Inhibition of Plasmodium falciparum plasmepsins by drugs targeting HIV-1 protease: A way forward for antimalarial drug discovery.

Plasmodium species are causative agents of malaria, a disease that is a serious global health concern. FDA-approved HIV-1 protease inhibitors (HIV-1 PIs) have been reported to be effective in reducing the infection by Plasmodium parasites in the population co-infected with both HIV-1 and malaria. However, the mechanism of HIV-1 PIs in mitigating Plasmodium pathogenesis during malaria/HIV-1 co-infection is not fully understood. In this study we demonstrate that HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum. Crystal structures of the complexes of PMII with both drugs have been determined. The inhibitors interact with PMII via multiple hydrogen bonding and hydrophobic interactions. The P4 moiety of RTV forms additional interactions compared to LPV and exhibits conformational flexibility in a large S4 pocket of PMII. Our study is also the first to report inhibition of P. falciparum PMX by RTV and the mode of binding of the drug to the PMX active site. Analysis of the crystal structures implies that PMs can accommodate bulkier groups of these inhibitors in their S4 binding pockets. Structurally similar active sites of different vacuolar and non-vacuolar PMs suggest the potential of HIV-1 PIs in targeting these enzymes with differential affinities. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing HIV-1 PIs as antimalarial drugs.

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

Polyclonal Antibody Generation against PvTRAg for the Development of a Diagnostic Assay for Plasmodium vivax.

The World Health Organization (WHO) has set forth a global call for eradicating malaria, caused majorly by the protozoan parasites Plasmodium falciparum and Plasmodium vivax. The lack of diagnostic biomarkers for P. vivax, especially those that differentiate the parasite from P. falciparum, significantly hinders P. vivax elimination. Here, we show that P. vivax tryptophan-rich antigen (PvTRAg) can be a diagnostic biomarker for diagnosing P. vivax in malaria patients. We report that polyclonal antibodies against purified PvTRAg protein show interactions with purified PvTRAg and native PvTRAg using Western blots and indirect enzyme-linked immunosorbent assay (ELISA). We also developed an antibody-antigen-based qualitative assay using biolayer interferometry (BLI) to detect vivax infection using plasma samples from patients with different febrile diseases and healthy controls. The polyclonal anti-PvTRAg antibodies were used to capture free native PvTRAg from the patient plasma samples using BLI, providing a new expansion range to make the assay quick, accurate, sensitive, and high-throughput. The data presented in this report provides a proof of concept for PvTRAg, a new antigen, for developing a diagnostic assay for P. vivax identification and differentiation from the rest of the Plasmodium species and, at a later stage, translating the BLI assay into affordable, point-of-care formats to make it more accessible.

Conservation of Importin α Function in Apicomplexans: Ivermectin and GW5074 Target Plasmodium falciparum Importin α and Inhibit Parasite Growth in Culture.

Signal-dependent transport into and out of the nucleus mediated by members of the importin (IMP) superfamily of nuclear transporters is critical to the eukaryotic function and a point of therapeutic intervention with the potential to limit disease progression and pathogenic outcomes. Although the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii both retain unique IMPα genes that are essential, a detailed analysis of their properties has not been performed. As a first step to validate apicomplexan IMPα as a target, we set out to compare the properties of P. falciparum and T. gondii IMPα (PfIMPα and TgIMPα, respectively) to those of mammalian IMPα, as exemplified by Mus musculus IMPα (MmIMPα). Close similarities were evident, with all three showing high-affinity binding to modular nuclear localisation signals (NLSs) from apicomplexans as well as Simian virus SV40 large tumour antigen (T-ag). PfIMPα and TgIMPα were also capable of binding to mammalian IMPß1 (MmIMPß1) with high affinity; strikingly, NLS binding by PfIMPα and TgIMPα could be inhibited by the mammalian IMPα targeting small molecules ivermectin and GW5074 through direct binding to PfIMPα and TgIMPα to perturb the α-helical structure. Importantly, GW5074 could be shown for the first time to resemble ivermectin in being able to limit growth of P. falciparum. The results confirm apicomplexan IMPα as a viable target for the development of therapeutics, with agents targeting it worthy of further consideration as an antimalarial.

Diverse Malaria Presentations across National Institutes of Health South Asia International Center for Excellence in Malaria Research Sites in India.

The Malaria Evolution in South Asia (MESA) International Center for Excellence in Malaria Research (ICEMR) was established by the US National Institutes of Health (US NIH) as one of 10 malaria research centers in endemic countries. In 10 years of hospital-based and field-based work in India, the MESA-ICEMR has documented the changing epidemiology and transmission of malaria in four different parts of India. Malaria Evolution in South Asia-ICEMR activities, in collaboration with Indian partners, are carried out in the broad thematic areas of malaria case surveillance, vector biology and transmission, antimalarial resistance, pathogenesis, and host response. The program integrates insights from surveillance and field studies with novel basic science studies. This is a two-pronged approach determining the biology behind the disease patterns seen in the field, and generating new relevant biological questions about malaria to be tested in the field. Malaria Evolution in South Asia-ICEMR activities inform local and international stakeholders on the current status of malaria transmission in select parts of South Asia including updates on regional vectors of transmission of local parasites. The community surveys and new laboratory tools help monitor ongoing efforts to control and eliminate malaria in key regions of South Asia including the state of evolving antimalarial resistance in different parts of India, new host biomarkers of recent infection, and molecular markers of pathogenesis from uncomplicated and severe malaria.

High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.

The role of upstream open reading frames in translation regulation in the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.

During their complex life cycles, the Apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii employ several layers of regulation of their gene expression. One such layer is mediated at the level of translation through upstream open reading frames (uORFs). As uORFs are found in the upstream regions of a majority of transcripts in both the parasites, it is essential that their roles in translational regulation be appreciated to a greater extent. This review provides a comprehensive summary of studies that show uORF-mediated gene regulation in these parasites and highlights examples of clinically and physiologically relevant genes, including var2csa in P. falciparum, and ApiAT1 in T. gondii, that exhibit uORF-mediated regulation. In addition to these examples, several studies that use bioinformatics, transcriptomics, proteomics and ribosome profiling also indicate the possibility of widespread translational regulation by uORFs. Further analysis of these genome-wide datasets, taking into account uORFs associated with each gene, will reveal novel genes involved in key biological pathways such as cell-cycle progression, stress-response and pathogenicity. The cumulative evidence from studies presented in this review suggests that uORFs will play crucial roles in regulating gene expression during clinical disease caused by these important human pathogens.

Protein Arrays for the Identification of Seroreactive Protein Markers for Infectious Diseases.

The protein array is a powerful platform to study humoral responses to infectious agents using small sample volumes [<3 µL]. Its success can be largely attributed to the development of new strategies for high-throughput cloning and expression, and improved manufacturing techniques for the construction of arrays. Here, we describe a method to hybridize protein arrays with malaria patients' sera in order to identify seroreactive antigens, some of which may have a high potential of conferring protection from severe forms of malaria.

Dually localised proteins found in both the apicoplast and mitochondrion utilize the Golgi-dependent pathway for apicoplast targeting in Toxoplasma gondii.

BACKGROUND INFORMATION: Like other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle - the apicoplast. Apicoplast proteins are nuclear encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi-independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, whereas a handful of reports show that a few proteins use the Golgi-dependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known. RESULTS: In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN/IRP) are trafficked through the Golgi, whereas proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast. CONCLUSIONS AND SIGNIFICANCE: Collectively, with these results, for the first time, we establish that the driver of the Golgi-dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.

Multiplexed quantitative proteomics provides mechanistic cues for malaria severity and complexity.

Management of severe malaria remains a critical global challenge. In this study, using a multiplexed quantitative proteomics pipeline we systematically investigated the plasma proteome alterations in non-severe and severe malaria patients. We identified a few parasite proteins in severe malaria patients, which could be promising from a diagnostic perspective. Further, from host proteome analysis we observed substantial modulations in many crucial physiological pathways, including lipid metabolism, cytokine signaling, complement, and coagulation cascades in severe malaria. We propose that severe manifestations of malaria are possibly underpinned by modulations of the host physiology and defense machinery, which is evidently reflected in the plasma proteome alterations. Importantly, we identified multiple blood markers that can effectively define different complications of severe falciparum malaria, including cerebral syndromes and severe anemia. The ability of our identified blood markers to distinguish different severe complications of malaria may aid in developing new clinical tests for monitoring malaria severity.

Messenger RNAs with large numbers of upstream open reading frames are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum.

The genome of Plasmodium falciparum has one of the most skewed base-pair compositions of any eukaryote, with an AT content of 80-90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 11 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream open reading frame (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation.In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case, the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to the codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole-genome data was analysed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

Comprehensive proteomics investigation of P. vivax-infected human plasma and parasite isolates.

BACKGROUND: In recent times, Plasmodium vivax (P. vivax) has become a serious threat to public health due to its ability to cause severe infection with fatal outcomes. Its unique biology makes it resilient to control measures that are otherwise effective against P. falciparum. A deeper understanding of P. vivax biology and pathogenesis is, therefore, essential for developing the right control strategies. Proteomics of P. falciparum has been helpful in studying disease biology and elucidating molecular mechanisms involved in the development of disease. However, unlike P. falciparum, proteomics data for P. vivax infection is minimal due to the absence of a continuous culture system. The dependence on clinical samples and animal models has drastically limited P. vivax research, creating critical knowledge gaps in our understanding of the disease. This study describes an in-depth proteomics analysis of P. vivax-infected human plasma and parasite isolates, to understand parasite biology, pathogenesis, and to identify new diagnostic targets for P. vivax malaria. METHODS: A mass-spectrometry- (MS) based proteomics approach (Q Exactive) was applied to analyze human plasma and parasite isolates from vivax malaria patients visiting a primary health centre in India. Additionally, a targeted proteomics assay was standardized for validating unique peptides of most recurring parasite proteins. RESULTS: Thirty-eight P. vivax proteins were detected in human plasma with high confidence. Several glycolytic enzymes were found along with hypothetical, cytoskeletal, ribosomal, and nuclear proteins. Additionally, 103 highly abundant P. vivax proteins were detected in parasite isolates. This represents the highest number of parasite proteins to be reported from clinical samples so far. Interestingly, five of these; three Plasmodium exported proteins (PVX_003545, PVX_003555 and PVX_121935), a hypothetical protein (PVX_083555) and Pvstp1 (subtelomeric transmembrane protein 1, PVX_094303) were found in both plasma and parasite isolates. CONCLUSIONS: A parasite proteomics investigation is essential to understand disease pathobiology and design novel interventions. Control strategies against P. vivax also depend on early diagnosis. This work provides deeper insights into the biology of P. vivax by identifying proteins expressed by the parasite during its complex life-cycle within the human host. The study also reports antigens that may be explored as diagnostic candidates.

An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii.

Toxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast, and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2.

Presence of Potential G-Quadruplex RNA-Forming Motifs at the 5'-UTR of PP2Acα mRNA Repress Translation.

RNA G-quadruplex (G4)-forming motifs present at the 5'-UTR of the protein phosphatase (PP2Ac) gene are the regulatory targets of the fragile X mental retardation protein (FMRP), which is weakly expressed in Fragile X patients. Herein, we report that the existence of such G4-forming sequence represses the translation of the PP2Acα gene. This study opens therapeutic avenues to design small molecule ligands that mimic the function of the FMRP.

Hospital-derived antibody profiles of malaria patients in Southwest India.

BACKGROUND: Naturally acquired immunity to malaria across the globe varies in intensity and protective powers. Many of the studies on immunity are from hyperendemic regions of Africa. In Asia, particularly in India, there are unique opportunities for exploring and understanding malaria immunity relative to host age, co-occurrence of Plasmodium falciparum and Plasmodium vivax infections, varying travel history, and varying disease severity. Variation in immunity in hospital settings is particularly understudied. METHODS: A US NIH ICEMR (South Asia) team examined the level of immunity in an Indian malaria patient population visiting or admitted to Goa Medical College and Hospital in Goa, India. Sera from 200 patients of different ages, in different seasons, infected with P. falciparum or P. vivax or both species, and with different clinical severity were applied to an established protein array system with over 1000 P. falciparum and P. vivax antigens. Differential binding of patient IgG to different antigens was measured. RESULTS: Even though Goa itself has much more P. vivax than P. falciparum, IgG reactivity towards P. falciparum antigens was very strong and comparable to that seen in regions of the world with high P. falciparum endemicity. Of 248 seropositive P. falciparum antigens, the strongest were VAR, MSP10, HSP70, PTP5, AP2, AMA1, and SYN6. In P. vivax patients, ETRAMPs, MSPs, and ApiAP2, sexual stage antigen s16, RON3 were the strongest IgG binders. Both P. falciparum and P. vivax patients also revealed strong binding to new antigens with unknown functions. Seropositives showed antigens unique to the young (HSP40, ACS6, GCVH) or to non-severe malaria (MSP3.8 and PHIST). CONCLUSION: Seroreactivity at a major hospital in Southwest India reveals antibody responses to P. falciparum and P. vivax in a low malaria transmission region with much migration. In addition to markers of transmission, the data points to specific leads for possible protective immunity against severe disease. Several, but not all, key antigens overlap with work from different settings around the globe and from other parts of India. Together, these studies confidently help define antigens with the greatest potential chance of universal application for surveillance and possibly for disease protection, in many different parts of India and the world.

Heterologous expression in Toxoplasma gondii reveals a topogenic signal anchor in a Plasmodium apicoplast protein.

Glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) is an antioxidant enzyme trafficked to the apicoplast, a secondary endosymbiotic organelle, in Plasmodium falciparum. Apicoplast trafficking signals usually consist of N-terminal signal and transit peptides, but the trafficking signal of PfTPxGl appears to exhibit important differences. As transfection is a protracted process in P. falciparum, we expressed the N terminus of PfTPxGl as a GFP fusion protein in a related apicomplexan, Toxoplasma gondii, in order to dissect its trafficking signals. We show that PfTPxGl possesses an N-terminal signal anchor that takes the protein to the endoplasmic reticulum in Toxoplasma-this is the first step in the apicoplast targeting pathway. We dissected the residues important for endomembrane system uptake, membrane anchorage, orientation, spacing, and cleavage. Protease protection assays and fluorescence complementation revealed that the C terminus of the protein lies in the ER lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered PfTPxGl targeting in Toxoplasma, in Plasmodium. This study has demonstrated that PfTPxGl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins. This work adds to the mounting evidence that the signals involved in the targeting of apicoplast membrane proteins may not be as straightforward as those of luminal proteins, and also highlights the usefulness of T. gondii as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.

Molecular basis for the lack of auto-inhibition of Plasmodium falciparum importin α.

Importin α is nuclear transport receptor that recognises nuclear localisation sequences (NLS). The protein has two domains: armadillo (ARM) repeats containing NLS-binding sites and the importin ß-binding (IBB) domain. The IBB domain mimics an NLS and can bind to the ARM repeats, preventing NLS binding. This phenomenon, called auto-inhibition, is a key regulatory feature for binding and release of NLS-containing cargo by importin α and mutants that lack auto-inhibition show impaired viability in Saccharomyces cerevisiae. The genome of the human malaria parasite, Plasmodium falciparum, contains a single gene for importin α and here we show that the native protein expressed by this gene lacks auto-inhibition, suggesting that P. falciparum parasites possess unconventional mechanisms for regulation of cargo binding and release. Mutation of a single SKR motif (conserved in Plasmodium species) to KRR in P. falciparum importin α restores auto-inhibition. This is the first report of a single-celled eukaryote that has evolved with a single importin α isoform lacking auto-inhibition.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.

Identification of Highly Expressed Plasmodium Vivax Proteins from Clinical Isolates Using Proteomics.

Plasmodium vivax is the most geographically widespread species responsible for malaria in humans. Our study focused on identifying highly expressed parasite proteins using a shotgun proteomics approach. Parasites (P. vivax) are isolated from seven patient samples using saponin lysis. Protein extracts from these parasites are processed and subjected to LC-MS/MS analysis. An overall proteome coverage of 605 P. vivax proteins along with 1670 human host proteins are obtained upon combining the data from LC-MS/MS runs. While a major proportion of the P. vivax proteins are either hypothetical or involved in basic cellular activities, few proteins such as tryptophan-rich antigen (Pv-fam-a; PVX_090265), Pv-fam-d protein (PVX_101520), Plasmodium exported protein (PVX_003545), Pvstp1 (PVX_094303) and hypothetical protein (PVX_083555) are detected in more than 80% of the clinical isolates and found to be unique to P. vivax without orthologs in P. falciparum. Our proteomics study on individual parasite isolates reveals highly expressed P. vivax proteins, few of which may be good candidates for vivax malaria diagnosis due to their abundance and absence in P. falciparum. This study represents the first step towards the identification of biomarkers for P. vivax malaria. In future, their clinical diagnostic values must be explored and validated on large patient cohorts.