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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. However, there is a little understanding about the biological role of G4s and G4-binding proteins. Here, we provide the first snapshot of G4-interactome in P. falciparum using DNA pull-down assay followed by LC-MS/MS. Interestingly, we identified ~24 potential G4-binding proteins (G4-BP) that bind to a stable G4FS (AP2_G4). Furthermore, we characterised the role of G-strand binding protein 2 (PfGBP2), a putative telomere-binding protein in P. falciparum. We validated the interaction of PfGBP2 with G4 in vitro as well as in vivo. PfGBP2 is expressed throughout the intra-erythrocytic developmental cycle and is essential for the parasites in the presence of G4-stabilising ligand, pyridostatin. Gene knockout studies showed the role of PfGBP2 in the expression of var genes. Taken together, this study suggests that PfGBP2 is a bona fide G4-binding protein, which is likely to be involved in the regulation of G4-related functions in these malarial parasites. In addition, this study sheds light on this understudied G4 biology in P. falciparum., (© 2020 John Wiley & Sons Ltd.)

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. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod-2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off-target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9-mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis., (© 2015 John Wiley & Sons Ltd.)

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 Maurer's 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., (© 2015 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)