Dataset Information

Differential roles of an Anopheline midgut GPI-anchored protein in mediating Plasmodium falciparum and Plasmodium vivax ookinete invasion.

ABSTRACT: Novel strategies to directly thwart malaria transmission are needed to maintain the gains achieved by current control measures. Transmission-blocking interventions (TBIs), namely vaccines and drugs targeting parasite or mosquito molecules required for vector-stage parasite development, have been recognized as promising approaches for preventing malaria transmission. However, the number of TBI targets is limited and their degree of conservation among the major vector-parasite systems causing human disease is unclear. Therefore, discovery and characterization of novel proteins involved in vector-stage parasite development of Plasmodium falciparum and Plasmodium vivax is paramount. We mined the recent Anopheles gambiae midgut lipid raft proteome for putative mosquito-derived TBI targets and characterized a secreted glycoconjugate of unknown function, AgSGU. We analyzed molecular variation in this protein among a range of anopheline mosquitoes, determined its transcriptomic and proteomic profiles, and conducted both standard and direct membrane feeding assays with P. falciparum (lab/field) and P. vivax (field) in An. gambiae and Anopheles dirus. We observed that ?-AgSGU antibodies significantly reduced midgut infection intensity for both lab and field isolates of P. falciparum in An. gambiae and An. dirus. However, no transmission-reducing effects were noted when comparable concentrations of antibodies were included in P. vivax-infected blood meals. Although antibodies against AgSGU exhibit transmission-reducing activity, the high antibody titer required for achieving 80% reduction in oocyst intensity precludes its consideration as a malaria mosquito-based TBI candidate. However, our results suggest that P. falciparum and P. vivax ookinetes use a different repertoire of midgut surface glycoproteins for invasion and that ?-AgSGU antibodies, as well as antibodies to other mosquito-midgut microvillar surface proteins, may prove useful as tools for interrogating Plasmodium-mosquito interactions.

SUBMITTER: Mathias DK

PROVIDER: S-EPMC4257886 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Differential roles of an Anopheline midgut GPI-anchored protein in mediating Plasmodium falciparum and Plasmodium vivax ookinete invasion.

Mathias Derrick K DK Jardim Juliette G JG Parish Lindsay A LA Armistead Jennifer S JS Trinh Hung V HV Kumpitak Chalermpon C Sattabongkot Jetsumon J Dinglasan Rhoel R RR Dinglasan Rhoel R RR

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20140611

Novel strategies to directly thwart malaria transmission are needed to maintain the gains achieved by current control measures. Transmission-blocking interventions (TBIs), namely vaccines and drugs targeting parasite or mosquito molecules required for vector-stage parasite development, have been recognized as promising approaches for preventing malaria transmission. However, the number of TBI targets is limited and their degree of conservation among the major vector-parasite systems causing huma ...[more]

PMID: 24929123

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Project description:Malaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. Plasmodium falciparum ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, P. gallinaceum, have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that P. gallinaceum PgCHT2 and its ortholog, P. falciparum PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in P. falciparum and P. gallinaceum ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with P. berghei ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric P. berghei parasites expressing PfCHT1 to replace PbCHT1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric P. berghei ookinetes express monomeric PfCHT1, but a HMW complex containing PfCHT1 is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.

Project description:BackgroundThe development of an effective malarial vaccine is an urgent need. Most glycosylphosphatidylinositol (GPI)-anchored proteins of Plasmodium parasites are exposed to neutralizing antibodies, and several are advanced vaccine candidates. In the present study, Plasmodium vivax Pv32 (PVX_084815) as a hypothetical, predicted GPI-anchored and cysteine-rich motif was identified from our previous findings with a focus on its antigenic profiling. The orthologue gene pv32, a predicted GPI anchor of P. falciparum PF3D7_1434400, has still not been well studied.MethodsThe gene information of pv32 was obtained from PlasmoDB. Recombinant Pv32 protein was expressed and purified using a wheat germ cell-free expression system and a glutathione-Sepharose column. Naturally acquired immune response to recombinant Pv32 protein was evaluated using a protein microarray with 96 parasite-infected patients and 96 healthy individuals. Antibodies against recombinant Pv32 proteins from immune animals were produced, used and analyzed for the subcellular localization of native Pv32 protein by an immunofluorescence assay. A total of 48 pv32 sequences from 11 countries retrieved from PlasmoDB were used to determine the genetic diversity, polymorphisms and genealogical relationships with DNAsp and NETWORK software packages.ResultsPv32 is encoded by a conserved gene with two introns that are located on chromosome 13 and expressed as a 32 kDa protein in mature asexual stage parasites. Immunofluorescence data showed that Pv32 localized on the merozoite surface in schizont-stage parasites. The recombinant Pv32 was recognized by 39.6% of antibodies from P. vivax-infected individuals compared with healthy individuals. Low levels of nucleotide diversity (π = 0.0028) and polymorphisms of pv32 were detected within worldwide isolates.ConclusionsThis study shows the identification and characterization of the hypothetical protein, Pv32. Pv32 provides important characteristics, including a merozoite surface protein, a predicted GPI motif and Cysteine-rich motif among Plasmodium species. These results suggested that Pv32 is immunogenic with a merozoite surface pattern to antibodies during natural infection in humans.

Project description:Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5-Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.

Project description:BackgroundElimination of malaria depends on mastering transmission and understanding the biological basis of Plasmodium infection in the vector. The first mosquito organ to interact with the parasite is the midgut and its transcriptomic characterization during infection can reveal effective antiplasmodial responses able to limit the survival of the parasite. The vector response to Plasmodium vivax is not fully characterized, and its specificities when compared with other malaria parasites can be of fundamental interest for specific control measures.MethodsExperimental infections were performed using a membrane-feeding device. Three groups were used: P. vivax-blood-fed, blood-fed on inactivated gametocytes, and unfed mosquitoes. Twenty-four hours after feeding, the mosquitoes were dissected and the midgut collected for transcriptomic analysis using RNAseq. Nine cDNA libraries were generated and sequenced on an Illumina HiSeq2500. Readings were checked for quality control and analysed using the Trinity platform for de novo transcriptome assembly. Transcript quantification was performed and the transcriptome was functionally annotated. Differential expression gene analysis was carried out. The role of the identified mechanisms was further explored using functional approaches.ResultsForty-nine genes were identified as being differentially expressed with P. vivax infection: 34 were upregulated and 15 were downregulated. Half of the P. vivax-related differentially expressed genes could be related to autophagy; therefore, the effect of the known inhibitor (wortmannin) and activator (spermidine) was tested on the infection outcome. Autophagic activation significantly reduced the intensity and prevalence of infection. This was associated with transcription alterations of the autophagy regulating genes Beclin, DRAM and Apg8.ConclusionsOur data indicate that P. vivax invasion of An. aquasalis midgut epithelium triggers an autophagic response and its activation reduces infection. This suggests a novel mechanism that mosquitoes can use to fight Plasmodium infection.

Dataset Information

Differential roles of an Anopheline midgut GPI-anchored protein in mediating Plasmodium falciparum and Plasmodium vivax ookinete invasion.

Publications

Differential roles of an Anopheline midgut GPI-anchored protein in mediating Plasmodium falciparum and Plasmodium vivax ookinete invasion.

Similar Datasets

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