Plasmodium berghei serine repeat antigen 3 (PbSERA3) is required for hepatic merozoite egress

TL;DR

The study shows that PbSERA3 is essential for malaria parasites to leave liver cells and start blood-stage infection, suggesting it could be a target for new therapies.

Contribution

The study demonstrates that PbSERA3 is required for hepatic merozoite egress and is processed in sporozoite and ookinete stages.

Findings

PbSERA3 is indispensable for blood-stage development and is localized on the surface of ookinetes and sporozoites.

PbSERA3 is processed in ookinete and sporozoite stages, similar to its processing in blood stages.

PbSERA3 mutants fail to initiate blood-stage infection despite normal liver development.

Abstract

The asexual replicating intracellular stages of Plasmodium reside within the parasitophorous vacuole (PV) and become extracellular through an active, protease-mediated process known as egress. While egress is critical for parasite viability and dissemination, the number of effector proteases involved in this process remains unknown. The genome of P. berghei, a rodent malaria parasite, encodes five serine repeat antigens (SERAs) that bear a central papain protease domain and a serine or cysteine residue in their active site. Of these, only Pbsera3 is indispensable for blood-stage development, thus limiting its functional investigation. Using a yeast-based Flp/FRT conditional mutagenesis system, we successfully obtained Pbsera3-edited sporozoites that invaded hepatocytes and developed normally in late liver stages but failed to initiate a blood-stage infection. Interestingly, we observed PbSERA3 localization on the surface of ookinetes and sporozoites under non-permeabilized conditions, likely indicating its extracellular nature. Further, PbSERA3 is processed in ookinete and sporozoite stages, yielding two bona fide products as reported in the blood stages. Given that PbSERA3 and its ortholog in P. falciparum (PfSERA6) are substrates for subtilisin-like protease (SUB1), proteolytic maturation of PfSERA6 may also be a conserved and critical event for hepatic egress. Developing inhibitors that prevent maturation of PbSERA3/PfSERA6 may have important therapeutic implications for preventing clinical malaria.

The intracellular stages of Plasmodium that replicate asexually reside within a vacuole delimited by a parasitophorous vacuolar membrane (PVM). A family of serine-rich antigens (SERAs), with a cysteine residue in its catalytic site, is implicated in liberating these parasites from PVM. In P. berghei, a rodent malaria parasite, PbSERA3, an ortholog of PfSERA6, is indispensable for the parasite. However, its maturation by another parasite protease called subtilisin 1 (SUB1) is critical for its effector functions. During EEF development, the processed PbSERA3 is translocated across the PVM and possibly implicated in hepatic takeover. A direct role of PbSERA3 in liver stages is lacking to date. Our study generated conditional mutants of PbSERA3 and demonstrated normal development of the mutant in hepatocytes, but an inability to cause blood-stage infection. These observations point to the role of PbSERA3 in hepatic egress. We further demonstrated the extracellular nature of PbSERA3 in the ookinete, midgut, and salivary gland sporozoite stages, with a bona fide processing pattern similar to that of blood stages. Our studies demonstrated the essentiality of PbSERA3 in liver stages, making it an attractive target for antimalarial therapy. As PbSERA3 mutants manifest a late developmental arrest in the liver, they have implications in eliciting cross-stage immunity, owing to a shared repertoire of antigens with blood stages.

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