Host-parasite-vector interactions during malaria transmission

National Institute Of Allergy And Infectious Diseases

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Abstract

Role of the mammalian fibrinolytic system during Plasmodium transmission. Plasmodium spp. must migrate across proteinaceous matrices to successfully infect the mosquito vector and the vertebrate host. While parasite motility is powered by a subpellicular actomyosin motor, the contribution of host factors to facilitate parasite migration is largely underexplored. Recruitment of the mammalian host fibrinolytic protease plasmin is a mechanism used by several pathogens to enhance invasion and dissemination through physical barriers. Plasminogen is an abundant zymogen in mammals and is activated into the serine protease plasmin by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Although the primary function of plasmin is the degradation of fibrin, it can also degrade extracellular matrix proteins that form physical barriers. Our data shows that Plasmodium sporozoites bind plasminogen, tPA and uPA from the host. Importantly, inhibition of plasminogen activation uncovered multiple requirements of plasmin activity for Plasmodium parasite infectivity: 1) for gamete motility and fertilization within the mosquito midgut blood bolus, 2) for sporozoite migration and escape from the skin, and 3) for sporozoite evasion of the complement system, and 4) for invasion of the liver. Thiago Luiz Alves e Silva, Andrea Radtke, Amanda Balaban, Tales Vicari Pascini, Zarna Rajeshkumar Pala, Alison Roth, Patricia H. Alvarenga, Yeong Je Jeong, Janet Olivas, Anil K. Ghosh, Hanhvy Bui, Brandon S. Pybus, Photini Sinnis, Marcelo Jacobs-Lorena and Joel Vega-Rodriguez. 2020. The fibrinolytic system enables the onset of Plasmodium infection in the mosquito vector and the mammalian host. Science Advances, 7:eabe3362. PMID: 33547079. Reiss T, Theis HI, Gonzalez-Delgado A, Vega-Rodriguez J, Zipfel PF, Skerka C, Pradel G. 2020. Acquisition of human plasminogen facilitates complement evasion by the malaria parasite Plasmodium falciparum. Eur J Immunol. PMID: 33022775. Pascini TV, Jeong YJ, Huang W, Pala ZR, Sá JM, Wells MB, Kizito C, Sweeney B, Alves E Silva TL, Andrew DJ, Jacobs-Lorena M, Vega-Rodríguez J. 2022. Transgenic Anopheles mosquitoes expressing human PAI-1 impair malaria transmission. Nature Communications 13:2949. PMID: 35618711. Parasite receptors for plasminogen and tPA. Our data shows that Plasmodium sporozoites and gametes can bind plasminogen, tPA and uPA which results in plasminogen activation at the parasite surface. These three proteins presumably bind to parasite receptors. Once identified, these receptors can be targeted with antibodies or small molecules to block their interaction with the fibrinolytic proteins and therefore, inhibit parasite transmission. Surface enolase has been described as a receptor for human plasminogen; and surface GAPDH works as a receptor for plasminogen in various pathogens including bacteria, fungi, and parasites. Some plasminogen receptors can also be co-receptors for the plasminogen activators, e.g., enolase. In Plasmodium, the cell surface receptor for tPA is currently unknown. We hypothesized that parasite enolase and GAPDH, previously reported to occur on the surface of P. falciparum, are receptors for plasminogen and tPA. We observed that both, enolase and GAPDH, are detected on the surface of several developmental stages of the parasite, including micro- and macrogametes, zygotes, ookinetes, and sporozoites. Furthermore, we found that tPA and plasminogen bind non-competitively to both enolase and GAPDH in vitro. This binding is mediated by the kringle domains of both tPA and plasminogen. We are currently testing the potential of enolase and GAPDH as transmission-blocking targets, either individually or in combination. Role of the mosquito saliva in the activation of plasminogen. Mosquito saliva is a complex mixture of proteins, enzymes, and other molecules that are injected into the host during blood feeding. These molecules help mosquitoes overcome the host's hemostatic and immune responses, allowing for successful blood feeding and pathogen transmission. These properties have important implications for disease transmission, as the modulation of the host's immune response can affect the outcome of infection. More recently, some salivary proteins from Anopheles mosquitoes have been shown to influence Plasmodium transmission. It is evident that mosquito salivary proteins are essential for successful transmission of malaria parasites however, the targeting these proteins for malaria interventions, including vaccines, remains underexplored. Understanding the complex interactions between mosquito saliva, the host, and the pathogens they transmit is critical for the development of effective strategies to control mosquito-borne diseases. Recently we showed that salivary apyrase from An. gambiae mosquitoes enhances fibrinolysis by activating tPA and inhibiting platelet activation and aggregation in the midgut. Therefore, we hypothesize that salivary apyrase enhances Plasmodium parasite transmission to the mosquito by reducing the blood bolus viscosity. It is well known that mosquito saliva is deposited at the bite site in the skin when the mosquito is probing in search for a blood vessel. However, while there is evidence that mosquitoes ingest their own saliva, the extent to which they ingest saliva and its effect on blood bolus biology is not well known. Immunohistochemistry with anti-apyrase antibodies developed in our laboratory, on blood-fed mosquito midguts dissected 30 min post feeding show that An. gambiae mosquitoes ingest a significant amount of apyrase during feeding. The intensity of apyrase staining in the blood bolus shows that the mosquito ingests a substantial amount of saliva while feeding on blood, and the pattern of staining shows that the blood meal is fully mixed with saliva. Using a competitive ELISA assay to measure D-dimer formation in the midgut blood bolus as a readout of fibrinolysis, we observed a significant increase in D-dimers in mosquitos fed on rAgApyrase supplemented blood, showing that apyrase ingestion enhanced fibrinolysis in the blood bolus. Furthermore, immunohistochemistry of mosquito blood boluses with an anti-P-selectin antibody to detect and quantify platelet activation showed a significant reduction in P-selectin staining in the midguts of mosquitoes fed on rAgApyrase supplemented mice when compared to the control mice. These results show that salivary apyrase ingested during blood feeding inhibits hemostasis by enhancing fibrinolysis and inhibiting platelet activation. We then studied the role of mosquito salivary apyrase on parasite transmission. Supplementation of P. berghei infected mouse blood with rAgApyrase significantly increased the number of oocysts in the mosquito, while heat-denatured recombinant protein failed to increase the infection. We then immunized mice with rAgApyrase. When An. gambiae mosquitoes fed on rAgApyrase immunized mice the oocyst intensity and infection prevalence where dramatically reduced when compared to mice immunized with adjuvant alone. We further studied whether salivary AgApyrase is required for sporozoite transmission from the mosquito to the mammalian host. Mice immunized with rAgApyrase were challenged with the bite of five An. stephensi mosquitoes infected with P. berghei. We obesrved a significant reduction of 68% in parasite infection in mice immunized with rAgApyrase compared to adjuvant treated mice. These findings show that salivary AgApyrase is a critical factor for successful gamete transmission to mosquitoes and for sporozoite transmission via the mosquito bite. Methods used: PCR, qPCR, DNA sequencing, Immunofluorescence, Western blot, Confocal microscopy, tissue culture, ELISA, DNA synthesis, standard membrane feeding assay, nucleic acid and protein isolation, protein expression, mosquito dissection, mosquito infection, mosquito rearing.