A very large C-loop in EGF domain IV is characteristic of the P28 family of ookinete surface proteins

The P28 family of proteins are 28 kDa proteins expressed on the surface of sexual stages—zygote, ookinete and young oocyst stages—of Plasmodium species when the parasite resides inside the mosquito midgut. Together with P25 proteins, P28 proteins protect the parasite from the harsh proteolytic environment prevailing inside the mosquito midgut. Vaccines against these proteins induce antibodies in vertebrate hosts that are capable of inhibiting parasite development in the mosquito midgut, thus preventing transmission of the parasite from the mosquito to another human host. These transmission-blocking vaccines are helpful in reducing the burden caused by malaria, which affects 300–600 million, and kills 1–3 million, people annually. The purpose of this study was to structurally characterise six members of the P28 family of ookinete surface proteins with the help of homology modelling, to compare these proteins in terms of transmission blocking and host parasite interactions, and to analyse phylogenetic relationships within the P28 family and with the P25 family. Our results indicate that all the members of the P28 family studied have four EGF domains arranged in triangular fashion with a very big C loop present in EGF domain IV, which could serve as a diagnostic feature of the P28 family as this loop is absent in the P25 family of ookinete surface proteins. The models of the P28 family of ookinete surface proteins obtained may help in understanding the biology of the parasite inside the mosquito midgut, and in designing transmission-blocking vaccines against malaria in the absence of experimentally determined structures of these important surface proteins. An erratum to this article can be found at     

Targeting molecular interactions essential for Plasmodium sexual reproduction

Malaria remains one of the most devastating infectious diseases, killing up to a million people every year. Whereas much progress has been made in understanding the life cycle of the parasite in the human host and in the mosquito vector, significant gaps of knowledge remain. Fertilization of malaria parasites, a process that takes place in the lumen of the mosquito midgut, is poorly understood and the molecular interactions (receptor–ligand) required for Plasmodium fertilization remain elusive. By use of a phage display library, we identified FG1 (Female Gamete peptide 1), a peptide that binds specifically to the surface of female Plasmodium berghei gametes. Importantly, FG1 but not a scrambled version of the peptide, strongly reduces P. berghei oocyst formation by interfering with fertilization. In addition, FG1 also inhibits P. falciparum oocyst formation suggesting that the peptide binds to a molecule on the surface of the female gamete whose structure is conserved. Identification of the molecular interactions disrupted by the FG1 peptide may lead to the development of novel malaria transmission‐blocking strategies.     

Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.     

An essential role of the basal body protein SAS‐6 in Plasmodium male gamete development and malaria transmission

Gametocytes are the sole Plasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the Plasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS‐6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in Plasmodium, we hypothesized that SAS‐6 disruption may cause gametogenesis defects and perturb transmission. We show that Plasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/SAS‐6 to malaria transmission blocking interventions.     

The development of malaria parasites in the mosquito midgut

The mosquito midgut stages of malaria parasites are crucial for establishing an infection in the insect vector and to thus ensure further spread of the pathogen. Parasite development in the midgut starts with the activation of the intraerythrocytic gametocytes immediately after take‐up and ends with traversal of the midgut epithelium by the invasive ookinetes less than 24 h later. During this time period, the plasmodia undergo two processes of stage conversion, from gametocytes to gametes and from zygotes to ookinetes, both accompanied by dramatic morphological changes. Further, gamete formation requires parasite egress from the enveloping erythrocytes, rendering them vulnerable to the aggressive factors of the insect gut, like components of the human blood meal. The mosquito midgut stages of malaria parasites are unprecedented objects to study a variety of cell biological aspects, including signal perception, cell conversion, parasite/host co‐adaptation and immune evasion. This review highlights recent insights into the molecules involved in gametocyte activation and gamete formation as well as in zygote‐to‐ookinete conversion and ookinete midgut exit; it further discusses factors that can harm the extracellular midgut stages as well as the measures of the parasites to protect themselves from any damage.     

Plasmodium berghei histamine‐releasing factor favours liver‐stage development via inhibition of IL‐6 production and associates with a severe outcome of disease

Plasmodium spp., which causes malaria, produces a histamine‐releasing factor (HRF), an orthologue of mammalian HRF. Histamine‐releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre‐erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito‐injected parasite form into the erythrocyte‐infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL‐6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti‐IL‐6 antibodies or infection in IL‐6‐deficient mice and parasite HRF is sufficient to decrease IL‐6 synthesis, indicating a direct role of parasite HRF in reducing host IL‐6. The target cells modulated by HRF for IL‐6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down‐regulate a cytokine with anti‐parasite activity. Our data also highlight the link between a prolonged transition from liver to blood‐stage infection and reduced incidence of experimental cerebral malaria.     

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.     

Affinity purification of Plasmodium ookinetes from in vitro cultures using extracellular matrix gel

Malaria transmission depends on the parasites' successful invasion of the mosquito. This is achieved by the ookinete, a motile zygote that forms in the blood bolus after the mosquito takes an infectious blood meal. The ookinete invades the midgut epithelium and strongly attaches to the basal lamina, differentiating into an oocyst that produces the vertebrate-invasive sporozoites. Despite their importance, the ookinete and the oocyst are the least studied stages of the parasite. Much of what we know about the ookinete comes from in vitro experiments, which are hindered by the concomitant contamination with blood cells and other parasite stages. Although methods to purify them exist, they vary in terms of yield, costs, and difficulty to perform. A method for ookinete purification taking advantage of their adhesive properties was herein developed. The method consists of covering any culture-suitable surface with extracellular matrix gel, after which the ookinete culture is incubated on the gel to allow for ookinete attachment. The contaminant cells are then simply washed away. This procedure results in purer and less stressed ookinete preparations, which, by the nature of the method, are ready for oocyst production. Furthermore, it allows for micro-purifications using only 1 μl of blood, opening the possibility to make axenic ookinete cultures without sacrificing mice     

Inhibition of Ca2+/calmodulin-dependent protein kinase blocks morphological differentiation of plasmodium gallinaceum zygotes to ookinetes

Once ingested by mosquitoes, malaria parasites undergo complex cellular changes. These include zygote formation, transformation of zygote to ookinete, and differentiation from ookinete to oocyst. Within the oocyst, the parasite multiplies into numerous sporozoites. Modulators of intracellular calcium homeostasis, MAPTAM, and TMB-8 blocked ookinete development as did the calmodulin (CaM) antagonists W-7 and calmidazolium. Ca(2+)/CaM-dependent protein kinase inhibitor KN-93 also blocked zygote elongation, while its ineffective analog KN-92 did not have such effect. In vitro both zygote and ookinete extracts efficiently phosphorylated autocamtide-2, a classic CaM kinase substrate, which could be blocked by calmodulin antagonists W-7 and calmidazolium and CaM kinase inhibitor KN-93. These results demonstrated the presence of calmodulin-dependent CaM kinase activity in the parasite. KN-93-treated parasites, however, expressed the ookinete-specific enzyme chitinase and the ookinete surface antigen Pgs28 normally, suggesting that the morphologically untransformed parasites are biochemically mature ookinetes. In mosquitoes, KN-93-treated parasites did not develop as oocysts, while KN-92-treated parasites produced similar numbers of oocysts as controls. These data suggested that in Plasmodium gallinaceum morphological development of zygote to ookinete, but not its biochemical maturation, relies on Ca(2+)/CaM-dependent protein kinase activity and demonstrated that the morphological differentiation is essential for the further development of the parasite in infected blood-fed mosquitoes.     

Minimum requirements for ookinete to oocyst transformation in Plasmodium

During their passage through a mosquito vector, malaria parasites undergo several developmental transformations including that from a motile zygote, the ookinete, to a sessile oocyst that develops beneath the basal lamina of the midgut epithelium. This transformation process is poorly understood and the oocyst is the least studied of all the stages in the malaria life cycle. We have used an in vitro culture system to monitor morphological features associated with transformation of Plasmodium berghei ookinetes and the role of basal lamina components in this process. We also describe the minimal requirements for transformation and early oocyst development. A defined sequence of events begins with the break-up of the inner surface membrane, specifically along the convex side of the ookinete, where a protrusion occurs. A distinct form, the transforming ookinete or took, has been identified in vitro and also observed in vivo. Contrary to previous suggestions, we have shown that no basal lamina components are required to trigger ookinete to oocyst transformation in vitro. We have demonstrated that transformation does not occur spontaneously; it is initiated in the presence of bicarbonate added to PBS, but it is not mediated by changes in pH alone. Transformation is a two-step process that is not completed unless a range of nutrients are also present. A minimal medium is defined which supports transformation and oocyst growth from 7.8 to 11.4microm by day 5 with 84% viability. We conclude that ookinete transformation is mediated by bicarbonate and occurs in a similar manner to the differentiation of sporozoite to the hepatic stage.     

Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double‐strand break repair

Malaria parasites survive through repairing a plethora of DNA double‐stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium Rad51 mediated homologous recombination (HR) mechanism and homology‐independent alternative end‐joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51^K143R mutant on Plasmodium DSB repair and host–parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51^K143R) failed to repair DSBs as evidenced by hypersensitivity to DNA‐damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51^K143R on HR, we used yeast as a surrogate model and established that the presence of PfRad51^K143R completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild‐type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria.     

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents^1-3 and humans^4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite^7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh¹⁰.Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur¹¹. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.Download video file.^{(85M, mov)}     

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (> 50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.     

A Plasmodium α/β‐hydrolase modulates the development of invasive stages

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/β‐hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46‐like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46‐like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo‐erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild‐type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood‐stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo‐erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive‐stage morphogenesis throughout the parasite life cycle.     

Feeling at home from arrival to departure: protein export and host cell remodelling during Plasmodium liver stage and gametocyte maturation

Obligate intracellular pathogens actively remodel their host cells to boost propagation, survival, and persistence. Plasmodium falciparum, the causative agent of the most severe form of malaria, assembles a complex secretory system in erythrocytes. Export of parasite factors to the erythrocyte membrane is essential for parasite sequestration from the blood circulation and a major factor for clinical complications in falciparum malaria. Historic and recent molecular reports show that host cell remodelling is not exclusive to P. falciparum and that parasite‐induced intra‐erythrocytic membrane structures and protein export occur in several Plasmodia. Comparative analyses of P. falciparum asexual and sexual blood stages and imaging of liver stages from transgenic murine Plasmodium species show that protein export occurs in all intracellular phases from liver infection to sexual differentiation, indicating that mammalian Plasmodium species evolved efficient strategies to renovate erythrocytes and hepatocytes according to the specific needs of each life cycle phase. While the repertoireof identified exported proteins is remarkably expanded in asexual P. falciparum blood stages, the putative export machinery and known targeting signatures are shared across life cycle stages. A better understanding of the molecular mechanisms underlying Plasmodium protein export could assist in designing novel strategies to interrupt transmission between Anopheles mosquitoes and humans.     

Ookinete antigens of Plasmodium berghei: a light and electron-microscope immunogold study of expression of the 21 kDa determinant recognized by a transmission-blocking antibody

The expression of a 21 kDa transmission-blocking determinant on the malarial parasite Plasmodium berghei was studied by using the immunogold method at the light, scanning-electron and transmission-electron microscope levels. The determinant was shown to be expressed exclusively on the macrogamete and its immediate progeny the zygote, ookinete and oocyst. It is first detected on the plasmalemma two hours after the escape of the parasite from the red blood cell, reaches a maximal density on the young ookinete some ten hours later, and is still found on the oocyst after six days. The antigen is distributed evenly over the entire surface of the zygote and ookinete, but is readily shed from the parasite surface. The general applicability of the silver-enhanced immunogold method in parasitological research is emphasized.     

Protein export in malaria parasites: an update

Symptomatic malaria is caused by the infection of human red blood cells (RBCs) with Plasmodium parasites. The RBC is a peculiar environment for parasites to thrive in as they lack many of the normal cellular processes and resources present in other cells. Because of this, Plasmodium spp. have adapted to extensively remodel the host cell through the export of hundreds of proteins that have a range of functions, the best known of which are virulence‐associated. Many exported parasite proteins are themselves involved in generating a novel trafficking system in the RBC that further promotes export. In this review we provide an overview of the parasite synthesized export machinery as well as recent developments in how different classes of exported proteins are recognized by this machinery.     

Vital functions of the malarial ookinete protein, CTRP, reside in the A domains

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.