We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. We investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose a new model, the lock-and-key theory of P. falciparum globalization. According to this model, we can think of Pfs47 as a key that allows the parasite to turn off the mosquito detection system by interacting with some mosquito receptor protein(s) (the lock). There are different haplotypes of this key, and the parasite needs to have the right key for the lock present in a given mosquito species to survive, continue to be transmitted, and become established in a new region. As humans migrate around the globe, parasites are selected as they adapt to local mosquitoes that are sometimes evolutionarily distant from those vectors present in their place of origin. Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. This work was published in PNAS We produced recombinant Pfs47 in E. coli and developed a refolding protocol. The protein is immunogenic and mouse polyclonal antibodies recognize Pfs47 in the membrane of female gametocytes. We generated 14 monoclonal antibodies and this allowed us to identify the regions associated with transmission blocking activity. We are currently refining the mapping of the target region to define the antigen to generate an effective transmission blocking vaccine. Pfs47 allows some African P. falciparum parasites (NF54 and GB4) to evade the immune system of a highly refractory mosquito strain (L3-5), while parasites from South America (7G8) are detected and eliminated. The predicted Pfs47 proteins from these strains only differ by four amino acids. We complemented the Pfs47KO line with Pfs47 variants in which a single amino acid in the African sequence was changed to that of the South American strain. We found that changing any individual amino acid results in parasite recognition and eliminations by the immune system, indicating that there is a highly specific interaction between Pfs47 and some receptor in the mosquito. One of this mutation had the opposite effect when A. albimanus mosquitoes were infected, enhancing the level of infection. This is in agreements with the proposed Lock and Key model that predicts that different haplotypes of P47 have been selected by their ability to interact with specific receptors in different anopheline mosquitoes. The manuscript describing this work is ready for submission. We also identified the mosquito Pfs47 receptor (P47-Rec) using far-Western binding assays of recombinant Pfs47 with mosquito midgut epithelial cell membrane extracts. We also expressed the mosquito P47-Rec as a recombinant protein and confirmed P47 binding to this receptor with ELISA and Biacore assays. Silencing P47-Rec significantly reduces P. falciparum infection, indicating that the receptor is required for parasite survival. The subcellular localization and mechanism of action of the receptor are under investigation. Malaria is endemic in the American continent and the Amazonian rainforest is the region with the highest risk of transmission. However, the lack of suitable experimental models to infect malaria vectors from the Americas has limited the progress to understand the biology of transmission in this region. Anopheles aquasalis, a major vector in coastal areas of South America, was found to be highly refractory to infection with two strains of Plasmodium falciparum (NF54 and 7G8) and with Plasmodium berghei (mouse malaria), even when the microbiota was eliminated with antibiotics and oxidative stress was reduced with uric acid. In contrast, An. aquasalis females treated with antibiotics and uric acid are highly susceptible to infection with a second murine parasite, Plasmodium yoelii nigeriensis N67 (PyN67). Anopheles albimanus, one of the main malaria vectors in Central America, Southern Mexico and the Caribbean, was more susceptible to infection with PyN67 than An. aquasalis, even in the absence of any pre-treatment, but was still less susceptible than Anopheles stephensi. Disruption of the complement-like system in Anopheles albimanus significantly enhanced PyN67 infection, indicating that the mosquito immune system is mounting effective antiplasmodial responses. Plasmodium yoelii nigeriensis has the ability to infect a broad range of anophelines and is an excellent model to study malaria transmission by South American vectors. The manuscript describing this work has been submitted for publication. Hemocytes are key players of the priming response. However, it is not clear how they enhance antiplasmodial immunity. To address this question, we labeled hemocytes in vivo with a lipophilic dye and analyzed the cells associated with the midgut. We were unable to find any intact hemoctyes. Instead, we found fluorescence-labeled microvesicles associated with Plasmodium-invaded midgut cells that were not present in uninfected controls. This indicates that hemocytes released vesicles that fuse with invaded midgut cells. We also found HDF injection increases the number of vesicles, while pre-injection of sephadex beads that are phagocytized by hemocytes prevents vesicle release and increases parasite survival. We know from previous studies that overactivation of the Toll pathway in hemocytes, by silencing the suppressor cactus, enhances antiplasmodial immunity. We recently found that this treatment also greatly enhances the number of midgut-associated vesicles. Conversely, when vesicle release is prevented by pre-injection this prevents activation of the mosquito complement-like system. Taken together, our results indicate that hemocytes associate with the basal side of the mosquito midgut in response to Plasmodium invasion and release microvesicles that are critical to mount an effective immune response by activating the mosquito complement-like system. The nature of the signals that trigger hemocyte vesicle release are under investigation.
| Canepa, Gaspar E; Molina-Cruz, Alvaro; Yenkoidiok-Douti, Lampouguin et al. (2018) Antibody targeting of a specific region of Pfs47 blocks Plasmodium falciparum malaria transmission. NPJ Vaccines 3:26 |
| Castillo, Julio César; Ferreira, Ana Beatriz Barletta; Trisnadi, Nathanie et al. (2017) Activation of mosquito complement antiplasmodial response requires cellular immunity. Sci Immunol 2: |
| Gomes, Fabio M; Hixson, Bretta L; Tyner, Miles D W et al. (2017) Effect of naturally occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission. Proc Natl Acad Sci U S A 114:12566-12571 |
| Molina-Cruz, Alvaro; Canepa, Gaspar E; Barillas-Mury, Carolina (2017) Plasmodium P47: a key gene for malaria transmission by mosquito vectors. Curr Opin Microbiol 40:168-174 |
| Ukegbu, Chiamaka Valerie; Giorgalli, Maria; Yassine, Hassan et al. (2017) Plasmodium berghei P47 is essential for ookinete protection from the Anopheles gambiae complement-like response. Sci Rep 7:6026 |
| Canepa, Gaspar E; Molina-Cruz, Alvaro; Barillas-Mury, Carolina (2016) Molecular Analysis of Pfs47-Mediated Plasmodium Evasion of Mosquito Immunity. PLoS One 11:e0168279 |
| Orfano, Alessandra S; Nacif-Pimenta, Rafael; Duarte, Ana P M et al. (2016) Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites. Malar J 15:394 |
| Molina-Cruz, Alvaro; Zilversmit, Martine M; Neafsey, Daniel E et al. (2016) Mosquito Vectors and the Globalization of Plasmodium falciparum Malaria. Annu Rev Genet 50:447-465 |
| Orfano, Alessandra S; Duarte, Ana Paula M; Molina-Cruz, Alvaro et al. (2016) Plasmodium yoelii nigeriensis (N67) Is a Robust Animal Model to Study Malaria Transmission by South American Anopheline Mosquitoes. PLoS One 11:e0167178 |
| Smith, Ryan C; Barillas-Mury, Carolina (2016) Plasmodium Oocysts: Overlooked Targets of Mosquito Immunity. Trends Parasitol 32:979-990 |
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