Structure and expression of an adhesive protein-like molecule of mosquito invasive-stage malarial parasite.
ABSTRACT: Invasion of the malarial parasite into a vector mosquito begins when the motile ookinete transverses the gut epithelium. Adhesive proteins that may mediate this invasive process have not been identified to date. We found that a molecule with an adhesive protein-like structure was expressed in the ookinete of Plasmodium berghei. This protein is structurally homologous to circumsporozoite protein and thrombospondin-related adhesive protein (TRAP)-related protein, CTRP, of Plasmodium falciparum. We named it P. berghei CTRP (PbCTRP) and report here its structure and manner of expression. PbCTRP has six integrin I region-like domains and seven thrombospondin-like domains in its putative extracellular region. This structure is similar to that of CTRP and TRAPs of malaria sporozoite. The putative transmembrane and cytoplasmic regions of PbCTRP, CTRP, and TRAP also have conserved amino acid sequences. PbCTRP is produced at least 10 h after fertilization when zygotes begin transformation to ookinetes. In the mature ookinete, PbCTRP is located mainly in the anterior cytoplasm. The staining pattern was also similar to TRAP in the sporozoite. We suggest that PbCTRP may play a role in ookinete invasive motility and belongs to a protein family together with TRAP and other structurally related proteins of apicomplexan parasites.
Project description:The transformation of malaria ookinetes into oocysts occurs in the mosquito midgut and is a major bottleneck for parasite transmission. The secreted ookinete surface protein, circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP), is essential for this transition and hence constitutes a potential target for malaria transmission blockade. CTRP is a modular multidomain protein containing six tandem von Willebrand factor A-like (A) domains and seven tandem thrombospondin type I repeat-like (TS) domains. Here we present, to our knowledge, the first structure-function analysis of CTRP using genetically modified Plasmodium berghei parasites expressing mutant versions of the ctrp gene. Our data show that the A domains of CTRP are critical for ookinete gliding motility and oocyst formation whilst, unexpectedly, its TS domains are fully redundant. These results may have important implications for the design of CTRP-based transmission blocking strategies.
Project description:In the apicomplexan protozoans motility and cell invasion are mediated by the TRAP/MIC2 family of transmembrane proteins, members of which link extracellular adhesion to the intracellular actomyosin motor complex. Here we characterize a new member of the TRAP/MIC2 family, named TRAP-Like Protein (TLP), that is highly conserved within the Plasmodium genus. Similar to the Plasmodium sporozoite protein, TRAP, and the ookinete protein, CTRP, TLP possesses an extracellular domain architecture that is comprised of von Willebrand factor A (vWA) and thrombospondin type 1 (TSP1) domains, plus a short cytoplasmic domain. Comparison of the vWA domain of TLP genes from multiple Plasmodium falciparum isolates showed relative low sequence diversity, suggesting that the protein is not under selective pressures of the host immune system. Analysis of transcript levels by quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that TLP is predominantly expressed in salivary gland sporozoites of P. falciparum and P. berghei. Targeted disruption of P. berghei TLP resulted in a decreased capacity for cell traversal by sporozoites, and reduced infectivity of sporozoites in vivo, whereas in vitro sporozoite motility and hepatocyte invasion were unaffected. These results indicate a role of TLP in cell traversal by sporozoites.
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.
Project description:Malaria infection is initiated when the insect vector injects Plasmodium sporozoites into a susceptible vertebrate host. Sporozoites rapidly leave the circulatory system to invade hepatocytes, where further development generates the parasite form that invades and multiplies within erythrocytes. Previous experiments have shown that the thrombospondin-related adhesive protein (TRAP) plays an important role in sporozoite infectivity for hepatocytes. TRAP, a typical type-1 transmembrane protein, has a long extracellular region, which contains two adhesive domains, an A-domain and a thrombospondin repeat. We have generated recombinant proteins of the TRAP adhesive domains. These TRAP fragments show direct interaction with hepatocytes and inhibit sporozoite invasion in vitro. When the recombinant TRAP A-domain was used for immunoprecipitation against hepatocyte membrane fractions, it bound to alpha2-Heremans-Schmid glycoprotein/fetuin-A, a hepatocyte-specific protein associated with the extracellular matrix. When the soluble sporozoite protein fraction was immunoprecipitated on a fetuin-A-adsorbed protein A column, TRAP bound this ligand. Importantly, anti-fetuin-A antibodies inhibited invasion of hepatocytes by sporozoites. Further, onset of malaria infection was delayed in fetuin-A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmodium berghei sporozoites. These data demonstrate that the extracellular region of TRAP interacts with fetuin-A on hepatocyte membranes and that this interaction enhances the parasite's ability to invade hepatocytes.
Project description:CD8? T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8? T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H(2b)-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20??? and PbTRAP??? respectively, as targets of CD8? T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20??? and PbTRAP??? elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP???-specific CD8? T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP???-specific, but not PbS20???-specific, CD8? T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8? T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8? T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8? T cells will allow interrogation of the development of immune responses against malaria liver stages.
Project description:Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.
Project description:Successful sporogony of Plasmodium berghei in vector mosquitoes requires expression of a family of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs). The LAPs share a subcellular localization in the crystalloid, a unique parasite organelle that forms during ookinete development. Here, LAP interactions in P. berghei were studied using a series of parasite lines stably expressing reporter-tagged LAPs combined with affinity purification and high accuracy label free quantitative mass spectrometry. Our results show that abundant complexes containing LAP1, LAP2 and LAP3 are formed in gametocytes through high avidity interactions. Following fertilization, LAP4, LAP5 and LAP6 are recruited to this complex, a process that is facilitated by LAP1 chiefly through its scavenger receptor cysteine-rich modules. These collective findings provide new insight into the temporal and molecular dynamics of protein complex formation that lead up to, and are required for, crystalloid biogenesis and downstream sporozoite transmission of malaria parasites.
Project description:The leading malaria vaccine candidate, RTS,S, based on the Plasmodium falciparum circumsporozoite protein (CSP), will likely be the first publicly adopted malaria vaccine. However, this and other subunit vaccines, such as virus-vectored thrombospondin-related adhesive protein (TRAP), provide only intermediate to low levels of protection. In this study, the Plasmodium berghei homologues of antigens CSP and TRAP are combined. TRAP is delivered using adenovirus- and vaccinia virus-based vectors in a prime-boost regime. Initially, CSP is also delivered using these viral vectors; however, a reduction of anti-CSP antibodies is seen when combined with virus-vectored TRAP, and the combination is no more protective than either subunit vaccine alone. Using an adenovirus-CSP prime, protein-CSP boost regime, however, increases anti-CSP antibody titers by an order of magnitude, which is maintained when combined with virus-vectored TRAP. This combination regime using protein CSP provided 100% protection in C57BL/6 mice compared to no protection using virus-vectored TRAP alone and 40% protection using adenovirus-CSP prime and protein-CSP boost alone. This suggests that a combination of CSP and TRAP subunit vaccines could enhance protection against malaria.
Project description:Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite-mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Deltapblap sporozoites, but not if the individual Deltapblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Deltapbs47) and male-deficient (Deltapbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Deltapblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.
Project description:Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.