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Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood.

ABSTRACT: Successful completion of the Plasmodium lifecycle in the mosquito vector is critical for malaria transmission. It has been documented that the fate of Plasmodium in the mosquito ultimately depends on a fine interplay of molecular mosquito factors that act as parasite agonists and antagonists. Here we investigate whether the cellular responses of the invaded midgut epithelium can also determine the parasite fate and development. We show that the parasite hood, an actin-rich structure formed around the ookinete as it exits the epithelium, is a local epithelial defence reaction observed around 60% of invading parasites. The hood co-localizes with WASP, a promoter of actin filament nucleation, suggesting that it is an active reaction of the invaded cell against invading parasites. Importantly, depletion of WASP by RNAi leads to a significant reduction in hood formation, which is consistent with the previously documented role of this gene as a potent parasite antagonist. Indeed, in mosquitoes that are either genetically selected or manipulated by RNAi to be refractory to Plasmodium, most dead parasites exhibit an actin hood. In these mosquitoes, invading ookinetes are killed by lysis or melanization while exiting the midgut epithelium. Silencing WASP in these mosquitoes inhibits the formation of the hood and allows many parasites to develop to oocysts. These data in conjunction with fine microscopic observations suggest that the presence of the hood is linked to ookinete killing through lysis.

SUBMITTER: Schlegelmilch T

PROVIDER: S-EPMC4073529 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood.

Schlegelmilch Timm T Vlachou Dina D

Pathogens and global health 20131201 8

Successful completion of the Plasmodium lifecycle in the mosquito vector is critical for malaria transmission. It has been documented that the fate of Plasmodium in the mosquito ultimately depends on a fine interplay of molecular mosquito factors that act as parasite agonists and antagonists. Here we investigate whether the cellular responses of the invaded midgut epithelium can also determine the parasite fate and development. We show that the parasite hood, an actin-rich structure formed aroun ...[more]

PMID: 24428832

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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: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 α-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 α-AgSGU antibodies, as well as antibodies to other mosquito-midgut microvillar surface proteins, may prove useful as tools for interrogating Plasmodium-mosquito interactions.

Project description:Malaria transmission-blocking (T-B) interventions are essential for malaria elimination. Small molecules that inhibit the Plasmodium ookinete-to-oocyst transition in the midgut of Anopheles mosquitoes, thereby blocking sporogony, represent one approach to achieving this goal. Chondroitin sulfate glycosaminoglycans (CS-GAGs) on the Anopheles gambiae midgut surface are putative ligands for Plasmodium falciparum ookinetes. We hypothesized that our synthetic polysulfonated polymer, VS1, acting as a decoy molecular mimetic of midgut CS-GAGs confers malaria T-B activity. In our study, VS1 repeatedly reduced midgut oocyst development by as much as 99% (P<0.0001) in mosquitoes fed with P. falciparum and Plasmodium berghei. Through direct-binding assays, we observed that VS1 bound to two critical ookinete micronemal proteins, each containing at least one von Willebrand factor A (vWA) domain: (i) circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) and (ii) vWA domain-related protein (WARP). By immunofluorescence microscopy, we observed that VS1 stains permeabilized P. falciparum and P. berghei ookinetes but does not stain P. berghei CTRP knockouts or transgenic parasites lacking the vWA domains of CTRP while retaining the thrombospondin repeat region. We produced structural homology models of the first vWA domain of CTRP and identified, as expected, putative GAG-binding sites on CTRP that align closely with those predicted for the human vWA A1 domain and the Toxoplasma gondii MIC2 adhesin. Importantly, the models also identified patches of electropositive residues that may extend CTRP's GAG-binding motif and thus potentiate VS1 binding. Our molecule binds to a critical, conserved ookinete protein, CTRP, and exhibits potent malaria T-B activity. This study lays the framework for a high-throughput screen of existing libraries of safe compounds to identify those with potent T-B activity. We envision that such compounds when used as partner drugs with current antimalarial regimens and with RTS,S vaccine delivery could prevent the transmission of drug-resistant and vaccine-breakthrough strains.

Dataset Information

Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood.

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Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood.

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