Plasmodium-infected Erythrocytes Research Articles

Comparative spatial proteomics of Plasmodium-infected erythrocytes

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

Unique Properties of Nutrient Channels on Plasmodium-Infected Erythrocytes.

Intracellular malaria parasites activate an ion and organic solute channel on their host erythrocyte membrane to acquire a broad range of essential nutrients. This plasmodial surface anion channel (PSAC) facilitates the uptake of sugars, amino acids, purines, some vitamins, and organic cations, but remarkably, it must exclude the small Na+ ion to preserve infected erythrocyte osmotic stability in plasma. Although molecular, biochemical, and structural studies have provided fundamental mechanistic insights about PSAC and advanced potent inhibitors as exciting antimalarial leads, important questions remain about how nutrients and ions are transported. Here, I review PSAC's unusual selectivity and conductance properties, which should guide future research into this important microbial ion channel.

The Plasmodium falciparum exported J domain proteins fine-tune human and malarial Hsp70s: pathological exploitation of proteostasis machinery.

Cellular proteostasis requires a network of molecular chaperones and co-chaperones, which facilitate the correct folding and assembly of other proteins, or the degradation of proteins misfolded beyond repair. The function of the major chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90), is regulated by a cohort of co-chaperone proteins. The J domain protein (JDP) family is one of the most diverse co-chaperone families, playing an important role in functionalizing the Hsp70 chaperone system to form a powerful protein quality control network. The intracellular malaria parasite, Plasmodium falciparum, has evolved the capacity to invade and reboot mature human erythrocytes, turning them into a vehicles of pathology. This process appears to involve the harnessing of both the human and parasite chaperone machineries. It is well known that malaria parasite-infected erythrocytes are highly enriched in functional human Hsp70 (HsHsp70) and Hsp90 (HsHsp90), while recent proteomics studies have provided evidence that human JDPs (HsJDPs) may also be enriched, but at lower levels. Interestingly, P. falciparum JDPs (PfJDPs) are the most prominent and diverse family of proteins exported into the infected erythrocyte cytosol. We hypothesize that the exported PfJPDs may be an evolutionary consequence of the need to boost chaperone power for specific protein folding pathways that enable both survival and pathogenesis of the malaria parasite. The evidence suggests that there is an intricate network of PfJDP interactions with the exported malarial Hsp70 (PfHsp70-x) and HsHsp70, which appear to be important for the trafficking of key malarial virulence factors, and the proteostasis of protein complexes of human and parasite proteins associated with pathology. This review will critically evaluate the current understanding of the role of exported PfJDPs in pathological exploitation of the proteostasis machinery by fine-tuning the chaperone properties of both human and malarial Hsp70s.

Potential application of the haematology analyser XN-31 prototype for field malaria surveillance in Kenya

BackgroundSimple and accurate diagnosis is a key component of malaria control programmes. Microscopy is the current gold standard, however it requires extensive training and the results largely rely on the skill of the microscopists. Malaria rapid diagnostic tests (RDT) can be performed with minimal training and offer timely diagnosis, but results are not quantitative. Moreover, some Plasmodium falciparum parasites have evolved and can no longer be detected by existing RDT. Developed by the Sysmex Corporation, the XN-31 prototype (XN-31p) is an automated haematology analyser capable of detecting Plasmodium-infected erythrocytes and providing species differentiation and stage specific parasite counts in venous blood samples without any preparation in approximately one minute. However, factors such as stable electricity supply in a temperature-controlled room, cost of the instrument and its initial set-up, and need for proprietary reagents limit the utility of the XN-31p across rural settings. To overcome some of these limitations, a hub and spoke diagnosis model was designed, in which peripheral health facilities were linked to a central hospital where detection of Plasmodium infections by the XN-31p would take place. To explore the feasibility of this concept, the applicability of capillary blood samples with the XN-31p was evaluated with respect to the effect of sample storage time and temperature on the stability of results.MethodsPaired capillary and venous blood samples were collected from 169 malaria-suspected outpatients in Homa Bay County Referral Hospital, Kenya. Malaria infections were diagnosed with the XN-31p, microscopy, RDT, and PCR. Capillary blood samples were remeasured on the XN-31p after 24 h of storage at either room (15–25 °C) or chilled temperatures (2–8 °C).ResultsIdentical results in malaria diagnosis were observed between venous and capillary blood samples processed immediately after collection with the XN-31p. Relative to PCR, the sensitivity and specificity of the XN-31p with capillary blood samples were 0.857 and 1.000, respectively. Short-term storage of capillary blood samples at chilled temperatures had no adverse impact on parasitaemia and complete blood counts (CBC) measured by the XN-31p.ConclusionThese results demonstrate the potential of the XN-31p to improve routine malaria diagnosis across remote settings using a hub and spoke model.

Plasmodium falciparum Molecular Chaperones: Guardians of the Malaria Parasite Proteome and Renovators of the Host Proteome.

Plasmodium falciparum is a unicellular protozoan parasite and causative agent of the most severe form of malaria in humans. The malaria parasite has had to develop sophisticated mechanisms to preserve its proteome under the changing stressful conditions it confronts, particularly when it invades host erythrocytes. Heat shock proteins, especially those that function as molecular chaperones, play a key role in protein homeostasis (proteostasis) of P. falciparum. Soon after invading erythrocytes, the malaria parasite exports a large number of proteins including chaperones, which are responsible for remodeling the infected erythrocyte to enable its survival and pathogenesis. The infected host cell has parasite-resident and erythrocyte-resident chaperones, which appear to play a vital role in the folding and functioning of P. falciparum proteins and potentially host proteins. This review critiques the current understanding of how the major chaperones, particularly the Hsp70 and Hsp40 (or J domain proteins, JDPs) families, contribute to proteostasis of the malaria parasite-infected erythrocytes.

Rosetting Responses of Plasmodium-infected Erythrocytes to Antimalarials.

ABSTRACT.In malaria, rosetting is a phenomenon involving the cytoadherence of uninfected erythrocytes to infected erythrocytes (IRBC) harboring the late erythrocytic stage of Plasmodium spp. Recently, artesunate-stimulated rosetting has been demonstrated to confer a survival advantage to P. falciparum late-stage IRBC. This study investigated the rosetting response of P. falciparum and P. vivax clinical isolates to ex vivo antimalarial treatments. Brief exposure of IRBC to chloroquine, mefloquine, amodiaquine, quinine, and lumefantrine increased the rosetting rates of P. falciparum and P. vivax. Furthermore, the ex vivo combination of artesunate with mefloquine and piperaquine also resulted in increased the rosetting rates. Drug-mediated rosette-stimulation has important implications for the therapeutic failure of rapidly cleared drugs such as artesunate. However, further work is needed to establish the ramifications of increased rosetting rates by drugs with longer half-lifves, such as chloroquine, mefloquine, and piperaquine.

MicroRNAs miR-451a and Let-7i-5p Profiles in Circulating Exosomes Vary among Individuals with Different Sickle Hemoglobin Genotypes and Malaria.

Sickle cell disease (SCD) occurs when two alleles of mutated hemoglobin (HbS or HbC) are inherited (HbSS and HbSC) rather than one (HbAS or HbAC), which indicates a person carries the sickle cell trait. The high prevalence of these two alleles in Africa have been associated with reduced malaria susceptibility. Recent in vitro research has been shown that microRNAs (miRNAs) miR-451a and let-7i-5p are differentially expressed in HbSS erythrocytes compared to healthy controls (HbAA) and are overexpressed in Plasmodium-infected malaria erythrocytes. However, these miRNAs have not been fully examined in the plasma of people with different sickle hemoglobin genotypes. Plasma circulating miRNAs are commonly encapsulated in extracellular vesicles, such as exosomes, and are thought to play a role in disease development. Circulating exosomal miR-451a and let-7i-5p were quantified from individuals with various hemoglobin genotypes (HbAA, HbAS, HbAC, HbSS, HbSC, and HbCC) with (+) and without (−) malaria. The results showed a higher level of exosomal let-7i-5p and miR-451a in HbSS-. Exosomal let-7i-5p and miR-451a levels were lower in HbSS+ compared to other genotypes. Based on the area under the curve (AUC) of the Receiver Operating Characteristics (ROCs), both exosomal miRNAs may be useful disease biomarkers for SCD with malaria. Finally, miR-451a and let-7i-5p modulate genes involved in inflammation, making them potential biomarkers of pathogenesis for both diseases.

Discovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin.

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4–5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57–65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

Comparing infrared spectroscopic methods for the characterization of Plasmodium falciparum-infected human erythrocytes

Malaria, caused by parasites of the species Plasmodium, is among the major life-threatening diseases to afflict humanity. The infectious cycle of Plasmodium is very complex involving distinct life stages and transitions characterized by cellular and molecular alterations. Therefore, novel single-cell technologies are warranted to extract details pertinent to Plasmodium-host cell interactions and underpinning biological transformations. Herein, we tested two emerging spectroscopic approaches: (a) Optical Photothermal Infrared spectroscopy and (b) Atomic Force Microscopy combined with infrared spectroscopy in contrast to (c) Fourier Transform InfraRed microspectroscopy, to investigate Plasmodium-infected erythrocytes. Chemical spatial distributions of selected bands and spectra captured using the three modalities for major macromolecules together with advantages and limitations of each method is presented here. These results indicate that O-PTIR and AFM-IR techniques can be explored for extracting sub-micron resolution molecular signatures within heterogeneous and dynamic samples such as Plasmodium-infected human RBCs.

Cover Feature: Chemoproteomics for Plasmodium Parasite Drug Target Discovery (ChemBioChem 16/2021)

Understanding drugs’ mechanism of action is integral to successful antimalarial drug development. Recent advances in chemoproteomic methods have created an avenue to globally profile drug targets in the Plasmodium parasites that cause malaria. Some of these strategies even allow direct measurement in living intracellular parasites. The cover feature highlights the achievement of probing the drug–protein interactome in an intact Plasmodium-infected erythrocyte. More information can be found in the review article by E. R. Derbyshire et al.

TLR4-Endothelin Axis Controls Syncytiotrophoblast Motility and Confers Fetal Protection in Placental Malaria.

Pregnancy-associated malaria is often associated with adverse pregnancy outcomes. Placental circulatory impairments are an intriguing and unsolved component of malaria pathophysiology. Here, we uncovered a Toll-like receptor 4 (TLR4)-TRIF-endothelin axis that controls trophoblast motility and is linked to fetal protection during Plasmodium infection. In a cohort of 401 pregnancies from northern Brazil, we found that infection during pregnancy reduced expression of endothelin receptor B in syncytiotrophoblasts, while endothelin expression was only affected during acute infection. We further show that quantitative expression of placental endothelin and endothelin receptor B proteins are differentially controlled by maternal and fetal TLR4 alleles. Using murine malaria models, we identified placental autonomous responses to malaria infection mediated by fetally encoded TLR4 that not only controlled placental endothelin gene expression but also correlated with fetal viability protection. In vitro assays showed that control of endothelin expression in fetal syncytiotrophoblasts exposed to Plasmodium-infected erythrocytes was dependent on TLR4 via the TRIF pathway but not MyD88 signaling. Time-lapse microscopy in syncytiotrophoblast primary cultures and cell invasion assays demonstrated that ablation of TLR4 or endothelin receptor blockade abrogates trophoblast collective motility and cell migration responses to infected erythrocytes. These results cohesively substantiate the hypothesis that fetal innate immune sensing, namely, the TRL4-TRIF pathway, exerts a fetal protective role during malaria infection by mediating syncytiotrophoblast vasoregulatory responses that counteract placental insufficiency.

Performance of a sensitive haemozoin\u2010based malaria diagnostic test validated for vivax malaria diagnosis in Brazilian Amazon

BackgroundVivax malaria diagnosis remains a challenge in malaria elimination, with current point of care rapid diagnostic tests (RDT) missing many clinically significant infections because of usually lower peripheral parasitaemia. Haemozoin-detecting assays have been suggested as an alternative to immunoassay platforms but to date have not reached successful field deployment. Haemozoin is a paramagnetic crystal by-product of haemoglobin digestion by malaria parasites and is present in the food vacuole of malaria parasite-infected erythrocytes. This study aimed to compare the diagnostic capability of a new haemozoin-detecting platform, the Gazelle™ device with optical microscopy, RDT and PCR in a vivax malaria-endemic region.MethodsA comparative, double-blind study evaluating symptomatic malaria patients seeking medical care was conducted at an infectious diseases reference hospital in the western Brazilian Amazon. Optical microscopy, PCR, RDT, and Gazelle™ were used to analyse blood samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Kappa values were calculated.ResultsOut of 300 patients, 24 test results were excluded from the final analysis due to protocol violation (6) and inconclusive and/or irretrievable results (18). Gazelle™ sensitivity was 96.1 % (91.3–98.3) and 72.1 % (65.0–78.3) when compared to optical microscopy and PCR, respectively whereas it was 83.9 % and 62.8 % for RDTs. The platform presented specificity of 100 % (97.4–100), and 99.0 % (94.8–99.9) when compared to optical microscopy, and PCR, respectively, which was the same for RDTs. Its correct classification rate was 98.2 % when compared to optical microscopy and 82.3 % for PCR; the test’s accuracy when compared to optical microscopy was 98.1 % (96.4–99.7), when compared to RDT was 95.2 % (93.0–97.5), and when compared to PCR was 85.6 % (82.1–89.1). Kappa (95 % CI) values for Gazelle™ were 96.4 (93.2–99.5), 88.2 (82.6–93.8) and 65.3 (57.0–73.6) for optical microscopy, RDT and PCR, respectively.ConclusionsThe Gazelle™ device was shown to have faster, easier, good sensitivity, specificity, and accuracy when compared to microscopy and was superior to RDT, demonstrating to be an alternative for vivax malaria screening particularly in areas where malaria is concomitant with other febrile infections (including dengue fever, zika, chikungunya, Chagas, yellow fever, babesiosis).

Performance of the hematology analyzer XN-31 prototype in the detection of Plasmodium infections in an endemic region of Colombia

Early and accurate diagnosis is critical in reducing the morbidity and mortality associated with malaria. Microscopy (MI) is the current diagnostic gold standard in the field; however, it requires expert personnel, is time-consuming, and has limited sensitivity. Although rapid diagnostic tests for antigen detection (RDTs) are an alternative to diagnosis, they also have limited sensitivity and produce false positive results in detecting recent past infection. The automated hematology analyzer XN-31 prototype (XN-31p) (Sysmex Corporation, Kobe, Japan) is able to identify plasmodium-infected erythrocytes, count parasitemia and perform complete blood-cell counts within one minute. The performance of the XN-31p in diagnosing malaria was evaluated and compared with real-time polymerase chain reaction (qPCR), MI and RDT in an endemic area of Colombia where Plasmodium falciparum and Plasmodium vivax are present. Acute febrile patients were enrolled from July 2018 to April 2019 in Quibdó, Colombia. Malaria diagnoses were obtained from MI and RDT in the field and later confirmed by qPCR. Venous blood samples in EDTA were processed with an XN-31p in the field. Sensitivity, specificity, positive/negative predictive values, and the likelihood ratios of positive and negative tests were calculated with respect to the results from qPCR, MI and RDT. The intraclass correlation coefficient (ICC) and Bland–Altman plot were used to evaluate the concordance in the parasitemia with respect to MI. A total of 1,754 subjects were enrolled. The mean age was 27.0 years (IQR 14–44); 89.6% were Afro-Colombians, 94.3% lived in urban areas and 0.91% were pregnant. With respect to qPCR, the XN-31p showed a sensitivity of 90% (95% CI 87.24–92.34) and a specificity of 99.83% (95% CI 99.38–99.98) in detecting Plasmodium spp.; both parameters were equivalent to those for MI and RDT. Using MI as the reference, the XN-31p showed a sensitivity of 98.09% (95% CI 96.51–99.08), a specificity of 99.83% (95% CI 99.4–99.98), an ICC of 0.85 (95% CI 0.83–0.87) and an average difference of − 3096 parasites/µL when compared with thick-smear MI and an ICC of 0.98 (95% CI 0.97–0.98) and an average difference of − 0.0013% when compared with thin-smear MI. The XN-31p offers a rapid and accurate alternative method for diagnosing malaria in clinical laboratories in areas where P. falciparum and P. vivax cocirculate.

Choline and PEG dually modified artemether nano delivery system targeting intra-erythrocytic Plasmodium and its pharmacodynamics in vivo

Objective The choline derivative (CD) and polyethylene-glycol (PEG) dually modified artemether (ARM) nanostructured lipid carriers (CD-PEG-ARM-NLC) have been designed to prolong the circulation of ARM in blood, as well as to develop targeting for new permeability pathways (NPPs) and erythrocyte choline carriers (ECCs) that are expressed on the Plasmodium-infected erythrocyte membrane. Significance The CD-PEG-ARM-NLC constructed in this study was found to be able to target endoerythrocytic Plasmodium by increasing the drug concentration and residence time in the infected erythrocytic microenvironment and minimizing toxicity and side effects. Methods CD-PEG-ARM-NLC was prepared using high-pressure homogenization followed by physicochemical characterization. The targeting ability of CD-PEG-NLC to infected erythrocytes probed by coumarin-6 was investigated by using fluorescence microscopy imaging. The SYBR Green I assay for parasite nucleic acid was adapted in order to assess the efficacy of inhibition against parasite growth in vitro. The antimalarial activity of ARM-loaded NLCs was evaluated by a Pearson four-day suppressive test in Pyy265BY-bearing mice. Results In vitro imaging indicated that the intracellular delivery of CD-PEG-ARM-NLC was efficiently taken up by the infected erythrocytes via ECCs and NPPs, which could be inhibited by addition of furosemide (an inhibitor of NPPs) and excessive choline (native substrate of ECCs). Moreover, in vitro and in vivo studies that evaluated antimalarial activity suggested that CD-PEG-ARM-NLC exhibited higher antimalarial activity in comparison to ARM-NLC and PEG-ARM-NLC. Conclusion These findings suggested that choline and PEG dually modified NLC could be promising preparations for the production of hydrophobic antimalarial drugs, particularly for ARM.

γδ T cells burst malaria's bubble.

New research demonstrates that γδ T cells recognize Plasmodium-infected erythrocytes via interaction of the T cell antigen receptor with the phosphoantigen sensor BTN3A1 and subsequently destroy infected cells through either cytotoxic molecule secretion or antibody-dependent phagocytosis.

Insights into the Membranolytic Activity of Antimalarial Drug-Cell Penetrating Peptide Conjugates.

Conjugation of TP10, a cell-penetrating peptide with intrinsic antimalarial activity, to the well-known antimalarial drugs chloroquine and primaquine has been previously shown to enhance the peptide’s action against, respectively, blood- and liver-stage malaria parasites. Yet, this was achieved at the cost of a significant increase in haemolytic activity, as fluorescence microscopy and flow cytometry studies showed the conjugates to be more haemolytic for non-infected than for Plasmodium-infected red blood cells. To gain further insight into how these conjugates distinctively bind, and likely disrupt, membranes of both Plasmodium-infected and non-infected erythrocytes, we used dynamic light scattering and surface plasmon resonance to study the interactions of two representative conjugates and their parent compounds with lipid model membranes. Results obtained are herein reported and confirm that a strong membrane-disruptive character underlies the haemolytic properties of these conjugates, thus hampering their ability to exert selective antimalarial action.

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes.

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.

Cholesterol-dependent enrichment of understudied erythrocytic stages of human Plasmodium parasites

For intracellular pathogens, the host cell provides needed protection and nutrients. A major challenge of intracellular parasite research is collection of high parasite numbers separated from host contamination. This situation is exemplified by the malaria parasite, which spends a substantial part of its life cycle inside erythrocytes as rings, trophozoites, and schizonts, before egress and reinvasion. Erythrocytic Plasmodium parasite forms refractory to enrichment remain understudied due to high host contamination relative to low parasite numbers. Here, we present a method for separating all stages of Plasmodium-infected erythrocytes through lysis and removal of uninfected erythrocytes. The Streptolysin O-Percoll (SLOPE) method is effective on previously inaccessible forms, including circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. SLOPE can be used on multiple parasite species, under multiple media formulations, and lacks measurable impacts on parasite viability. We demonstrate erythrocyte membrane cholesterol levels modulate the preferential lysis of uninfected host cells by SLO, and therefore modulate the effectiveness of SLOPE. Targeted metabolomics of SLOPE-enriched ring stage samples confirms parasite-derived metabolites are increased and contaminating host material is reduced compared to non-enriched samples. Due to consumption of cholesterol by other intracellular bacteria and protozoa, SLOPE holds potential for improving research on organisms beyond Plasmodium.

Reduction of Sphingosine Kinase 1 Phosphorylation and Activity in Plasmodium-Infected Erythrocytes.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator is involved in an array of biological processes and linked to pathological manifestations. Erythrocyte is known as the major reservoir for S1P as they lack S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) and harbor sphingosine kinase-1 (SphK-1) essential for sphingosine conversion to S1P. Reduced S1P concentration in serum was correlated with disease severity in patients with Plasmodium falciparum and Plasmodium vivax infections. Herein, we aimed to identify the underlying mechanism and contribution of host erythrocytes toward depleted S1P levels in Plasmodium-infected patients vs. healthy individuals. The level and activity of SphK-1 were measured in vitro in both uninfected and cultured P. falciparum-infected erythrocytes. Infected erythrocytes demonstrated a significant decrease in SphK-1 level in a time-dependent manner. We found that 10–42 h post invasion (hpi), SphK1 level was predominantly reduced to ∼50% in rings, trophozoites, and schizonts compared to uninfected erythrocytes. We next analyzed the phosphorylation status of SphK-1, a modification responsible for its activity and S1P production, in both uninfected control and Plasmodium-infected erythrocytes. Almost ∼50% decrease in phosphorylation of SphK-1 was observed that could be corroborated with significant reduction in the production and release of S1P in infected erythrocytes. Serum S1P levels were studied in parallel in P. falciparum (N = 15), P. vivax (N = 36)-infected patients, and healthy controls (N = 6). The findings revealed that S1P concentration was significantly depleted in uncomplicated malaria cases and was found to be lowest in complicated malaria and thrombocytopenia in both P. falciparum and P. vivax-infected groups (∗∗p < 0.01). The lower serum S1P level could be correlated with the reduced platelet count defining the role of S1P level in platelet formation. In conclusion, erythrocyte SphK-1 and S1P levels were studied in Plasmodium-infected individuals and erythrocytes that helped in characterizing the complications associated with malaria and thrombocytopenia, providing insights into the contribution of host erythrocyte biology in malaria pathogenesis. Finally, this study proposes the use of S1P and its analog as a novel adjunct therapy for malaria complications.

Association of EPCR Polymorphism rs867186-GG With Severity of Human Malaria

BackgroundCerebral malaria (CM) is characterized by the sequestration of Plasmodium-infected erythrocytes (pRBCs) to host brain microvasculature beds via P. falciparum erythrocyte membrane protein 1 (PfEMP1). Under normal conditions, activated protein C (APC) bound to endothelial protein C receptor (EPCR) has cytoprotective properties via the activation of protease-activated receptor 1 (PAR1). During malaria infection, pRBCs transports PfEMP1 to the membranes to bind EPCR in the same region as APC. As a result, APC is less capable of inducing cytoprotective effects via PAR1. Two studies involving adult malaria patients revealed that EPCR rs867186-GG allele is associated with protection against severe malaria, while three other studies involving child malaria patients could not show association between EPCR rs867186-GG genotype and severe malaria or increased mortality among children with CM.MethodsWe examined the association between the EPCR rs867186-GG genotype and the protection against cerebral malaria. Peripheral blood samples were collected from 47 malaria patients and 34 healthy individuals from a study conducted from 2004 to 2007 at the NSCB Medical College Hospital in India. CM and malaria-associated complications were defined based on WHO criteria. Genomic DNA was isolated from the peripheral blood mononuclear cells. Primer sequences were designed to contain rs867186 of the PROCR gene (NM 006404) and were used to amplify a 660 bp product as described before. PCR products were purified, and DNA sequences were determined by Sanger Sequencing (Genewiz, NJ). Nonparametric tests were used to compare the groups. To analyze differences in allele frequencies, we used chi-squared or Fisher's exact tests for categorical variables if the expected values were less than 5. P-value <0.05 was considered statistically significant.ResultsOur results showed significantly higher rates of AG and GG genotypes in CM patients compared to mild malaria (P = 0.0034). ConclusionOur results indicate that rs867186-GG or rs867186-AG genotypes are not associated with protection against HCM.