Gene cloning,bacterial expression,and purification of calreticulin from Anopheles stephensi (AsCRT)

During the invasion of Plasmodium ookinetes to the mosquito midgut epithelium, several proteins or glycoproteins are involved. Recent study has shown that the calreticulin (CRT) of the midgut from Anopheles albimanus can bind to the protein receptor Pvs25 on surface of Plasmodium vivax ookinetes. Thus, in order to get more insight into the potential roles of Anopheles stephensi calreticulin (AsCRT) in the midgut, we amplified and cloned the full‐length of calreticulin coding sequence from Anopheles stephensi. The AsCRT consists of 1221 bp nucleic acids with one open reading frame (ORF) encoding 406 amino acids and an apparent molecular weight around 46 KDa. Subsequently, the recombinant calreticulin as Glutathione S‐transferase (GST) fusion in pGEX ?6p‐1 expression vector (GST‐AsCRT) was produced in the prokaryotic system under optimum conditions. GST‐AsCRT fusion protein has a molecular weight around 73 KDa. The recombinant protein was detected by Western blotting using a rabbit anti‐GST polyclonal antibody. Here, we report via single protein purification procedure using MagneGST beads, 25 mg of the recombinant protein was obtained per liter of bacterial culture. This is the first report describing the heterologous expression of Anopheles stephensi calreticulin in the prokaryotic system. The production of this recombinant protein will now allow us to further investigate AsCRT molecular protein analyses, characterization of physiochemical properties, as well as interaction between calreticulin and plasmodium protein surface.     

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.     

Plasmodium berghei: mechanisms and sites of resistance to sporogonic development in different mosquitoes

An experimental study of the mechanisms and patterns of resistance to Plasmodium berghei in different mosquito species revealed a diversity of factors which prevent or inhibit sporogonic development in its different phases and in different sites in the mosquito vector. The experiments showed that Culex salinarius was a totally resistant species in which exflagellation and ookinete formation are prevented. In Aedes aegypti, ookinetes in small or moderate numbers are formed but penetration of the mosquito midgut wall is blocked and oocysts are not formed. In the three experimental vectors, Anopheles quadrimaculatus, Anopheles aztecus, and Anopheles stephensi grades of enhanced susceptibility are recognized. They are expressed in lesser numbers of abnormal and degenerative oocysts, in higher numbers of sporozoites in the salivary gland, and greater viability and invasiveness of these sporozoites. In Anopheles dureni, the natural vector of rodent malaria, one observes both in nature and under experimental conditions the highest adaptation and most pronounced grade of susceptibility to P. berghei.     

A Plant-Produced Pfs25 VLP Malaria Vaccine Candidate Induces Persistent Transmission Blocking Antibodies against Plasmodium falciparum in Immunized Mice

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based ‘launch’ vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20–30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the ‘launch’ vector technology for the production of VLP-based recombinant vaccines against infectious diseases.     

Transmission Blocking Immunity in the Malaria Non-Vector Mosquito Anopheles quadriannulatus Species A

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.     

Anopheles Midgut FREP1 Mediates Plasmodium Invasion

Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission.     

Plasmodium chabaudi: Expression of active recombinant chabaupain-1 and localization studies in Anopheles sp.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction.Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.     

Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes

The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2. Antibodies against Anopheles gambiae serpin-2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.     

Ookinete-interacting proteins on the microvillar surface are partitioned into detergent resistant membranes of Anopheles gambiae midguts

Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.     

Scanning electron microscope observations of Plasmodium berghei ookinetes in primary mosquito cell cultures

Plasmodium berghei-infected blood from mice was inoculated into primary cell cultures (PCC) obtained from the mosquito Anopheles stephensi. Immature and mature ookinetes of Plasmodium berghei, which developed in these cultures were studied with the scanning electron microscope. Immature ookinetes had a bulbous-like structure at the posterior end and a slightly wrinkled surface. Mature ookinetes were smoother in appearance and somewhat longer than immature forms. Shallow spiraling waves were observed on the surface of some ookinetes, especially in the anterior half of the body. Such waves may be involved in ookinete locomotion. Penetration of cultured cells by ookinetes was not observed. Infected red cells, which were present in the inoculum, had small depressions on the red cell surface, whereas some uninfected red cells had accentuated concavities. Mouse blood cells adhered closely to PCC cells; some attached red cells were irregular in shape.     

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called “regenerative” cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

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.     

Plasmodium berghei serine/threonine protein phosphatase PP5 plays a critical role in male gamete fertility

Sexual development in malaria parasites involves multiple signal transduction pathways mediated by reversible protein phosphorylation. Here, we functionally characterised a protein phosphatase, Ser/Thr protein phosphatase 5 (PbPP5), during sexual development of the rodent malaria parasite Plasmodium berghei. The recombinant protein phosphatase domain displayed obvious protein phosphatase activity and was sensitive to PP1/PP2A inhibitors including cantharidic acid (IC₅₀ = 122.2 nM), cantharidin (IC₅₀ = 74.3 nM), endothall (IC₅₀ = 365.5 nM) and okadaic acid (IC₅₀ = 1.3 nM). PbPP5 was expressed in both blood stages and ookinetes with more prominent expression during sexual development. PbPP5 was localised in the cytoplasm of the parasite and highly concentrated beneath the parasite plasma membrane in free merozoites and ookinetes. Targeted deletion of the pbpp5 gene had no influence on asexual blood-stage parasite multiplication or the survival curve of the infected hosts. However, male gamete formation and fertility were severely affected, resulting in almost complete blockade of ookinete conversion and oocyst development in the Δpbpp5 lines. This sexual development defect was rescued by crossing Δpbpp5 with the female defective Δpbs47 parasite line, but not with the male defective Δpbs48/45 line, thus confirming the essential function of the pbpp5 gene in male gamete fertility. Furthermore, the aforementioned PP1/PP2A inhibitors all had inhibitory effects on exflagellation of male gametocytes and ookinete conversion. In particular, endothall, a selective inhibitor of PP2A, completely blocked exflagellation and ookinete conversion at ～548.3 nM. This study elucidated an essential function of PbPP5 during male gamete development and fertility.     

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.     

新型疟疾传播阻断疫苗候选抗原PbPSOP7基因截短片段的克隆及表达特点的研究

传播阻断疫苗(transmission-blocking vaccines,TBVs)可以有效地阻断疟原虫从蚊媒向人的传播,是控制疟疾流行的关键,但目前的TBVs候选抗原十分有限,迫切需要寻找有效的候选抗原。动合子分泌蛋白7(putative secreted ookinete protein 7,PSOP7)在疟原虫有性生殖阶段发挥着至关重要的作用,本研究对伯氏疟原虫抗原PSOP7(Pb PSOP7)进行简要的生物信息学分析,并应用原核表达系统高效表达纯化了截短的重组Pb PSOP7蛋白(r Pb PSOP7),免疫BALB/c小鼠后,获得小鼠高滴度多克隆抗体。经Western Blot方法证实该多克隆抗体可识别疟原虫抗原。间接免疫荧光实验显示,Pb PSOP7主要表达于疟原虫的合子与动合子表面。这些特点符合TBVs的基本设计理念,为确认和证实Pb PSOP7蛋白具有疟疾TBVs候选抗原的潜能奠定基础。     

Plasmodium vivax transmission-blocking vaccines: Progress,challenges and innovation

Existing control measures have significantly reduced malaria morbidity and mortality in the last two decades, although these reductions are now stalling. Significant efforts have been undertaken to develop malaria vaccines. Recently, extensive progress in malaria vaccine development has been made for Plasmodium falciparum. To date, only the RTS,S/AS01 vaccine has been tested in Phase 3 clinical trials and is now under implementation, despite modest efficacy. Therefore, the development of a malaria transmission-blocking vaccine (TBV) will be essential for malaria elimination. Only a limited number of TBVs have reached pre-clinical or clinical development with several major challenges impeding their development, including low immunogenicity in humans. TBV development efforts against P. vivax, the second major cause of malaria morbidity, lag far behind those for P. falciparum. In this review we summarize the latest progress, challenges and innovations in P. vivax TBV research and discuss how to accelerate its development.