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Clain, J.

Publications and source records attributed to Clain, J..

7 recordsLinked to original sources

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↗

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↗

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↗

Splenic clearance of rigid erythrocytes as an inherited mechanism for splenomegaly and natural resistance to malaria

In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. In Benin, West Africa, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe genetic background of individuals influences their susceptibility to infectious diseases. Specific human groups, like the Fulani in Africa, react to malaria parasites (named Plasmodium) in a specific way. Upon infection, Fulani develop a grossly enlarged spleen, and high levels of anti-Plasmodium antibodies in their blood. They also carry smaller numbers of parasites in their blood, and thus are considered partially protected against malaria. The mechanisms underlying this natural protection, different from other natural protective mechanisms such as the sickle cell trait, are not well understood. Malaria impairs the deformability of red blood cells and the spleen is a key organ to controlling red blood cell quality. We have recently demonstrated that red blood cells containing live malaria parasites accumulate intensely in the spleen of subjects with long term exposure to these parasites. Enhanced retention of infected and uninfected red blood cells in the spleen would explain why the spleen is larger and why lower numbers of parasites are left in circulation. We thus explored whether the retention of infected and uninfected red blood cells could explain why Fulani are partially protected against malaria. Because it is unethical to perform spleen puncture or biopsies for research purposes, our explorations were indirect by carefully analyzing the properties of circulating red blood cells in a large number of subjects and by assessing whether observations could be explained by their genetic make-up. Added value of this studyIn more than 500 subjects, we confirmed the high frequency of large spleens in Fulani and, through 2 different methods, we demonstrated an enhanced deformability of their circulating red blood cells, that likely stems from the more efficient removal of the less deformable ones. This enhanced deformability was found to be inheritable based on carefully collected family links and refined analysis of genetic markers. Implications of all the available evidenceOur findings indicate that genes potentially driving the filtration of red blood cells by the spleen likely influence how subjects in specific groups in Africa and elsewhere react to malaria. While most previous hypotheses pointed to conventional immunological mechanisms as the trigger, we propose that a simple physiological mechanism that controls the quality of red blood cells may drive natural protection from malaria even before the intervention of immunological cells. A better understanding of these processes is of great importance in the context of malaria elimination efforts. These findings may also have an impact on the understanding of other red blood cell-related disorders, such as inherited red cell diseases, in which splenic filtration of abnormal red blood cells may precipitate splenic complications.

physiology↗

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↗

Plasmodium falciparum sexual parasites regulate infected erythrocyte permeability

To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These New Permeation Pathways (NPP) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPP has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPP are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPP are regulated by cyclic AMP (cAMP) signaling cascade during sexual parasite stages, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPP facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPP and increase drug uptake in mature gametocyte-infected erythrocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.

microbiology↗

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↗