Abstract
BackgroundFor the success of the malaria control and eradication programme it is essential to reduce parasite transmission by mosquito vectors. In the midguts of mosquitoes fed with parasite-infected blood, sexual-stage parasites fertilize to develop into motile ookinetes that traverse midgut epithelial cells and reside adjacent the basal lamina. Therefore, the ookinete is a promising target of transmission-blocking vaccines to break the parasite lifecycle in mosquito vectors. However, the molecular mechanisms of ookinete formation and invasion of epithelial cells have not been fully elucidated. A unique structure called the crystalloid body has been identified in the ookinete cytoplasm by electron microscopy, but its biological functions remain unclear.MethodsA recombinant protein of a novel molecule, designated as crystalloid body specific PH domain-containing protein of Plasmodium yoelii (PyCryPH), was synthesized using a wheat germ cell-free system. Specific rabbit antibodies against PyCryPH were obtained to characterize the expression and localization of PyCryPH during sexual-stage parasite development. In addition, PyCryPH knockout parasites were generated by targeted gene disruption to examine PyCryPH function in mosquito-stage parasite development.ResultsWestern blot and immunofluorescence assays using specific antibodies showed that PyCryPH is specifically expressed in zygotes and ookinetes. By immunoelectron microscopy it was demonstrated that PyCryPH is localized within crystalloid bodies. Parasites with a disrupted PyCryPH gene developed normally into ookinetes and formed oocysts on the basal lamina of midguts. In addition, the number of sporozoites residing in salivary glands was comparable to that of wild-type parasites.ConclusionsCryPH, containing a signal peptide and PH domain, is predominantly expressed in zygotes and ookinetes and is localized to crystalloid bodies in P. yoelii. CryPH accumulates in vesicle-like structures prior to the appearance of typical crystalloid bodies. Unlike other known crystalloid body localized proteins, CryPH does not appear to have a multiple domain architecture characteristic of the LAP/CCp family proteins. Although CryPH is highly conserved among Plasmodium, Babesia, Theileria, and Cryptosporidium, PyCryPH is dispensable for the development of invasive ookinetes and sporozoites in mosquito bodies.
Highlights
For the success of the malaria control and eradication programme it is essential to reduce parasite transmission by mosquito vectors
In this study crystalloid body specific protein containing pleckstrin homology (PH) domain (CryPH) was described as a PH domaincontaining protein, which localized to crystalloid bodies in ookinetes of P. yoelii
CryPH is dispensable for erythrocytic- and mosquito-stage parasite development, as demonstrated by the observation that genetically modified CryPH-deficient P. yoelii normally developed into ookinetes, oocysts, sporozoites as well as intra-erythrocyticstage parasites
Summary
For the success of the malaria control and eradication programme it is essential to reduce parasite transmission by mosquito vectors. Members of the LCCL lectin adhesive-like protein (LAP) family, which share a conserved architecture of multiple predicted adhesive domains including the LCCL (Limulus coagulation factor C, Coch-5b2 and Lgl1) domain, have been demonstrated to localize to crystalloid bodies of Plasmodium berghei ookinetes [14, 26, 27] These molecules are categorized as the CCp family in Plasmodium falciparum and were shown to form multi-protein complexes in sexual-stage parasites [28,29,30,31,32]. In the course of screening for novel secreted or membrane proteins of sexual-stage parasites based on microarray data of P. falciparum stage-specific gene expression [38] and P. berghei AP2-G2 KO gametocyte gene expression [39], a protein with a pleckstrin homology (PH) domain, which is localized in the crystalloid bodies of ookinetes, was identified In this study, this protein, designated as CryPH, was characterized in the rodent malaria parasite line Plasmodium yoelii 17XNL (PyXNL). The role of PyCryPH during parasite development in mosquito bodies was examined by generating PyCryPH gene-disrupted parasites
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