Abstract
Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.
Highlights
Transmission of the malaria parasite to a vertebrate host is initiated by the bite of an infected Anopheline mosquito
To address the question of whether subtilisin-like protease 1 (SUB1) is expressed in P. berghei liver stages, we produced a rabbit polyclonal antibody specific for the catalytic domain of P. berghei SUB1 (PbSUB1)
Examination of P. berghei blood stage schizont extracts by Western blot using the antibody produced signals likely corresponding to the full-length and processed forms of PbSUB1 (Supplemental Figure S1A in Text S1), by analogy with the maturation profile previously observed with recombinant forms of SUB1 from P. berghei and three other Plasmodium species [37,38,39]
Summary
Transmission of the malaria parasite to a vertebrate host is initiated by the bite of an infected Anopheline mosquito. After an initial period of non-replicative development, which lasts around 24 h in the rodent malaria species Plasmodium berghei, the intracellular parasite - known as an exoerythrocytic form (EEF) - initiates an asexual replicative program. This begins with several rounds of nuclear division to form a multinucleated syncytium or schizont, concomitant with a large increase in the size of the PV to accommodate the growing parasite. Whilst not itself associated with any pathology, the liver stage and other pre-erythrocytic stages are a prerequisite to the asexual blood-stage cycle in a natural malarial infection, and so are potential targets for prophylactic immune-based or chemotherapeutic interventions designed to prevent disease
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