Identification of the HSP70-II gene in Leishmania braziliensis HSP70 locus: genomic organization and UTRs characterization

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

Sequence analysis of the 3-untranslated region of HSP70 (type I) genes in the genus Leishmania: Its usefulness as a molecular marker for species identification

Background: The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. Methods: In the present study, we analyzed the 3-untranslated region (UTR) of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. Results: It was observed that there was a remarkable degree of sequence conservation in this region, evenbetween species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex) typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. Conclusions: Sequence and hylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination. © 2012 Requena et al.; licensee BioMed Central Ltd.Ministerio de Ciencia y Tecnología (BFU2009-08986); Fondo de Investigaciones Sanitarias (ISCIII-RETIC RD06/0021/0008-FEDER and ISCIII-RETIC RD06/0021/0009-FEDER); Agencia Española de Cooperación Internacional para el Desarrollo (AECID, A/024740/09); Fundación Ramón ArecesPeer Reviewe

A dataset of proteins associated with Trypanosoma cruzi LYT1 mRNAs

Post-transcriptional gene regulation in Trypanosoma cruzi, the etiological agent of Chagas disease, plays a critical role in ensuring that the parasite successfully completes its life cycle in both of its obligate hosts: insect vector and mammals. This regulation is basically governed by RNA binding proteins (RBPs) through their interactions with cis-elements located in the UTRs of their mRNA targets. LYT1 gene, coding for a virulence factor of T. cruzi, is expressed into two isoforms: kLYT1 and mLYT1, which play different functions according to their cellular location and parasite life-cycle stages. Whereas kLYT1 exhibits a regulatory role during the epimastigote-to-metacyclic trypomastigote stage transition, mLYT1 acts as a pore-forming protein, relevant for host cell invasion and parasite intracellular survival. Considering the LYT1 biological relevance and the fact that this is a protein exclusive of T. cruzi, the protein and its mechanisms regulating the alternative gene expression products are promising targets for therapeutic intervention. In this work, an experimental approach consisting of pull-downs assays followed by proteomic analyzes was carried out to identify the proteins interacting with the different LYT1 mRNAs. The dataset presented here was obtained through three biological replicates using all the different UTRs characterized in the LYT1 mRNAs (i.e., 5´UTR kLYT1, 5´UTR mLYT1, and I and II-type 3´UTRs) as baits, and protein extracts from epimastigotes and trypomastigotes of the 058 PUJ (DTU I) strain. Bound proteins were analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). As a control of non-specificity, the same protein extracts were incubated with Leishmania braziliensis rRNA and the bound proteins also identified by LC/MS. In all, 1,557 proteins were identified, 313 of them were found in at least two replicates and 18 proteins were exclusively associated with the LYT1 baits. Of these, six proteins have motifs related to RNA binding, and seven remain annotated as hypothetical proteins. Remarkably, three of these hypothetical proteins also contain nucleic acid binding motifs. This knowledge, beside expanding the known T. cruzi proteome, gains insight into putative regulatory proteins responsible for alternative LYT1 mRNAs processing. Raw mass spectrometry data are available via MassIVE proteome Xchange with identifier PXD027371

SARS-CoV-2 infection: The role of cytokines in COVID-19 disease.

COVID-19 disease, caused by infection with SARS-CoV-2, is related to a series of physiopathological mechanisms that mobilize a wide variety of biomolecules, mainly immunological in nature. In the most severe cases, the prognosis can be markedly worsened by the hyperproduction of mainly proinflammatory cytokines, such as IL-1, IL-6, IL-12, IFN-γ, and TNF-α, preferentially targeting lung tissue. This study reviews published data on alterations in the expression of different cytokines in patients with COVID-19 who require admission to an intensive care unit. Data on the implication of cytokines in this disease and their effect on outcomes will support the design of more effective approaches to the management of COVID-19

Chromosomal localization of the KMP-11 genes in the KP1(+) and KP1(-) strains of Trypanosoma rangeli

Genes encoding for the KMP-11 protein were localized on the chromosomes of Trypanosoma rangeli. These genes were located in two chromosomes of 3,100 and 3,400 kb in the KP1(-) strain whereas in the KP1(+) H14 and Choachí strains, the genes are located in a chromosome of 1,600 kb. The Choachí strain presents an additional band of 1,400 kb. In the Shubacbarina and Munanta strains of Trypanosoma cruzi, the KMP-11 genes are located on a chromosomal band of 1,490 kb. Therefore, the chromosomal localization of the KMP-11 genes presents a potential tool to differentiate among these parasites.En este trabajo se determinó la localización cromosómica de los genes codificantes para la proteína 11 de membrana de los kinetoplástidos en Trypanosoma rangeli. Los resultados indican que estos genes se localizan en dos cromosomas de 3.100 y 3.400 kb en la cepa Tre, KP1(-) mientras que en las cepas KP1(+), H14 y Choachí se ubican en 1.600 kb; la cepa Choachí presenta una banda adicional de 1.400 kb. En las cepas Shubacbarina y Munantá de Trypanosoma cruzi, los genes KMP-11 se localizaron en una banda cromosómica de 1.490 kb. Estos resultados sugieren la potencialidad de la localización cromosómica de los genes kmp-11 para diferenciar estos parásitos.Este estudió fue financiado por Colciencias contrato No. 190-2000.Peer reviewe

Extracción de ADN de Trypanosoma cruzi mediante tratamiento con bromuro de hexadecil-trimetil-amonio

The present work describes a fast, simple and efficient method of isolating pure and easily handling of genomic DNAfrom Trypanosoma cruzi This protocol is based on parasite lysis with SDS and the removal of proteins by digestion with proteinase K, followed by polysaccharides and remaining proteins' selective precipitation with CTAB. Finally, the DNA is extracted with chloroform-isoamyl alcohol and is recovered from the aqueous supernatant by isopropanol precipitation.En el presente trabajo se describe un método rápido, sencillo y eficaz para la obtención de ADN genómico de Trypanosoma cruzi, libre de impurezas y fácil de manipular. Dicho procedimiento se basa en la lisis del parásito con SDS y remoción de proteínas mediante la digestión con proteinasa K, seguida de la precipitación selectiva de carbohidratos y proteínas residuales con bromuro de hexadecil-trimetil-amonio (CTAB). Finalmente, el ADN se extrae con cloroformo: alcohol isoamílico y se recupera de la fase acuosa mediante precipitación con isopropanol

Molecular characterization of TC964, a novel antigenic protein from trypanosoma cruzi

The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosisThis research was funded by Ministerio de Ciencia,Tecnología e Innovación (Minciencias) and Pontificia Universidad Javeriana, research project ID PPTA 120356933228 granted to C.J.P. The article publication was funded by the Vicerrectoría de Investigación from the Pontificia Universidad Javeriana, code 120813F0401200. The Network of Tropical Diseases Research RICET (RD16/0027/0008, Instituto de Salud Carlos III and co-funded by FEDER) to J.M.R., and grants from the Spanish Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación RTC-2017-6494-1 and RTI2018-094434-B-I00 (MCIU/AEI/FEDER, UE) to P.G.-P., E.R.-M and E.R.R. were supported by Minciencias convocatoria doctorados nacionales 647-2014 and convocatoria jóvenes investigadores e innovadores 706-2015, respectivel