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. In collaboration with Dr. Gabriele Pradel from the RWT Aachen University in Germany, we explored the role of plasmin for complement evasion of Plasmodium asexual stages (Reiss et al., 2020). We found that plasminogen bound to infected RBCs (iRBCs) was partially activated into plasmin in the absence of tPA, while complete activation to plasmin was observed in the presence of tPA. Plasmin bound to the iRBCs degrades complement C3b, reducing the formation of the terminal complement complex (TCC) on the iRBC surface. Supplementation of P. falciparum cultures with plasminogen enhanced parasite development, whereas plasminogen depletion had the opposite effect. We propose that plasminogen acquisition enhances blood stages by promoting C3b inactivation, allowing the parasite to evade the complement system. 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. Increasing evidence show that the saliva of mosquitoes and other insect vectors, contains factors that enhance the virulence of the pathogens they transmit. Initial data shows that the saliva of Anopheles female mosquitoes contain a protein that activates tPA. Our hypothesis is that activation of tPA by the saliva will result in an increased activation of plasminogen at the biding site and consequently, an enhancement of sporozoite infectivity. The inactive t-PA exists in the form of a single chain molecule and the active t-PA is a cleaved product and results in two-chain t-PA. Using a fluorogenic substrate for tPA (D-Val-Leu-Lys-7-amido-4-methyl coumarin) we can detect activation of t-PA when incubated with salivary gland extracts and with saliva from Anopheles mosquitoes. Activation of tPA was lost when the salivary gland extract was pre-incubated at 65 C or 100 C, and Western blotting analysis shows that saliva induces cleavage of inactive single-chain tPA into the active two-chain form, pointing that the tPA activator is a protease. In collaboration with Dr. Eric Calvo, we fractionated saliva collected from female Anopheles mosquitoes by high-performance liquid chromatography (HPLC) and identified a fraction that activates tPA. This fraction will be further studied to identify the saliva tPA activator. Our data show that mosquito saliva activates tPA which could enhance plasminogen activation at the biting site or in the blood bolus inside the mosquito midgut. Transgenic mosquitoes expressing Human PAI to block malaria transmission: We have developed transgenic Anopheles mosquitoes that secrete human plasminogen activator inhibitor 1 (PAI-1) in their midgut and saliva. In humans, PAI-1 inhibits the activation of plasminogen by blocking the activity of tPA and uPA. Data from our lab shows that inhibition of plasminogen activation by PAI-1 inhibits parasite development in the mosquito midgut and transmission of sporozoites to the mammalian host. The rationale is that PAI-1 produced by these mosquitoes will inhibit plasminogen activation in the mosquito midgut lumen and at the biting site, thereby preventing parasite development and transmission to a new host. Each of the transgenic lines were analyzed for fitness, infectivity, and compared with its respective parental lines and a wild-type control (WT). No difference in survival was observed between transgenic and WT mosquitoes. The transgenic mosquitoes expressing human PAI-1 strongly inhibit Plasmodium berghei, Plasmodium falciparum and Plasmodium vivax parasite development in the mosquito and transmission of malaria.