Article

Malaria-Infected Erythrocyte-Derived Microvesicles Mediate Cellular Communication within the Parasite Population and with the Host Immune System

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Abstract

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system.

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... EVs from iRBCs have also been shown to contain a functional microRNA-argonaute 2 complex that can modulate gene expression and alter barrier function [52,53] when transferred to endothelial cells after vesicle uptake. Such EVs do not only affect endothelial cells but are also able to induce pro-inflammatory responses, particularly the activation of macrophages, monocytes as well as other immune cells through the upregulation of cytokines [54,55] . Interestingly, when these EVs were compared to their mother cells, they were able to activate inflammation and immune activation to a greater degree [54,55] . ...
... Such EVs do not only affect endothelial cells but are also able to induce pro-inflammatory responses, particularly the activation of macrophages, monocytes as well as other immune cells through the upregulation of cytokines [54,55] . Interestingly, when these EVs were compared to their mother cells, they were able to activate inflammation and immune activation to a greater degree [54,55] . EVs from iRBCs also contain small RNAs and genomic DNA. ...
... [49] . (2) EVs from iRBCs have been suggested to act not only as a decoy, by providing alternative targets expressing PfEMP-1 for immune cells to attack [51] , but also promote secretion of increased levels of pro-inflammatory cytokines, notably IFN-γ, LT-α and IL-12 once internalised by immune cells [54,55] . These cytokines have also been shown to stimulate astrocytes, which then respond by secreting additional cytokines and chemokines of their own [13] . ...
Article
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Malaria infections due to the Plasmodium parasite remains a major global health problem. Plasmodium falciparum is responsible for majority of the severe cases, resulting in more than 400,000 deaths per annum. Extracellular vesicles (EVs) released by vascular cells, including parasitised erythrocytes, have been detected with increased levels in patients with malaria. EVs are thought to be involved in the pathogenesis of severe malaria, particularly cerebral malaria, and represent a unique molecular signature for different forms of the infection. In this review, we will cover the known effects of EVs on the vasculature and discuss their potential use as a biomarker of disease severity.
... In vitro studies demonstrated the role of RBC EVs in intercellular communication between parasite populations. Parasite genetic material is transferred via RBC EVs and internalized into iRBCs, influencing the transmission stage of parasites [25,29]. Obviously, parasite growth in vitro has limited maximum growth capacity. ...
... EVs play a key role in intercellular communication by transferring bioactive molecules such as proteins and genetic materials from parasites to host cells and parasites to parasites, thereby modifying the properties of recipient cells [25,27,34,50,[54][55][56]. EVs in plasma have several cellular origins. ...
... For example, MSP-1 (an essential protein in the parasite's initial contact), proteins involved in the apical orientation step (EBA-175, PfRh1, PfRh2a, PfRh2b, and PfRh3), and AMA1 (a crucial protein in tight junction formation) were all found. Moreover, these parasite proteins are common in RBC EVs derived from P. falciparum culture supernatants [25,27,30,50,56,63]. Other interesting parasite proteins that were identified by proteomics include two proteins linked to the iRBC surface membrane, RESA and KAHRP. ...
Article
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In infectious diseases, extracellular vesicles (EVs) released from a pathogen or pathogen-infected cells can transfer pathogen-derived biomolecules, especially proteins, to target cells and consequently regulate these target cells. For example, malaria is an important tropical infectious disease caused by Plasmodium spp. Previous studies have identified the roles of Plasmodium falciparum-infected red blood cell-derived EVs (Pf-EVs) in the pathogenesis, activation, and modulation of host immune responses. This study investigated the proteomic profiles of Pf-EVs isolated from four P. falciparum strains. We also compared the proteomes of EVs from (i) different EV types (microvesicles and exosomes) and (ii) different parasite growth stages (early- and late-stage). The proteomic analyses revealed that the human proteins carried in the Pf-EVs were specific to the type of Pf-EVs. By contrast, most of the P. falciparum proteins carried in Pf-EVs were common across all types of Pf-EVs. As the proteomics results revealed that Pf-EVs contained invasion-associated proteins, the effect of Pf-EVs on parasite invasion was also investigated. Surprisingly, the attenuation of parasite invasion efficiency was found with the addition of Pf-MVs. Moreover, this effect was markedly increased in culture-adapted isolates compared with laboratory reference strains. Our evidence supports the concept that Pf-EVs play a role in quorum sensing, which leads to parasite growth-density regulation.
... Another consequence of P. falciparum infection is the production of plasma membrane vesicles known as microparticles (MPs) [17][18][19]. P. falciparum infection has been shown to induce a strong production of MPs from i-RBCs, a feature also observed when RBCs are exposed to bacterial toxins [20] and other stressors in pathological contexts [21,22]. Although MPs from leukocytes, platelets, RBCs, and endothelial cells are involved in thrombosis, hemostasis, and inflammation [23,24], the potential role of RBCs derived MPs on eATP regulation has not been studied so far. ...
... Recent studies have reported enhanced production of MPs both in the circulation of malaria patients and in i-RBCs cultures. MPs are important not only for cell-cell communication [17][18][19]57,58] but also contribute to vascular dysfunction during malaria infection [59]. Coinciding with previous reports, we show that the production of MPs was also significantly enhanced during infection. ...
... As reported before for RBCs exposed to various stimuli causing MP production [17,18,57], the membranes of the resulting particles will not necessarily display an identical composition to that of the originating RBCs. Nevertheless, to evaluate the contribution of MPs to eATP regulation we checked whether MPs membranes exhibited ectoATPase activity. ...
Article
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Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs). ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space. Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases. RBCs were cultured with P. falciparum for 24–48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs. Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP. Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria. The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.
... In-vitro studies of P. falciparum demonstrated the active uptake of P. falciparum-infected RBC derived EVs (pf-iREVs) by monocyte-derived macrophages. The pf-iREVs not only had a potent proinflammatory effect on macrophages, they also induced the expression of anti-inflammatory cytokines [100]. Mantel and colleagues showed that, while pf-iREVs are also capable of activating neutrophils, human monocytes are the main target immune cells [100]. ...
... The pf-iREVs not only had a potent proinflammatory effect on macrophages, they also induced the expression of anti-inflammatory cytokines [100]. Mantel and colleagues showed that, while pf-iREVs are also capable of activating neutrophils, human monocytes are the main target immune cells [100]. ...
... Microglia are the resident macrophages of the brain. Hence, this finding that pf-iREVs induce an immunomodulatory response in microglia is in line with that of Mantel and colleagues [100]. An imbalance in the inflammatory response that tips the scale toward excessive production of pro-inflammatory cytokines constitutes neuroinflammation [15]. ...
Article
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Malaria is a complex parasitic disease, caused by Plasmodium spp. More than a century after the discovery of malaria parasites, this disease continues to pose a global public health problem and the pathogenesis of the severe forms of malaria remains incompletely understood. Extracellular vesicles (EVs), including exosomes and microvesicles, have been increasingly researched in the field of malaria in a bid to fill these knowledge gaps. EVs released from Plasmodium -infected red blood cells and other host cells during malaria infection are now believed to play key roles in disease pathogenesis and are suggested as vital components of the biology of Plasmodium spp. Malaria-derived EVs have been identified as potential disease biomarkers and therapeutic tools. In this review, key findings of malaria EV studies over the last 20 years are summarized and critically analysed. Outstanding areas of research into EV biology are identified. Unexplored EV research foci for the future that will contribute to consolidating the potential for EVs as agents in malaria prevention and control are proposed.
... Malaria parasites, including P. falciparum and P. vivax, use EV to manipulate their host's immune response and to send genetic material to neighboring infected red blood cells (iRBC) [2][3][4][5][6]. In particular, it has been demonstrated that EV derived from P. falciparum-infected RBCs (Pf-EV) promote differentiation to gametocyte stages, which increases parasite survival within the host and the probability of transmission to the mosquito vectors [7][8][9]. Moreover, DNA from Pf-EV activates host cytosolic DNA sensors that in turn modify monocyte gene expression and chemokine production [10]. ...
... Different methods have been applied to isolate EV from cells, and have been systematized according to the Minimal Information for studies of extracellular vesicles 2018 (MISEV018) [16]. Pf-EV can be purified by differential centrifugation [9,14], size-exclusion chromatography [5], density gradient flotation [7][8][9][10], or a combination of these methods [9] to remove cellular debris and concentrate the nanoparticles present in the samples. In our experience, differential centrifugation is the procedure that delivers the highest yield of Pf-EV from a relatively low volume of cultured cells. ...
... Different methods have been applied to isolate EV from cells, and have been systematized according to the Minimal Information for studies of extracellular vesicles 2018 (MISEV018) [16]. Pf-EV can be purified by differential centrifugation [9,14], size-exclusion chromatography [5], density gradient flotation [7][8][9][10], or a combination of these methods [9] to remove cellular debris and concentrate the nanoparticles present in the samples. In our experience, differential centrifugation is the procedure that delivers the highest yield of Pf-EV from a relatively low volume of cultured cells. ...
Chapter
Malaria is one the most devastating infectious diseases in the world: of the five malaria-associated parasites, Plasmodium falciparum and P. vivax are the most pathogenic and widespread, respectively. P. falciparum invades human red blood cells (RBCs), releasing extracellular vesicles (Pf-EV) carrying DNA, RNA and protein cargo components involved in host-pathogen communications in the course of the disease. Different strategies have been used to analyze Pf-EV biophysically and chemically. Atomic force microscopy (AFM) stands out as a powerful tool for rendering high quality images of extracellular vesicles. In this technique, a sharp tip attached to a cantilever reconstructs the topographic surface of the extracellular vesicles and probes their nano-mechanical properties based on force–distance curves. Here, we describe a method to separate Pf-EV using differential ultracentrifugation, followed by nanoparticle tracking analysis (NTA) to quantify and estimate the size distribution. Finally, the AFM imaging procedure on Pf-EV adsorbed on a Mg2+-modified mica surface is detailed.
... Malaria-infected RBCs secrete EVs containing parasitederived DNA, RNA and protein cargo (Sisquella et al, 2017;Toda et al, 2020;Avalos-Padilla et al, 2021;Dekel et al, 2021;. These secreted EVs have been implicated in host pathogen crosstalk with cells of the immune system (Mantel et al, 2013;Sisquella et al, 2017;Sampaio et al, 2018;Ye et al, 2018;Demarta-Gatsi et al, 2019;Ofir-Birin et al, 2021), in parasite-parasite communication (Mantel et al, 2013;Regev-Rudzki et al, 2013), in host RBC priming (Dekel et al, 2021) and in erythropoiesis (Neveu et al, 2020). Mature RBCs lack internal organelles, endocytosis and exocytosis machinery (Moras et al, 2017), indeed it has been demonstrated that the Plasmodium parasite utilizes its own protein network to release EVs (Regev-Rudzki et al, 2013;Avalos-Padilla et al, 2021). ...
... Malaria-infected RBCs secrete EVs containing parasitederived DNA, RNA and protein cargo (Sisquella et al, 2017;Toda et al, 2020;Avalos-Padilla et al, 2021;Dekel et al, 2021;. These secreted EVs have been implicated in host pathogen crosstalk with cells of the immune system (Mantel et al, 2013;Sisquella et al, 2017;Sampaio et al, 2018;Ye et al, 2018;Demarta-Gatsi et al, 2019;Ofir-Birin et al, 2021), in parasite-parasite communication (Mantel et al, 2013;Regev-Rudzki et al, 2013), in host RBC priming (Dekel et al, 2021) and in erythropoiesis (Neveu et al, 2020). Mature RBCs lack internal organelles, endocytosis and exocytosis machinery (Moras et al, 2017), indeed it has been demonstrated that the Plasmodium parasite utilizes its own protein network to release EVs (Regev-Rudzki et al, 2013;Avalos-Padilla et al, 2021). ...
Article
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Malaria is the most serious mosquito-borne parasitic disease, caused mainly by the intracellular parasite Plasmodium falciparum. The parasite invades human red blood cells and releases extracellular vesicles (EVs) to alter its host responses. It becomes clear that EVs are generally composed of sub-populations. Seeking to identify EV subpopulations, we subject malaria-derived EVs to size-separation analysis, using asymmetric flow field-flow fractionation. Multi-technique analysis reveals surprising characteristics: we identify two distinct EV subpopulations differing in size and protein content. Small EVs are enriched in complement-system proteins and large EVs in proteasome subunits. We then measure the membrane fusion abilities of each subpopulation with three types of host cellular membranes: plasma, late and early endosome. Remarkably, small EVs fuse to early endosome liposomes at significantly greater levels than large EVs. Atomic force microscope imaging combined with machine-learning methods further emphasizes the difference in biophysical properties between the two subpopulations. These results shed light on the sophisticated mechanism by which malaria parasites utilize EV subpopulations as a communication tool to target different cellular destinations or host systems.
... Mass spectrometric analysis of extracellular microvesicles (MVs) derived from a P. falciparum-infected Kenyan child identified about 153 enriched parasite proteins associated with cytoadhesion, virulence, and host cell remodeling [79]. This result significantly overlaps with another proteomic study which reported 84 parasite proteins modulating the host-parasite and parasite-parasite interactions [80]. These proteins can act as possible biomarkers for MVs released by the P. falciparum-infected RBCs. ...
... Enriched host and parasite proteins associated with cytoadhesion, and virulence [80] Blood transcriptomics study in Gabonese CM patients Microarray hybridization, real-time PCR ...
Article
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Background Cerebral malaria is often pronounced as a major life-threatening neurological complication of Plasmodium falciparum infection. The complex pathogenic landscape of the parasite and the associated neurological complications are still not elucidated properly. The growing concerns of drugresistant parasite strains along with the failure of anti-malarial drugs to subdue post-recovery neuro-cognitive dysfunctions in cerebral malaria patients have called for a demand to explore novel biomarkers and therapeutic avenues. Due course of the brain infection journey of the parasite, events such as sequestration of infected RBCs, cytoadherence, inflammation, endothelial activation, and blood–brain barrier disruption are considered critical. Methods In this review, we briefly summarize the diverse pathogenesis of the brain-invading parasite associated with loss of the blood-brain barrier integrity. In addition, we also discuss proteomics, transcriptomics, and bioinformatics strategies to identify an array of new biomarkers and drug candidates. Conclusion A proper understanding of the parasite biology and mechanism of barrier disruption coupled with emerging state-of-art therapeutic approaches could be helpful to tackle cerebral malaria.
... Likewise, during infection by the malaria parasite Plasmodium falciparum, EVs purified from IRBCs could transfer drug-resistance plasmids into drug sensitive strains and increase survival of the parasite under drug pressure (Regev-Rudzki et al., 2013; Figure 5). In addition, IRBC-derived EVs could regulate the transmission stage production of parasites in vivo (Mantel et al., 2013; Figure 5). The EVs could promote gametocyte formation in the parasite population; gametocytes are the form of the parasite transmitted to the mosquito vector. ...
... The EVs could promote gametocyte formation in the parasite population; gametocytes are the form of the parasite transmitted to the mosquito vector. It was proposed that EVs released from IRBCs serve as a sensor for parasite density, signaling a switch to increased gametocyte formation (Mantel et al., 2013). ...
Article
Full-text available
Extracellular vesicles (EVs) represent a prominent mechanism of transport and interaction between cells, especially microbes. Increasing evidence indicates that EVs play a key role in the physiological and pathological processes of pathogens and other symbionts. Recent research has focused on the specific functions of these vesicles during pathogen-host interactions, including trans-kingdom delivery of small RNAs, proteins and metabolites. Much current research on the function of EVs is focused on immunity and the interactions of microbes with human cells, while the roles of EVs during plant-microbe interactions have recently emerged in importance. In this review, we summarize recent research on the biogenesis of these vesicles and their functions in biology and pathology. Many key questions remain unclear, including the full structural and functional diversity of EVs, the roles of EVs in communication among microbes within microbiomes, how specific cargoes are targeted to EVs, whether EVs are targeted to specific destinations, and the full scope of EVs’ transport of virulence effectors and of RNA and DNA molecules.
... The majority of what is known has come from extensive studies of the biology of the parasites' asexual blood stages and how infected RBCs (iRBCs) directly interact with various host cells, as orchestrated by P. falciparum virulence factors [4][5][6][7][8]. More recently, studies have shown that indirect interaction plays a vital role in the pathophysiology of malaria, whereby communication is not only between P. falciparum iRBCs and other host cells [9][10][11][12][13][14][15][16] but also between populations of P. falciparum [10,17]. These studies have been extensively and goat anti-rabbit (ab97080). ...
... The majority of what is known has come from extensive studies of the biology of the parasites' asexual blood stages and how infected RBCs (iRBCs) directly interact with various host cells, as orchestrated by P. falciparum virulence factors [4][5][6][7][8]. More recently, studies have shown that indirect interaction plays a vital role in the pathophysiology of malaria, whereby communication is not only between P. falciparum iRBCs and other host cells [9][10][11][12][13][14][15][16] but also between populations of P. falciparum [10,17]. These studies have been extensively and goat anti-rabbit (ab97080). ...
Article
Full-text available
Malaria is caused by obligate intracellular parasites belonging to the genus Plasmodium. Red blood cells (RBCs) infected with different stages of Plasmodium spp. release extracellular vesicles (EVs). Extensive studies have recently shown that these EVs are involved in key aspects of the parasite’s biology and disease pathogenesis. However, they are yet to be fully characterized. The blood stages of Plasmodium spp., namely the rings, trophozoites and schizonts, are phenotypically distinct, hence, may induce the release of characteristically different EVs from infected RBCs. To gain insights into the biology and biogenesis of malaria EVs, it is important to characterize their biophysical and biochemical properties. By differential centrifugation, we isolated EVs from in vitro cultures of RBCs infected with different stages of Plasmodium falciparum. We performed a preliminary characterization of these EVs and observed that important EV markers were differentially expressed in EVs with different sedimentation properties as well as across EVs released from ring-, trophozoite- or schizont-infected RBCs. Our findings show that RBCs infected with different stages of malaria parasites release EVs with distinct protein expression profiles.
... They induce macrophage activation via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) pathways, resulting in an increase in CD40 expression and tumor necrosis factor alpha (TNF-α) secretion [55]. Blood-stage P. falciparum-derived MVs (Pf MVs) express parasite antigens capable of stimulating human peripheral blood mononuclear cells (PBMCs) and macrophages, upregulating pro-inflammatory cytokine secretion and activating neutrophil migration [20]. In P. vivaxinfected patients, circulating pMVs can adhere to human splenic cells by interacting with intercellular adhesion molecule-1 (ICAM-1) [56]. ...
... PMVs isolated from P. falciparumand P. vivax-infected patients have been shown to transport miRNA and parasite proteins [14,58]. Such vesicles can be internalized by iRBCs, resulting in the transfer of genetic information inside exosome-like vesicles, and these vesicles have been found to promote gametocytogenesis [20,59]. In addition, pMVs derived from human brain endothelial cells stimulate the proliferation/activation of T cells [60,61]. ...
Article
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Malaria, caused by Plasmodium species (spp.), is a deadly parasitic disease that results in approximately 400,000 deaths per year globally. Autophagy pathways play a fundamental role in the developmental stages of the parasite within the mammalian host. They are also involved in the production of Plasmodium-derived extracellular vesicles (EVs), which play an important role in the infection process, either by providing nutrients for parasite growth or by contributing to the immunopathophysiology of the disease. For example, during the hepatic stage, Plasmodium-derived EVs contribute to parasite virulence by modulating the host immune response. EVs help in evading the different autophagy mechanisms deployed by the host for parasite clearance. During cerebral malaria, on the other hand, parasite-derived EVs promote an astrocyte-mediated inflammatory response, through the induction of a non-conventional host autophagy pathway. In this review, we will discuss the cross-talk between Plasmodium-derived microvesicles and autophagy, and how it influences the outcome of infection.
... Many proteomic studies have demonstrated the release of virulence factors by various protozoan parasites into the host cell environment, suggesting that these proteins could play an important role for parasite survival in the host [20,21,[81][82][83][84][85][86][87][88][89][90][91]. Several studies have shown that Leishmania modifies the epigenetic state of the host cell upon invasion, but little is known about the virulence factors that regulate this subversion mechanism [92][93][94][95][96]. Interestingly, the four histones H2A, H2B, H3, and H4, and a nucleosome assembly protein (NAP1)-like protein have been identified among the secreted proteins in Leishmania, suggesting potential epigenetic-related mechanisms by which the parasite could subvert its host cell [20,[82][83][84]. ...
... The secretion of histones and other epigenetic proteins appears to be a widespread phenomenon in pathogenic parasites. The other four histones have also been detected as secreted proteins in various parasites such as Trypanosoma [21,87] and Plasmodium [81,88], as well as fungal pathogens such as Histoplasma and Cryptococcus [89,90], and the helminthic parasitic Echinococcus [91]. We can therefore speculate that some parasites may use stratagems targeting host chromatin by exploiting their secreted chromatin proteins, including the parasitic-human hybrid nucleosome strategy, to maximize their survival in the host. ...
Article
Full-text available
In eukaryotes, genomic DNA is bound with histone proteins and packaged into chromatin. The nucleosome, a fundamental unit of chromatin, regulates the accessibility of DNA to enzymes involved in gene regulation. During the past few years, structural analyses of chromatin architectures have been limited to evolutionarily related organisms. The amino acid sequences of histone proteins are highly conserved from humans to yeasts, but are divergent in the deeply branching protozoan groups, including human parasites that are directly related to human health. Certain large DNA viruses, as well as archaeal organisms, contain distant homologs of eukaryotic histone proteins. The divergent sequences give rise to unique and distinct nucleosome architectures, although the fundamental principles of histone folding and DNA contact are highly conserved. In this article, we review the structures and biophysical properties of nucleosomes containing histones from the human parasites Giardia lamblia and Leishmania major, and histone-like proteins from the Marseilleviridae amoeba virus family. The presented data confirm the sharing of the overall DNA compaction system among evolutionally distant species and clarify the deviations from the species-specific nature of the nucleosome.
... I. Homeostasis follows to recover erythrocytes. (Mantel et al., 2013). ...
... This also shows that parasites can sense their environment (Mantel et al., 2013;Regev-Rudzki et al., 2013). AP2-G is a major transcription factor that regulates gametogenesis, while the regulation of epigenetics is crucial for controlling sexual differentiation (Kafsack et al., 2014). ...
Thesis
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Evidence of Plasmodium resistance to some of the current antimalarial agents makes it imperative to search for newer, and effective drugs to combat this challenge, and combination therapy remains key in the quest to address this problem. Therefore, this study evaluated the interaction that exist when xylopic acid-artesunate and xylopic acid-amodiaquine are co-administered in managing malaria in mice. Antiplasmodial effect of xylopic acid (XA: 3, 10, 30, 100, 150 mg kg-1), artesunate (ART: 1, 2, 4, 8, 16 mg kg-1), and amodiaquine (AQ: 1.25, 2.5, 5, 10, 20 mg kg-1) were evaluated in mice infected with Plasmodium berghei ANKA and treated for 5 days with the respective drugs p.o. Using iterative curve-fitting of the log-dose responses, the respective ED50s for the three compounds were determined. XA and ART, and XA and AQ were subsequently administered in a fixed-dose combination of their ED50s (1:1) along with the combination fractions of (1/2, 1/4, 1/8, 1/16, and 1/32) to get the experimental ED50S (Zexp). An isobologram was constructed to determine the nature of interaction between xylopic acid —artesunate, and xylopic acid —amodiaquine combinations by comparing Zexp with the theoretical ED50 (Zadd). Also, the treatments ability to relieve other signs of malaria such as weight loss and pyrexia were assessed. All animals were continuously observed further after treatment in a 30-day survival test. ED50s for xylopic acid, artesunate, and amodiaquine were 9.0±3.2, 1.61±0.6, and 3.1±0.8 mg/kg. The Zadd and Zexp for xylopic acid and artesunate co-administration (XA—ART) was 5.3±2.61 and 1.98±0.25, respectively, with an interaction index of 0.37. The xylopic acid and amodiaquine combination therapy (XA—AQ) gave Zadd and Zexp of 6.05±2.0 and 1.69±0.42, respectively, with an interaction index of 0.28. The Zexp for both combination therapies lied significantly (p<0.001) below the additive isoboles showing that xylopic acid acts synergistically with both artesunate and amodiaquine in clearing the parasites. XA/AQ and the high dose XA/ART combination significantly (p<0.05) increased the survival days of infected mice and also reduced their weight loss.
... The generation of extracellular particles (EPs) (100 to 1,000 nm) by erythrocytes has been associated with the pathogenesis of human and rodent CM (32,33). Also, vesicles released from P. falciparum IE activate expression of proinflammatory cytokines (i.e., TNF and IFNβ) in innate immune cells (19,34). We asked whether EPs obtained from the serum of P. berghei-infected mice or from IE cell cultures can induce type I IFN signaling in primary cultures of BECs. ...
... IE-derived EPs were prepared from the supernatant of IE cultures. EPs were isolated adapting a previously described procedure (34) (Fig. 5A). Briefly, the cell suspension was first centrifuged at 600 × g to remove IEs and non-IEs and then centrifuged at 1,600 × g for 15 min to remove cell debris. ...
Article
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Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnβ1 transcription in the brain of mice infected with Plasmodium berghei ANKA ( Pba ). This STING1/IFNβ-mediated response increases brain CXCL10 governing the extent of brain leukocyte infiltration and blood–brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN–driven brain inflammation was demonstrated in brain endothelial–specific IFNβ-reporter and STING1-deficient Pba -infected mice, which were significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba -infected erythrocytes activated the STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNβ in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments, and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin, triggering brain endothelial inflammatory responses via the STING1/IFNβ/CXCL10 axis crucial to CM pathogenesis and lethality.
... Invasion of erythrocytes by merozoites occur as the parasite latches itself on the erythrocyte plasma membrane, initially invaginating then sealing to form a parasitophorous vacuole, which is both a secondary barrier for the parasite as well as a hub for communication and exchange with the host [58]. Indeed, malaria-infected red blood cells (iRBCs) have been demonstrated to quantitatively release EVs, particularly microvesicles, which can be transferred between parasites, enabling them to regulate formation of transmission stages [59]. ...
... tsRNAs, specifically those that are released in EVs, appear to play a common role in stage conversion across different protozoan parasite species. tsRNAcontaining EVs released by malaria-infected RBCs and E. histolytica were shown to guide the stage-transformation decision of parasite populations [59]. More interestingly, tsRNAs in EVs can contribute to intercellular communication among not only the same population but also across other species. ...
Article
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Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are newly identified non-coding small RNAs that have recently attracted attention due to their functional significance in both prokaryotes and eukaryotes. tsRNAs originated from the cleavage of precursor or mature tRNAs by specific nucleases. According to the start and end sites, tsRNAs can be broadly divided into tRNA halves (31–40 nucleotides) and tRNA-derived fragments (tRFs, 14–30 nucleotides). tsRNAs have been reported in multiple organisms to be involved in gene expression regulation, protein synthesis, and signal transduction. As a novel regulator, tsRNAs have also been identified in various protozoan parasites. The conserved biogenesis of tsRNAs in early-branching eukaryotes strongly suggests the universality of this machinery, which requires future research on their shared and potentially disparate biological functions. Here, we reviewed the recent studies of tsRNAs in several representative protozoan parasites including their biogenesis and the roles in parasite biology and intercellular communication. Furthermore, we discussed the remaining questions and potential future works for tsRNAs in this group of organisms.
... This reproductive restraint can be lifted under certain circumstances, resulting in greatly enhanced sexual commitment of parasites and conversion rates above 30% under specific in vitro conditions (Neveu et al., 2020). These include, but are not restricted to, high parasite densities (Bruce et al., 1990;Delves et al., 2016), exposure to P. falciparumconditioned (nutrient-depleted) medium (Williams, 1999;Dyer and Day, 2003;Fivelman et al., 2007;Brancucci et al., 2015), endoplasmic reticulum stress (Chaubey et al., 2014) or the uptake of extracellular vesicles derived from infected RBCs (iRBCs) (Mantel et al., 2013;Regev-Rudzki et al., 2013). These observations contributed to the appreciation that intraerythrocytic parasites are able to interact with and respond to their environment. ...
... n=3; error bars represent the standard error of the mean. Mantel et al., 2013;Regev-Rudzki et al., 2013;Chaubey et al., 2014;Brancucci et al., 2017;Portugaliza et al., 2020), the impact of therapeutic interventions on gametocyte production and malaria transmission is still a matter of debate. Differences in drugsusceptibility between P. falciparum asexual parasites and gametocytes, combined with the long period of sexual differentiation, render an evaluation of drug-induced effects on gametocytogenesis a challenging endeavor. ...
Article
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Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum , the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo . These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro . Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we did not observe evidence for mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro . Our data hence does not support scenarios in which currently applied therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.
... During their life-cycle, Pf parasites secrete EVs while growing inside human red blood cells (RBCs), which deliver multiple cargo components (Regev-Rudzki et al., 2013;Mantel et al., 2016;Sisquella et al., 2017;Ye et al., 2018;Avalos-Padilla et al., 2021;Dekel et al., 2021;Ofir-Birin et al., 2021). These EVs modulate different host cells by promoting parasitic invasion (Dekel et al., 2021), endothelial cell modulation (Mantel et al., 2016) and immune cell alteration (Mantel et al., 2013;Sisquella et al., 2017;Ye et al., 2018;Ofir-Birin et al., 2021). For instance, it was shown that Pf-derived EVs deliver parasitic genomic DNA (gDNA), recognized by the STING-dependent cytosolic DNA sensing pathway in recipient monocytes (Sisquella et al., 2017). ...
... In addition, it was demonstrated that uptake of Pfderived EVs into host primary Natural Killer (NK) cells serves as a delivery mode of parasitic RNA into the cytosol of these cells (Ye et al., 2018). Pf-derived EVs have also been demonstrated to be efficiently internalized by macrophages, inducing a strong inflammatory response, including activation of proinflammatory cytokines IL-6, IL-12 and IL-1b and the antiinflammatory cytokine IL-10 in a dose-dependent manner (Mantel et al., 2013). ...
Article
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Extracellular vesicles (EVs) are produced by across almost all the living kingdoms and play a crucial role in cell-cell communication processes. EVs are especially important for pathogens, as Plasmodium falciparum (Pf) parasite, the leading causing species in human malaria. Malaria parasites are able to modulate the host immune response from a distance via delivering diverse cargo components inside the EVs, such as proteins and nucleic acids. We have previously shown that imaging flow cytometry (IFC) can be effectively used to monitor the uptake of different cargo components of malaria derived EVs by host human monocytes. Here, we take this approach one step further and demonstrate that we can directly investigate the dynamics of the cargo distribution pattern over time by monitoring its distribution within two different recipient cells of the immune system, monocytes vs macrophages. By staining the RNA cargo of the vesicles and monitor the signal we were able to evaluate the kinetics of its delivery and measure different parameters of the cargo’s distribution post internalization. Interestingly, we found that while the level of the EV uptake is similar, the pattern of the signal for RNA cargo distribution is significantly different between these two recipient immune cells. Our results demonstrate that this method can be applied to study the distribution dynamics of the vesicle cargo post uptake to different types of cells. This can benefit significantly to our understanding of the fate of cargo components post vesicle internalization in the complex interface between pathogen-derived vesicles and their host recipient cells.
... Extracellular vesicles can also mediate transfer of DNA between infected red blood cells in P. falciparum co-cultures resulting in the transfer of drug-resistance genes [68]. Additionally, signalling through extracellular vesicles can promote differentiation of Plasmodium parasites to transmissible gametocyte stages in culture [69] such that, in combination, virulence or drug-resistance genes can be exchanged between strains in a co-infection, helping new variants to be generated and transmitted. More recently extracellular vesicles containing lactate dehydrogenase derived from density-stressed P. falciparum cultures were found to limit the growth of low-density parasite populations in vitro by triggering apoptotic events, suggesting an intercellular signalling mechanism that could regulate parasite population density [70]. ...
Article
Laboratory studies of pathogens aim to limit complexity in order to disentangle the important parameters contributing to an infection. However, pathogens rarely exist in isolation, and hosts may sustain co-infections with multiple disease agents. These interact with each other and with the host immune system dynamically, with disease outcomes affected by the composition of the community of infecting pathogens, their order of colonization, competition for niches and nutrients, and immune modulation. While pathogen-immune interactions have been detailed elsewhere, here we examine the use of ecological and experimental studies of trypanosome and malaria infections to discuss the interactions between pathogens in mammal hosts and arthropod vectors, including recently developed laboratory models for co-infection. The implications of pathogen co-infection for disease therapy are also discussed.
... Investigations into the various vesicle types, from both host and parasite origin, have revealed important roles for EVs in disease pathogenesis and susceptibility, as well as in cell-cell communication and immune responses (Marcilla et al., 2014;Robbins and Morelli, 2014;Sampaio et al., 2017). In malaria, the transfer of membrane and cytosolic proteins, lipids, DNA, and RNA through EVs modulate the immune response (Couper et al., 2010) and cellular communication between parasites including factors that triggers differentiation to transmission stages (Mantel et al., 2013;Regev-Rudzki et al., 2013). Importantly, for what we believe is the first time, the physiological role of EVs in malaria demonstrated that they facilitate intrasplenic infections in P. vivax , now known to represent the largest cryptic parasite biomass during chronic infections (Kho et al., 2021a;Kho et al., 2021b). ...
Article
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Plasmodium vivax is the most widely distributed human malaria parasite with 7 million annual clinical cases and 2.5 billion people living under risk of infection. There is an urgent need to discover new antigens for vaccination as only two vaccine candidates are currently in clinical trials. Extracellular vesicles (EVs) are small membrane-bound vesicles involved in intercellular communication and initially described in reticulocytes, the host cell of P. vivax , as a selective disposal mechanism of the transferrin receptor (CD71) in the maturation of reticulocytes to erythrocytes. We have recently reported the proteomics identification of P. vivax proteins associated to circulating EVs in P. vivax patients using size exclusion chromatography followed by mass spectrometry (MS). Parasite proteins were detected in only two out of ten patients. To increase the MS signal, we have implemented the direct immuno-affinity capture (DIC) technique to enrich in EVs derived from CD71-expressing cells. Remarkably, we identified parasite proteins in all patients totaling 48 proteins and including several previously identified P. vivax vaccine candidate antigens (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as membrane, cytosolic and exported proteins. Notably, a member of the Plasmodium helical interspersed sub-telomeric (PHIST-c) family and a member of the Plasmodium exported proteins, were detected in five out of six analyzed patients. Humoral immune response analysis using sera from vivax patients confirmed the antigenicity of the PHIST-c protein. Collectively, we showed that enrichment of EVs by CD71-DIC from plasma of patients, allows a robust identification of P. vivax immunogenic proteins. This study represents a significant advance in identifying new antigens for vaccination against this human malaria parasite.
... Human RBCEVs carrying α-synuclein isolated from patients with Parkinson's disease can cross the blood-brain barrier and impair glutamate uptake via an interaction between excitatory amino acid transporter 2 and oligomeric α-synuclein at astrocytic endfeet, leading to reduced synaptophysin levels in the striatum in a mouse model (158). In addition, increased numbers of RBCEVs in blood circulation can indicate hemolytic disorders such as autoimmune hemolytic anemia, complement-mediated hemolysis, malaria, and hereditary erythrocyte membrane disorders, whereas reduced counts were observed in Scott syndrome (cellular calcium abnormality) (21,(159)(160)(161)(162). RBCEVs containing miRNA are potential biomarkers for several specific diseases such as cancers, malaria, sickle cell anemia, multiple sclerosis, and diabetes (46). ...
Article
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Recently, red blood cell-derived extracellular vesicles (RBCEVs) have attracted attention for clinical applications because of their safety and biocompatibility. RBCEVs can escape macrophages through the binding of CD47 to inhibitory receptor signal regulatory protein α. Furthermore, genetic materials such as siRNA, miRNA, mRNA, or single-stranded RNA can be encapsulated within RBCEVs and then released into target cells for precise treatment. However, their side effects, half-lives, target cell specificity, and limited large-scale production under good manufacturing practice remain challenging. In this review, we summarized the biogenesis and composition of RBCEVs, discussed the advantages and disadvantages of RBCEVs for drug delivery compared with synthetic nanovesicles and non-red blood cell-derived EVs, and provided perspectives for overcoming current limitations to the use of RBCEVs for clinical applications.
... De plus, les monocytes ont la capacité de phagocyter des vésicules extracellulaires contenant des antigènes parasitaires libérés par les GRi. Ces dernières vont alors pouvoir modifier les propriétés des monocytes en stimulant notamment les réponses pro-inflammatoires et la production de TNFα(80,(307)(308)(309)(310).Les concentrations plasmatiques de cytokines pro-inflammatoires telles que l'IFNγ, le TNFα, l'IL1-β, ou l'IL-12 ont souvent été décrites comme étant augmentées lors d'infections palustres à P. falciparum. Comme décrit précédemment, l'IFNγ et l'IL-12 sont des cytokines clés de la réponse immunitaire précoce face au Plasmodium, produites entre autres par les lymphocytes (IFNγ) et les monocytes/macrophages (IL-12), et permettent notamment l'activation de cellules de immunité innée comme les neutrophiles et les cellules NK (138, 143). ...
Thesis
En 2020, le paludisme reste un problème majeur de santé publique. Malgré des avancées scientifiques capitales, il est encore responsable de plus de 400 000 décès chaque année, principalement chez les enfants en Afrique Sub-saharienne. Le neuropaludisme est l'une des formes les plus sévères du paludisme. Sa physiopathologie est complexe, impliquant l'adhésion des érythrocytes infectés par Plasmodium falciparum à l'endothélium vasculaire dans le cerveau. Ce mécanisme conduit à une obstruction du flux sanguin, une inflammation locale, une altération de la barrière hémato-encéphalique et à un éventail de réponses cellulaires visant à résoudre le processus de neuroinflammation. Parmi ces cellules, les monocytes/macrophages jouent un rôle clé de par leur capacité à s'adapter à leur microenvironnement en fonction des différents signaux qu'ils perçoivent, modulant ainsi les balances pro/anti-inflammatoire et pro/antioxydante, essentielles à la résolution du neuropaludisme. Les monocytes sont composés de trois sous-populations dont le rôle dans la physiopathologie du neuropaludisme a été peu étudié et reste à éclaircir. Pour compléter ces connaissances, une approche combinant modèle murin et études chez des patients béninois a été menée. Une première étude a été conduite chez des enfants béninois présentant un paludisme simple, une anémie sévère palustre, ou un neuropaludisme dans le but de déterminer si les paramètres monocytaires constituent des facteurs de sévérité et/ou de risque de décès au cours d'un paludisme grave. Les pourcentages et phénotypes des sous-populations monocytaires obtenus chez les trois groupes d'enfants lors de leur admission dans les centres de santé (J0) ont été comparés. Les résultats ont permis de mettre en lumière l'implication des monocytes non classiques dans la protection vis-à-vis des formes sévères de paludisme et de confirmer l'importance du récepteur CD36 des monocytes comme facteur protecteur de sévérité et de survenue de décès. Pour aller plus loin, un travail a ensuite été mené sur un modèle murin de neuropaludisme mis en place pour se rapprocher des conditions réelles, à savoir un traitement tardif, et débouchant sur une résolution de l'infection. Des souris C57BL/6 ont été infectées par P. berghei ANKA (J0) et traitées tardivement à la chloroquine. La cinétique d'évolution des sous-populations monocytaires a été suivie en termes de pourcentage et d'expression protéique et génique, à la fois dans le sang, la rate et le cerveau, jusqu'à la résolution de l'infection (J12). Les résultats suggèrent une implication des monocytes classiques et intermédiaires dans la neuroinflammation, au contraire des monocytes non classiques. A J12 était observé un nouvel afflux de monocytes intermédiaires vers le cerveau, suggérant également un rôle de cette sous-population dans la résolution tardive de la neuroinflammation. Les analyses protéique et génique ont mis en évidence une implication favorable de Nrf2, CD36, HO-1, CMH-II, COX-2 et de la 12/15-LOX dans la résolution de l'infection. Une deuxième étude a ensuite été menée chez des enfants béninois, avec un plus grand nombre de patients et un suivi à J3 et J30. Deux groupes de sujets ont été inclus, présentant un paludisme simple (n = 94) ou un neuropaludisme (n = 65). Dans l'ensemble, les résultats indiquent que les monocytes non classiques et intermédiaires étaient respectivement impliqués dans l'amélioration clinique et la survie des patients. L'expression de CD14, CD16, CD36 et HLA-DR était un facteur protecteur vis-à-vis du neuropaludisme. De manière intéressante, nos résultats montrent également une association entre une réponse pro-oxydante moins active, la sévérité du paludisme, et la survenue de décès. L'ensemble de ces résultats apporte un nouvel éclairage sur l'implication des monocytes/macrophages au cours du neuropaludisme.
... These nanovesicles initially described from reticulocytes [73,74], the host cell of P. vivax, are currently recognized by their remarkable role in intercellular communication in normal and pathological conditions [72] [75], including parasitic diseases [76]. Of note, their role as intercellular communicators in malaria was first described for P. falciparum where microvesicles and exosome-like vesicles of parasitic origin were reported to induce the formation of gametocytes [77,78]. It was also recently shown that EVs obtained from P. falciparum in vitro cultures delayed erythroid differentiation, thus allowing gametocyte maturation [79]. ...
Article
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Human malaria caused by Plasmodium vivax infection (vivax malaria) is a major global health issue. It is the most geographically widespread form of the disease, accounting for 7 million annual clinical cases, the majority of cases in America and Asia and an estimation of over 2.5 billion people living under risk of infection. The general perception towards vivax malaria has shifted recently, following a series of reports, from being viewed as a benign infection to the recognition of its potential for more severe manifestations including fatal cases. However, the underlying pathogenic mechanisms of vivax malaria remain largely unresolved. Asymptomatic carriers of malaria parasites are a major challenge for malaria elimination. In the case of P. vivax, it has been widely accepted that the only source of cryptic parasites is hypnozoite dormant stages. Here, we will review new evidence indicating that cryptic erythrocytic niches outside the liver, in particular in the spleen and bone marrow, can represent a major source of asymptomatic infections. The origin of such parasites is being controversial and many key gaps in the knowledge of such infections remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Last, we will glimpse into the role of reticulocyte-derived exosomes, extracellular vesicles of endocytic origin, as intercellular communicators likely involved in the formation of such cryptic erythrocytic infections.
... The stimuli that causes this adjustment is poorly understood. P. falciparum parasitized red blood cellderived microvesicles promote sexual differentiation (Mantel et al., 2013;Regev-Rudzki et al., 2013), however little is known about sensing and signaling pathways and factors that trigger this process. Similarly, there is evidence that the density of uninfected mosquito bites increases in at the start of transmission season (Paul et al., 2004). ...
Article
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Malaria parasites can adjust the proportion of parasites that develop into gametocytes, and thus the probability for human-to-vector transmission, through changes in the gametocyte conversion rate. Understanding the factors that impact the commitment of malaria parasites to transmission is required to design better control interventions. Plasmodium spp. persist across countries with vast differences in transmission intensities, and in sites where transmission is highly seasonal. Mounting evidence shows that Plasmodium spp. adjusts the investment in transmission according to seasonality of vector abundance, and transmission intensity. Various techniques to determine the investment in transmission are available, i.e., short-term culture, where the conversion rate can be measured most directly, genome and transcriptome studies, quantification of mature gametocytes, and mosquito feeding assays. In sites with seasonal transmission, the proportion of gametocytes, their densities and infectivity are higher during the wet season, when vectors are plentiful. When countries with pronounced differences in transmission intensity were compared, the investment in transmission was higher when transmission was low, thus maximizing the parasite’s chances to be transmitted to mosquitoes. Increased transmissibility of residual infections after a successful reduction of malaria transmission levels need to be considered when designing intervention measures.
... One study showed malaria-infected red blood cells use exosome-like vesicles to communicate which promotes differentiation to sexual forms [39]. Another study showed malaria antigens are enriched in microvesicles released from infected red blood cells which activate host monocytes and neutrophils [40]. Toxoplasma is acquired by ingestion of raw or uncooked meat and these parasites are common in virtually all species of warm-blooded vertebrates. ...
Chapter
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Extracellular vesicles (EVs) are carriers of various biomolecules including bioactive enzymes, lipids, proteins, nucleic acids, and metabolites. EVs are classified into three main types based on their size, biogenesis, and cargo. Exosomes originate from endosomal membranes and are the smallest type of EV. Microvesicles (MVs) or microparticles are larger in size, and like apoptotic bodies which represent the largest type of EVs, both of these vesicles originate from outward budding of the plasma membrane. As discussed in this chapter, cargo loading of EVs and their release into the extracellular space where they can be taken up by neighboring or distant cells plays an important role in physiology and pathophysiology. This chapter will outline specific mechanisms involved in the loading and enrichment of miRNAs, proteins, and lipids within EVs. As explained here, various external and biological stimuli play a role in EV release. Finally, recent studies have shown that the biogenesis, cargo loading, and release of EVs are governed by circadian rhythms. Although EVs were once thought to serve as garbage disposals of cells, the numerous roles they serve in physiology and pathophysiology are now being appreciated.
... These medium-or low-affinity antibodies are polyreactive, recognizing Pf and human antigens simultaneously [151]. The generation of autoantibodies has been associated with malaria, not only due to persistent infection, but to Pf extracellular vesicles as well as to various parasite molecules, which mimic host antigens [152][153][154][155][156]. This reflects the weakness of the tolerance mechanism to self-antigens in the host during malaria infection. ...
Article
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Malaria reflects not only a state of immune activation, but also a state of general immune defect or immunosuppression, of complex etiology that can last longer than the actual episode. Inhabitants of malaria-endemic regions with lifelong exposure to the parasite show an exhausted or immune regulatory profile compared to non- or minimally exposed subjects. Several studies and experiments to identify and characterize the cause of this malaria-related immunosuppression have shown that malaria suppresses humoral and cellular responses to both homologous (Plasmodium) and heterologous antigens (e.g., vaccines). However, neither the underlying mechanisms nor the relative involvement of different types of immune cells in immunosuppression during malaria is well understood. Moreover, the implication of the parasite during the different stages of the modulation of immunity has not been addressed in detail. There is growing evidence of a role of immune regulators and cellular components in malaria that may lead to immunosuppression that needs further research. In this review, we summarize the current evidence on how malaria parasites may directly and indirectly induce immunosuppression and investigate the potential role of specific cell types, effector molecules and other immunoregulatory factors.
... Plasmodium falciparum-infected RBCs release extracellular vesicles that contain RNAs, which could be transferred to endothelial cells where they regulate vascular function [98]. In addition, an in vitro experiment showed that the extracellular vesicles released by P. falciparum-infected RBCs could activate the innate immune response [99]. Thus, we can conclude that erythrocyte EVs, including microvesicles, ectosomes, and exosomes, activate lymphocyte immune reactions mainly through cytokines to increase T-cell proliferation and inhibit Figure 3: Red blood cell-derived exosome effects on immune cells. ...
Article
Sepsis is a health issue that affects millions of people worldwide. It was assumed that erythrocytes were affected by sepsis. However, in recent years, a number of studies have shown that erythrocytes affect sepsis as well. When a pathogen invades the human body, it infects the blood and organs, causing infection and sepsis-related symptoms. Pathogens change the internal environment, increasing the levels of reactive oxygen species, influencing erythrocyte morphology, and causing erythrocyte death, i.e., eryptosis. Characteristics of eryptosis include cell shrinkage, membrane blebbing, and surface exposure of phosphatidylserine (PS). Eryptotic erythrocytes increase immune cell proliferation, and through PS, attract macrophages that remove the infected erythrocytes. Erythrocyte-degraded hemoglobin derivatives and heme deteriorate infection; however, they could also be metabolized to a series of derivatives. The result that erythrocytes play an anti-infection role during sepsis provides new perspectives for treatment. This review focuses on erythrocytes during pathogenic infection and sepsis.
... Parasites appear to respond to metabolic challenges during growth, although most cellular mechanisms remain to be identified [25]. An increase in gametocytogenesis may be triggered by cellular stress involving redox perturbation [26,27], by adding low doses of the antimalarial artemisinin at the early trophozoite growth stage [28], or by adding parasiteconditioned culture medium [29] or extracellular vesicles from such medium [30]. Notably, gametocyte conversion rates in cultured lines are suppressed by lysophosphatidylcholine (LysoPC) within serum, or by adding choline as a supplement in serum-free medium [31,32]. ...
Preprint
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Asexual blood stage malaria parasites must produce sexual progeny in order to infect mosquitoes. It is therefore important to understand the scope of variation in sexual commitment rates of the most important human parasite Plasmodium falciparum , and to assess responses to conditions that may affect this commitment. First, using a genetically modified line with inducible elevated sexual commitment we compared two different methods of measuring the rates, either staining the Pfs16 marker in very early gametocytes or counting gametocytes after further development. The methods showed correlated estimates in evaluation across multiple independent replicate assays, with higher sensitivity and precision being achieved by Pfs16 marker detection in early gametocytes, so this method was used to survey a wide range of P. falciparum lines. A panel of six recently culture-established clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3.3% to 12.2%, with some significant differences among isolates as well as variation among biological replicates of each. Comparing 13 long-term laboratory-adapted lines of diverse origins, eight had sexual commitment rates similar to those of the recent clinical isolates, with means across multiple biological replicates for each line ranging from 4.7% to 14.5%, while four lines had significantly lower rates with means ranging from 0.3 to 1.6%, and one line with a non-functional ap2-g gene always showed zero sexual commitment. Among a subset of parasite lines, adding choline to suppress commitment had quantitatively variable effects, although these were significant in most assays of lines that had relatively high rates. This study indicates the importance of multiple assay replicates and comparisons of diverse isolates to understand natural and culture-induced variation in this key reproductive trait, relevant to investigating potential effects of parasite adaptation on transmission. Author Summary Only sexual malaria parasites are transmitted from humans to mosquitoes, so it is vital to understand variation in sexual commitment rates of blood stage malaria parasites, and responses to conditions that affect this. We compared two different methods of measuring the rates in a Plasmodium falciparum line with engineered sexual commitment variation, demonstrating higher sensitivity and precision by detection of an early differentiation marker, and this method was then used to survey diverse P. falciparum lines in culture with extensive biological replicate measurements. Recent clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3% to 12%, with significant differences among isolates as well as variation among biological replicates. Long-term laboratory-adapted lines of diverse origins had a wide range, most having rates similar to those of the clinical isolates, while a minority consistently had much lower or zero rates. There was quantitative variation among lines in the effects of adding choline to suppress commitment, although most assays of lines that had relatively high rates showed significant effects. Performing multiple biological replicates and comparisons of recent parasite isolates is vital to understand intra-specific variation in this important reproductive trait, and move towards investigating direct effects on disease transmission.
... Currently, the functions and potential applications of exosomes derived from host cells and pathogens in progression of CM diseases had evoked increasing interest (Sampaio et al., 2017;de Souza and Barrias, 2020;Debs et al., 2019). Previous studies had demonstrated that exosomes or exosome-like vesicles derived from erythrocytes (or reticulocytes) infected with P. falciparum had multiple biological functions, including enhancing parasite transmission (Mantel et al., 2013) and altering vascular function (Mantel et al., 2016). In addition, immunization with exosomes from a non-lethal strain of P. yoelii 17X-infected reticulocytes plus CpG ODN 1826 protected mice from a lethal strain of P. yoelii 17XL infection, as indicated by eliciting IgG2a and IgG2b antibodies, promoting host survival time and clearance of parasites (Martin-Jaular et al., 2011). ...
Article
Cerebral malaria (CM) is the most severe neurological complication caused by Plasmodium falciparum infection. The accumulating evidence demonstrated that mast cells (MCs) and its mediators played a critical role in mediating malaria severity. Earlier studies identified that exosomes were emerging as key mediators of intercellular communication and can be released from several kinds of MCs. However, the potential functions and pathological mechanisms of MCs-derived exosomes (MCs-Exo) impacting on CM pathogenesis remain largely unknown. Herein, we utilized an experimental CM (ECM) model (C57BL/6 mice infected with P. berghei ANKA strain), and then intravenously (i.v.) injected MCs-Exo into P. berghei ANKA-infected mice to unfold this mechanism and investigate the effect of MCs-Exo on ECM pathogenies. We also used an in vitro model by investigating the pathogenesis development of brain microvascular endothelial cells line (bEnd.3 cells) co-cultured with P. berghei ANKA blood-stage soluble antigen (PbAg) after MCs-Exo treatment. The higher numbers of MCs and levels of MCs degranulation were observed in skin, cervical lymph node, and brain of ECM mice than those of the uninfected mice. Exosomes were successfully isolated from culture supernatants of mouse MCs line (P815 cells) and characterized by spherical vesicles with the diameter of 30-150 nm, and expression of typical exosomal markers (e.g., CD9, CD63, and CD81). The i.v. injection of MCs-Exo dramatically elevated incidence of ECM in the P. berghei ANKA-infected mice, exacerbated liver and brain histopathological damage, promoted Th1 cytokine response, aggravated brain vascular endothelial activation and blood brain barrier breakdown in ECM mice. In addition, the treatment of MCs-Exo led to the decrease of cells viability and mRNA levels of Ang-1, ZO-1, and Claudin-5, but increase of mRNA levels of Ang-2, CCL2, CXCL1, and CXCL9 in bEnd.3 cells co-cultured with PbAg in vitro. Taken together, our data indicated that MCs-Exo could worsen pathogenesis of ECM in mice.
... In cellular microenvironments enriched in cytokines (as in the case of several inflammatory conditions), the RMVs are supposed to offer a substrate for the production of bioactive lipids (such as arachidonic acid and lysophosphatidic acid) through phosphatidylserine exposure, that have a negative impact on endothelial function and vascular integrity [181]. Additionally, in the malaria infection, engulfment of RMVs by macrophages or infected RBCs induces changes in their inflammatory phenotype and in the developmental stage of the parasite, respectively [82]. Finally, enrichment of RMVs from Parkinson disease patients in the toxic protein a-synuclein constitutes them as potential biomarkers of disease progression and classification [86,87,182]. ...
Article
Full-text available
Microvesicles or ectosomes represent a major type of extracellular vesicles that are formed by outward budding of the plasma membrane. Typically, they are bigger than exosomes but smaller than apoptotic vesicles, although they may overlap with both in size and content. Their release by cells is a means to dispose redundant, damaged, or dangerous material; to repair membrane lesions; and, primarily, to mediate intercellular communication. By participating in these vital activities, microvesicles may impact a wide array of cell processes and, consequently, changes in their concentration or components have been associated with several pathologies. Of note, microvesicles released by leukocytes, red blood cells, and platelets, which constitute the vast majority of plasma microvesicles, change under a plethora of diseases affecting not only the hematological, but also the nervous, cardiovascular, and urinary systems, among others. In fact, there is evidence that microvesicles released by blood cells are significant contributors towards pathophysiological states, having inflammatory and/or coagulation and/or immunomodulatory arms, by either promoting or inhibiting the relative disease phenotypes. Consequently, even though microvesicles are typically considered to have adverse links with disease prognosis, progression, or outcomes, not infrequently, they exert protective roles in the affected cells. Based on these functional relations, microvesicles might represent promising disease biomarkers with diagnostic, monitoring, and therapeutic applications, equally to the more thoroughly studied exosomes. In the current review, we provide a summary of the features of microvesicles released by blood cells and their potential implication in hematological and non-hematological diseases.
... After antimalaria treatment, these MV levels decreased rapidly (Nantakomol et al., 2011). In 2013, Mantel et al. (2013) found that MVs derived from erythrocytes infected with P. falciparum directly mediated communication between parasites through EVs. ...
Article
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Protozoan diseases seriously affect the health of human beings, livestock and poultry and lead to high economic and medical costs. Extracellular vesicles (EVs) are membranous structures formed through biological processes that play important roles in immune regulation. Studies have shown that parasites transmit information to hosts through EVs to modulate host immune responses. The major roles played by EVs released from parasites involve facilitating parasitization of the host. In this review, we discuss relevant recently obtained data on EVs secreted by different kinds of protozoa, including their molecular mechanisms, and discuss the roles played by EVs in the occurrence and development of parasitic diseases.
... They correspond to proteins targeted to the erythrocyte membrane and are transported through a vesicle-independent route involving chaperone-associated transport complexes known as J-dots. Once at the membrane, some proteins are packed into exosomes-like vesicles and released into de bloodstream, providing a mechanism for infected erythrocytes to communicate and modify the host immune response (Mantel et al., 2013;Regev-Rudzki et al., 2013). ...
Thesis
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Plasmodium is a genus of protozoan parasites causing malaria. The team demonstrated, in vitro, the import of exogenous tRNA and identified a potential transporter: tRip (tRNA import protein). In vitro, tRip binds all tRNAs by recognizing their structure. In vivo, tRip is transmembrane protein and its tRNA binding domain is exposed outside the parasite. tRip is not essential, but its deletion slows down parasite multiplication and protein synthesis. In addition to its role in tRNA import, tRip interacts with 3 aminoacyl-ARNt synthetases: glutamyl- (ERS), glutaminyl- (QRS) and methionyl-ARNt synthetase (MRS), suggesting that tRip allows the organization of a multi tRNA synthetase complex (MARS) localized at the membrane. During my thesis, I demonstrated that assembly of the complex is mediated by GST domains appended to the N-terminus of each partner. I identified, reconstituted and characterize in solution 2 distinct complexes: complex Q (tRip:ERS:QRS) and complex M (tRip:ERS:MRS). The resolution of the crystal structure of ERS GST domain as well as point mutation experiments allowed me to propose a model for these complexes MARS.
... In P. falciparum, rigidity of infected RBCs and their adherence to the capillary blood vessels was attributed, in part, to increased RBC phospholipid levels with increased ratio of unsaturated to saturated fatty acids. In addition, production of micro-vesicles blebs out of the lipid rafts of the cell membrane of infected RBCs promote cellular signaling for gametocyte development [49,50] . ...
... In the context of the parasitic diseases that these organisms cause, EVs are known to play a major role in intercellular communication between the parasite and the host. Importantly, EVs can modulate the host immune response, increase parasite invasion, and alter the integrity and function of cells and tissues, resulting in different disease outcomes [20,28,[30][31][32][33][34]. Moreover, the EV's capacity to mediate immune evasion through a broad type of mechanisms contributes to the exacerbation of infection [15]. ...
Article
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Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is a neglected tropical disease and a major public health problem affecting more than 6 million people worldwide. Many challenges remain in the quest to control Chagas disease: the diagnosis presents several limitations and the two available treatments cause several side effects, presenting limited efficacy during the chronic phase of the disease. In addition, there are no preventive vaccines or biomarkers of therapeutic response or disease outcome. Trypomastigote form and T. cruzi-infected cells release extracellular vesicles (EVs), which are involved in cell-to-cell communication and can modulate the host immune response. Importantly, EVs have been described as promising tools for the development of new therapeutic strategies, such as vaccines, and for the discovery of new biomarkers. Here, we review and discuss the role of EVs secreted during T. cruzi infection and their immunomodulatory properties. Finally, we briefly describe their potential for biomarker discovery and future perspectives as vaccine development tools for Chagas Disease.
... Indeed, this suggestion was based on previous findings reporting that P. falciparum can be transformed by the spontaneous take-up of erythrocytes' cytoplasmic DNA [22]. More interestingly, infected erythrocytes communicate via microvesicles and exosome-like vesicles, which promote differentiation to the sexual form of the parasite [23,24]. Additionally, exosome-like vesicles derived from erythrocytes upon infection with transgenic P. falciparum are able to deliver DNA encoding for a drug resistance marker and can support the growth of normal P. falciparum under conditions of drug selection [24]. ...
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Extracellular vesicle (EV) secretion is a highly conserved evolutionary trait in all organisms in the three domains of life. The packaging and release of EVs appears to be a bulk-flow process which takes place mainly under extreme conditions. EVs participate in horizontal gene transfer, which supports the survival of prokaryotic and eukaryotic microbes. In higher eukaryotes, almost all cells secrete a heterogeneous population of EVs loaded with various biomolecules. EV secretion is typically higher in cancer microenvironments, promoting tumor progression and metastasis. EVs are now recognized as additional mediators of autocrine and paracrine communication in health and disease. In this context, proteins and RNAs have been studied the most, but extracellular vesicle DNA (EV-DNA) has started to gain in importance in the last few years. In this review, we summarize new findings related to the loading mechanism(s), localization, and post-shedding function of EV-DNA. We also discuss the feasibility of using EV-DNA as a biomarker when performing a liquid biopsy, at the same time emphasizing the lack of data from clinical trials in this regard. Finally, we outline the potential of EV-DNA uptake and its interaction with the host genome as a promising tool for understanding the mechanisms of cancer evolution.
... Red blood cell (RBC)-derived EVs (RBC-EVs) are secreted during erythropoiesis, cellular aging, such as stored blood (Bosman et al., 2008;Kriebardis et al., 2008;Thangaraju et al., 2020), or in response to activated conditions, such as an increase of intracellular Ca 2+ or activation of protein kinase C (PKC) (Nguyen et al., 2016(Nguyen et al., , 2017Bernhardt et al., 2020), and disease conditions (Alaarg et al., 2013;Mantel et al., 2013;Leal et al., 2018). Under physiological and pathological conditions, RBC-EVs could be loaded with proteins, lipids, and miRNAs might be vital for homeostasis. ...
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Extracellular vesicles (EVs) are cell-derived membrane particles that include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies, and other EV subsets. EVs are involved in intercellular communication and the transport of macromolecules between cells. Here, we propose and test the ability of red blood cell (RBC)-derived EVs (RBC-EVs) as putative drug carriers. EVs were produced by treating RBCs with Phorbol-12-myristate-13-acetate (PMA) and separating from the cells by differential centrifugation steps. RBC-EVs were characterized by size determination, flow cytometry, and scanning electron microscopy (SEM). EVs were loaded with DNA plasmids coding for the green fluorescent protein (GFP) by electroporation. The DNA-loaded EVs (DNA-EVs) were used to transfect THP-1-derived macrophages and analyzed by fluorescence microscopy and flow cytometry. The results showed that RBC-EVs had an almost spherical shape and a polydispersity in their size with an average of 197 ± 44 nm and with a zeta potential of −36 ± 8 mV. RBC-EVs were successfully loaded with DNA but associated with an increase of the polydispersity index (PdI) and showed a positive signal with Picogreen. DNA-EVs were almost completely taken up by macrophages within 24 h, however, resulting in the expression of the GFP in a subpopulation of macrophages. As the way, we designed that RBC-EVs could be potential nucleic acid carriers when the immune system was addressed. This study may contribute to the understanding of the role of EVs in the development of microvesicle-based vehicles.
... To address the mechanism of malaria-induced APOBEC3A expression in B cells, we stimulated B cells from healthy donors with extracellular vesicles (EV) of P. falciparum-infected erythrocytes. Such vesicles display the same molecules on their surface as P. falciparum-infected erythrocytes and are known to activate immune cells in human and rodent malaria, thus, mimicking the interaction of B cells with infected erythrocytes in malaria patients [32][33][34][35]. Vesicles from noninfected erythrocytes served as a control. ...
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Endemic Burkitt lymphoma (eBL) is characterized by an oncogenic IGH/c-MYC translocation and Epstein-Barr virus (EBV) positivity, and epidemiologically linked to Plasmodium falciparum malaria. Both EBV and malaria are thought to contribute to eBL by inducing the expression of activation-induced cytidine deaminase (AID), an enzyme involved in the IGH/c-MYC translocation. AID/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) family enzymes have recently emerged as potent mutagenic source in a variety of cancers, but apart from AID, their involvement in eBL and their regulation by EBV and P. falciparum is unknown. Here, we show that upon inoculation with EBV, human B cells strongly upregulate the expression of enzymatically active APOBEC3B and APOBEC3G. In addition, we found significantly increased levels of APOBEC3A in B cells of malaria patients, which correlated with parasite load. Interestingly, despite the fact that APOBEC3A, APOBEC3B and APOBEC3G caused c-MYC mutations when overexpressed in HEK293T cells, a mutational enrichment in eBL tumors was only detected in AID motifs. This suggests that even though the EBV- and P. falciparum-directed immune response triggers the expression and activity of several AID/APOBEC members, only the upregulation of AID has oncogenic consequences, while the induction of the APOBEC3 subfamily may primarily have immunoprotective functions. This article is protected by copyright. All rights reserved.
... In addition, erythrocyte miRNAs play an essential role in communication between the IEs, endothelial and immune cells [239,240] (figure 5b). IEs produce EVs containing miRNAs (EVs-miRNAs), including hsa-miR-451a and hsalet-7b, and functional miRNA-AGO2 complexes that are internalized by ECs where they modulate the vascular function through regulation of the expression of target genes and barrier properties [240]. ...
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MicroRNAs (miRNAs) are a group of small non-coding RNAs present in a wide diversity of organisms. MiRNAs regulate gene expression at a post-transcriptional level through their interaction with the 3′ untranslated regions of target mRNAs, inducing translational inhibition or mRNA destabilization and degradation. Thus, miRNAs regulate key biological processes, such as cell death, signal transduction, development, cellular proliferation and differentiation. The dysregulation of miRNAs biogenesis and function is related to the pathogenesis of diseases, including parasite infection. Moreover, during host–parasite interactions, parasites and host miRNAs determine the probability of infection and progression of the disease. The present review is focused on the possible role of miRNAs in the pathogenesis of diseases of clinical interest caused by parasitic protists. In addition, the potential role of miRNAs as targets for the design of drugs and diagnostic and prognostic markers of parasitic diseases is also discussed.
... Some factors such as high parasitemia and exposure to drugs are associated with increased conversion to gametocyte production. It has been found in vitro that extracellular vesicles containing protein, RNA and DNA trafficking between parasites provide a means of cell-cell communication which increases the production of gametocytes (136,137). Additionally, epigenetic regulation (the process by which the activity of a particular gene is controlled by the structure of nearby chromatin) is essential for the control of sexual differentiation, and the transcription factor AP2-G is a primary regulator of gametocytogenesis (138). ...
Thesis
Epidemiology of Plasmodium spp. infections among school-age children living in rural and urban areas of Kinshasa in Democratic Republic of Congo Background and rationale Malaria is a threatening tropical disease that still causes morbidity and mortality in middle- and low-income countries despite remarkable efforts done for its control. The recent World Health Organization (WHO) report showed an estimated 229 million cases and 409 000 deaths globally in 2019 of which the Democratic Republic of Congo (DRC) accounted for 12% of cases and 11% of deaths ranged in second place in the world after Nigeria. However, Malaria is preventable and treatable as many patients easily recovered from the infection when correctly treated with effective antimalarial drug or individuals are protected when correctly applied preventive measures. Thus, the promotion of malaria control and surveillance strategy requires political will, inter collaboration of local governments, stakeholders, civil organizations, and research communities. For malaria control and prevention, WHO recommends prompt diagnosis using microscopic examination and/or malaria rapid diagnostic tests (mRDTs), and effective treatment for suspected malaria case management, use of insecticide-treated nets and indoor residual spraying, preventive chemotherapy including chemoprophylaxis for travelers, intermittent preventive treatment for infants (children under 12 months living in high-transmission areas of Africa) and pregnant women living in moderate-to-high transmission areas, seasonal malaria chemoprevention for preschool children living in areas of the Sahel sub-region of Africa and mass drug administration. However, most of these recommendations mostly target children under five and pregnant women. School-age children are a neglected group, not usually covered by household-based cluster surveys and/or malaria interventions and so represent an untreated demographic that may harbour a significant parasite reservoir thus posing a major challenge for malaria control, surveillance, and elimination strategies. Even tough school-age children have low risk to develop complicated forms of malaria, they are subjects of chronic malaria with major health consequences including anemia, cognitive disorders absenteeism, poor performance and dropouts. Therefore, the understanding of the burden of malaria among school-aged children using school-based malaria survey is essential to justify the impact of capturing schools and school-age children in national malaria control program on malaria control and elimination strategies in the country. Also, the evaluation of PfHRP2-based RDTs, the mostly diagnostic method used, and continuous molecular evaluation of the current antimalaria drugs as well as the evaluation of the current national malaria control strategy will significantly contribute to the improvement of malaria control policy decision-making in the country. Objectives The main objective of this thesis was to collect and analyse data that may contribute to an improved malaria control strategy in the DRC. The specific objectives were: a) to determine malaria parasite species composition of Plasmodium infections among asymptomatic and symptomatic school-age children in rural and urban areas of Kinshasa, DRC; b) to identify the Plasmodium falciparum parasites lacking P. falciparum histidine-rich protein 2 and 3 (pfhrp2/3) genes in isolates collected from them and evaluate the performance PfHRP2-based RDTs; c) to identify polymorphisms in P. falciparum Kelch 13 (pfK13), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and chloroquine resistance transporter (pfcrt) gene mutations in isolates and d) to identify unmet needs of the current national malaria control. strategy in DRC by showing the importance of capturing schools and school-age children onto national malaria survey useful for malaria control and elimination. Methods A cross-sectional study was undertaken between October and November 2019 among school-age children aged 6 to 14 years at primary schools and health facilities in the rural area of Mont-Ngafula 2 Health Zone (HZ) and the urban area of Selembao HZ in Kinshasa, DRC. A total of 634 samples were collected from 427 asymptomatic children in selected schools and 217 symptomatic children in selected health facilities. Microscopy, mRDTs, and filter papers spotted blood were performed in Kinshasa. The DNA samples were extracted in laboratory of Institute of Tropical Medicine at Nagasaki University in Nagasaki city and PCRs were performed for genotyping of Plasmodium species, pfhrp2/3 and genotyping and sequencing of P. ovale spp., pfk13, pfmdr1, pfdhfr, pfdhps and pfcrt using specific primers. Major findings The overall prevalence of Plasmodium spp. was 33%, 42% and 62% among asymptomatic children and 59%, 64% and 94% in symptomatic children by microscopy, RDT and PCR, respectively. In asymptomatic carriages, P. falciparum, P. malariae and P. ovale spp. accounted for 77%, 31% and 17% in rural area and 40%, 9% and 5% in urban settings, respectively. Among symptomatic carriers P. falciparum, P. malariae and P. ovale spp. accounted for 96%, 8% and 18% in rural area and 90%, 18% and 14% in urban settings, respectively. Residence in the rural area was an approximately five times greater risk of asymptomatic carriage of malaria parasites as opposed to the urban area. The prevalence of the pfhrp2 gene deletion was 2% while it was 1% for the pfhrp3 gene among RDT positive results. None of RDT negative result was pfhrp2/3 gene deleted. The prevalence of pfcrt K76T, pfdhps K540E and pfmdr1 N86Y was low with 27%, 20% and 9%, respectively. None of the isolates showed artemisinin derivates gene resistance and two new Kelch 13 mutations C532S and Q613E were discovered in the country. The evaluation of the malaria control policy in the DRC showed that the WHO recommendations are still partially implemented by the national malaria control program in DRC and that could be the root of the delay in reducing the burden of malaria in the country.
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Plasmodium vivax is the major cause of human malaria in the Americas. How P. vivax infection can lead to poor clinical outcomes, despite low peripheral parasitaemia remains a matter of intense debate. Estimation of total P. vivax biomass based on circulating markers indicates existence of a predominant parasite population outside of circulation. In this study we investigate associations between both peripheral and total parasite biomass and host response in vivax malaria. We analysed parasite and host signatures in a cohort of uncomplicated vivax malaria patients from Manaus, Brazil, combining clinical and parasite parameters, multiplexed analysis of host responses and ex vivo assays. Patterns of clinical features, parasite burden and host signatures measured in plasma across the patient cohort were highly heterogenous. Further data deconvolution revealed two patient clusters, here termed Vivax low and Vivax high . These patient subgroups were defined based on differences in total parasite biomass but not peripheral parasitaemia. Overall Vivax low patients clustered with healthy donors and Vivax high patients showed more profound alterations in haematological parameters, endothelial cell (EC) activation and glycocalyx breakdown and levels of cytokines regulating different haematopoiesis pathways compared to Vivax low . Vivax high patients presented more severe thrombocytopenia and lymphopenia, along with enrichment of neutrophils in the peripheral blood and increased neutrophil-to-lymphocyte ratio (NLCR). When patients' signatures were combined, high association of total parasite biomass with a subset of markers of EC activation, thrombocytopenia and lymphopenia severity was observed. Finally, machine learning models defined a combination of host parameters measured in the circulation that could predict the extent of parasite infection outside of circulation. Altogether, our data show that total parasite biomass is a better predictor of perturbations in host homeostasis in P. vivax patients than peripheral parasitaemia. This supports the emerging paradigm of a P. vivax tissue reservoir, in particular in the hematopoietic niche of bone marrow and spleen.
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Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum , the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo . These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro . Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we provide conclusive evidence for the absence of mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro . Our data hence contradict scenarios in which therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.
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Reproducible induction of gametocytes of Plasmodium falciparum in vitro is crucial for performing various experimental analyses to understand gametocyte cellular and molecular biology and immunology, and for the evaluation of antigametocidal agents and vaccine development. In this protocol, we present specific procedures for the enrichment, synchronous production and separation of developmental stages of P. falciparum gametocytes from culture-adapted field isolates.
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In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell–derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13–4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281–503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127–200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3–166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.
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