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

Variación antigénica de la cepa Munantá de Trypanosoma cruzí después de pase por ratón

This study evaluated the isoenzyme and antigenic changes of Trypanosoma cruzfs Munanta strain after two consecutive passages in mice, using isoenzyme electrophoresis, SDS-PAGE electrophoresis and immunoblot. The results obtained show significant statistical differences within the strain before and after animal passages, which suggests that the mouse is not a recommended model for obtaining the parasite's Munanta strain's trypomastigote forms destined for study of the immune response.Este estudio evaluó los cambios en el perfil isoenzimático y antigénico de la cepa Munantá de Trypanosoma cruzidespués de dos pases consecutivos por ratón, mediante electrofóresis isoenzimática, electrofóresis en PAGE-SDS e immunoblot. Los resultados obtenidos muestran diferencias estadísticamente significativas entre la cepa antes y después de los pases en los animales, lo cual sugiere que el ratón no es un modelo recomendable para obtener las formas tripomastigotas de la cepa Munanta del parásito destinadas a estudios de la respuesta inmune

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

Intermediate monocytes and cytokine production associated with severe forms of chagas disease

Q1Monocytes are classified according to their CD14 and CD16 expression into classical (reparative), intermediate (inflammatory), and non-classical. This study assessed the frequency of monocyte and the relationship between monocyte subset percentages and the levels of blood cytokines in Colombian chagasic patients with different clinical forms. This study included chagasic patients in different clinical stages: indeterminate (IND) n = 14, chronic chagasic cardiomyopathy (CCC) n = 14, and heart transplant chagasic (HTCC) n = 9; controls with non-chagasic cardiopathy (NCC) n = 15, and healthy individuals (HI) n = 15. Peripheral blood mononuclear cells (PBMCs) were isolated, labeled for CD14, CD16, and HLA-DR, and analyzed by flow cytometry. Cytokines were measured with a bead-based immunoassay. Percentages of total CD14+ CD16+ and CD14+ HLA-DR+ monocytes were higher in patients with heart involvement (CCC, HTCC, and NCC) than controls. Percentages of intermediate monocytes increased in symptomatic chagasic patients (CCC and HTCC) compared to asymptomatic chagasic patients (IND) and controls (HI). Asymptomatic chagasic patients (IND) had higher percentages of classical monocytes, an increased production of CCL17 chemokine compared to chagasic symptomatic patients (CCC), and their levels of CCL17 was positively correlated with the percentage of classical monocyte subset. In CCC, the percentages of intermediate and classical monocytes were positively correlated with IL-6 levels, which were higher in this group compared to HI, and negatively with IL-12p40 concentration, respectively. Remarkably, there also was an important increased of classical monocytes frequency in three chronic chagasic patients who underwent cardiac transplant, of which one received anti-parasitic treatment. Our findings suggest that cardiac chagasic patients have an increased percentage of inflammatory monocytes and produce more IL-6, a biomarker of heart failure and left ventricular dysfunction, whereas asymptomatic chagasic individuals present a higher percentage of reparative monocytes and CCL17.N/

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