Project description:The sexual phase of the malaria parasite Plasmodium falciparum is accompanied by the coordinated expression of stage-specific adhesive proteins. Among these are six secreted proteins with multiple adhesion domains, termed P. falciparum LCCL domain-containing protein (PfCCp) proteins, which are expressed in the parasitophorous vacuole of the differentiating gametocytes and which are later associated with macrogametes. Although the majority of the PfCCp proteins are implicated in parasite development in the mosquito vector, their functions remain unknown. In the present study we investigated the molecular interactions between the PfCCp proteins during gametocyte development and emergence. Using five different gene-disruptant parasite lines, we show that the lack of one PfCCp protein leads to the loss of other PfCCp family members. Co-immunoprecipitation assays on gametocyte lysates revealed formation of complexes involving all PfCCp proteins, and affinity chromatography co-elution binding assays with recombinant PfCCp domains further indicated direct binding between distinct adhesion domains. PfCCp-coated latex beads bind to newly formed macrogametes but not to gametocytes or older macrogametes 6 or 24 h post-activation. In view of these data, we propose that the PfCCp proteins form multi-protein complexes that are exposed during gametogenesis, thereby mediating cell contacts of macrogametes.
Project description:The evolution of hematophagy involves a series of adaptations that allow blood-feeding insects to access and consume blood efficiently while managing and circumventing the host's hemostatic and immune responses. Mosquito, and other insects, utilize salivary proteins to regulate these responses at the bite site during and after blood feeding. We investigated the function of Anopheles gambiae salivary apyrase (AgApyrase) in regulating hemostasis in the mosquito blood meal and in Plasmodium transmission. Our results demonstrate that salivary apyrase, a known inhibitor of platelet aggregation, interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protease that degrades fibrin and facilitates Plasmodium transmission. We show that mosquitoes ingest a substantial amount of apyrase during blood feeding, which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. AgApyrase significantly enhanced Plasmodium infection in the mosquito midgut, whereas AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammalian host, underscoring the potential for strategies to prevent malaria transmission.
Project description:To identify the putative salivary tPA activator, we fractionated salivary proteins by size-exclusion chromatography and identified fraction Z8 as the strongest tPA activator, whereas the adjacent fractions Z7 and Z9 activated tPA at lower levels. Mass spectrometry analysis of these fractions identified a total of 152 unique proteins.
Project description:BackgroundPlasmodium knowlesi, identified as the fifth human malaria parasite, has rapidly spread across various Southeast Asian countries, yet uncertainties persist regarding its human-mosquito-human transmission. Therefore, this study aims to explore the transmission potential of P. knowlesi from human blood to mosquitoes.MethodsA direct membrane-feeding assay was conducted by infecting laboratory-reared female Anopheles dirus mosquitoes with P. knowlesi-infected human blood from a single patient presenting with febrile malaria. Mosquitoes were dissected 7 days post-infection under a stereomicroscope to detect oocysts in the midgut, stained with mercurochrome. Salivary glands were examined 14 days post-infection for the presence of sporozoites. Malaria diagnosis employed microscopy by expert microscopists and nested PCR assays.ResultsUpon dissecting 745 out of 1439 blood-fed An. dirus mosquitoes on day 7 post-infection, two oocysts were identified in the midguts of two mosquitoes (0.27%). An additional 694 mosquitoes were dissected for salivary glands on day 14 post-infection, with three mosquitoes (0.43%) exhibiting sporozoites. Further confirmation by nested-PCR assay verified these sporozoites as belonging to the P. knowlesi species.ConclusionsThe findings underscore the potential transmission of P. knowlesi from human blood to mosquitoes. The significance of these findings necessitates further investigation, such as repeating similar experiments among natural vectors, to gain deeper insights into the transmission dynamics of P. knowlesi in Southeast Asia.
Project description:Centrins are small calcium-binding proteins that have a variety of roles and are universally associated with eukaryotic centrosomes. Rapid proliferation of the malaria-causing parasite Plasmodium falciparum in the human blood depends on a particularly divergent and acentriolar centrosome, which incorporates several essential centrins. Their precise mode of action, however, remains unclear. In this study calcium-inducible liquid-liquid phase separation is revealed as an evolutionarily conserved principle of assembly for multiple centrins from P. falciparum and other species. Furthermore, the disordered N-terminus and calcium-binding motifs are defined as essential features for reversible biomolecular condensation, and we demonstrate that certain centrins can form co-condensates. In vivo analysis using live cell STED microscopy shows liquid-like dynamics of centrosomal centrin. Additionally, implementation of an inducible protein overexpression system reveals concentration-dependent formation of extra-centrosomal centrin assemblies with condensate-like properties. The timing of foci formation and dissolution suggests that centrin assembly is regulated. This study thereby provides a new model for centrin accumulation at eukaryotic centrosomes.
Project description:Malaria hotspots have been the focus of public health managers for several years due to the potential elimination gains that can be obtained from targeting them. The identification of hotspots must be accompanied by the description of the overall network of stable and unstable hotspots of malaria, especially in medium and low transmission settings where malaria elimination is targeted. Targeting hotspots with malaria control interventions has, so far, not produced expected benefits. In this work we have employed a mechanistic-stochastic algorithm to identify clusters of super-spreader houses and their related stable hotspots by accounting for mosquito flight capabilities and the spatial configuration of malaria infections at the house level. Our results show that the number of super-spreading houses and hotspots is dependent on the spatial configuration of the villages. In addition, super-spreaders are also associated to house characteristics such as livestock and family composition. We found that most of the transmission is associated with winds between 6pm and 10pm although later hours are also important. Mixed mosquito flight (downwind and upwind both with random components) were the most likely movements causing the spread of malaria in two out of the three study areas. Finally, our algorithm (named MALSWOTS) provided an estimate of the speed of malaria infection progression from house to house which was around 200-400 meters per day, a figure coherent with mark-release-recapture studies of Anopheles dispersion. Cross validation using an out-of-sample procedure showed accurate identification of hotspots. Our findings provide a significant contribution towards the identification and development of optimal tools for efficient and effective spatio-temporal targeted malaria interventions over potential hotspot areas.
Project description:Cyclic nucleotide-dependent phosphodiesterases (PDEs) play essential roles in regulating the malaria parasite life cycle, suggesting that they may be promising antimalarial drug targets. PDE inhibitors are used safely to treat a range of noninfectious human disorders. Here, we report three subseries of fast-acting and potent Plasmodium falciparum PDEβ inhibitors that block asexual blood-stage parasite development and that are also active against human clinical isolates. Two of the inhibitor subseries also have potent transmission-blocking activity by targeting PDEs expressed during sexual parasite development. In vitro drug selection experiments generated parasites with moderately reduced susceptibility to the inhibitors. Whole-genome sequencing of these parasites detected no mutations in PDEβ but rather mutations in downstream effectors: either the catalytic or regulatory subunits of cyclic adenosine monophosphate-dependent protein kinase (PKA) or in the 3-phosphoinositide-dependent protein kinase that is required for PKA activation. Several properties of these P. falciparum PDE inhibitor series make them attractive for further progression through the antimalarial drug discovery pipeline.
Project description:Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.
Project description:Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials.
Project description:BackgroundDespite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination.Methods13,533 small molecules with known activity against Plasmodium falciparum asexual parasites were screened for additional transmission-blocking activity in an ex vivo Plasmodium berghei ookinete development assay. Active molecules were then counterscreened in dose response against HepG2 cells to determine their activity/cytotoxicity window and selected non-toxic representative molecules were fully profiled in a range of transmission and mosquito infection assays. Furthermore, the entire dataset was compared to other published screens of the same molecules against P. falciparum gametocytes and female gametogenesis.Results437 molecules inhibited P. berghei ookinete formation with an IC50 < 10 μM. of which 273 showed >10-fold parasite selectivity compared to activity against HepG2 cells. Active molecules grouped into 49 chemical clusters of three or more molecules, with 25 doublets and 94 singletons. Six molecules representing six major chemical scaffolds confirmed their transmission-blocking activity against P. falciparum male and female gametocytes and inhibited P. berghei oocyst formation in the standard membrane feeding assay at 1 μM. When screening data in the P. berghei development ookinete assay was compared to published screens of the same library in assays against P. falciparum gametocytes and female gametogenesis, it was established that each assay identified distinct, but partially overlapping subsets of transmission-blocking molecules. However, selected molecules unique to each assay show transmission-blocking activity in mosquito transmission assays.ConclusionThe P. berghei ookinete development assay is an excellent high throughput assay for efficiently identifying antimalarial molecules targeting early mosquito stage parasite development. Currently no high throughput transmission-blocking assay is capable of identifying all transmission-blocking molecules.