Jose-Juan Lopez-Rubio

Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system

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. Additionally, an artemisinin-resistant strain was generated by introducing a previously implicated polymorphism, thus illustrating the value of efficient genome editing in malaria research.

Genome-wide Analysis of Heterochromatin Associates Clonally Variant Gene Regulation with Perinuclear Repressive Centers in Malaria Parasites

Clonally variant gene families underlie phenotypic plasticity in Plasmodium falciparum, a process indispensable for survival of the pathogen in its human host. Differential transcription of one of these gene families in clonal parasite lineages has been associated with chromatin modifications. Here, we determine the genome-wide distribution in P. falciparum of a histone mark of heterochromatin, trimethylation of histone H3 lysine 9 (H3K9me3), using high-resolution ChIP-chip analysis. We show that H3K9me3 is specifically associated with clonally variant gene families, which are clustered on subtelomeric and some chromosome internal regions. High levels of H3K9me3 correlate with genes localized to the nuclear periphery, implying chromosome loop formation. Disruption of the histone deacetylase PfSir2 causes changes in H3K9me3 that are discontinuous along chromosomes and associated with disrupted monoallelic transcription. Our data point to the existence of perinuclear repressive centers associated with control of expression of malaria parasite genes involved in phenotypic variation and pathogenesis.

A Critical Role of Perinuclear Filamentous Actin in Spatial Repositioning and Mutually Exclusive Expression of Virulence Genes in Malaria Parasites

Many microbial pathogens, including the malaria parasite Plasmodium falciparum, vary surface protein expression to evade host immune responses. P. falciparium antigenic variation is linked to var gene family-encoded clonally variant surface protein expression. Mututally exclusive var gene expression is partially controlled by spatial positioning; silent genes are retained at distinct perinuclear sites and relocated to transcriptionally active locations for monoallelic expression. We show that var introns can control this process and that var intron addition relocalizes episomes from a random to a perinuclear position. This var intron-regulated nuclear tethering and repositioning is linked to an 18 bp nuclear protein-binding element that recruits an actin protein complex. Pharmacologically induced F-actin formation, which is restricted to the nuclear periphery, repositions intron-carrying episomes and var genes and disrupts mutually exclusive var gene expression. Thus, actin polymerization relocates var genes from a repressive to an active perinuclear compartment, which is crucial for P. falciparium phenotypic variation and pathogenesis.

Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway

BackgroundProtein 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 parasites 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.ResultsWe now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro.ConclusionsOur reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.

Comprehensive Histone Phosphorylation Analysis and Identification of Pf14-3-3 Protein as a Histone H3 Phosphorylation Reader in Malaria Parasites

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. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes

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. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence‐associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co‐exported with PfEMP1 into the host cell via vesicle‐like structures called Maurers clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface.

Genome-wide Chromatin Immunoprecipitation-Sequencing in Plasmodium

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.

Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.

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. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

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. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia. Methodology/Principal Findings The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int. Conclusions/Significance Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.