Biology subjects

Coppee, R.

Publications and source records attributed to Coppee, R..

8 recordsLinked to original sources

bioRxiv2026

Dual-stage inhibition of Plasmodium falciparum by a Skeletocutis derived fungal metabolite targeting Pyruvate Kinase II

Plasmodium falciparum resistance to current first line treatments is threatening at-risk populations and underscores the urgent need for novel therapeutic targets and drugs. P. falciparum pyruvate kinases I and II are two essential enzymes with distinct roles and subcellular localizations within the parasite. PfPyrKI is cytosolic, while PfPyrKII is found in the apicoplast, a specific organelle of Apicomplexa, where it is required for the production of (d)NTPs essential for apicoplast maintenance. We identify skeletocutin E, a Basidiomycete-derived metabolite, as a specific inhibitor of PfPyrKII. Skeletocutin E inhibits in vitro the activity of PfPyrKII with an IC50 of 0.52 {+/-} 0.08 {micro}M through a mixed inhibition mechanism and does not affect the activities of three human pyruvate kinases. Structure-activity relationship analyses using synthetic skeletocutin E analogues allowed us to identify the molecular determinants of this inhibition. Furthermore, determination of the quaternary structure of PfPyrKII by mass photometry, showed that this enzyme exists as monomers, dimers, and tetramers in equal proportions, revealing its singularity compared to other pyruvate kinases. Interestingly, skeletocutin E does not alter the distribution of the complexes, indicating that it does not interact at the subunit interfaces. Importantly, skeletocutin E inhibits P. falciparum growth in both blood and liver stages, with IC values of 3.56 {+/-} 0.50 {micro}M in red blood cells and 3.70 {+/-} 0.74 {micro}M in primary human hepatocytes. Together, these findings establish PfPyrKII as a druggable antimalarial target and identify skeletocutin E as a promising lead compound for the rational development of dual-stage antimalarial therapies.

microbiology↗

bioRxiv2024

CONSTRUCT: an algorithmic tool for identifying functional or structurally important regions in protein tertiary structure

In the realm of protein-coding genes, evolutionary rates show considerable variability. Essential or highly expressed proteins evolve more slowly, and within a protein, different amino acid sites evolve at different rates. Accurately modeling this variation is critical for identifying structurally or functionally important amino acid sites. Standard methods such as Rate4Site assume independent substitution rates across sites, and the most conserved ones are widely distributed in protein tertiary structure. This is biologically unrealistic because functional sites often cluster together. Here, we developed CONSTRUCT, an improved strategy for identifying functional and structurally important regions in protein tertiary structure. Given a set of orthologous sequences, CONSTRUCT first calculates site-specific substitution rates using the Rate4site model, which are then weighted by the rates of neighboring amino acid sites within a range of window sizes. The optimal window size is determined by the strongest spatial correlation, if present. This method can be analyzed using either C atoms or the center of mass of amino acid sites to account for side chain orientation. Validation on 14 functionally characterized proteins of different size, conservation level and kingdom/domain of species has demonstrated the relevance of CONSTRUCT.

bioinformatics↗

bioRxiv2024

Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation

The rapid emergence of artemisinin resistance (ART-R) poses a challenge to global malaria control efforts. ART potency is triggered by ferrous iron- and/or heme-mediated cleavage of the endoperoxide bond to generate reactive heme-ART alkoxy radicals and covalent heme-ART adducts that alkylate parasite targets or inhibit the detoxification of heme into {beta}-hematin crystals; both of which lead to parasite death. Mutations in the P. falciparum Kelch-containing protein Kelch13 (PfKekch13) confer clinical ART-R, in which the resistant parasites exhibit impaired hemoglobin uptake, reduced heme yield, and thus decreased ART activation. However, a more direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that recombinant, purified PfKelch13 wild-type (WT) protein displays measurable binding affinity for both iron and heme, the main effectors for ART activation. Comparative biochemical analyses further indicate weaker heme-binding affinities in the two Southeast Asian ART-R PfKelch13 mutants C580Y and R539T compared to the ART-sensitive WT and A578S mutant proteins, which ultimately translates into reduced yield of heme-ART derivatives. In conclusion, this study provides the first evidence for regulated ART activation via the heme-binding propensity of PfKelch13, which may contribute towards modulating the level of ART-R in malaria parasites with PfKelch13 mutations.

biochemistry↗

bioRxiv2023

Genetic profiling of Plasmodium ovale wallikeri relapses with microsatellite markers and whole-genome sequencing

Like Plasmodium vivax, both Plasmodium ovale curtisi and Plasmodium ovale wallikeri have the ability to cause relapse in humans, defined as recurring asexual parasitaemia originating from liver dormant forms subsequent to a primary infection. Here, we investigated relapse patterns in P. ovale wallikeri infections from a cohort of travelers who were exposed to the parasite in Sub-Saharan Africa and then experienced relapses after their return to France. Using a novel set of eight highly polymorphic microsatellite markers, we genotyped 15 P. ovale wallikeri relapses. For most relapses, the paired primary and relapse infections were highly genetically related (with 12 being homologous), an observation that was confirmed by whole-genome sequencing for the four relapses we further studied. This is, to our knowledge, the first genetic evidence of relapses in P. ovale spp.

microbiology↗

bioRxiv2021

Plasmodium sporozoites require the protein B9 to invade hepatocytes

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite in the liver. This leads to the release of thousands of merozoites into the blood circulation and initiation of the pathogenic blood stages of malaria. Merozoite invasion of erythrocytes has been well characterized at the molecular and structural levels. In sharp contrast, the molecular mechanisms of sporozoite invasion of hepatocytes are poorly characterized. Here we report a new role during sporozoite entry for the B9 protein, a member of the 6-cysteine domain protein family. Using genetic tagging and gene deletion approaches in rodent malaria parasites, we show that B9 is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. Structural modelling indicates that the N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an invasion complex with Rh5 and RIPR in P. falciparum merozoites. We provide evidence that the beta-propeller domain of B9 is essential for protein function during sporozoite entry and interacts with P36 and P52, both also essential for productive invasion of hepatocytes. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

microbiology↗

bioRxiv2021

5WBF: A low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates

BackgroundWhole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programs. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7-8 m and exhibit some deformability, we hypothesized that cheap and commercially available 5 m filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 m Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS. MethodsWhole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitemia range: 0.04-5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitemia range: 0.02-1.1%). These whole blood samples (50 to 400 L) were diluted in RPMI 1640 medium or PBS 1X buffer and filtered with syringes connected to a 5 m commercial filter. DNA was extracted from filtered and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, we also applied to the same unprocessed whole blood samples the selective whole-genome amplification (sWGA) method which does not rely on blood filtration. ResultsAfter applying 5WBF, qPCR indicated an average of 2-fold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4,096- to 65,536-fold loss of human template DNA (human {beta} actin gene). WGS analyses revealed that > 95% of the nuclear genome and the entire whole organellar genomes were covered at [≥] 10x depth for all samples tested. In sWGA counterparts, none of the organellar genomes were covered, and from 47.7 to 82.1% of the nuclear genome was covered at [≥] 10x depth depending on parasitemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5,460 genes; mean coverage: 91x; median coverage: 93x; 5th percentile: 70x; 95th percentile: 103x), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as we observed a much more heterogeneous distribution of reads among gene sequences (mean coverage: 80x; median coverage: 51x; 5th percentile: 7x; 95th percentile: 245x). ConclusionsThe novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized, 5 m filters which cost from 1.0 to 1.7{euro} per unit, depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum DNA from minute amounts of whole blood even with parasitemias as low as 0.02%.

genomics↗

bioRxiv2020

Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytic cells.

Sporozoite forms of the malaria parasite Plasmodium are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection. IMPORTANCEMalaria is caused by Plasmodium parasites and remains one of the deadliest parasitic diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first invades the liver for an initial, obligatory and silent round of replication. The liver infection is an attractive target for antimalarial vaccine strategies, however the molecular mechanisms of parasite invasion of hepatocytes remain to be fully elucidated. Two hepatocyte surface proteins are known to be important for parasite entry into hepatocytes, the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1). These receptors constitute independent gateways depending on the Plasmodium species. Here, we identified the structural determinants of SR-B1, an important hepatocyte entry factor for human-infecting P. vivax. This study paves the way toward a better characterization of the molecular interactions underlying the crucial early stages of infection, a pre-requisite for the development of novel malaria vaccine strategies.

microbiology↗

bioRxiv2018

Delineating functional sites in the K13 antimalarial resistance protein through evolutionary and structural analyses

K13 is an essential Plasmodium falciparum protein that plays a key role in malaria resistance to artemisinins. Although K13 resembles BTB- and Kelch/propeller-containing proteins involved in ubiquitin ligase complexes, its functional sites remain uncharacterized. Using evolutionary and structural information, we searched for the most conserved K13 sites across Apicomplexa species evolution to identify sub-regions of K13 that are likely functional. An amino acid electropositive patch in the K13 propeller domain has a dense concentration of extraordinarily conserved positions located at a shallow pocket, suggesting a role as binding surface. When applied to experimentally-characterized BTB-Kelch proteins, our strategy successfully identifies the validated substrate-binding residues within their own propeller shallow pocket. Another patch of slowly evolving sites is identified in the K13 BTB domain which partially overlaps the surface that binds to Cullin proteins in BTB-Cullin complexes. We provide candidate binding sites in K13 propeller and BTB domains for functional follow-up studies.

evolutionary biology↗