Jose M Requena | Universidad Autónoma de Madrid (original) (raw)

Papers by Jose M Requena

Leishmania infantum gene coding for Kinesin-13

Genes

Leishmania infantum is one of the causative agents of visceral leishmaniases, the most severe for... more 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 ...

Leishmaniases, a group of parasitic diseases caused by species of the genus Leishmania, a"ic... more Leishmaniases, a group of parasitic diseases caused by species of the genus Leishmania, a"ict millions of people across the globe and cause signi#cant morbidity and mortality. Unfortunately, vaccine and chemotherapy options are limited. $e advances in whole genome sequencing have led to renewed impetus in identifying druggable targets for future development of more e%ective treatments. Hence, the last decade has witnessed a revolution in our understanding of the Leishmania genomes through the completion of an increasing number of genome sequencing projects for several species and strains. However, the completion of a genome sequence is not the #nal product, rather it is just the beginning towards the objective of linking the wealth of data encoded in millions of bases to the biological processes of an organism. Moreover, the genome features (genomics) is only a part of the problem to be solved: the genome yields on transcription, the transcriptome, which in turn yields the prot...

Scientific reports, Jan 22, 2017

Leishmania parasites are the causative of leishmaniasis, a group of potentially fatal human disea... more Leishmania parasites are the causative of leishmaniasis, a group of potentially fatal human diseases. Control strategies for leishmaniasis can be enhanced by genome based investigations. The publication in 2005 of the Leishmania major genome sequence, and two years later the genomes for the species Leishmania braziliensis and Leishmania infantum were major milestones. Since then, the L. infantum genome, although highly fragmented and incomplete, has been used widely as the reference genome to address whole transcriptomics and proteomics studies. Here, we report the sequencing of the L. infantum genome by two NGS methodologies and, as a result, the complete genome assembly on 36 contigs (chromosomes). Regarding the present L. infantum genome-draft, 495 new genes have been annotated, a hundred have been corrected and 75 previous annotated genes have been discontinued. These changes are not only the result of an increase in the genome size, but a significant contribution derives from t...

Leishmania infantum is causative of visceral leishmaniasis (kala-azar), the most severe form of l... more Leishmania infantum is causative of visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, lethal if untreated. Few years ago, re-sequencing and de novo assembling of the L. infantum (strain JPCM5) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we have performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2,352 proteins based on the search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We have detected many proteins belonging to organelles such as glycosomes, mitochondria or flagellum, as well as many metabolic enzymes, and a large number of putative RNA binding proteins and molecular chaperones. Moreover, we listed the proteins presenting post-translational modifications, such as phosphorylat...

Prokaryotic and eukaryotic organisms respond to potentially damaging stimuli such as elevated tem... more Prokaryotic and eukaryotic organisms respond to potentially damaging stimuli such as elevated temperature by increasing the synthesis of a family of proteins collectively known as heat shock proteins (HSPs) or stress proteins. HSPs are among the most highly conserved and abundant proteins found in nature. Much interest has been generated in HSPs since they were described as dominant antigens of infectious microorganisms. It has been suggested that this immune recognition of pathogen HSPs serves as a first line of defense as well as a means by which autoimmune cascades may develop due to the inappropriate cross reactivity with self-HSPs. As a consequence of their chaperone functions, HSPs are associated with a broad spectrum of peptides derived from the cells from which they are isolated. Hence, it is not surprising that HSP-peptide complexes have been described as tumor specific antigens. Remarkably, it has been demonstrated that vaccination with these HSP-peptide complexes elicit p...

Trypanosomatids represent one of the earlier groups of eukaryotic organisms in evolution. During ... more Trypanosomatids represent one of the earlier groups of eukaryotic organisms in evolution. During their long evolutionary history, the trypanosomatids have developed an impressive variety of life styles and adaptations to parasitism. The parasitic cycle of these organisms is characterized by a succesion of different forms adapted to the different environments they encounter in their mammalian hosts and insect vectors. Perhaps as a reflection of their ancient origin, these organisms posses a variety of unusual molecular mechanisms that result in regulated gene expression. In this context, a general feature of gene expression in these protozoan parasites is its high regulation at post-transcriptional level. In this work, some recent advances in understanding the mechanisms of gene expression in trypanosomatids and its regulation are discussed.

In this work we describe the characterization of five antigenic proteins of Leishmania infantum, ... more In this work we describe the characterization of five antigenic proteins of Leishmania infantum, parasite whose infection cause canine visceral leishmaniosis in Spain. The following antigenic proteins were characterized: the histones H2A and H3, and three members of the acidic ribosomal protein family, LiP2a, LiP2b and LiP0. All of them were isolated after isolation of their cDNAs by immunoscreening of a L. infantum egt11 expression library with the sera from dogs naturally infected with the parasite. Aminoacid sequence analysis showed that all of them were conserved relative to the eukaryotic equivalent proteins but also that they possessed divergent regions. Epitope mapping demonstrated that the antigenic determinants were always located in these specific regions of the parasite proteins. In order to develop a serodiagnosis test for canine leishmaniasis these antigenic regions were expressed and purified as independient polypeptides. The usefulness of these recombinant proteins in...

Parasitology, 1997

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and seque... more The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree o...

Parasitology, 2006

Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermo... more Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermore, heat shock genes are extensively studied as gene models to decipher mechanisms of gene regulation in kinetoplastids. Here, we describe the organization and expression of the HSP70 loci in representative Leishmania species (L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis and L. braziliensis). With the exception of L. braziliensis, the organization of the HSP70 loci was found to be well conserved among the other Leishmania species. Two types of genes, HSP70-I and HSP70-II, were found to be present in these Leishmania species except for L. braziliensis that lacks HSP70-II gene. Polymorphisms in the HSP70 locus allow the differentiation of the Old and New World species within the subgenus Leishmania. A notable discrepancy between our data and those of the L. major genome database in relation to the gene copy number composing the L. major HSP70 locus was revealed. The temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among the different Leishmania species with the exception of L. braziliensis. In spite of these differences, analysis of the HSP70 synthesis indicated that the HSP70 mRNAs are also preferentially translated during heat shock in L. braziliensis.

Parasite Immunology, 2006

It has been reported that the level of protection provided by vaccines against murine visceral le... more It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10(3), 10(5) and 10(6) parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti-Leishmania antibodies, up-regulation of spleen arginase activity, balance between IFN-gamma and IL-10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 10(5) parasites should prove useful for vaccine studies against VL.

Molecular and Biochemical Parasitology, 1992

The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures... more The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures and growth conditions has been analyzed by Northern blotting. We show that only one size class of hsp70 mRNA, of about 2.2 kb, is transcribed from the hsp70 cluster and that its transcription is constitutive at 28°C. However, after a heat shock treatment at 37°C for 2 h of logarithmically growing parasites, the abundance of the hsp70 mRNA increased about 4-fold. A similar increase was observed at 28~'C when the parasite culture reached the stationary phase of growth. On the other hand, a heat shock at 42°C did not change the steady state level of the 2.2-kb size class of hsp70 mRNA. However, accumulation of transcripts of high molecular weight was detected when stationary growing parasites were cultured at 42°C for 2 h. Also at 37°C the steady state level of the a-and [/-tubulin mRNAs of logarithmically growing parasites exhibited a slight increase but only after a period of 24 h. Analysis by one-dimensional immunoblots of the Hsp70 levels showed that at 37°C the abundance of the protein was 4fold higher than at 28°C. lmmunoblots of high-resolution two-dimensional gel electrophoresis showed, moreover, that various isoforms of this protein are constitutively expressed at 28~C and that some of them have a specific pattern of induction at 37°C. We observed, moreover, that the heat, shock induces the expression of a series of proteins while it causes repression of others.

Clinical and Experimental Immunology, 1999

SUMMARY In this study we show that sera from dogs naturally infected with Leishmania infantum con... more SUMMARY In this study we show that sera from dogs naturally infected with Leishmania infantum contain antibodies that specifically react against the parasite H2B and H4 histones. The Leishmania H2B and the amino-terminal region of the histone H4, expressed as fusion proteins, when confronted with sera from canine viscerocutaneous leishmaniasis (VCL) dogs, were recognized by 63% and 47%, respectively. No reactivity was detected when sera from dogs naturally infected with pathogens other than Leishmania were used. Using a collection of synthetic peptides covering the complete sequence of both proteins, we have determined that the main linear antigenic determinants are located in the amino-terminal domains of these histones. The humoral response against histones H2B and H4 induced during canine leishmaniasis was found to be specific for Leishmania histones, since no cross-reactivity of the VCL sera with mammal histones was observed. Also, a comparative study of the prevalence of antibo...

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994

The isolation of a Leishmania cDNA clone coding for an antigen identified as the histone H3 is de... more The isolation of a Leishmania cDNA clone coding for an antigen identified as the histone H3 is described. The nucleotide sequence of the cDNA predicts that the Leishmania histone H3 contains 129 residues and that it has a molecular mass of 14 620 Da. Comparison of the amino acid sequence with the consensus sequence of the eukaryotic histone H3 shows that the Leishmania protein has a highly conserved globular region and an extremely divergent amino-terminal portion.

Memórias do Instituto Oswaldo Cruz, 1997

During recent years, several Leishmania infantum genes have been cloned and characterized. Here, ... more During recent years, several Leishmania infantum genes have been cloned and characterized. Here, we have summarized the available information on the gene organization and expression in this protozoan parasite. From a comparative analysis, the following outstanding features were found to be common to most of the genes characterized: tandemly organized genes with conserved coding regions and divergent untranslated regions, polycistronic transcription and post-transcriptional regulation of gene expression. The analysis of chromosomes of L. infantum by pulsed-field electrophoresis showed the existence of both size and number polymorphisms such that each strain has a distinctive molecular karyotype. Despite this variability, highly conserved physical linkage groups exists among different strains of L. infantum and even among Old World Leishmania species. Gene mapping on the L. infantum molecular karyotype evidenced a bias in chromosomal distribution of, at least, the evolutionary conserved genes.

Clinical and Experimental Immunology, 2008

SUMMARY In this work we show that in the sera from dogs naturally infected with the protozoan par... more SUMMARY In this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas’ disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.

Briefings in Bioinformatics, 2021

NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio)... more NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio) have revolutionized the sequencing field over the last decade improving multiple genomic applications like de novo genome assemblies. However, their error rate, mostly involving insertions and deletions (indels), is currently an important concern that requires special attention to be solved. Multiple algorithms are available to fix these sequencing errors using short reads (such as Illumina), although they require long processing times and some errors may persist. Here, we present Accurate long-Reads Assembly correction Method for Indel errorS (ARAMIS), the first NGS long-reads indels correction pipeline that combines several correction software in just one step using accurate short reads. As a proof OF concept, six organisms were selected based on their different GC content, size and genome complexity, and their PacBio-assembled genomes were corrected thoroughly by this pipeline. We fo...

Journal of Biological Chemistry, 1997

Microorganisms

Leishmania amazonensis parasites are etiological agents of cutaneous leishmaniasis in the New Wor... more Leishmania amazonensis parasites are etiological agents of cutaneous leishmaniasis in the New World. BALB/c mice are highly susceptible to L. amazonensis challenge due to their inability to mount parasite-dependent IFN-γ-mediated responses. Here, we analyzed the capacity of a single administration of the LiΔHSP70-II genetically-modified attenuated L. infantum line in preventing cutaneous leishmaniasis in mice challenged with L. amazonensis virulent parasites. In previous studies, this live attenuated vaccine has demonstrated to induce long-protection against murine leishmaniasis due to Old World Leishmania species. Vaccinated mice showed a reduction in the disease evolution due to L. amazonensis challenge, namely reduction in cutaneous lesions and parasite burdens. In contrast to control animals, after the challenge, protected mice showed anti-Leishmania IgG2a circulating antibodies accompanied to the induction of Leishmania-driven specific IFN-γ systemic response. An analysis perfo...

International Journal of Molecular Sciences

The Tc964 protein was initially identified by its presence in the interactome associated with the... more 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 pep...