Comparative structural and evolutionary analyses predict functional sites in the artemisinin resistance malaria protein K13

Numerous mutations in the Plasmodium falciparum Kelch13 (K13) protein confer resistance to artemisinin derivatives, the current front-line antimalarial drugs. K13 is an essential protein that contains BTB and Kelch-repeat propeller (KREP) domains usually found in E3 ubiquitin ligase complexes that target substrate protein(s) for ubiquitin-dependent degradation. K13 is thought to bind substrate proteins, but its functional/interaction sites and the structural alterations associated with artemisinin resistance mutations remain unknown. Here, we screened for the most evolutionarily conserved sites in the protein structure of K13 as indicators of structural and/or functional constraints. We inferred structure-dependent substitution rates at each amino acid site of the highly conserved K13 protein during the evolution of Apicomplexa parasites. We found two solvent-exposed patches of extraordinarily conserved sites likely involved in protein-protein interactions, one in BTB and the other one in KREP. The conserved patch in K13 KREP overlaps with a shallow pocket that displays a differential electrostatic surface potential, relative to neighboring sites, and that is rich in serine and arginine residues. Comparative structural and evolutionary analyses revealed that these properties were also found in the functionally-validated shallow pocket of other KREPs including that of the cancer-related KEAP1 protein. Finally, molecular dynamics simulations carried out on PfK13 R539T and C580Y artemisinin resistance mutant structures revealed some local structural destabilization of KREP but not in its shallow pocket. These findings open new avenues of research on one of the most enigmatic malaria proteins with the utmost clinical importance

Étude bioinformatique des protéines PfK13 et PfCRT impliquées dans la résistance de Plasmodium falciparum aux antipaludiques

Malaria is an infectious parasitic disease caused by Plasmodium species. P. falciparum is the most prevalent species and is responsible for the majority of the disease burden. Currently, malaria is treated using artemisinin-based combination therapies (ACTs), coupling an artemisinin derivative (ARTD) to another antimalarial drug. After a decade of use, ARTD-resistant parasites have emerged in Southeast Asia; they are currently not found in Africa. Resistance to ARTD is defined as parasites exhibiting in vivo a delayed clearance time following an artemisinin-based treatment, and is conferred by non-synonymous mutations localized in the Kelch-repeat propeller (KREP) domain of the P. falciparum K13 protein (PfK13). Similarly, multiple non-synonymous mutations conferring resistance to chloroquine (CQ) or piperaquine (PPQ, used as partner drug in one ACT) have been reported on the P. falciparum chloroquine resistance transporter (PfCRT). The physiological role of these proteins, essential at least for the intra-erythrocytic development of the parasite, remains poorly understood. This thesis aims to better characterize the PfK13 and PfCRT proteins by: i) predicting the protein regions and positions that would be involved in protein-substrate interactions; and ii) studying structural and/or physicochemical alterations induced by resistance mutations. According to concepts related to the theory of molecular evolution, mutations affecting sites exerting a critical function within an essential protein are eliminated by purifying selection. These sites are therefore more conserved than the rest of the protein. Using bioinformatics approaches combining sets of PfK13 and PfCRT orthologous sequences - respectively at Apicomplexa and Plasmodium scales - and information from protein tertiary structures, we highlighted extremely conserved regions within these two proteins. By comparing these results with those obtained for some proteins / domains belonging to the same structural families and validated at the experimental level, we identified several PfK13 and PfCRT amino acid sites that we propose as candidates for protein-substrate interactions. Remarkably, ARTD resistance mutations are not localized at the interaction surface that we predicted in the KREP domain of PfK13. Molecular dynamics simulations carried out on KREP domains carrying resistance mutations (C580Y and R539T) indicated that these mutations are associated with local structural destabilizations of the KREP domain. We assume that these resistance mutations may disrupt the stability of the KREP domain and, by extension, the whole PfK13 protein whose cellular abundance would be decreased. Regarding PfCRT, using the two tertiary structure models we predicted by structural homology, the majority of CQ and PPQ resistance mutations were found located in a binding pocket at the core of the transporter. Furthermore, these mutations strongly altered the electrostatic potential at the surface of this pocket, from a neutral to an electronegative potential. This change in physicochemical property of the pocket is probably a major determinant in the acquisition of the transport property of di-protonated CQ from the digestive vacuole to the cytoplasm of the parasite. Candidate functional sites of PfCRT and PfK13 identified in our work should be validated by experimental approaches. Here, we initiated differential pull-down biochemical experiments for the KREP domain of PfK13. First, the wild-type PfK13 domains, fused to glutathione S-transferase (GST), were expressed in Escherichia coli, purified, and then incubated with parasite lysate to characterize PfK13-interacting proteins. These experiments are still in progress. In a second step, the importance of candidate functional sites will be tested through genetic approaches directly in P. falciparum parasites using transfection and gene editing techniques.Le paludisme est une maladie infectieuse parasitaire causée par diverses espèces de Plasmodium, P. falciparum étant l'espèce la plus répandue et responsable des cas mortels de la maladie. Les traitements actuels reposent sur des combinaisons thérapeutiques à base d'artémisinine (CTA), couplant un dérivé d'artémisinine (ARTD) à une autre molécule antipaludique. Des parasites résistants aux ARTDs ont émergé en Asie du sud-est ; ceux-ci sont encore non détectés en Afrique. La résistance se traduit par une durée d'élimination des parasites allongée, et est conférée par des mutations non-synonymes localisées sur le domaine Kelch-repeat propeller (KREP) de la protéine P. falciparum K13 (PfK13). Similairement, de multiples mutations non-synonymes sur le transporteur P. falciparum Chloroquine Resistance Transporter (PfCRT) confèrent la résistance à la chloroquine (CQ, ancien traitement) et à la pipéraquine (PPQ, une des molécules partenaires dans les CTAs actuels). Le rôle physiologique de ces protéines, essentielles durant le développement du parasite, demeure mal connu. Ce travail de thèse a donc pour objectif de mieux caractériser PfK13 et PfCRT : i) en prédisant les positions qui seraient impliquées dans des interactions protéine-substrat ; et ii) en étudiant les altérations structurales et physico-chimiques induites par les mutations de résistance. Selon les concepts liés à la théorie de l'évolution moléculaire, les mutations touchant des sites exerçant une fonction critique au sein d'une protéine essentielle sont éliminées par la sélection purificatrice. Ces sites sont donc plus conservés que le reste de la protéine. Par des approches bioinformatiques couplant évolution et structure tertiaire, nous avons mis en évidence des régions extrêmement conservées au sein de PfK13 et PfCRT. En comparant ces résultats avec les données expérimentales de protéines / domaines appartenant aux mêmes familles structurales, nous avons identifié plusieurs sites de PfK13 et PfCRT que nous proposons comme candidats pour des interactions protéine-substrat. À notre surprise, les mutations de résistance aux ARTDs ne sont pas localisées à la surface d'interaction que nous avons prédite sur le domaine KREP de PfK13. Les dynamiques moléculaires que nous avons réalisées sur deux mutations de résistance (C580Y et R539T) ont révélé des déstabilisations structurales locales du domaine KREP. Nous supposons que ces mutations pourraient perturber la stabilité du domaine KREP et ainsi diminuer l'abondance cellulaire de PfK13. Concernant PfCRT, les deux modèles de structure tertiaire, prédits par homologie structurale, montrent que la majorité des mutations de résistance à la CQ et à la PPQ sont localisées au niveau d'une poche probable de liaison que nous avons identifiée au coeur du transporteur. Ces mutations altèrent fortement le potentiel électrostatique à la surface de cette poche, passant d'un potentiel neutre à un potentiel électronégatif. Ce changement de propriété physico-chimique de la poche du transporteur est probablement un déterminant majeur de l'acquisition de la propriété de transport de la CQ di-protonée de la vacuole digestive vers le cytoplasme du parasite. Les sites fonctionnels candidats de PfCRT et PfK13 identifiés doivent maintenant être validés par des approches expérimentales. Nous avons initié des approches biochimiques dites de pull-down différentiels pour le domaine KREP de PfK13. Dans un premier temps, certains domaines PfK13, fusionnés à la glutathion S-transférase (GST), ont été exprimés dans la bactérie Escherichia coli, purifiés, puis incubés avec un lysat parasitaire total afin de caractériser des protéines interagissant avec PfK13. Les mises au point de ces expériences se poursuivent. Dans un second temps, des approches génétiques directement chez le parasite, par des techniques de transfection et d'édition de gènes, seront mises en place pour tester l'importance des sites fonctionnels candidats

Étude bioinformatique des protéines PfK13 et PfCRT impliquées dans la résistance de Plasmodium falciparum aux antipaludiques

Malaria is an infectious parasitic disease caused by Plasmodium species. P. falciparum is the most prevalent species and is responsible for the majority of the disease burden. Currently, malaria is treated using artemisinin-based combination therapies (ACTs), coupling an artemisinin derivative (ARTD) to another antimalarial drug. After a decade of use, ARTD-resistant parasites have emerged in Southeast Asia; they are currently not found in Africa. Resistance to ARTD is defined as parasites exhibiting in vivo a delayed clearance time following an artemisinin-based treatment, and is conferred by non-synonymous mutations localized in the Kelch-repeat propeller (KREP) domain of the P. falciparum K13 protein (PfK13). Similarly, multiple non-synonymous mutations conferring resistance to chloroquine (CQ) or piperaquine (PPQ, used as partner drug in one ACT) have been reported on the P. falciparum chloroquine resistance transporter (PfCRT). The physiological role of these proteins, essential at least for the intra-erythrocytic development of the parasite, remains poorly understood. This thesis aims to better characterize the PfK13 and PfCRT proteins by: i) predicting the protein regions and positions that would be involved in protein-substrate interactions; and ii) studying structural and/or physicochemical alterations induced by resistance mutations. According to concepts related to the theory of molecular evolution, mutations affecting sites exerting a critical function within an essential protein are eliminated by purifying selection. These sites are therefore more conserved than the rest of the protein. Using bioinformatics approaches combining sets of PfK13 and PfCRT orthologous sequences - respectively at Apicomplexa and Plasmodium scales - and information from protein tertiary structures, we highlighted extremely conserved regions within these two proteins. By comparing these results with those obtained for some proteins / domains belonging to the same structural families and validated at the experimental level, we identified several PfK13 and PfCRT amino acid sites that we propose as candidates for protein-substrate interactions. Remarkably, ARTD resistance mutations are not localized at the interaction surface that we predicted in the KREP domain of PfK13. Molecular dynamics simulations carried out on KREP domains carrying resistance mutations (C580Y and R539T) indicated that these mutations are associated with local structural destabilizations of the KREP domain. We assume that these resistance mutations may disrupt the stability of the KREP domain and, by extension, the whole PfK13 protein whose cellular abundance would be decreased. Regarding PfCRT, using the two tertiary structure models we predicted by structural homology, the majority of CQ and PPQ resistance mutations were found located in a binding pocket at the core of the transporter. Furthermore, these mutations strongly altered the electrostatic potential at the surface of this pocket, from a neutral to an electronegative potential. This change in physicochemical property of the pocket is probably a major determinant in the acquisition of the transport property of di-protonated CQ from the digestive vacuole to the cytoplasm of the parasite. Candidate functional sites of PfCRT and PfK13 identified in our work should be validated by experimental approaches. Here, we initiated differential pull-down biochemical experiments for the KREP domain of PfK13. First, the wild-type PfK13 domains, fused to glutathione S-transferase (GST), were expressed in Escherichia coli, purified, and then incubated with parasite lysate to characterize PfK13-interacting proteins. These experiments are still in progress. In a second step, the importance of candidate functional sites will be tested through genetic approaches directly in P. falciparum parasites using transfection and gene editing techniques.Le paludisme est une maladie infectieuse parasitaire causée par diverses espèces de Plasmodium, P. falciparum étant l'espèce la plus répandue et responsable des cas mortels de la maladie. Les traitements actuels reposent sur des combinaisons thérapeutiques à base d'artémisinine (CTA), couplant un dérivé d'artémisinine (ARTD) à une autre molécule antipaludique. Des parasites résistants aux ARTDs ont émergé en Asie du sud-est ; ceux-ci sont encore non détectés en Afrique. La résistance se traduit par une durée d'élimination des parasites allongée, et est conférée par des mutations non-synonymes localisées sur le domaine Kelch-repeat propeller (KREP) de la protéine P. falciparum K13 (PfK13). Similairement, de multiples mutations non-synonymes sur le transporteur P. falciparum Chloroquine Resistance Transporter (PfCRT) confèrent la résistance à la chloroquine (CQ, ancien traitement) et à la pipéraquine (PPQ, une des molécules partenaires dans les CTAs actuels). Le rôle physiologique de ces protéines, essentielles durant le développement du parasite, demeure mal connu. Ce travail de thèse a donc pour objectif de mieux caractériser PfK13 et PfCRT : i) en prédisant les positions qui seraient impliquées dans des interactions protéine-substrat ; et ii) en étudiant les altérations structurales et physico-chimiques induites par les mutations de résistance. Selon les concepts liés à la théorie de l'évolution moléculaire, les mutations touchant des sites exerçant une fonction critique au sein d'une protéine essentielle sont éliminées par la sélection purificatrice. Ces sites sont donc plus conservés que le reste de la protéine. Par des approches bioinformatiques couplant évolution et structure tertiaire, nous avons mis en évidence des régions extrêmement conservées au sein de PfK13 et PfCRT. En comparant ces résultats avec les données expérimentales de protéines / domaines appartenant aux mêmes familles structurales, nous avons identifié plusieurs sites de PfK13 et PfCRT que nous proposons comme candidats pour des interactions protéine-substrat. À notre surprise, les mutations de résistance aux ARTDs ne sont pas localisées à la surface d'interaction que nous avons prédite sur le domaine KREP de PfK13. Les dynamiques moléculaires que nous avons réalisées sur deux mutations de résistance (C580Y et R539T) ont révélé des déstabilisations structurales locales du domaine KREP. Nous supposons que ces mutations pourraient perturber la stabilité du domaine KREP et ainsi diminuer l'abondance cellulaire de PfK13. Concernant PfCRT, les deux modèles de structure tertiaire, prédits par homologie structurale, montrent que la majorité des mutations de résistance à la CQ et à la PPQ sont localisées au niveau d'une poche probable de liaison que nous avons identifiée au coeur du transporteur. Ces mutations altèrent fortement le potentiel électrostatique à la surface de cette poche, passant d'un potentiel neutre à un potentiel électronégatif. Ce changement de propriété physico-chimique de la poche du transporteur est probablement un déterminant majeur de l'acquisition de la propriété de transport de la CQ di-protonée de la vacuole digestive vers le cytoplasme du parasite. Les sites fonctionnels candidats de PfCRT et PfK13 identifiés doivent maintenant être validés par des approches expérimentales. Nous avons initié des approches biochimiques dites de pull-down différentiels pour le domaine KREP de PfK13. Dans un premier temps, certains domaines PfK13, fusionnés à la glutathion S-transférase (GST), ont été exprimés dans la bactérie Escherichia coli, purifiés, puis incubés avec un lysat parasitaire total afin de caractériser des protéines interagissant avec PfK13. Les mises au point de ces expériences se poursuivent. Dans un second temps, des approches génétiques directement chez le parasite, par des techniques de transfection et d'édition de gènes, seront mises en place pour tester l'importance des sites fonctionnels candidats

Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance

International audiencePartial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13

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

Abstract Background Whole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. 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 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm 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 µm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS. Methods Whole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitaemia 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 parasitaemia range: 0.02–1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated 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, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples. Results After applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4096- to 65,536-fold loss of human template DNA (human β actin gene). WGS analyses revealed that > 95% of the  parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×). Conclusions The novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%

Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

International audienceSporozoite forms of the Plasmodium parasite, the causative agent of malaria, 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 hepatocytes. 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

Comparison of biofilm formation and motility processes in arsenic-resistant Thiomonas spp. strains revealed divergent response to arsenite

International audienceBacteria of the genus Thiomonas are found ubiquitouslyin arsenic contaminated waters such as acidmine drainage (AMD), where they contribute to theprecipitation and the natural bioremediation ofarsenic. In these environments, these bacteria havedeveloped a large range of resistance strategiesamong which the capacity to form particular biofilmstructures. The biofilm formation is one of the mostubiquitous adaptive response observed in prokaryotesto various stresses, such as those induced inthe presence of toxic compounds. This studyfocused on the process of biofilm formation in threeThiomonas strains (CB1, CB2 and CB3) isolatedfrom the same AMD. The results obtained hereshow that these bacteria are all capable of formingbiofilms, but the architecture and the kinetics of formationof these biofilms differ depending onwhether arsenite is present in the environment andfrom one strain to another. Indeed, two strainsfavoured biofilm formation, whereas one favouredmotility in the presence of arsenite. To identify theunderlying mechanisms, the patterns of expressionof some genes possibly involved in the process of biofilm formation were investigated in Thiomonassp. CB2 in the presence and absence of arsenite,using a transcriptomic approach (RNA-seq). Thefindings obtained here shed interesting light onhow the formation of biofilms, and the motility processescontribute to the adaptation of Thiomonasstrains to extreme environments

Genomic diversity of mpox virus in Paris area (France) during the 2022 outbreak

International audienceAbstract In May 2022, several countries reported mpox cases from patients without history of traveling to endemic areas. France was one of the most affected European countries by this outbreak. In this study, the clinical characteristics of mpox cases in France were described, and the genetic diversity of the virus was studied. Patients diagnosed with mpox infection (quantitative polymerase chain reaction c t < 28) between May 21, and July 4, 2022 and between 16th August and 10th September 2022 were included to this study. Twelve amplicons corresponding to the most polymorphic regions of the mpox genome and covering ~30 000 nucleotides were generated and sequenced using the S5 XL Ion Torrent technology to evaluate the genetic diversity of mpox sequences. One hundred and forty‐eight patients were diagnosed with mpox‐infection. 95% were men, 5% transgender (M‐to‐F), 50% were taking human immunodeficiency virus (HIV) pre‐exposure prophylaxis, and 25% were HIV seropositive. One hundred and sixty‐two samples (some patients had two samples) were sequenced and compared to GenBank sequences. Overall, low genetic diversity of mpox sequences was found compared with pre‐epidemic Western‐African sequences, with 32 distinct mutational patterns. This study provides a first glance at the mutational landscape of early mpox 2022 circulating strains in Paris (France)

Pneumocystis Cytochrome b Mutants Associated With Atovaquone Prophylaxis Failure as the Cause of Pneumocystis Infection Outbreak Among Heart Transplant Recipients

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From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases

International audienceBackground PfEMP1 is the major protein from parasitic origin involved in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability is endless and current publicly available protein repositories do not reflect the high diversity of the sequences of PfEMP1 proteins. The identification of PfEMP1 protein sequences expressed with samples remains challenging. The aim of our study is to identify the different PfEMP1 proteins variants expressed within patient samples, and therefore identify PfEMP1 proteins domains expressed by patients presenting uncomplicated malaria or severe malaria in malaria endemic setting in Cotonou, Benin. Methods We performed a multi-omic approach to decipher PfEMP1 expression at the patient's level in different clinical settings. Using a combination of whole genome sequencing approach and RNA sequencing, we were able to identify new PfEMP1 sequences and created a new custom protein database. This database was used for protein identification in mass spectrometry analysis. Results The differential expression analysis of RNAsequencing data shows an increased expression of the var domains transcripts DBL alpha 1.7, DBL alpha 1.1, DBL alpha 2 and DBL beta 12 in samples from patients suffering from Cerebral Malaria compared to Uncomplicated Malaria. Our approach allowed us to attribute PfEMP1 sequences to each sample and identify new peptides associated to PfEMP1 proteins in mass spectrometry. Conclusion We highlighted the diversity of the PfEMP1 sequences from field sample compared to reference sequences repositories and confirmed the validity of our approach. These findings should contribute to further vaccine development strategies based on PfEMP1 proteins