Publications by authors named "Jose Juan Lopez-Rubio"

Various origin mapping approaches have enabled genome-wide identification of origins of replication (ORI) in model organisms, but only a few studies have focused on divergent organisms. By employing three complementary approaches we provide a high-resolution map of ORIs in Plasmodium falciparum, the deadliest human malaria parasite. We profiled the distribution of origin of recognition complex (ORC) binding sites by ChIP-seq of two PfORC subunits and mapped active ORIs using NFS and SNS-seq.

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Long regulatory elements (LREs), such as CpG islands, polydA:dT tracts or AU-rich elements, are thought to play key roles in gene regulation but, as opposed to conventional binding sites of transcription factors, few methods have been proposed to formally and automatically characterize them. We present here a computational approach named DExTER (Domain Exploration To Explain gene Regulation) dedicated to the identification of candidate LREs (cLREs) and apply it to the analysis of the genomes of P. falciparum and other eukaryotes.

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Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinin, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress-responsive genes in a poised state and regulates their expression under stress conditions.

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Guanine-quadruplexes (G4s) are non-canonical DNA structures that can regulate key biological processes such as transcription, replication and telomere maintenance in several organisms including eukaryotes, prokaryotes and viruses. Recent reports have identified the presence of G4s within the AT-rich genome of Plasmodium falciparum, the protozoan parasite causing malaria. In Plasmodium, potential G4-forming sequences (G4FS) are enriched in the telomeric and sub-telomeric regions of the genome where they are associated with telomere maintenance and recombination events within virulence genes.

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To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These new permeation pathways (NPPs) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPPs has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation.

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Epigenetic mechanisms such as histone acetylation and deacetylation participate in regulation of the genes involved in encystation of Entamoeba invadens. However, the histones and target residues involved, and whether the acetylation and deacetylation of the histones leads to the regulation of gene expression associated with the encystation of this parasite, remain unknown. In this study, we found that E.

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Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness.

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Plasmodium falciparum gametocytes, the sexual stage responsible for malaria parasite transmission from humans to mosquitoes, are key targets for malaria elimination. Immature gametocytes develop in the human bone marrow parenchyma, where they accumulate around erythroblastic islands. Notably though, the interactions between gametocytes and this hematopoietic niche have not been investigated.

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Background: For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms.

Objectives: We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo.

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CRISPR/Cas9 approaches are revolutionizing our ability to perform functional genomics across a wide range of organisms, including the Plasmodium parasites that cause malaria. The ability to deliver single point mutations, epitope tags and gene deletions at increased speed and scale is enabling our understanding of the biology of these complex parasites, and pointing to potential new therapeutic targets. In this review, we describe some of the biological and technical considerations for designing CRISPR-based experiments, and discuss potential future developments that broaden the applications for CRISPR/Cas9 interrogation of the malaria parasite genome.

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Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P.

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Plasmodium falciparum gametocytes, the sexual stages responsible for malaria parasite transmission, develop in the human bone marrow parenchyma in proximity to the erythroblastic islands. Yet, mechanisms underlying gametocytes interactions with these islands are unknown. Here, we have investigated whether gametocyte-infected erythrocytes (GIE) adhere to erythroid precursors, and whether a putative adhesion may be mediated by a mechanism similar to the adhesion of erythrocytes infected with P.

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G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T.

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Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P.

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Background: The asexual intra-erythrocytic multiplication of the malaria parasite Plasmodium falciparum is regulated by various molecular mechanisms. In eukaryotic cells, protein kinases are known to play key roles in cell cycle regulation and signaling pathways. The activity of cAMP-dependent protein kinase (PKA) depends on A-kinase anchoring proteins (AKAPs) through protein interactions.

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Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR-Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator.

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Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance.

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Plasmodium falciparum virulence is linked to its ability to sequester in post-capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9-96.

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Genome manipulation in the malaria parasite Plasmodium falciparum remains largely intractable and improved genomic tools are needed to further understand pathogenesis and drug resistance. We demonstrated the CRISPR-Cas9 system for use in P. falciparum by disrupting chromosomal loci and generating marker-free, single-nucleotide substitutions with high efficiency.

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Background: Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets.

Results: To gain insights into the genome-wide distribution of NATs in P.

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The Plasmodium falciparum histone deacetylase Sir2a localizes at telomeric regions where it contributes to epigenetic silencing of clonally variant virulence genes. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. Here we investigate the nucleolar function of PfSir2a and demonstrate that PfSir2a fine-tunes ribosomal RNA gene transcription.

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The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs.

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Background: Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes.

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Chromatin immunoprecipitation (ChIP) studies have been used extensively in recent years to study the functional role of histone marks, variant histones, and other chromatin factors in gene expression in the human malaria parasite, Plasmodium falciparum. In this chapter, we present a ChIP-sequencing protocol optimized for blood-stage forms of this parasite. The processing of the immunoprecipitated DNA prior to high-throughput sequencing is performed in a way to minimize amplification biases due to the high genomic AT-content of the parasite.

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Background: Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2.

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