Archaea and Eukarya domains. This superfamily of proteins has been split into three major branches that include Archaea proteins and two eukaryotic-specific branches: the Rpp25/Mdp2 group and the Rpp20/Pop7 group. In Leishmania infantum and Trypanosoma brucei the Alba-proteins of Rpp25/Mdp2 group are involved in translation repression and in the regulation of gene expression during protozoan development. In T. cruzi, there is evidence that TcAlba30 protein may aggregate into cytoplasmic foci in parasites submitted to nutritional stress. This protein interacts with β-amastin390 mRNA and its overexpression resulted in the decrease of amastin mRNA levels, suggesting that this protein regulates the β-amastin390 expression. However, the role of other proteins of Rpp25/Mdp2 group and the proteins of Rpp20/Pop7 group identified in the genome of T. cruzi are still poorly understood. Therefore, the aim of the present work was to characterize the Alba protein family of T. cruzi and its RNA-binding capacity as an approach to uncover its functionality. In silico analyzes revealed that the genome of T. cruzi strains Brazil and Dm28c (TcI), Y (TcII), CL Brener Esmeraldo-like and TCC (TcVI) has four Alba genes, two for Rpp20/Pop7 group (TcAlba1 and TcAlba2) and two for Rpp25/Mdp2 group (TcAlba30 and TcAlba40). These genes code for highly conserved proteins in T. cruzi, but also among other Trypanosomatidae family members. The four proteins have physicochemical and structural characteristics compatible with RBPs. Particularly, the TcAlba30 and TcAlba40 proteins bind in silico to two RNA motifs: the Musashi binding element (MBE) and the Sxl binding site (SBS). In vitro interaction RNA-Protein assays using a representative of each Alba-family group (TcAlba2 and TcAlba40) showed that TcAlba40 protein, but not the TcAlba2, directly interacted with the 5' UTR region of the kLYT1 transcript, where a MBE element is found. Likewise, transcription and translation expression of each protein were analyzed in the three T. cruzi stages, and the results showed that TcAlba2 and TcAlba40 mRNA are expressed in all stages. Nevertheless, expression of both genes is higher in the trypomastigote form compared to the epimastigote and amastigote forms, being approximately 2-fold for TcAlba2 and 3-fold for TcAlba40. Also, TcAlba proteins family were detected in all three stages, suggesting a continuous expression throughout T. cruzi 's life cycle. TcAlba1/2 were constitutively expressed, whereas TcAlba30/40 were mainly expressed in the parasite's replicative stages. Our results suggesting that TcAlba30/40 proteins from Rpp25/Mdp2 group are RBPs and them function may be related to biological processes associated with stages of development of T. cruzi. [ABSTRACT FROM AUTHOR]

Background: Alpha tubulin is a fundamental component of the cytoskeleton which is responsible for cell shape and is involved in cell division, ciliary and flagellar motility and intracellular transport. Alpha tubulin gene expression varies according to the morphological changes suffered by Leishmania in its life cycle. However, the objective of studying the mechanisms responsible for the differential expression has resulted to be a difficult task due to the complex genome organization of tubulin genes and to the non-conventional mechanisms of gene regulation operating in Leishmania. Results: We started this work by analyzing the genomic organization of α-tubulin genes in the Leishmania braziliensis genome database. The genomic organization of L. braziliensis α-tubulin genes differs from that existing in the L. major and L. infantum genomes. Two loci containing α-tubulin genes were found in the chromosomes 13 and 29, even though the existence of sequence gaps does not allow knowing the exact number of genes at each locus. Southern blot assays showed that α-tubulin locus at chromosome 13 contains at least 8 gene copies, which are tandemly organized with a 2.08-kb repetition unit; the locus at chromosome 29 seems to contain a sole α-tubulin gene. In addition, it was found that L. braziliensis α-tubulin locus at chromosome 13 contains two types of α-tubulin genes differing in their 3' UTR, each one presumably containing different regulatory motifs. It was also determined that the mRNA expression levels of these genes are controlled by post-transcriptional mechanisms tightly linked to the growth temperature. Moreover, the decrease in the α-tubulin mRNA abundance observed when promastigotes were cultured at 35°C was accompanied by parasite morphology alterations, similar to that occurring during the promastigote to amastigote differentiation. Conclusions: Information found in the genome databases indicates that α-tubulin genes have been reorganized in a drastic manner along Leishmania speciation. In the L. braziliensis genome database, two loci containing α-tubulin sequences were found, but only the locus at chromosome 13 contains the prototypic α-tubulin genes, which are repeated in a head-to-tail manner. Also, we determined that the levels of α-tubulin mRNAs are down-regulated drastically in response to heat shock by a post-transcriptional mechanism which is dependent upon active protein synthesis. [ABSTRACT FROM AUTHOR]

A genomic library of Leishmania amazonensis in expression vector of eukaryote cells (pEF1HisA, pEF1HisB, pEF1HisC) was prepared. Also two subgenomic libraries having each 500 clones approximately were created and BALB/c mice were immunized with 50 mg/0,1 mL of DNA from each. Two immunizations were administered intramuscularly at 15-day interval. Groups of control mice were immunized with DNA from empty plasmid pEF1His, with soluble parasite antigen (100 mg/0,1 mL) and saline solution. The size of lesions was measured for 12 weeks and at the end of the experiment, the parasite load at lesion sites was determined by plaque microtitration method. In mice immunized with subgenomic library DNAI and with soluble antigens,the size of lesions was controlled, which reached an statistical difference (p< 0,05) in relation to the rest of groups whose lesions increased. The parasite load found in lesion sites confirmed the previous results; the number of promastigots was significantly lower in those mice already protected. It was concluded that in subgenomic library DNA1 there should be genes or gene fragments whose in vivo expression induces protective immune response against the challenge in the murine model used.