Plasmodium Proteins Research Articles

Preparedness for malaria elimination in the wake of climate change in the State of Uttarakhand (India).

Climate change is an emerging issue particularly in the context of vector-borne diseases. A study was undertaken in Nainital and Almora districts of Uttarakhand to provide evidences of changing climatic conditions, abundance of vectors, and knocking of malaria in hilly areas. Longitudinal data on temperature and relative humidity were procured from Tussar Silk Centre, Bhimtal, India as well as generated using HOBO device. Monthly density of malaria vectors, their positivity for sporozoite proteins of malaria parasite and fever surveys were conducted as per the standard procedures from 2010 to 2013. Epidemiological data were procured from the State Programme Officer of Uttarakhand state. It was found that the temperature has increased since 1990 resulting in extension in windows of malaria transmission, temporal distribution as well as man hour density of Anopheles culicifacies and An. fluviatilis in hilly districts of Uttarakhand state. Both the vectors were found in high density up to a maximum man hour density of 110 (An. culicifacies) and 69 (An. fluviatilis) as compared to 32 and 33, respectively during 1998. The field collected vector species were also found positive for sporozoite proteins of malaria parasites in the month of October and November. Evidence of occurrence of malaria cases was also found in areas hitherto free from malaria. The findings reveal that Himalayan region needs attention to strengthen surveillance for malaria to identify emerging new foci of malaria transmission in view of climate change. Health education to communities about preventive measures to contain breeding of vectors and seeking timely treatment should be imparted so as to achieve the goal of malaria elimination in category-1 in the first instance.

Plasmodium PK9 Inhibitors Promote Growth of Liver-Stage Parasites

There is a scarcity of pharmacological tools to interrogate protein kinase function in Plasmodium parasites, the causative agent of malaria. Among Plasmodium's protein kinases, those characterized as atypical represent attractive drug targets as they lack sequence similarity to human proteins. Here, we describe takinib as a small molecule to bind the atypical P.falciparum protein kinase 9 (PfPK9). PfPK9 phosphorylates the Plasmodium E2 ubiquitin-conjugating enzyme PfUBC13, which mediates K63-linkage-specific polyubiquitination. Takinib is a potent human TAK1 inhibitor, thus we developed the Plasmodium-selective takinib analog HS220. We demonstrate that takinib and HS220 decrease K63-linked ubiquitination in P.falciparum, suggesting PfPK9 inhibition in cells. Takinib and HS220 induce a unique phenotype where parasite size in hepatocytes increases, yet high compound concentrations decrease the number of parasites. Our studies highlight the role of PK9 in regulating parasite development and the potential of targeting Plasmodium kinases for malaria control.

Essentiality of Plasmodium falciparum plasmepsin V.

The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested early in the ring-to-trophozoite transition in the erythrocytic cycle following gene excision. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway.

The Micronemal Plasmodium Proteins P36 and P52 Act in Concert to Establish the Replication-Permissive Compartment Within Infected Hepatocytes.

Within the liver, Plasmodium sporozoites traverse cells searching for a “suitable” hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.

Interaction with complement proteins and dendritic cells implicates LCCL domain-containing proteins (CCps) of malaria parasites in immunomodulation.

The evasion of host immune defense is critical for pathogens to invade, establish infection and perpetuate in the host. The complement system is one of the first lines of innate immune defense in humans that destroys pathogens in the blood circulation. Activation of the complement system through direct encounter with pathogens or some other agents leads to osmolysis of pathogens, clearance of soluble immune complexes and recruitment of lymphocytes at the site of activation. Although malaria parasites are not exposed to the complement system owing to their intracellular development for most part of their life cycle in the human host, the extracellular stages must face the complement system of human or mosquito or both. In a recent issue of the Biochemical Journal, Sharma et al. reported that Plasmodiumfalciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells, which implicate PfCCp1 and related proteins in immunomodulation of the host that likely benefits the parasite. PfCCp1 belongs to a multi-domain protein family that exists as multimeric protein complexes. It needs to be investigated whether PfCCp1 or its multimeric protein complexes have an immunomodulatory effect in vivo and on the mosquito complement system.

Genetic diversity in two Plasmodium vivax protein ligands for reticulocyte invasion.

The interaction between Plasmodium vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) has been described as critical for the invasion of human reticulocytes, although increasing reports of P. vivax infections in Duffy-negative individuals questions its unique role. To investigate the genetic diversity of the two main protein ligands for reticulocyte invasion, PvDBP and P. vivax Erythrocyte Binding Protein (PvEBP), we analyzed 458 isolates collected in Cambodia and Madagascar from individuals genotyped as Duffy-positive. First, we observed a high proportion of isolates with multiple copies PvEBP from Madagascar (56%) where Duffy negative and positive individuals coexist compared to Cambodia (19%) where Duffy-negative population is virtually absent. Whether the gene amplification observed is responsible for alternate invasion pathways remains to be tested. Second, we found that the PvEBP gene was less diverse than PvDBP gene (12 vs. 33 alleles) but provided evidence for an excess of nonsynonymous mutations with the complete absence of synonymous mutations. This finding reveals that PvEBP is under strong diversifying selection, and confirms the importance of this protein ligand in the invasion process of the human reticulocytes and as a target of acquired immunity. These observations highlight how genomic changes in parasite ligands improve the fitness of P. vivax isolates in the face of immune pressure and receptor polymorphisms.

Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains

Acquired antibodies directed towards antigens expressed on the surface of merozoites and infected erythrocytes play an important role in protective immunity to Plasmodium falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major parasite component of the infected erythrocyte surface, has been implicated in malaria pathology, parasite sequestration and host immune evasion. However, the extent to which unique PfEMP1 domains interact with host immune response remains largely unknown. In this study, we sought to comprehensively understand the naturally acquired antibody responses targeting different Duffy binding-like (DBL), and Cysteine-rich interdomain region (CIDR) domains in a Ugandan cohort. Consequently, we created a protein library consisting of full-length DBL (n = 163) and CIDR (n = 108) domains derived from 62-var genes based on 3D7 genome. The proteins were expressed by a wheat germ cell-free system; a system that yields plasmodial proteins that are comparatively soluble, intact, biologically active and immunoreactive to human sera. Our findings suggest that all PfEMP1 DBL and CIDR domains, regardless of PfEMP1 group, are targets of naturally acquired immunity. The breadth of the immune response expands with children’s age. We concurrently identified 10 DBL and 8 CIDR domains whose antibody responses were associated with reduced risk to symptomatic malaria in the Ugandan children cohort. This study highlights that only a restricted set of specific domains are essential for eliciting naturally acquired protective immunity in malaria. In light of current data, tandem domains in PfEMP1s PF3D7_0700100 and PF3D7_0425800 (DC4) are recommended for extensive evaluation in larger population cohorts to further assess their potential as alternative targets for malaria vaccine development.

A tiling array-based comparative genomic hybridization approach to predict copy number variations between Plasmodium falciparum field isolates from the Indian Sub-continent

Background: There are several techniques to analyse copy number variation in both research and clinical settings, such as whole genome amplification (sWGA), SNP arrays and one of the most commonly used techniques, array based comparative genomic hybridization (aCGH). In the latter, copy number comparison is obtained between differentially labelled target and reference DNAs by measuring ratio of fluorescence intensity of probes indicating loss or gain in the chromosomal region. Methods: Here we carry out a comparative analysis between two Plasmodium falciparum parasite isolates (Pf-isolate-2 and Pf-isolate-1) causing malaria using array CGH. The array contains approximately 418,577, 60mer custom-designed probes with an average probe spacing of 56 bp. The significant major variations (amplifications and deletions) copy number variations (CNV) in Pf-isolate-2 (Pf-2) in comparison with Pf-isolate-1 (Pf-1), are reported. Results: CNVs have been seen in all the chromosomes in Pf-2, most of the deletions have been seen mostly in sub-telomeric and telomeric regions of the chromosomes that comprises of variant surface antigen family genes. Apart from the subtelomeric regions other parts of the chromosomes have also shown CNVs. Novel variations , like continuous amplification of 28kb region (249817-278491) of chromosome-8, which covers for 3 genes two of which codes for conserved Plasmodium proteins with unknown function (MAL8P1.139, PF08_0122) and tRNA pseudouridine synthase, putative (PF08_0123). Amplifications in regions harboring genes like GTP cyclohydrolase I (GCH-1, PFL1155W) and ribosomal protein, L24, putative (PFL1150C) of chromosome 12 were seen. Conclusion: Other than known variations reported earlier, some novel variations have also been seen in the chromosomes of Pf-2. This is an experimental case study reporting major amplifications and deletions in Pf-isolate-2 in comparison with Pf-isolate-1 using a tiling array based comparative genomic hybridization approach.

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice.

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.

Surface antibody maturation

Immunology Affinity maturation in B cells generates antibodies with increasingly enhanced antigen-binding properties. Imkeller et al. investigated the maturation of human B cells that express protective antibodies against the circumsporozoite protein of the malaria-causing parasite Plasmodium

Specific β-Turns Precede PPIIL Structures Binding to Allele-Specific HLA-DRβ1* PBRs in Fully-Protective Malaria Vaccine Components.

The 3D structural analysis of 62 peptides derived from highly pathogenic Plasmodium falciparum malaria parasite proteins involved in host cell invasion led to finding a striking association between particular β-turn types located in the N-terminal peripheral flanking residue region (preceding the polyproline II left-handed structures fitting into the HLA-DRβ* allele family) and modified immune protection-inducing protein structure induced long-lasting protective immunity. This is the first time association between two different secondary structures associated with a specific immunological function has been described: full, long-lasting protective immunity.

Study on the species composition and ecology of anophelines in Addis Zemen, South Gondar, Ethiopia

BackgroundMalaria is a public health problem in Ethiopia and its transmission is generally unstable and seasonal. For the selection of the most appropriate vector control measures, knowledge on the ecology of the vector is necessary at a local level. Therefore, the objectives of this study were to document the species composition, breeding habitat characteristics and occurrence of anopheline larva in Sheni stream and the vectorial role of the prevailing Anopheles in relation to malaria transmission in Addis Zemen, Ethiopia.MethodsImmature anophelines were sampled from breeding habitats and characteristics, such as water temperature, turbidity, water current, water pH and other variables, of the habitats were measured from October 2011 to February 2012. Adult anophelines were sampled inside human dwellings using space spray and Center for Disease Control light traps. Artificial pit shelters and clay pots were also used for outdoor adult collections. Anophelines collected were identified using morphological key. The enzyme-linked immunosorbent assay was applied to detect circumsporozoite proteins of Plasmodium and source of blood meals.ResultsA total of 6258 Anopheles larvae were collected and identified morphologically. Five anopheline species were found: An. gambiae (s.l.), An. cinereus, An. demeilloni, An. christi and An. pretoriensis. Anopheles gambiae (s.l.) existed in most of the habitats investigated. Only the former three species were captured in the adult collections. Sun-lit Sheni stream, rain pools, hoof prints, drainage and irrigation canals were found to be habitats of larvae. Anopheles gambiae (s.l.) larvae were most abundantly sampled from sand mining and natural sand pools of Sheni stream. Multiple regression analysis showed that clear, permanent and temporary habitats devoid of mats of algae were the best predictors of An. gambiae (s.l.) larval abundance. It is also the responsible malaria vector in the study area and exhibits anthropophilic and endophagic behaviour.ConclusionsThe malaria vector An. gambiae (s.l.) was found in Addis Zemen throughout the study period from both adult and larval collections. Sheni stream is the main larval habitat responsible for the occurrence of anopheline larvae during the dry season of the study area when other breeding sites perish.

Proteome-wide mapping of immune features onto Plasmodium protein three-dimensional structures

Humoral immune responses against the malaria parasite are an important component of a protective immune response. Antibodies are often directed towards conformational epitopes, and the native structure of the antigenic region is usually critical for antibody recognition. We examined the structural features of various Plasmodium antigens that may impact on epitope location, by performing a comprehensive analysis of known and modelled structures from P. falciparum. Examining the location of known polymorphisms over all available structures, we observed a strong propensity for polymorphic residues to be exposed on the surface and to occur in particular secondary structure segments such as hydrogen-bonded turns. We also utilised established prediction algorithms for B-cell epitopes and MHC class II binding peptides, examining predicted epitopes in relation to known polymorphic sites within structured regions. Finally, we used the available structures to examine polymorphic hotspots and Tajima’s D values using a spatial averaging approach. We identified a region of PfAMA1 involving both domains II and III under a high degree of balancing selection relative to the rest of the protein. In summary, we developed general methods for examining how sequence-based features relate to one another in three-dimensional space and applied these methods to key P. falciparum antigens.

KSHV oral shedding and plasma viremia result in significant changes in the extracellular tumorigenic miRNA expression profile in individuals infected with the malaria parasite.

Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS). Both KSHV and HIV infections are endemic in Uganda, where KS is among the most common cancers in HIV-infected individuals. Recent studies examined the use of small RNAs as biomarkers of disease, including microRNAs (miRNAs), with viral and tumor-derived miRNAs being detected in exosomes from individuals with KSHV-associated malignancies. In the current study, the host and viral extracellular mature miRNA expression profiles were analyzed in blood of KS-negative individuals in Uganda, comparing those with or without KSHV detectable from the oropharynx. We observed increased levels of cellular oncogenic miRNAs and decreased levels of tumor-suppressor miRNAs in plasma of infected individuals exhibiting oral KSHV shedding. These changes in host oncomiRs were exacerbated in people co-infected with HIV, and partially reversed after 2 years of anti-retroviral therapy. We also detected KSHV miRNAs in plasma of KSHV infected individuals and determined that their expression levels correlated with KSHV plasma viremia. Deep sequencing revealed an expected profile of small cellular RNAs in plasma, with miRNAs constituting the major RNA biotype. In contrast, the composition of small RNAs in exosomes was highly atypical with high levels of YRNA and low levels of miRNAs. Mass spectrometry analysis of the exosomes revealed eleven different peptides derived from the malaria parasite, Plasmodium falciparum, and small RNA sequencing confirmed widespread plasmodium co-infections in the Ugandan cohorts. Proteome analysis indicated an exosomal protein profile consistent with erythrocyte and keratinocyte origins for the plasma exosomes. A strong correlation was observed between the abundance of Plasmodium proteins and cellular markers of malaria. As Plasmodium falciparum is an endemic pathogen in Uganda, our study shows that co-infection with other pathogens, such as KSHV, can severely impact the small RNA repertoire, complicating the use of exosome miRNAs as biomarkers of disease.

A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation.

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti‐PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission‐blocking vaccine target candidate.

Converting a Liability to an Asset: Using the Clearance of a Malaria Parasite Protein From the Blood of Infected Subjects to Predict the Outcome of Treatment.

Converting a Liability to an Asset: Using the Clearance of a Malaria Parasite Protein From the Blood of Infected Subjects to Predict the Outcome of Treatment.

Repetitive sequences in malaria parasite proteins.

Five species of parasite cause malaria in humans with the most severe disease caused by Plasmodium falciparum. Many of the proteins encoded in the P. falciparum genome are unusually enriched in repetitive low-complexity sequences containing a limited repertoire of amino acids. These repetitive sequences expand and contract dynamically and are among the most rapidly changing sequences in the genome. The simplest repetitive sequences consist of single amino acid repeats such as poly-asparagine tracts that are found in approximately 25% of P. falciparum proteins. More complex repeats of two or more amino acids are also common in diverse parasite protein families. There is no universal explanation for the occurrence of repetitive sequences and it is possible that many confer no function to the encoded protein and no selective advantage or disadvantage to the parasite. However, there are increasing numbers of examples where repetitive sequences are important for parasite protein function. We discuss the diverse roles of low-complexity repetitive sequences throughout the parasite life cycle, from mediating protein-protein interactions to enabling the parasite to evade the host immune system.

Identification of Secretory Proteins of Malaria Parasite by Feature Selection Technique

Identification of Secretory Proteins of Malaria Parasite by Feature Selection Technique

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.

Protein O-fucosylation in Plasmodium falciparum ensures efficient infection of mosquito and vertebrate hosts

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.