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The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/β-hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46-like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46-like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo-erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild-type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood-stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo-erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive-stage morphogenesis throughout the parasite life cycle., (© 2015 John Wiley & Sons Ltd.)

After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Plasmodium sporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasite Plasmodium yoelii. SSP3 is a putative type I transmembrane protein unique to Plasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion of SSP3 adversely affected in vitro sporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasion in vitro, nor infectivity in vivo. Together, these data reveal a previously unappreciated complexity of the Plasmodium sporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)