Inhibition of malaria parasite development in mosquitoes by anti-mosquito-midgut antibodies.

The mosquito midgut plays a central role in the development and subsequent transmission of malaria parasites. Using a rodent malaria parasite, Plasmodium berghei, and the mosquito vector Anopheles stephensi, we investigated the effect of anti-mosquito-midgut antibodies on the development of malaria parasites in the mosquito. In agreement with previous studies, we found that mosquitoes that ingested antimidgut antibodies along with infectious parasites had significantly fewer oocysts than mosquitoes in the control group. We also found that the antimidgut antibodies inhibit the development and/or translocation of the sporozoites. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.     

Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo.

The effect of induction of an acute-phase response and its mediators on the development of liver schizonts of the rodent malaria parasite Plasmodium berghei was investigated in Brown Norway rats. Subcutaneous injection of turpentine oil 24 h or 5 min before inoculation of sporozoites resulted in 80% and 35% reduction of schizont development, respectively. Turpentine oil induced high plasma levels of interleukin-6 (IL-6). Intraperitoneal administration of IL-1, IL-6 or both, significantly reduced liver schizont development. This reduction was also present if IL-6 had been administered 24 h after sporozoite inoculation. Inhibition induced by IL-1 could be prevented by simultaneous administration of polyclonal anti-IL-6. Administration of polyclonal anti-IL-6 without IL-1 resulted in a 40% increase of liver schizonts compared to control animals. We conclude that induction of an acute-phase response during experimental Plasmodium berghei infections in Brown Norway rats, strongly inhibits liver schizont development and that IL-6 is a key mediator in this process.     

Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.     

Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formation-suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.     

Strategic use of antimalarial drugs that block falciparum malaria parasite transmission to mosquitoes to achieve local malaria elimination

The ultimate aim of malaria chemotherapy is not only to treat symptomatic infection but also to reduce transmission potential. With the absence of clinically proven vaccines, drug-mediated blocking of malaria transmission gains growing interest in the research agenda for malaria control and elimination. In addition to the limited arsenal of antimalarials available, the situation is further complicated by the fact that most commonly used antimalarials are being extensively resisted by the parasite and do not assist in blocking its transmission to vectors. Most antimalarials do not exhibit gametocytocidal and/ or sporontocidal activity against the sexual stages of Plasmodium falciparum but may even enhance gametocytogenesis and gametocyte transmissibility. Artemisinin derivatives and 8-aminoquinolines are useful transmission-blocking antimalarials whose optimal actions are on different stages of gametocytes. Transmission control interventions that include gametocytocides covering the spectrum of gametocyte development should be used to reduce and, if possible, stop transmission and infectivity of gametocytes to mosquitoes. Potent gametocytocidal drugs could also help deter the spread of antimalarial drug resistance. Novel proof-of-concept compounds with gametocytocidal activity, such as trioxaquines, synthetic endoperoxides, and spiroindolone, should be further tested for possible clinical utility before investigating the possibility of integrating them in transmission-reducing interventions. Strategic use of potent gametocytocides at appropriate timing with artemisinin-based combination therapies should be given attention, at least, in the short run. This review highlights the role that antimalarials could play in blocking gametocyte transmission and infectivity to mosquitoes and, hence, in reducing the potential of falciparum malaria transmissibility and drug resistance spread.     

Avian malaria infections in western European mosquitoes

In the complex life cycle of avian malaria parasites (Plasmodium sp.), we still have a poor understanding on the vector-parasite relationships. This study described the community of potential avian malaria vectors in four Portuguese reedbeds. We tested if their geographical distribution differed, and investigated on their Plasmodium infections. The mosquitoes' feeding preferences were evaluated using CO(2), mice, and birds as baits. The most abundant species were Culex pipiens, Culex theileri, and Ochlerotatus caspius (and, in one site, Coquillettidia richiardii). Plasmodium lineages SGS1 and SYAT05 were found in unengorged Cx. pipiens and Cx. theileri, respectively, suggesting that these mosquitoes were competent vectors of those lineages. The species' abundance was significantly different among sites, which may help to explain the observed differences in the prevalence of SGS1. At the study sites, SGS1 was detected in the most abundant mosquito species and reached a high prevalence in the most abundant passerine species. Probably, this parasite needs abundant hosts in all phases of its cycle to keep a good reservoir of infection in all its stages. Cq. richiardii showed an opportunistic feeding behavior, while Cx. pipiens appeared to be more mammophilic than previously described, perhaps because the used avian bait was not its preferential target. In one of the study sites, mosquitoes seem to be attracted to the Spotless Starling Sturnus unicolor, an abundant bird species that may be an important local reservoir of avian malaria infections. To our knowledge, this is the first report of detection of avian Plasmodium DNA from European mosquitoes.     

Proteobacteria-like ferrochelatase in the malaria parasite

A gene encoding the heme biosynthetic enzyme ferrochelatase (FC) was found in the genomic DNA databases of Plasmodium spp. The predicted amino acid sequence of malarial FC is highly conserved and fairly well conserved by comparison with other orthologues. The FC genes of P. falciparum and P. yoelii are transcribed and the mRNAs are processed to encode polypeptides of the expected amino acid sequence. The cloned cDNA for the FC of P. falciparum successfully rescued a FC-null mutant of Escherichia coli, indicating that it encodes an active enzyme. Unlike eukaryotic FCs, the malarial enzyme lacks a characteristic extension at the C-terminus. In addition, the sequence of the malarial FC resembles proteobacterial orthologues rather than eukaryotic enzymes. Strikingly, the malarial FC lacks a bipartite presequence at its N-terminus, unlike delta-aminolevulinic acid dehydratase of the same organism. This suggests an unusual intracellular distribution of heme biosynthetic enzymes, involving multiple subcellular compartments.     

Inhibition of malaria parasite development by a cyclic peptide that targets the vital parasite protein SERA5

The serine repeat antigen (SERA) proteins of the malaria parasites Plasmodium spp. contain a putative enzyme domain similar to that of papain family cysteine proteases. In Plasmodium falciparum parasites, more than half of the SERA family proteins, including the most abundantly expressed form, SERA5, have a cysteine-to-serine substitution within the putative catalytic triad of the active site. Although SERA5 is required for blood-stage parasite survival, the occurrence of a noncanonical catalytic triad casts doubt on the importance of the enzyme domain in this function. We used phage display to identify a small (14-residue) disulfide-bonded cyclic peptide (SBP1) that targets the enzyme domain of SERA5. Biochemical characterization of the interaction shows that it is dependent on the conformation of both the peptide and protein. Addition of this peptide to parasite cultures compromised development of late-stage parasites compared to that of control parasites or those incubated with equivalent amounts of the carboxymethylated peptide. This effect was similar in two different strains of P. falciparum as well as in a transgenic strain where the gene encoding the related serine-type parasitophorous vacuole protein SERA4 was deleted. In compromised parasites, the SBP1 peptide crosses both the erythrocyte and parasitophorous vacuole membranes and accumulates within the parasitophorous vacuole. In addition, both SBP1 and SERA5 were identified in the parasite cytosol, indicating that the plasma membrane of the parasite was compromised as a result of SBP1 treatment. These data implicate an important role for SERA5 in the regulation of the intraerythrocytic development of late-stage parasites and as a target for drug development.     

Effects of magainins and cecropins on the sporogonic development of malaria parasites in mosquitoes.

Magainins and cecropins are families of peptides with broad antimicrobial and antiparasitic activities derived respectively from the skin of frogs or from giant silk moths. In insects, cecropins function as part of an inducible immune system against a number of bacterial infections. When injected into anopheline mosquitoes previously infected with a variety of Plasmodium species, both magainins and cecropins disrupt sporogonic development by aborting the normal development of oocysts; sporozoites are not formed and the vector cannot transmit the parasite to another host. It may be possible to induce effective transmission-blocking immunity in the mosquito vector by the introduction and expression of genes coding for magainins, cecropins, or similarly acting parasiticidal peptides into the mosquito genome.     

Entomological study on transmission of avian malaria parasites in a zoological garden in Japan: bloodmeal identification and detection of avian malaria parasite DNA from blood-fed mosquitoes

Several species of captive and wild birds have been found to be infected with various avian blood protozoa in Japan. We investigated the prevalence and transmission of avian malaria parasite and determined the bloodmeal hosts of mosquitoes collected in a zoological garden in Tokyo, Japan, by using the polymerase chain reaction. In total, 310 unfed and 140 blood-fed mosquitoes of seven species were collected by using sweep nets and CDC traps. Bloodmeal identification indicated that mosquitoes had fed on 17 avian and five mammalian species, including captive animals. The results of avian malaria parasite detection from mosquitoes with avian bloodmeals indicated that Culex pipiens pallens Coquillet is a main vector of avian Plasmodium in the current study site and that some captive and wild birds could be infected with avian malaria parasites. Furthermore, the distances between the collection site of blood-fed mosquitoes and the locations of their blood-source captive animals were estimated. Most females with fresh bloodmeals were found within 40 m of caged animals, whereas half-gravid and gravid females were found between 10 and 350 m from caged host animals. We demonstrated that blood-fed mosquitoes can provide useful information regarding the mosquito vector species of avian malaria parasites and allows for noninvasive detection of the presence of avian malaria parasites in bird populations.     

Blood meal identification and prevalence of avian malaria parasite in mosquitoes collected at Kushiro wetland, a subarctic zone of Japan

In Japan, the prevalence of avian Plasmodium in birds and mosquitoes has been partially examined in the temperate and subtropical zones; however, mosquitoes in the Japanese subarctic zone have not been adequately investigated. In this study, mosquito collections and avian Plasmodium detections from the mosquito samples were carried out to demonstrate the avian Plasmodium transmission between vector mosquitoes and birds inhabiting in Kushiro Wetland, subarctic zone of Japan. A total of 5657 unfed mosquitoes from 18 species and 320 blood-fed mosquitoes from eight species was collected in summer 2008, 2009, and 2010. Three Aedes esoensis that fed on Hokkaido Sika Deer and one unfed Culex pipiens group were found to be positive for avian Plasmodium by polymerase chain reaction. This is the first report of the detection of avian Plasmodium DNA from mosquitoes distributing in the subarctic zone of Japan. The blood meals were successfully identified to captive or wild animals, including seven mammalian species, four bird species, and one amphibian species. These results indicated that infected birds with avian Plasmodium inhabited and direct contacts occurred between the infected birds and mosquitoes in Kushiro Wetland, Hokkaido, Japan.     

The insulin signaling cascade from nematodes to mammals: insights into innate immunity of Anopheles mosquitoes to malaria parasite infection

As revealed over the past 20 years, the insulin signaling cascade plays a central role in regulating immune and oxidative stress responses that affect the life spans of mammals and two model invertebrates, the nematode Caenorhabitis elegans and the fruit fly Drosophila melanogaster. In mosquitoes, insulin signaling regulates key steps in egg maturation and immunity and likely affects aging, although the latter has yet to be examined in detail. Reproduction, immunity and aging critically influence the capacity of mosquitoes to effectively transmit malaria parasites. Current work has demonstrated that molecules from the invading parasite and the blood meal elicit functional responses in female mosquitoes that are regulated through the insulin signaling pathway or by cross-talk with interacting pathways. Defining the details of these regulatory interactions presents significant challenges for future research, but will increase our understanding of mosquito/malaria parasite transmission and of the conservation of insulin signaling as a key regulatory nexus in animal biology.     

Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum

The regulation of intracellular Ca(2+) in the intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, was investigated using parasites 'isolated' from their host cells by saponin-permeabilisation of the erythrocyte membrane. The isolated parasites maintained tight control over their resting cytosolic Ca(2+) concentration which ranged from approximately 100 nM in the absence of extracellular Ca(2+) to approximately 700 nM in the presence of 1 mM extracellular Ca(2+). The parasite has two functionally discrete intracellular Ca(2+) stores. One is an 'endoplasmic reticulum (ER)-like' store, the other an 'acidic store'. The ER-like store was discharged by cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) of animal and plant cells, but not by thapsigargin (TG), a more specific inhibitor of SERCAs of animal cells. The acidic store was discharged by nigericin and by NH(4)(+). The amount of Ca(2+) in the ER-like store increased with increasing extracellular Ca(2+) concentration, whereas the amount of Ca(2+) in the acidic store did not. Ca(2+) released from the ER-like store by CPA was cleared from the parasite cytosol by uptake into the acidic store (over a range of extracellular Ca(2+) concentrations), consistent with the acidic store serving as a Ca(2+) reservoir within the intracellular parasite.     

Gene targeting in the rodent malaria parasite Plasmodium yoelii

It is anticipated that the sequencing of Plasmodium falciparum genome will soon be completed. Rodent models of malaria infection and stable transformation systems provide powerful means of using this information to study gene function in vivo. To date, gene targeting has only been developed for one rodent malaria species, Plasmodium berghei. Another rodent species, Plasmodium yoelii, however, is favored to study the mechanisms of protective immunity to the pre-erythrocytic stages of infection and vaccine development. In addition, it offers the opportunity to investigate unique aspects of pathogenesis of blood stage infection. Here, we report on the stable transfection and gene targeting of P. yoelii. Purified late blood stage schizonts were used as targets for electroporation with a plasmid that contains a pyrimethamine-resistant form of the P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) fused to green fluorescent protein (gfp) gene. After drug selection, fluorescent parasites contained intact, non-rearranged plasmids that remain stable under drug-pressure. In addition, we used another dhfr-ts/gfp based plasmid to disrupt the P. yoelii trap (thrombospondin-related anonymous protein) locus by site-specific integration. The phenotype of P. yoelii TRAP knockout was identical to that previously reported for the P. berghei TRAP knockout. In the absence of TRAP, the erythrocytic cycle, gametocyte and oocyst development of the mutant parasites were indistinguishable from wild type (WT). Although the sporozoites appeared morphologically normal, they failed to glide and to invade the salivary glands of mosquitoes.     

Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5-6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-Ch2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.     

Redox sensing and signaling by malaria parasite in vertebrate host

Plasmodium parasites, which is responsible to cause malaria, are also exceedingly receptive to oxidative stress during their intraerythrocytic life stage as they devour haemoglobin inside their food vacuoles and engender toxic haem moieties and reactive oxygen species (ROS). Other than, several studies suggest that the generation of reactive oxygen and nitrogen species (ROS and RNS) associated with oxidative stress, plays a decisive role in the ripeness of systemic complications caused by malaria. Malaria infection provokes the generation of hydroxyl radicals (OH^?), which most probably is the main reason for the induction of oxidative stress and apoptosis. In this study, it has been described to understand how redox molecules and NO carry out their diverse functions in both parasites and host. It is very important to understand the chemical reactions that produce those outcomes and how its regulation carried out by parasite during erythrocytic phase.

     

Insecticide resistance in malaria vector mosquitoes in a gold mining town in Ghana and implications for malaria control

Malaria control programmes in Africa, for the most part, address only treatment of the disease and supply of insecticide treated bed nets. The impact of these restricted programmes has been limited and new approaches are being advocated, including integrated vector management strategies and partnerships with industry. Mosquito surveys were carried out for AngloGold/Ashanti in preparation for their implementation of an integrated malaria control programme at the Obuasi gold mine in Ghana. Malaria vectors that were collected inside houses were identified to species and molecular forms by PCR, and tested for insecticide resistance using standard WHO bioassays and molecular target site insensitivity (kdr) assays. Species were identified as An. funestus s.s. and An. gambiae S and M forms. The An. gambiae S form samples showed resistance to DDT, pyrethroids and carbamates while An. funestus was resistant to DDT and carbamates. The An. gambiae M form occurred in very low numbers and could not be assessed reliably for resistance. The standard PCR assay for detection of the kdr mutation in An. gambiae S form showed little association with pyrethroid resistance. Subsequent sequencing of the II56 domain containing the kdr mutation from nine surviving mosquitoes showed that eight were homozygous resistant and one heterozygous. This correlated with the bioassay results and with previous studies on West African An. gambiae, but raised concerns about the reliability of the PCR assay for detection of the kdr mutation. As a result of these investigations AngloGold/Ashanti are implementing, in addition to treatment and case management, a vector control programme that includes insecticide resistance management by alternation of various classes of insecticides for house spraying, supply of ITNs, screening of houses and environmental management where appropriate, i.e. integrated vector management.