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 expressionThis work was supported by grants (to B.A. and J.M.R.) from Ministerio de Economía, Industria y Competitividad, project number SAF2017-86965-R (co-funded with FEDER funds), and by the Network of Tropical Diseases Research RICET (RD16/0027/0008), co-funded with FEDER funds. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santande

The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis

<p>Abstract</p> <p>Background</p> <p>RNA-binding proteins of the PUF family share a conserved domain consisting of tandemly repeated 36-40 amino acid motifs (typically eight) known as Puf repeats. Proteins containing tandem repeats are often dominant targets of humoral responses during infectious diseases. Thus, we considered of interest to analyze whether <it>Leishmania </it>PUF proteins result antigenic during visceral leishmaniasis (VL).</p> <p>Findings</p> <p>Here, employing whole-genome databases, we report the composition, and structural features, of the PUF family in <it>Leishmania infantum</it>. Additionally, the 10 genes of the <it>L. infantum </it>PUF family were cloned and used to express the <it>Leishmania </it>PUFs in bacteria as recombinant proteins. Finally, the antigenicity of these PUF proteins was evaluated by determining levels of specific antibodies in sera from experimentally infected hamsters. The <it>Leishmania </it>PUFs were all recognized by the sera, even though with different degree of reactivity and/or frequency of recognition. The reactivity of hamster sera against recombinant LiPUF1 and LiPUF2 was particularly prominent, and these proteins were subsequently assayed against sera from human patients. High antibody responses against rLiPUF1 and rLiPUF2 were found in sera from VL patients, but these proteins resulted also recognized by sera from Chagas' disease patients.</p> <p>Conclusion</p> <p>Our results suggest that <it>Leishmania </it>PUFs are targets of the humoral response during <it>L. infantum </it>infection and may represent candidates for serodiagnosis and/or vaccine reagents; however, it should be kept in mind the cross-reactivity of LiPUFs with antibodies induced against other trypanosomatids such as <it>Trypanosoma cruzi</it>.</p

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.This research was supported by the Spanish Ministerio de Ciencia, Innovación (MICINN), Agencia Estatal de Investigación (AEI), grant number PID2020-117916RB-I00, and Instituto de Salud Carlos III, grant CB21/13/00018 (CIBERINFEC). An institutional grant from Fundacion Ramon Areces is also acknowledged.S

Effects of the disruption of the HSP70-II gene on the growth, morphology, and virulence of Leishmania infantum promastigotes

The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Deltahsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Deltahsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Deltahsp70-II mutant was determined by flow cytometry. Furthermore, Deltahsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Deltahsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship

<p>Abstract</p> <p>Background</p> <p>Protozoan parasites of the genus <it>Leishmania </it>are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three <it>Leishmania </it>species has generated invaluable information enabling a direct analysis of genome structure and organization.</p> <p>Results</p> <p>By using DNA macroarrays, made with <it>Leishmania infantum </it>genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of <it>L. infantum</it>, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different <it>L. infantum </it>chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the <it>Leishmania major </it>genome; thus, we adopted this nomenclature for the <it>Leishmania </it>elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in <it>L. major </it>and <it>Leishmania braziliensis </it>chromosomes 32. While SIDER2 elements are highly conserved both in number and location between <it>L. infantum </it>and <it>L. major</it>, no such conservation exists when comparing with SIDER2s in <it>L. braziliensis </it>chromosome 32.</p> <p>Conclusion</p> <p>SIDER2 elements constitute a relevant piece in the <it>Leishmania </it>genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in <it>Leishmania</it>. Apart from a proved involvement in post-trancriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.</p

Immunity to Visceral Leishmaniasis

Leishmaniasis is a major vector-borne parasitic disease affecting 12 million people worldwide. With a broad range of clinical manifestations, ranging from self-healing skin ulcers to disfiguring mucosal lesions to life-threatening infections of visceral organs (liver and spleen), the disease has become a serious human health issue, particularly in developing countries. Among all of its forms, visceral leishmaniasis (VL, also known as kala-azar), caused by the Leishmania donovani complex (i.e., L. donovani and L. infantum in Old World and L. chagasi in New World), is often fatal in the absence of treatment. Although humans are the principal hosts for L. donovani, canine species are the main reservoirs of L. infantum. Canine VL affects millions of dogs and is associated with outbreaks of human VL and hence has become a major public health issue. The lack of vaccines to prevent and/or treat these infections, as well as the emergence of drug resistant parasites, is serious impediments to control leishmaniasis. Therefore, developing new prophylactic and therapeutic strategies against this disease is urgently required. However, for this to occur, a better understanding of the complex immune mechanisms generated in response to infection and defining those involved in resistance to infection is required. In this special issue on “Immunity to visceral leishmaniasis”, several selected papers will discuss these issues.Peer reviewe

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

<p>Abstract</p> <p>Background</p> <p>Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy.</p> <p>Results</p> <p>In this study, we tested the ability of a <it>Leishmania infantum </it>deletion mutant, lacking both <it>HSP70-II </it>alleles (ΔHSP70-II), to provide protection against <it>Leishmania </it>infection in the <it>L. major</it>-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-<it>Leishmania </it>IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (<it>Mesocricetus auratus</it>), infected with mutant parasites did not develop any sign of pathology.</p> <p>Conclusions</p> <p>The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.</p

The Leishmania HSP20 Is Antigenic during Natural Infections, but, as DNA Vaccine, It does not Protect BALB/c Mice against Experimental L. amazonensis Infection

Protozoa of the genus Leishmania are causative agents of leishmaniasis, an important health problem in both human and veterinary medicine. Here, we describe a new heat shock protein (HSP) in Leishmania, belonging to the small HSP (sHSP) family in kinetoplastids. The protein is highly conserved in different Leishmania species, showing instead significant divergence with sHSP's from other organisms. The humoral response elicited against this protein during Leishmania infection has been investigated in natural infected humans and dogs, and in experimentally infected hamsters. Leishmania HSP20 is a prominent antigen for canine hosts; on the contrary, the protein seems to be a poor antigen for human immune system. Time-course analysis of appearance of anti-HSP20 antibodies in golden hamsters indicated that these antibodies are produced at late stages of the infection, when clinical symptoms of disease are patent. Finally, the protective efficacy of HSP20 was assessed in mice using a DNA vaccine approach prior to challenge with Leishmania amazonensis

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 kinetoplastidsThis work was supported by grants (to B.A. and J.M.R.) from Proyecto del Ministerio de Economía, Industria y Competitividad SAF2017-86965-R, and by the Network of Tropical Diseases Research RICET (RD16/0027/0008); both grants are co-funded with FEDER funds. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santande