In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Identification of basal complex protein that is essential for maturation of transmission-stage malaria parasites.

Malaria remains a global driver of morbidity and mortality. To generate new antimalarials, one must elucidate the fundamental cell biology of Plasmodium falciparum, the parasite responsible for the deadliest cases of malaria. A membranous and proteinaceous scaffold called the inner membrane complex (IMC) supports the parasite during morphological changes, including segmentation of daughter cells during asexual replication and formation of transmission-stage gametocytes. The basal complex lines the edge of the IMC during segmentation and likely facilitates IMC expansion. It is unknown, however, what drives IMC expansion during gametocytogenesis. We describe the discovery of a basal complex protein, PfBLEB, which we find to be essential for gametocytogenesis. Parasites lacking PfBLEB harbor defects in IMC expansion and are unable to form mature gametocytes. This article demonstrates a role for a basal complex protein outside of asexual division, and, importantly, highlights a potential molecular target for the ablation of malaria transmission.

Declines in prevalence alter the optimal level of sexual investment for the malaria parasite Plasmodium falciparum.

Successful infectious disease interventions can result in large reductions in parasite prevalence. Such demographic change has fitness implications for individual parasites and may shift the parasite's optimal life history strategy. Here, we explore whether declining infection rates can alter Plasmodium falciparum's investment in sexual versus asexual growth. Using a multiscale mathematical model, we demonstrate how the proportion of polyclonal infections, which decreases as parasite prevalence declines, affects the optimal sexual development strategy: Within-host competition in multiclone infections favors a greater investment in asexual growth whereas single-clone infections benefit from higher conversion to sexual forms. At the same time, drug treatment also imposes selection pressure on sexual development by shortening infection length and reducing within-host competition. We assess these models using 148 P. falciparum parasite genomes sampled in French Guiana over an 18-y period of intensive intervention (1998 to 2015). During this time frame, multiple public health measures, including the introduction of new drugs and expanded rapid diagnostic testing, were implemented, reducing P. falciparum malaria cases by an order of magnitude. Consistent with this prevalence decline, we see an increase in the relatedness among parasites, but no single clonal background grew to dominate the population. Analyzing individual allele frequency trajectories, we identify genes that likely experienced selective sweeps. Supporting our model predictions, genes showing the strongest signatures of selection include transcription factors involved in the development of P. falciparum's sexual gametocyte form. These results highlight how public health interventions impose wide-ranging selection pressures that affect basic parasite life history traits.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media.

What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 - 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.

Antiparasitic Ovalicin Derivatives from Pseudallescheria boydii, a Mutualistic Fungus of French Guiana Termites.

Social insects are in mutualism with microorganisms, contributing to their resistance against infectious diseases. The fungus Pseudallescheria boydii SNB-CN85 isolated from termites produces ovalicin derivatives resulting from the esterification of the less hindered site of the ovalicin epoxide by long-chain fatty acids. Their structures were elucidated using spectroscopic analysis and semisynthesis from ovalicin. For ovalicin, these compounds displayed antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei, with IC50 values of 19.8 and 1.1 µM, respectively, for the most active compound, i.e., ovalicin linoleate. In parallel, metabolomic profiling of a collection of P. boydii strains associated with termites made it possible to highlight this class of compounds together with tyroscherin derivatives in all strains. Finally, the complete genome of P. boydii strains was obtained by sequencing, and the cluster of potential ovalicin and ovalicin biosynthesis genes was annotated. Through these metabolomic and genomic analyses, a new ovalicin derivative named boyden C, in which the 6-membered ring of ovalicin was opened by oxidative cleavage, was isolated and structurally characterized.

Dramatic Consequences of Reducing Erythrocyte Membrane Cholesterol on Plasmodium falciparum.

Cholesterol is the most abundant lipid in the erythrocyte. During its blood-stage development, the malaria parasite establishes an active cholesterol gradient across the various membrane systems within the infected erythrocyte. Interestingly, some antimalarial compounds have recently been shown to disrupt cholesterol homeostasis in the intraerythrocytic stages of Plasmodium falciparum. These studies point to the importance of cholesterol for parasite growth. Previously, reduction of cholesterol from the erythrocyte membrane by treatment with methyl-ß-cyclodextrin (MßCD) was shown to inhibit parasite invasion and growth. In addition, MßCD treatment of trophozoite-stage P. falciparum was shown to result in parasite expulsion from the host cell. We have revisited these phenomena by using live video microscopy, ultrastructural analysis, and response to antimalarial compounds. By using time-lapse video microscopy of fluorescently tagged parasites, we show that MßCD treatment for just 30 min causes dramatic expulsion of the trophozoite-stage parasites. This forceful expulsion occurs within 10 s. Remarkably, the plasma membrane of the host cell from which the parasite has been expelled does not appear to be compromised. The parasitophorous vacuolar membrane (PVM) continued to surround the extruded parasite, but the PVM appeared damaged. Treatment with antimalarial compounds targeting PfATP4 or PfNCR1 prevented MßCD-mediated extrusion of the parasites, pointing to a potential role of cholesterol dynamics underlying the expulsion phenomena. We also confirmed the essential role of erythrocyte plasma membrane cholesterol for invasion and growth of P. falciparum. This defect can be partially complemented by cholesterol and desmosterol but not with epicholesterol, revealing stereospecificity underlying cholesterol function. Overall, our studies advance previous observations and reveal unusual cell biological features underlying cholesterol depletion of the infected erythrocyte plasma membrane. IMPORTANCE Malaria remains a major challenge in much of the world. Symptoms of malaria are caused by the growth of parasites belonging to Plasmodium spp. inside the red blood cells (RBCs), leading to their destruction. The parasite depends upon its host for much of its nutritional needs. Cholesterol is a major lipid in the RBC plasma membrane, which is the only source of this lipid for malaria parasites. We have previously shown that certain new antimalarial compounds disrupt cholesterol homeostasis in P. falciparum. Here, we use live time-lapse video microscopy to show dramatic expulsion of the parasite from the host RBC when the cholesterol content of the RBC is reduced. Remarkably, this expulsion is inhibited by the antimalarials that disrupt lipid homeostasis. We also show stereospecificity of cholesterol in supporting parasite growth inside RBC. Overall, these results point to a critical role of cholesterol in the physiology of malaria parasites.

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes.

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

The transcriptional regulator HDP1 controls expansion of the inner membrane complex during early sexual differentiation of malaria parasites.

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their limited number of transcriptional and chromatin regulators, and the molecular mediators of these changes remain largely unknown. We recently identified homeodomain protein 1 (HDP1) as a DNA-binding protein, first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation. The discovery of HDP1 marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. Here, using molecular biology, biochemistry and microscopy techniques, we show that HDP1 is essential for gametocyte maturation, facilitating the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes their characteristic shape.

Six-year trend analysis of malaria prevalence at University of Gondar Specialized Referral Hospital, Northwest Ethiopia, from 2014 to 2019.

Globally, malaria is the major public health disease caused by plasmodium species and transmitted by the bite of the female anopheles mosquito. Assessment of the trend of malaria prevalence is important in the control and prevention of the disease. Therefore, the objective of this study was to assess the six year trend of malaria prevalence at the University of Gondar Comprehensive Specialized Hospital, northwest Ethiopia, from 2014 to 2019. A retrospective laboratory registration logbook review study was conducted on the malaria blood film examination results at the University of Gondar Comprehensive Specialized Hospital. The data was collected by using a data extraction tool and entered into SPSS version 20 for analysis. Descriptive statistics were used to summarize the socio-demographic characteristics of study participants and presented by graphs, tables and texts. The binary logistic regression was also used to test the association the trend of malaria prevalence and different factors like sex, age, year, and season. From a total of 17,500 malaria blood film examinations, 1341 (7.7%) were confirmed for malaria parasites. Of the confirmed malaria cases, 47.2%, 45.6% and 7.2% were P. vivax, P. falciparum and mixed infection, respectively. The proportion of P. vivax was the predominant species in the first three study years (2014-2016) and P. falciparum became the predominant species in the last three study years (2017-2019). The odds of malaria prevalence was lower by 68%, 60% and 69% in the year 2017, 2018 and 2019 compared to 2014, respectively. It was also 1.41 times higher in males than in females. Moreover, the odds of malaria prevalence were 1.60, 1.64, 2.45 and 1.82 times higher in the age group of < 5, 5-14, 15-24 and 25-54 years old compared to the older age groups (> 54 years old), respectively. Even there was a significant declining in prevalence trend; malaria is still a major public health problem. The study showed that there was high seasonal fluctuation from year to year. Moreover, males and the younger age groups were more affected than females and old age groups, respectively. Therefore, malaria prevention and control activities should be strengthened and require extra efforts by considering these variability.

Combined Transcriptome and Proteome Profiling for Role of pfEMP1 in Antimalarial Mechanism of Action of Dihydroartemisinin.

Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation. Artemisinin combination therapies are the first-line antiplasmodials in countries of endemicity. However, the mechanism of action of artemisinin is unclear, and drug resistance decreases long-term efficacy. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. Optical microscopy and scanning electron microscopy showed that DHA can cause morphological variation in the iRBC membrane. We identified 125 differentially expressed membrane proteins, and functional analysis indicated structural molecule activity and protein export as key biological functions of the two omics studies. DHA treatment decreased the expression of var gene variants PF3D7_0415700 and PF3D7_0900100 dose-dependently. Western blotting and immunofluorescence analysis showed that DHA treatment downregulates the var gene encoding P. falciparum erythrocyte membrane protein-1 (pfEMP1). pfEMP1 knockout significantly increased artemisinin sensitivity. Results showed that pfEMP1 might be involved in the antimalarial mechanism of action of DHA and pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new drugs targeting iRBC membrane. IMPORTANCE Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation, with artemisinin combination therapies as the first-line treatments. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. We found that DHA can cause morphological changes of iRBC membrane. Structural molecule activity and protein export are considered to be the key biological functions based on the two omics studies. pfEMP1 might be involved in the DHA mechanism of action. pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new antimalarial drugs targeting iRBC membrane.

Deletion of Plasmodium falciparum ubc13 increases parasite sensitivity to the mutagen, methyl methanesulfonate and dihydroartemisinin.

The inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene (ubc13) of Plasmodium falciparum to study its role in ubiquitylation and the functional consequence during the parasite asexual blood stage. Deletion resulted in a significant reduction of parasite growth in vitro, reduced ubiquitylation of the Lys63 residue of ubiquitin attached to protein substrates, and an increased sensitivity of the parasite to both the mutagen, methyl methanesulfonate and the antimalarial drug dihydroartemisinin (DHA), but not chloroquine. The parasite was also sensitive to the UBC13 inhibitor NSC697923. The data suggest that this gene does code for an ubiquitin conjugating enzyme responsible for K63 ubiquitylation, which is important in DNA repair pathways as was previously demonstrated in other organisms. The increased parasite sensitivity to DHA in the absence of ubc13 function indicates that DHA may act primarily through this pathway and that inhibitors of UBC13 may both enhance the efficacy of this antimalarial drug and directly inhibit parasite growth.

Derivatives of Dictyostelium differentiation-inducing factors suppress the growth of Plasmodium parasites in vitro and in vivo.

Malaria, which is caused by protozoa of the genus Plasmodium, remains a major endemic public health problem worldwide. Since artemisinin combination therapies are used as a first-line treatment in all endemic regions, the emergence of parasites resistant to these regimens has become a serious problem. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone originally found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivatives exhibit a range of biological activities. In the present study, we investigated the effects of 41 DIF derivatives on the growth of Plasmodium falciparum in vitro using four laboratory strains and 12 field isolates. Micromolar concentrations of several DIF derivatives strongly suppressed the growth of the four laboratory strains, including strains that exhibited resistance to chloroquine and artemisinin, as well as strains that were susceptible to these drugs. In addition, DIF-1(+2), the most potent derivative, strongly suppressed the growth of 12 field isolates. We also examined the effects of DIF-1(+2) on the activity of the rodent malarial parasite Plasmodium berghei in mice. Intraperitoneal administration of DIF-1(+2) over 4 days (50 or 70 mg/kg/day) significantly suppressed the growth of the parasite in the blood with no apparent adverse effects, and a dose of 70 mg/kg/day significantly prolonged animal survival. These results suggest that DIF derivatives, such as DIF-1(+2), could serve as new lead compounds for the development of antimalarial agents.

Osteopontin and malaria: no direct effect on parasite growth, but correlation with P. falciparum-specific B cells and BAFF in a malaria endemic area.

BACKGROUND: The dysregulation of B cell activation is prevalent during naturally acquired immunity against malaria. Osteopontin (OPN), a protein produced by various cells including B cells, is a phosphorylated glycoprotein that participates in immune regulation and has been suggested to be involved in the immune response against malaria. Here we studied the longitudinal concentrations of OPN in infants and their mothers living in Uganda, and how OPN concentrations correlated with B cell subsets specific for P. falciparum and B cell activating factor (BAFF). We also investigated the direct effect of OPN on P. falciparum in vitro. RESULTS: The OPN concentration was higher in the infants compared to the mothers, and OPN concentration in infants decreased from birth until 9 months. OPN concentration in infants during 9 months were independent of OPN concentrations in corresponding mothers. OPN concentrations in infants were inversely correlated with total atypical memory B cells (MBCs) as well as P. falciparum-specific atypical MBCs. There was a positive correlation between OPN and BAFF concentrations in both mothers and infants. When OPN was added to P. falciparum cultured in vitro, parasitemia was unaffected regardless of OPN concentration. CONCLUSIONS: The concentrations of OPN in infants were higher and independent of the OPN concentrations in corresponding mothers. In vitro, OPN does not have a direct effect on P. falciparum growth. Our correlation analysis results suggest that OPN could have a role in the B cell immune response and acquisition of natural immunity against malaria.

Identification of first-in-class plasmodium OTU inhibitors with potent anti-malarial activity.

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

Common host variation drives malaria parasite fitness in healthy human red cells.

The replication of Plasmodium falciparum parasites within red blood cells (RBCs) causes severe disease in humans, especially in Africa. Deleterious alleles like hemoglobin S are well-known to confer strong resistance to malaria, but the effects of common RBC variation are largely undetermined. Here, we collected fresh blood samples from 121 healthy donors, most with African ancestry, and performed exome sequencing, detailed RBC phenotyping, and parasite fitness assays. Over one-third of healthy donors unknowingly carried alleles for G6PD deficiency or hemoglobinopathies, which were associated with characteristic RBC phenotypes. Among non-carriers alone, variation in RBC hydration, membrane deformability, and volume was strongly associated with P. falciparum growth rate. Common genetic variants in PIEZO1, SPTA1/SPTB, and several P. falciparum invasion receptors were also associated with parasite growth rate. Interestingly, we observed little or negative evidence for divergent selection on non-pathogenic RBC variation between Africans and Europeans. These findings suggest a model in which globally widespread variation in a moderate number of genes and phenotypes modulates P. falciparum fitness in RBCs.

Contrasting effects of the alkaloid ricinine on the capacity of Anopheles gambiae and Anopheles coluzzii to transmit Plasmodium falciparum.

BACKGROUND: Besides feeding on blood, females of the malaria vector Anopheles gambiae sensu lato readily feed on natural sources of plant sugars. The impact of toxic secondary phytochemicals contained in plant-derived sugars on mosquito physiology and the development of Plasmodium parasites remains elusive. The focus of this study was to explore the influence of the alkaloid ricinine, found in the nectar of the castor bean Ricinus communis, on the ability of mosquitoes to transmit Plasmodium falciparum. METHODS: Females of Anopheles gambiae and its sibling species Anopheles coluzzii were exposed to ricinine through sugar feeding assays to assess the effect of this phytochemical on mosquito survival, level of P. falciparum infection and growth rate of the parasite. RESULTS: Ricinine induced a significant reduction in the longevity of both Anopheles species. Ricinine caused acceleration in the parasite growth rate with an earlier invasion of the salivary glands in both species. At a concentration of 0.04 g l-1 in An. coluzzii, ricinine had no effect on mosquito infection, while 0.08 g l-1 ricinine-5% glucose solution induced a 14% increase in An. gambiae infection rate. CONCLUSIONS: Overall, our findings reveal that consumption of certain nectar phytochemicals can have unexpected and contrasting effects on key phenotypic traits that govern the intensity of malaria transmission. Further studies will be required before concluding on the putative role of ricinine as a novel control agent, including the development of ricinine-based toxic and transmission-blocking sugar baits. Testing other secondary phytochemicals in plant nectar will provide a broader understanding of the impact which plants can have on the transmission of vector-borne diseases.

A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.

Plasmodium species have a single mitochondrion that is essential for their survival and has been successfully targeted by antimalarial drugs. Most mitochondrial proteins are imported into this organelle, and our picture of the Plasmodium mitochondrial proteome remains incomplete. Many data sources contain information about mitochondrial localization, including proteome and gene expression profiles, orthology to mitochondrial proteins from other species, coevolutionary relationships, and amino acid sequences, each with different coverage and reliability. To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight data sets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 445 proteins with a sensitivity of 87% and a specificity of 97%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. Mitochondrial localization of seven Plasmodium berghei orthologs was confirmed by epitope labeling and colocalization with a mitochondrial marker protein. One of these belongs to a newly identified apicomplexan mitochondrial protein family that in P. falciparum has four members. With the experimentally validated mitochondrial proteins and the complete ranked P. falciparum proteome, which we have named PlasmoMitoCarta, we present a resource to study unique proteins of Plasmodium mitochondria. IMPORTANCE The unique biology and medical relevance of the mitochondrion of the malaria parasite Plasmodium falciparum have made it the subject of many studies. However, we actually do not have a comprehensive assessment of which proteins reside in this organelle. Many omics data are available that are predictive of mitochondrial localization, such as proteomics data and expression data. Individual data sets are, however, rarely complete and can provide conflicting evidence. We integrated a wide variety of available omics data in a manner that exploits the relative strengths of the data sets. Our analysis gave a predictive score for the mitochondrial localization to each nuclear encoded P. falciparum protein and identified 445 likely mitochondrial proteins. We experimentally validated the mitochondrial localization of seven of the new mitochondrial proteins, confirming the quality of the complete list. These include proteins that have not been observed mitochondria before, adding unique mitochondrial functions to P. falciparum.

Effectiveness of seasonal malaria chemoprevention (SMC) treatments when SMC is implemented at scale: Case-control studies in 5 countries.

BACKGROUND: Seasonal malaria chemoprevention (SMC) has shown high protective efficacy against clinical malaria and severe malaria in a series of clinical trials. We evaluated the effectiveness of SMC treatments against clinical malaria when delivered at scale through national malaria control programmes in 2015 and 2016. METHODS AND FINDINGS: Case-control studies were carried out in Mali and The Gambia in 2015, and in Burkina Faso, Chad, Mali, Nigeria, and The Gambia in 2016. Children aged 3-59 months presenting at selected health facilities with microscopically confirmed clinical malaria were recruited as cases. Two controls per case were recruited concurrently (on or shortly after the day the case was detected) from the neighbourhood in which the case lived. The primary exposure was the time since the most recent course of SMC treatment, determined from SMC recipient cards, caregiver recall, and administrative records. Conditional logistic regression was used to estimate the odds ratio (OR) associated with receipt of SMC within the previous 28 days, and SMC 29 to 42 days ago, compared with no SMC in the past 42 days. These ORs, which are equivalent to incidence rate ratios, were used to calculate the percentage reduction in clinical malaria incidence in the corresponding time periods. Results from individual countries were pooled in a random-effects meta-analysis. In total, 2,126 cases and 4,252 controls were included in the analysis. Across the 7 studies, the mean age ranged from 1.7 to 2.4 years and from 2.1 to 2.8 years among controls and cases, respectively; 42.2%-50.9% and 38.9%-46.9% of controls and cases, respectively, were male. In all 7 individual case-control studies, a high degree of personal protection from SMC against clinical malaria was observed, ranging from 73% in Mali in 2016 to 98% in Mali in 2015. The overall OR for SMC within 28 days was 0.12 (95% CI: 0.06, 0.21; p < 0.001), indicating a protective effectiveness of 88% (95% CI: 79%, 94%). Effectiveness against clinical malaria for SMC 29-42 days ago was 61% (95% CI: 47%, 72%). Similar results were obtained when the analysis was restricted to cases with parasite density in excess of 5,000 parasites per microlitre: Protective effectiveness 90% (95% CI: 79%, 96%; P<0.001), and 59% (95% CI: 34%, 74%; P<0.001) for SMC 0-28 days and 29-42 days ago, respectively. Potential limitations include the possibility of residual confounding due to an association between exposure to malaria and access to SMC, or differences in access to SMC between patients attending a clinic and community controls; however, neighbourhood matching of cases and controls, and covariate adjustment, attempted to control for these aspects, and the observed decline in protection over time, consistent with expected trends, argues against a major bias from these sources. CONCLUSIONS: SMC administered as part of routine national malaria control activities provided a very high level of personal protection against clinical malaria over 28 days post-treatment, similar to the efficacy observed in clinical trials. The case-control design used in this study can be used at intervals to ensure SMC treatments remain effective.

Non-ribosomal insights into ribosomal P2 protein in Plasmodium falciparum-infected erythrocytes.

The enormous complexity of the eukaryotic ribosome has been a real challenge in unlocking the mechanistic aspects of its amazing molecular function during mRNA translation and many non-canonical activities of ribosomal proteins in eukaryotic cells. While exploring the uncanny nature of ribosomal P proteins in malaria parasites Plasmodium falciparum, the 60S stalk ribosomal P2 protein has been shown to get exported to the infected erythrocyte (IE) surface as an SDS-resistant oligomer during the early to the mid-trophozoite stage. Inhibiting IE surface P2 either by monoclonal antibody or through genetic knockdown resulted in nuclear division arrest of the parasite. This strange and serendipitous finding has led us to explore more about un-canonical cell biology and the structural involvement of P2 protein in Plasmodium in the search for a novel biochemical role during parasite propagation in the human host.