Human Malaria Parasite Plasmodium Research Articles

Genome-wide profiling of histone modifications in Plasmodium falciparum using CUT&RUN.

We recently adapted a CUT&RUN protocol for genome-wide profiling of chromatin modifications in the human malaria parasite Plasmodium Using the step-by-step protocol described below, we were able to generate high-quality profiles of multiple histone modifications using only a small fraction of the cells required for ChIP-seq. Using antibodies against two commonly profiled histone modifications, H3K4me3 and H3K9me3, we show here that CUT&RUN profiling is highly reproducible and closely recapitulates previously published ChIP-seq-based abundance profiles of histone marks. Finally, we show that CUT&RUN requires substantially lower sequencing coverage for accurate profiling compared with ChIP-seq.

Population genomics in neglected malaria parasites.

Malaria elimination includes neglected human malaria parasites Plasmodium vivax, Plasmodium ovale spp., and Plasmodium malariae. Biological features such as association with low-density infection and the formation of hypnozoites responsible for relapse make their elimination challenging. Studies on these parasites rely primarily on clinical samples due to the lack of long-term culture techniques. With improved methods to enrich parasite DNA from clinical samples, whole-genome sequencing of the neglected malaria parasites has gained increasing popularity. Population genomics of more than 2200 P. vivax global isolates has improved our knowledge of parasite biology and host-parasite interactions, identified vaccine targets and potential drug resistance markers, and provided a new way to track parasite migration and introduction and monitor the evolutionary response of local populations to elimination efforts. Here, we review advances in population genomics for neglected malaria parasites, discuss how the rich genomic information is being used to understand parasite biology and epidemiology, and explore opportunities for the applications of malaria genomic data in malaria elimination practice.

Five-Year Trend Analysis of Malaria Cases in East Shawa Zone, Ethiopia.

Malaria is an infectious disease caused by Plasmodium parasites. Of the five human malaria parasites Plasmodium falciparum and Plasmodium vivax are the two co-endemic predominant and widely distributed species in Ethiopia, with major public health importance. Even though enormous effort has been made countrywide to reduce the disease burden little was reported about trends of malaria transmission in the several localities of malarious areas like East Shawa Zone, Ethiopia. Thus, the present study was aimed at assessing fiveyear (2016-2020) trends of malaria transmission at Adama, Boset and Lume districts of East Shawa Zone of Oromia Regional State, Ethiopia. Retrospective data was extracted from the central surveillance database of East Shawa Zone Health Office. The data collected was analyzed from September 2020 to December 2020 to examine trends of malaria epidemiology in three malarious districts in the Zone. The results of the present study showed a remarkable decrease in slide positivity rate (SPR) from 16.3 to 1.4% from 2016 to 2018 in the areas. However, a recent slight increase of malaria SPR was observed. On the other hand, as age increases more male individuals were infected with malaria compared to female of similar age groups. Falciparum, vivax and mixed malaria infection accounted for 53%, 41% and 6% respectively. Even though, an overall reduction of malaria incidence was revealed in the study areas, an increase in malaria SPR was observed in 2019 and 2020. Such inconsistency in reduction of malaria cases in the study area demands due attention of health planners.

Complex infections in vivax malaria: the more you look, the more you find

The human malaria parasite Plasmodium vivax commonly causes complex multiclonal infections. Recently, Dia et al. have developed innovative methods for single-cell sequencing (SCS) of P. vivax infections by adapting an approach used previously for Plasmodium falciparum. Their studies provide fascinating new insights into P. vivax intrahost diversity and evolution.

Home Sweet Home: Plasmodium vivax-Infected Reticulocytes-The Younger the Better?

After a century of constant failure to produce an in vitro culture of the most widespread human malaria parasite Plasmodium vivax, recent advances have highlighted the difficulties to provide this parasite with a healthy host cell to invade, develop, and multiply under in vitro conditions. The actual level of understanding of the heterogeneous populations of cells—framed under the name ‘reticulocytes’—and, importantly, their adequate in vitro progression from very immature reticulocytes to normocytes (mature erythrocytes) is far from complete. The volatility of its individual stability may suggest the reticulocyte as a delusory cell, particularly to be used for stable culture purposes. Yet, the recent relevance gained by a specific subset of highly immature reticulocytes has brought some hope. Very immature reticulocytes are characterized by a peculiar membrane harboring a plethora of molecules potentially involved in P. vivax invasion and by an intracellular complexity dynamically changing upon its quick maturation into normocytes. We analyze the potentialities offered by this youngest reticulocyte subsets as an ideal in vitro host cell for P. vivax.

A Humanized Mouse Model for Plasmodium vivax to Test Interventions that Block Liver Stage to Blood Stage Transition and Blood Stage Infection.

SummaryThe human malaria parasite Plasmodium vivax remains vastly understudied, mainly due to the lack of suitable laboratory models. Here, we report a humanized mouse model to test interventions that block P. vivax parasite transition from liver stage infection to blood stage infection. Human liver-chimeric FRGN huHep mice infected with P. vivax sporozoites were infused with human reticulocytes, allowing transition of exo-erythrocytic merozoites to reticulocyte infection and development into all erythrocytic forms, including gametocytes, in vivo. In order to test the utility of this model for preclinical assessment of interventions, the invasion blocking potential of a monoclonal antibody targeting the essential interaction of the P. vivax Duffy Binding Protein with the Duffy antigen receptor was tested by passive immunization. This antibody inhibited invasion by over 95%, providing unprecedented in vivo evidence that PvDBP constitutes a promising blood stage vaccine candidate and proving our model highly suitable to test blood stage interventions.

Development of DNA Aptamers Against Plasmodium falciparum Blood Stages Using Cell-Systematic Evolution of Ligands by EXponential Enrichment.

New biomarkers have to be developed in order to increase the performance of current antigen-based malaria rapid diagnosis. Antibody production often involves the use of laboratory animals and is time-consuming and costly, especially when the target is Plasmodium, whose variable antigen expression complicates the development of long-lived biomarkers. To circumvent these obstacles, we have applied the Systematic Evolution of Ligands by EXponential enrichment method to the rapid identification of DNA aptamers against Plasmodium falciparum-infected red blood cells (pRBCs). Five 70 b-long ssDNA sequences, and their shorter forms without the flanking PCR primer-binding regions, have been identified having a highly specific binding of pRBCs versus non-infected erythrocytes. Structural analysis revealed G-enriched sequences compatible with the formation of G-quadruplexes. The selected aptamers recognized intracellular epitopes with apparent Kds in the μM range in both fixed and non-fixed saponin-permeabilized pRBCs, improving >30-fold the pRBC detection in comparison with aptamers raised against Plasmodium lactate dehydrogenase, the gold standard antigen for current malaria diagnostic tests. In thin blood smears of clinical samples the aptamers reported in this work specifically bound all P. falciparum stages versus non-infected erythrocytes, and also detected early and late stages of the human malaria parasites Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. The results are discussed in the context of their potential application in future malaria diagnostic devices.

Field assessment of 4-hydroxycoumarin as an attractant for anthropophilic Anopheles spp. vectors of malaria in Madagascar

Mosquito-borne diseases like malaria are a major public health problem in tropical countries, such as Madagascar. Female Anopheles mosquito vectors the human malaria parasites (Plasmodium spp.) and is important indicator in malaria surveillance activities. Among the various means of vector control in Madagascar, the use of attractants for mass trapping of target species could be an alternative to insecticides. The aim of this study is to evaluate whether 4-hydroxycoumarin can be used as an attractant for anthropophilic Anopheles spp. vectors of malaria. For this, a field study was conducted using CDC light traps in the village of Ambohidray, Madagascar. 16 days of trapping was conducted and four replicates nights were performed for each product tested. 4-hydroxycoumarin, octenol and two types of blend of these products were tested. The results showed that 4-hydroxycoumarin (2 mg) have a significant attractive effect on Anopheles spp. and significant selectivity towards Anopheles gambiae s.l, and Anopheles mascarensis which are both significant malaria vectors in Madagascar. A synergy of 4-hydroxycoumarin with octenol was found to attract these mosquito vectors. A significant decrease in vector populations was observed during this experiment. These results suggest that 4-hydroxycoumarin could be useful for malaria surveillance and the control of vector populations.

Increasingly inbred and fragmented populations of Plasmodium vivax associated with the eastward decline in malaria transmission across the Southwest Pacific.

The human malaria parasite Plasmodium vivax is more resistant to malaria control strategies than Plasmodium falciparum, and maintains high genetic diversity even when transmission is low. To investigate whether declining P. vivax transmission leads to increasing population structure that would facilitate elimination, we genotyped samples from across the Southwest Pacific region, which experiences an eastward decline in malaria transmission, as well as samples from two time points at one site (Tetere, Solomon Islands) during intensified malaria control. Analysis of 887 P. vivax microsatellite haplotypes from hyperendemic Papua New Guinea (PNG, n = 443), meso-hyperendemic Solomon Islands (n = 420), and hypoendemic Vanuatu (n = 24) revealed increasing population structure and multilocus linkage disequilibrium yet a modest decline in diversity as transmission decreases over space and time. In Solomon Islands, which has had sustained control efforts for 20 years, and Vanuatu, which has experienced sustained low transmission for many years, significant population structure was observed at different spatial scales. We conclude that control efforts will eventually impact P. vivax population structure and with sustained pressure, populations may eventually fragment into a limited number of clustered foci that could be targeted for elimination.

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate

BackgroundTechnical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated.ResultsDetailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity.ConclusionsThe MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

BackgroundThe Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax.MethodsWe used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19).Principal findings/ConclusionsWe found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10−4 and 6.2 × 10−4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data.

The Presence, Persistence and Functional Properties of Plasmodium vivax Duffy Binding Protein II Antibodies Are Influenced by HLA Class II Allelic Variants.

BackgroundThe human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked.Methodology/Principal FindingsA community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses.Conclusions/SignificanceThe current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II molecules linked with good or poor antibody responses might lead to the development of strategies for controlling the type of helper T cells activated in response to DBPII.

Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi

Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical. We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes.

The role of the human Duffy antigen receptor for chemokines in malaria susceptibility: current opinions and future treatment prospects.

The Duffy antigen receptor for chemokine (DARC) is a nonspecific receptor for several proinflammatory cytokines. It is homologous to the G-protein chemokine receptor superfamily, which is suggested to function as a scavenger in many inflammatory-and proinflammatory-related diseases. G-protein chemokine receptors are also known to play a critical role in infectious diseases; they are commonly used as entry vehicles by infectious agents. A typical example is the chemokine receptor CCR5 or CXCR4 used by HIV for infecting target cells. In malaria, DARC is considered an essential receptor that mediates the entry of the human and zoonotic malaria parasites Plasmodium vivax and Plasmodium knowlesi into human reticulocytes and erythrocytes, respectively. This process is mediated through interaction with the parasite ligand known as the Duffy binding protein (DBP). Most therapeutic strategies have been focused on blocking the interaction between DBP and DARC by targeting the parasite ligand, while strategies targeting the receptor, DARC, have not been intensively investigated. The rapid increase in drug resistance and the lack of new effective drugs or a vaccine for malaria constitute a major threat and a need for novel therapeutics to combat disease. This review explores strategies that can be used to target the receptor. Inhibitors of DARC, which block DBP–DARC interaction, can potentially provide an effective strategy for preventing malaria caused by P. vivax.

Maintenance of phenotypic diversity within a set of virulence encoding genes of the malaria parasite Plasmodium falciparum.

Infection by the human malaria parasite Plasmodium falciparum results in a broad spectrum of clinical outcomes, ranging from severe and potentially life-threatening malaria to asymptomatic carriage. In a process of naturally acquired immunity, individuals living in malaria-endemic regions build up a level of clinical protection, which attenuates infection severity in an exposure-dependent manner. Underlying this shift in the immunoepidemiology as well as the observed range in malaria pathogenesis is the var multigene family and the phenotypic diversity embedded within. The var gene-encoded surface proteins Plasmodium falciparum erythrocyte membrane protein 1 mediate variant-specific binding of infected red blood cells to a diverse set of host receptors that has been linked to specific disease manifestations, including cerebral and pregnancy-associated malaria. Here, we show that cross-reactive immune responses, which minimize the within-host benefit of each additionally expressed gene during infection, can cause selection for maximum phenotypic diversity at the genome level. We further show that differential functional constraints on protein diversification stably maintain uneven ratios between phenotypic groups, in line with empirical observation. Our results thus suggest that the maintenance of phenotypic diversity within P. falciparum is driven by an evolutionary trade-off that optimizes between within-host parasite fitness and between-host selection pressure.

Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection.

BackgroundFruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.MethodsA. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses.ResultsThe genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission.ConclusionsThese observations suggest that, as in mammals, innate immunity and mitochondrial responses are integrated in mosquitoes and that AsP38 MAPK-dependent signaling facilitates mosquito survival during parasite infection, a fact that may attest to the relatively longer evolutionary relationship of these parasites with their invertebrate compared to their vertebrate hosts. On a practical level, improved understanding of the balances and trade-offs between resistance and metabolism could be leveraged to generate fit, resistant mosquitoes for malaria control.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1016-x) contains supplementary material, which is available to authorized users.

Plasmodium simium, a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.

Structure, function and inhibition of the phosphoethanolamine methyltransferases of the human malaria parasites Plasmodium vivax and Plasmodium knowlesi.

Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.

Contrasting infection susceptibility of the Japanese macaques and cynomolgus macaques to closely related malaria parasites, Plasmodium vivax and Plasmodium cynomolgi

Although the human malaria parasite Plasmodium vivax is closely related to Asian Old World monkey malaria parasites, there are no reports of P. vivax infections in macaques. In this study, we compared the infectivity of P. vivax and Plasmodium cynomolgi in Japanese macaques (Macaca fuscata) and in cynomolgus macaques (Macaca fascicularis). The Japanese macaques were highly susceptible to P. cynomolgi but not to P. vivax, whereas cynomolgus macaques showed mild/limited P. cynomolgi infection and were, also, not susceptible to P. vivax. Serotyping and amino acid sequence comparison of erythrocyte surface Duffy antigen/receptor for chemokines (DARC) indicate that the Japanese macaque DARC sequence is nearly identical to that of rhesus (Macaca mulatta) and cynomolgus macaques. This suggests that the macaques share a common mechanism for preventing P. vivax infection. Comparison of amino acid sequences of the Duffy-binding-like (DBL) domain from several different Plasmodium species suggests that P. vivax DBLs will not bind to macaque DARCs, which can explain the lack of P. vivax infectivity. The DBL sequence analyses also suggest that P. cynomolgi DBLs may target Japanese macaque erythrocytes through a DARC-independent interaction.

Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target

Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.