Plasmodium female gamete surface HSP90 is a key determinant for fertilization.

Plasmodium fertilization, an essential step for the development of the malaria parasite in the mosquito, is a prime target for blocking pathogen transmission. Using phage peptide display screening, we identified MG1, a peptide that binds to male gametes and inhibits fertilization, presumably by competing with a female gamete ligand. Anti-MG1 antibodies bind to the female gamete surface and, by doing so, also inhibit fertilization. We determined that this antibody recognizes HSP90 on the surface of Plasmodium female gametes. Our findings establish Plasmodium HSP90 as a prime target for the development of a transmission-blocking vaccine.IMPORTANCEMalaria kills over half a million people every year and this number has not decreased in recent years. The development of new tools to combat this disease is urgently needed. In this article, we report the identification of a key molecule-HSP90-on the surface of the parasite's female gamete that is required for fertilization to occur and for the completion of the parasite cycle in the mosquito. HSP90 is a promising candidate for the development of a transmission-blocking vaccine.

Plasmodium sporozoite phospholipid scramblase interacts with mammalian carbamoyl-phosphate synthetase 1 to infect hepatocytes.

After inoculation by the bite of an infected mosquito, Plasmodium sporozoites enter the blood stream and infect the liver, where each infected cell produces thousands of merozoites. These in turn, infect red blood cells and cause malaria symptoms. To initiate a productive infection, sporozoites must exit the circulation by traversing the blood lining of the liver vessels after which they infect hepatocytes with unique specificity. We screened a phage display library for peptides that structurally mimic (mimotope) a sporozoite ligand for hepatocyte recognition. We identified HP1 (hepatocyte-binding peptide 1) that mimics a ~50 kDa sporozoite ligand (identified as phospholipid scramblase). Further, we show that HP1 interacts with a ~160 kDa hepatocyte membrane putative receptor (identified as carbamoyl-phosphate synthetase 1). Importantly, immunization of mice with the HP1 peptide partially protects them from infection by the rodent parasite P. berghei. Moreover, an antibody to the HP1 mimotope inhibits human parasite P. falciparum infection of human hepatocytes in culture. The sporozoite ligand for hepatocyte invasion is a potential novel pre-erythrocytic vaccine candidate.

FMRF-related peptides in Aedes aegypti midgut: neuromuscular connections and enteric nervous system.

FMRFamide-related peptides (FaRPs) are a class of neuropeptides that participate in a variety of physiological processes in invertebrates. They occur in nerves of stomatogastric ganglia and enteroendocrine cells of the insect digestive tract, where they may control muscle functions. However, their direct involvement in muscle function has never been shown in situ. We studied the relationship between FaRPs and midgut muscle during larval-pupal transition of the mosquito Aedes aegypti. In late L4, FaRP-positive neuronal extensions attach to the bundles of the external circular muscle layer, and muscle stem cells start to undergo mitosis in the internal circular layer. Thereafter, the external muscle layer degenerates, disappearing during early pupal development, and is completely absent in the adult mosquito. Our results indicate that FaRP-based neural signals are involved in the reorganization of the muscle fibers of the mosquito midgut during the larval-pupal transition. In addition to confirming FaRP involvement in muscle function, we show that the mosquito midgut muscles are largely innervated, and that circular and longitudinal muscle have specific neuron bodies associated with them.

CXCR4 regulates Plasmodium development in mouse and human hepatocytes.

The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca2+ levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.

Identification of GAPDH on the surface of Plasmodium sporozoites as a new candidate for targeting malaria liver invasion.

Malaria transmission begins when an infected mosquito delivers Plasmodium sporozoites into the skin. The sporozoite subsequently enters the circulation and infects the liver by preferentially traversing Kupffer cells, a macrophage-like component of the liver sinusoidal lining. By screening a phage display library, we previously identified a peptide designated P39 that binds to CD68 on the surface of Kupffer cells and blocks sporozoite traversal. In this study, we show that the P39 peptide is a structural mimic of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the sporozoite surface and that GAPDH directly interacts with CD68 on the Kupffer cell surface. Importantly, an anti-P39 antibody significantly inhibits sporozoite liver invasion without cross-reacting with mammalian GAPDH. Therefore, Plasmodium-specific GAPDH epitopes may provide novel antigens for the development of a prehepatic vaccine.

Antimalarial iron chelator FBS0701 blocks transmission by Plasmodium falciparum gametocyte activation inhibition.

Reducing the transmission of the malarial parasite by Anopheles mosquitoes using drugs or vaccines remains a main focus in the efforts to control malaria. Iron chelators have been studied as potential antimalarial drugs due to their activities against different stages of the parasite. The iron chelator FBS0701 affects the development of Plasmodium falciparum early gametocytes and lowers blood-stage parasitemia. Here, we tested the effect of FBS0701 on stage V gametocyte infectivity for mosquitoes. The incubation of stage V gametocytes for up to 3 days with increasing concentrations of FBS0701 resulted in a significant dose-related reduction in mosquito infectivity, as measured by the numbers of oocysts per mosquito. The reduction in mosquito infectivity was due to the inhibition of male and female gametocyte activation. The preincubation of FBS0701 with ferric chloride restored gametocyte infectivity, showing that the inhibitory effect of FBS0701 was quenched by iron. Deferoxamine, another iron chelator, also reduced gametocyte infectivity but to a lesser extent. Finally, the simultaneous administration of drug and gametocytes to mosquitoes without previous incubation did not significantly reduce the numbers of oocysts. These results show the importance of gametocyte iron metabolism as a potential target for new transmission-blocking strategies.

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.

Surface-expressed enolases of Plasmodium and other pathogens

Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito.

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a gamma-glutamylcysteine synthetase (gamma-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for gamma-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and gamma-GCS by simultaneous disruption of gr and gamma-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.

The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission.

Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that gamma-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs(-) parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter.

Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.

Induction of actin gene expression in the mosquito midgut by blood ingestion correlates with striking changes of cell shape.

Ingestion of a blood meal by the female mosquito Anopheles gambiae (L., Diptera: Culicidae), results in a dramatic distention of the midgut epithelium. Here, we report that these events correlate with a transient increase of actin mRNA and protein abundance. The newly synthesized actin may provide a pool of actin protein needed to remodel epithelial cell cytoarchitecture. We also document changes in midgut epithelial cell morphology. Upon blood ingestion, the columnar cells flatten accompanied by the loss of microvilli on the lumenal side and the unfolding of the labyrinth on the basal side. These changes correlate with the large increase of epithelial surface area needed to accommodate the blood meal. Actin gene expression, actin synthesis and cell morphology all return to the pre-feeding state by 24 h after blood intake.

Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti.

Immuno-screening of an adult Aedes aegypti midgut cDNA expression library with anti-peritrophic matrix antibodies identified cDNAs encoding a novel peritrophic matrix protein, termed Ae. aegypti Adult Peritrophin 50 (Ae-Aper50), and the epithelial cell-surface membrane protein, AEG12. Both genes are expressed exclusively in the midguts of adult female mosquitoes and their expression is strongly induced by blood feeding. Ae-Aper50 has a predicted secretory signal peptide and five chitin-binding domains with intervening mucin-like domains. Localization of Ae-Aper50 to the peritrophic matrix was demonstrated by immuno-electron microscopy. Recombinant Ae-Aper50 expressed in baculovirus-infected insect cells binds chitin in vitro. Site-directed mutagenesis was used to study the role that cysteine residues from a single chitin-binding domain play in the binding to a chitin substrate. Most of the cysteine residues proved to be critical for binding. AEG12 has a putative secretory signal peptide at the amino-terminus and a putative glycosyl-phosphatidylinositol (GPI) anchor signal at its carboxyl-terminus and the protein was localized by immuno-electron microscopy to the midgut epithelial cell microvilli.

Transgenic mosquitoes for malaria control: progresses and challenges / Mosquitos transgênicos para o controle da malária: progressos e desafios

Malaria kills millions of people every year and the current strategies to control the disease, such as insecticides and drugs have not been completely efficient. Because of that, novel means to fight against malaria are of utmost importance. Advances in the study of the mosquito vector and its interactions with the malaria parasite made scientists think that it is possible to genetically manipulate the mosquitoes to make them inefficient vectors. Here we review the advances on the introduction of foreign genes into the mosquito germ line, the characterization of tissue-specific promoters, the identification of gene products that block development of the parasite in the mosquito, and we discuss the recent generation of transgenic mosquitoes impaired for malaria transmission. While much progress has been made, many years of research are still needed before transgenic mosquitoes can be used in the field.

A malária mata milhões de pessoas a cada ano e as estratégias atuais de controle da doença, como inseticidas e drogas não têm sido tão eficientes. Por este motivo, novos meios para o combate à malária são de extrema importância. Avanços no estudo do mosquito vetor e sua interação com o parasito da malária fizeram os cientistas pensarem que é possível a manipulação genética dos mosquitos para torná-los vetores ineficientes. Neste artigo, revisamos os avanços na introdução de genes exógenos na linhagem germinativa de mosquitos, a caracterização de promotores específicos de certos tecidos, a identificação de produtos gênicos que bloqueiam o parasita no mosquito, bem como discutimos a recente geração de mosquitos transgênicos, menos eficientes na transmissão de malária. Enquanto muitos progressos foram obtidos, muitos anos de pesquisa são ainda necessários para que mosquitos transgênicos possam ser utilizados na natureza.

Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite.

Malaria is estimated to cause 0.7 to 2.7 million deaths per year, but the actual figures could be substantially higher owing to under-reporting and difficulties in diagnosis. If no new control measures are developed, the malaria death toll is projected to double in the next 20 years. Efforts to control the disease are hampered by drug resistance in the Plasmodium parasites, insecticide resistance in mosquitoes, and the lack of an effective vaccine. Because mosquitoes are obligatory vectors for malaria transmission, the spread of malaria could be curtailed by rendering them incapable of transmitting parasites. Many of the tools required for the genetic manipulation of mosquito competence for malaria transmission have been developed. Foreign genes can now be introduced into the germ line of both culicine and anopheline mosquitoes, and these transgenes can be expressed in a tissue-specific manner. Here we report on the use of such tools to generate transgenic mosquitoes that express antiparasitic genes in their midgut epithelium, thus rendering them inefficient vectors for the disease. These findings have significant implications for the development of new strategies for malaria control.