Datasets of Iso-Seq transcripts for decoding transcriptome complexity in four Leishmania species.

The Iso-Seq technology, based on PacBio sequencing, enables the generation of high-quality, full-length transcripts, providing insights into transcriptome complexity. In this study, total RNA from promastigotes of four Leishmania species (Leishmania braziliensis, Leishmania donovani, Leishmania infantum and Leishmania major) was sequenced using Single Molecule, Real-Time (SMRT) Sequencing (PacBio) methodology. The Iso-seq transcripts were categorized as either complete or truncated according to the presence or absence of the Spliced-Leader (SL) sequence at their 5'-end, respectively. Moreover, only transcripts having a poly-A+ at their 3'-end were considered. Supplied datasets represent valuable information that may help to uncover novel transcripts and alternative splicing events in a parasite that regulates its gene expression at the post-transcriptional level. A better knowledge of gene expression regulation in Leishmania will open avenues for the development of new drugs to treat leishmaniasis, a devastating disease that has worldwide distribution. Additionally, the bioinformatics pipeline followed here may guide the analysis of Iso-Seq data derived from related trypanosomatids like Trypanosoma cruzi (Chagas disease agent) and Trypanosoma brucei (sleeping disease). © 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Refinement of Leishmania donovani Genome Annotations in the Light of Ribosome-Protected mRNAs Fragments (Ribo-Seq Data).

Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses (e.g., transcriptomics, proteomics, and phylogenetics). In this study, we improved the annotation of the Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their potential role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies.

Leishmania infantum (JPCM5) Transcriptome, Gene Models and Resources for an Active Curation of Gene Annotations.

Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe form of leishmaniasis. An improved assembly for the L. infantum genome was published five years ago, yet delineation of its transcriptome remained to be accomplished. In this work, the transcriptome annotation was attained by a combination of both short and long RNA-seq reads. The good agreement between the results derived from both methodologies confirmed that transcript assembly based on Illumina RNA-seq and further delimitation according to the positions of spliced leader (SAS) and poly-A (PAS) addition sites is an adequate strategy to annotate the transcriptomes of Leishmania, a procedure previously used for transcriptome annotation in other Leishmania species and related trypanosomatids. These analyses also confirmed that the Leishmania transcripts boundaries are relatively slippery, showing extensive heterogeneity at the 5'- and 3'-ends. However, the use of RNA-seq reads derived from the PacBio technology (referred to as Iso-Seq) allowed the authors to uncover some complex transcription patterns occurring at particular loci that would be unnoticed by the use of short RNA-seq reads alone. Thus, Iso-Seq analysis provided evidence that transcript processing at particular loci would be more dynamic than expected. Another noticeable finding was the observation of a case of allelic heterozygosity based on the existence of chimeric Iso-Seq reads that might be generated by an event of intrachromosomal recombination. In addition, we are providing the L. infantum gene models, including both UTRs and CDS regions, that would be helpful for undertaking whole-genome expression studies. Moreover, we have built the foundations of a communal database for the active curation of both gene/transcript models and functional annotations for genes and proteins.

Assembly of a Large Collection of Maxicircle Sequences and Their Usefulness for Leishmania Taxonomy and Strain Typing.

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named "non-JPC like", may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability.

The Astonishing Large Family of HSP40/DnaJ Proteins Existing in Leishmania.

Abrupt environmental changes are faced by Leishmania parasites during transmission from a poikilothermic insect vector to a warm-blooded host. Adaptation to harsh environmental conditions, such as nutrient deprivation, hypoxia, oxidative stress and heat shock needs to be accomplished by rapid reconfiguration of gene expression and remodeling of protein interaction networks. Chaperones play a central role in the maintenance of cellular homeostasis, and they are responsible for crucial tasks such as correct folding of nascent proteins, protein translocation across different subcellular compartments, avoiding protein aggregates and elimination of damaged proteins. Nearly one percent of the gene content in the Leishmania genome corresponds to members of the HSP40 family, a group of proteins that assist HSP70s in a variety of cellular functions. Despite their expected relevance in the parasite biology and infectivity, little is known about their functions or partnership with the different Leishmania HSP70s. Here, we summarize the structural features of the 72 HSP40 proteins encoded in the Leishmania infantum genome and their classification into four categories. A review of proteomic data, together with orthology analyses, allow us to postulate cellular locations and possible functional roles for some of them. A detailed study of the members of this family would provide valuable information and opportunities for drug discovery and improvement of current treatments against leishmaniasis.

Live attenuated vaccines, a favorable strategy to provide long-term immunity against protozoan diseases.

The control of diseases caused by protozoan parasites is one of the United Nations' Sustainable Development Goals. In recent years much research effort has gone into developing a new generation of live attenuated vaccines (LAVs) against malaria, Chagas disease and leishmaniasis. However, there is a bottleneck related to their biosafety, production, and distribution that slows downs further development. The success of irradiated or genetically attenuated sporozoites against malaria, added to the first LAV against leishmaniasis to be evaluated in clinical trials, is indicative that the drawbacks of LAVs are gradually being overcome. However, whether persistence of LAVs is a prerequisite for sustained long-term immunity remains to be clarified, and the procedures necessary for clinical evaluation of vaccine candidates need to be standardized.

Protective Efficacy in a Hamster Model of a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin) Consisting of the KMP11, LEISH-F3+, and LJL143 Antigens in Virosomes, Plus GLA-SE Adjuvant.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, fatal if untreated. Vaccination is the most cost-effective approach to disease control; however, to date, no vaccines against human VL have been made available. This work examines the efficacy of a novel vaccine consisting of the Leishmania membrane protein KMP11, LEISH-F3+ (a recombinant fusion protein, composed of epitopes of the parasite proteins nucleoside hydrolase, sterol-24-c-methyltransferase, and cysteine protease B), and the sand fly salivary protein LJL143, in two dose ratios. The inclusion of the TLR4 agonist GLA-SE as an adjuvant, and the use of virosomes (VS) as a delivery system, are also examined. In a hamster model of VL, the vaccine elicited antigen-specific immune responses prior to infection with Leishmania infantum. Of note, the responses were greater when higher doses of KMP11 and LEISH-F3+ proteins were administered along with the GLA-SE adjuvant and/or when delivered within VS. Remarkably, hamsters immunized with the complete combination (i.e., all antigens in VS + GLA-SE) showed significantly lower parasite burdens in the spleen compared to those in control animals. This protection was underpinned by a more intense, specific humoral response against the KMP11, LEISH-F3+, and LJL143 antigens in vaccinated animals, but a significantly less intense antibody response to the pool of soluble Leishmania antigens (SLA). Overall, these results indicate that this innovative vaccine formulation confers protection against L. infantum infection, supporting the advancement of the vaccine formulation into process development and manufacturing and the conduction of toxicity studies towards future phase I human clinical trials.

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

Identification of the interactomes associated with SCD6 and RBP42 proteins in Leishmania braziliensis.

Leishmania are protozoan parasites responsible for leishmaniasis. These parasites present a precise gene regulation that allows them to survive different environmental conditions during their digenetic life cycle. This adaptation depends on the regulation of the expression of a wide variety of genes, which occurs, mainly at the post-transcriptional level. This differential gene expression is achieved by mechanisms based mainly in RNA binding proteins that regulate the translation and/or stability of mRNA targets by interaction with cis elements principally located in the untranslated regions (UTR). In recent studies, our group identified and characterized two proteins, SCD6 and RBP42, as RNA binding proteins in Leishmania braziliensis. To find clues about the cellular processes in which these proteins are involved, this work was aimed to determine the SCD6- and RBP42-interacting proteins (interactome) in L. braziliensis promastigotes. For this purpose, after an in vivo UV cross-linking, cellular extracts were used to immunoprecipitated, by specific antibodies, protein complexes in which SCD6 or RBP42 were present. Protein mass spectrometry analysis of the immunoprecipitated proteins identified 96 proteins presumably associated with SCD6 and 173 proteins associated with RBP42. Notably, a significant proportion of the identified proteins were shared in both interactomes, indicating a possible functional relationship between SCD6 and RBP42. Remarkably, many of the proteins identified in the SCD6 and RBP42 interactomes are related to RNA metabolism and translation processes, and many of them have been described as components of ribonucleoprotein (RNP) granules in Leishmania and related trypanosomatids. Thus, these results support a role of SCD6 and RBP42 in the assembly and/or function of mRNA-protein complexes, participating in the fate (decay/accumulation/translation) of L. braziliensis transcripts. SIGNIFICANCE: Parasites of the Leishmania genus present a particular regulation of gene expression, operating mainly at the post-transcriptional level, surely aimed to modulate quickly both mRNA and protein levels to survive the sudden environmental changes that occur during a parasite's life cycle as it moves from one host to another. This regulation of gene expression processes would be governed by the interaction of mRNA with RNA binding proteins. Nevertheless, the entirety of protein networks involved in these regulatory processes is far from being understood. In this regard, our work is contributing to stablish protein networks in which the L. braziliensis SCD6 and RBP42 proteins are involved; these proteins, in previous works, have been described as RNA binding proteins and found to participate in gene regulation in different cells and organisms. Additionally, our data point out a possible functional relationship between SCD6 and RBP42 proteins as constituents of mRNA granules, like processing bodies or stress granules, which are essential structures in the regulation of gene expression. This knowledge could provide a new approach for the development of therapeutic targets to control Leishmania infections.

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations.

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome.

Leishmania Mitochondrial Genomes: Maxicircle Structure and Heterogeneity of Minicircles.

The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In Leishmania and other trypanosomatids, the mtDNA (also referred to as kinetoplast DNA or kDNA) is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.

Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major.

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expression.

Complete assembly of the Leishmania donovani (HU3 strain) genome and transcriptome annotation.

Leishmania donovani is a unicellular parasite that causes visceral leishmaniasis, a fatal disease in humans. In this study, a complete assembly of the genome of L. donovani is provided. Apart from being the first published genome of this strain (HU3), this constitutes the best assembly for an L. donovani genome attained to date. The use of a combination of sequencing platforms enabled to assemble, without any sequence gap, the 36 chromosomes for this species. Additionally, based on this assembly and using RNA-seq reads derived from poly-A + RNA, the transcriptome for this species, not yet available, was delineated. Alternative SL addition sites and heterogeneity in the poly-A addition sites were commonly observed for most of the genes. After a complete annotation of the transcriptome, 2,410 novel transcripts were defined. Additionally, the relative expression for all transcripts present in the promastigote stage was determined. Events of cis-splicing have been documented to occur during the maturation of the transcripts derived from genes LDHU3_07.0430 and LDHU3_29.3990. The complete genome assembly and the availability of the gene models (including annotation of untranslated regions) are important pieces to understand how differential gene expression occurs in this pathogen, and to decipher phenotypic peculiarities like tissue tropism, clinical disease, and drug susceptibility.

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome.

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs.

Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (SbIII, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.

Analysis of the Antigenic and Prophylactic Properties of the Leishmania Translation Initiation Factors eIF2 and eIF2B in Natural and Experimental Leishmaniasis.

Different members of intracellular protein families are recognized by the immune system of the vertebrate host infected by parasites of the genus Leishmania. Here, we have analyzed the antigenic and immunogenic properties of the Leishmania eIF2 and eIF2B translation initiation factors. An in silico search in Leishmania infantum sequence databases allowed the identification of the genes encoding the α, ß, and γ subunits and the α, ß, and δ subunits of the putative Leishmania orthologs of the eukaryotic initiation factors F2 (LieIF2) or F2B (LieIF2B), respectively. The antigenicity of these factors was analyzed by ELISA using recombinant versions of the different subunits. Antibodies against the different LieIF2 and LieIF2B subunits were found in the sera from human and canine visceral leishmaniasis patients, and also in the sera from hamsters experimentally infected with L. infantum. In L. infantum (BALB/c) and Leishmania major (BALB/c or C57BL/6) challenged mice, a moderate humoral response against these protein factors was detected. Remarkably, these proteins elicited an IL-10 production by splenocytes derived from infected mice independently of the Leishmania species employed for experimental challenge. When DNA vaccines based on the expression of the LieIF2 or LieIF2B subunit encoding genes were administered in mice, an antigen-specific secretion of IFN-γ and IL-10 cytokines was observed. Furthermore, a partial protection against murine CL development due to L. major infection was generated in the vaccinated mice. Also, in this work we show that the LieIF2α subunit and the LieIF2Bß and δ subunits have the capacity to stimulate IL-10 secretion by spleen cells from naïve mice. B-lymphocytes were identified as the major producers of this anti-inflammatory cytokine. Taking into account the data found in this study, it may be hypothesized that these proteins act as virulence factors implicated in the induction of humoral responses as well as in the production of the down-regulatory IL-10 cytokine, favoring a pathological outcome. Therefore, these proteins might be considered markers of disease.

Dipeptidyl peptidase 3, a novel protease from Leishmania braziliensis.

The increase of leishmaniasis cases worldwide and the emergence of Leishmania strains resistant to current treatments make necessary to find new therapeutic targets. Proteases are appealing drug targets because they play pivotal roles in facilitating parasite survival and promoting pathogenesis. Enzymes belonging to the dipeptidyl peptidase 3 (DPP3) group have been described in different organisms such as mammals, insects and yeast, in which these enzymes have been involved in both protein turnover and protection against oxidative damage. The aim of this work was to characterize the structure and function of the Leishmania braziliensis DPP3 (LbDPP3) protein as the first step to elucidate its suitability as a potential drug target. Sequence alignment showed 43% of identity between LbDPP3 and its human orthologous (hDPP3) enzyme. Although the modeled protein adopted a globally conserved three-dimensional (3D) structure, structural differences were found in the vicinity of the active site and the substrate binding-cleft. In addition, the Leishmania protein was expressed as a soluble recombinant protein and its kinetics parameters were determined using the z-Arginine-Arginine-AMC substrate. The LbDPP3 activity was maximal at pH values between 8.0-8.5. Interestingly, classical enzyme inhibitors such as the tynorphin and its derivative peptide IVYPW were found to actively inhibit the LbDPP3 activity. Moreover, these DPP3 inhibitors showed a detrimental effect upon parasite survival, decreasing the viability of promastigotes by up to 29%. Finally, it was observed that LbDPP3 was equally expressed along the in vitro differentiation from promastigotes to axenic amastigotes. In conclusion, these findings suggest that the L. brazileinsis DPP3 could be a promising drug target.

Leishmania braziliensis SCD6 and RBP42 proteins, two factors with RNA binding capacity.

BACKGROUND: The study of RNA binding proteins (RBPs) is of great relevance for understanding processes like post-transcriptional control of gene expression. The post-transcriptional mechanisms are particularly important in Leishmania parasites and related trypanosomatids since transcriptional regulation is almost absent in them. Thus, RBPs should be essential during the development of these parasites and for survival strategies against the adverse conditions that they face during their life-cycle. This work was aimed to do a structural and biochemical characterization of two Leishmania braziliensis proteins, which were previously found in pull-down assays using an HSP70 RNA as bait. At that time, these proteins were annotated as hypothetical proteins (LbrM.25.2210 and LbrM.30.3080) in the GeneDB database. RESULTS: Structural analysis indicated that these two proteins belong to evolutionarily conserved families; thus, they have been renamed accordingly as LbSCD6 (LbrM.25.2210) and LbRBP42 (LbrM.30.3080). We have demonstrated experimentally that these proteins are RBPs, in agreement with their structural features. Both proteins were able to bind to the complete 3' UTR-II region of HSP70-type II mRNA, and to an A + U rich element (ARE) present in that UTR. Cellular localization assays suggested that both proteins are mainly distributed in the cytoplasm of promastigotes growing at 26 °C, but they accumulate in foci around the nucleus when the parasites are under heat-shock conditions. Also, our study showed that steady-state levels of LbSCD6 and LbRBP42 transcripts decreased significantly during incubation of L. braziliensis promastigotes at heat-shock temperatures. However, in these conditions, the cellular content of both proteins remained unaltered. CONCLUSIONS: Our data suggest that LbSCD6 and LbRBP42, as occurs for their orthologues in other organisms, are involved in mRNA regulation, and probably they have a relevant role facing the stress conditions that L. braziliensis encounters during insect-to-mammalian transmission.