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. 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. Plasmodium parasites utilize plasmin to evade complement. Multiple pathogenic microorganisms evade the complement system by recruiting plasmin to degrade surface-bound complement proteins (i.e., C3b). Several Plasmodium stages are constantly exposed to the human complement system and previous reports have shown that the parasite can evade complement attack. We hypothesize that Plasmodium parasites co-opt host plasmin for complement evasion during multiple stages of its life cycle. Plasmin and factor H degrade complement on the gamete and sporozoite surface. IFA revealed the deposition of both C3b and C5b-9 on the gamete and sporozoite surface, confirming that these parasite stages are targeted by complement. Binding of the complement regulator factor H to gametes and sporozoites was also detected. We showed that complement C3b is degraded on the surface of gametes and sporozoites only in the presence of either plasminogen and tPA or plasmin, suggesting that active plasmin is required to degrade C3b. Using a live/death cell flow cytometry assay we found that gamete lysis significantly increased when plasminogen or factor H were depleted from plasma, and the rate of gamete lysis was even higher when both plasminogen and factor H were simultaneously depleted. Supplementation of plasminogen and/or factor H reverted complement-mediated lysis. Our data suggest that Plasmodium gametes can use plasminogen and factor H to evade complement attack during development within the blood bolus in the mosquito midgut. Role of the mosquito saliva in the activation of plasminogen. Mosquito saliva can alter the hemostatic response. The immunomodulatory & anti-inflammatory role of mosquito saliva at the bite site in the host skin has been extensively studied but a significant gap remains in understanding its role in the mosquito midgut. We aim to investigate whether mosquito saliva activates the fibrinolytic system & its impact on Plasmodium infection in the mosquito. Using a fluorescence substrate, we observed that mosquito saliva or salivary gland (SG) extract activates tPA. Fractionation of SG extract & mass spectrometry analysis of active fractions identified an apyrase as the saliva tPA activator. We showed that apyrase-activated tPA enhances plasminogen activation & apyrase also inhibited ADP-mediated platelet aggregation, therefore reducing clotting in the midgut blood bolus. Intravenous injection of recombinant An. gambiae apyrase (rAgApy) in mice reduced fibrin polymerization in the mosquito midgut blood meal & enhanced P. berghei mosquito infection. Immunizing mice with rAgApy increased fibrin polymerization in the mosquito midgut blood meal & significantly inhibited parasite development. Sporozoite infection was also highly impaired in mice immunized with mosquito apyrase. Our data highlights the importance of mosquito saliva in malaria transmission by showing a novel role for salivary apyrase in enhancing fibrinolysis & inhibiting coagulation in the mosquito midgut thus, facilitating Plasmodium development in mosquitoes. Understanding the role of arthropod saliva proteins in pathogen transmission will widen our horizons for developing new intervention strategies for the treatment of several infectious diseases.