<i>Plasmodium berghei</i> rhoptry neck protein 6 maintains parasite infectivity and virulence

Apicomplexan parasites possess unique secretory organelles called rhoptries, which are reservoirs for rhoptry neck proteins (RONs) and rhoptry bulb proteins (ROPs) that aid in host cell attachment, invasion, and intracellular proliferation. In <i>Plasmodium falciparum</i>, the RON6 (<i>Pf</i>RON6) locus is indispensable, limiting its functional investigation. Using a rodent malaria model, we show the role of <i>P. berghei</i> RON6 (PbRON6), a putative ortholog of <i>Pf</i>RON6, in the invasion of RBC, hepatocytes, and during liver stage development. <i>Pb</i>RON6 localizes to the sporozoite membrane and has an extracellular C-terminal domain. RON6-depleted parasites fail to maintain infectivity and virulence, leading to prolonged survival of mice. The mutants induce chronic malaria and hyper-reactive malarial splenomegaly, characterized by decreased B and T lymphocytes concurring with loss of lymphoid follicles. Our findings provide a rationale for targeting <i>Pb</i>RON6 in pre-erythrocytic stages to prevent clinical malaria and also for understanding the basis of hyper-reactive splenomegaly in mice using the <i>PbΔron6</i> mutant model.IMPORTANCE<i>Plasmodium</i> sporozoites are infective to mammalian hepatocytes. Prior to entry into the cell, sporozoites release proteins from their apical cell organelles called micronemes and rhoptries. The secreted proteins contact the hepatocyte membrane to create a structure called a moving junction (MJ) that progressively invaginates inside the cell, utilizing the parasite's actomyosin molecular motor. This activity finally culminates in establishing an intracellular vacuole that harbors the parasite. As MJ is crucial for intracellular infection, targeting the components of this complex has implications in reducing malaria infection. We show that a rhoptry resident protein, RON6, is required for the invasion of merozoites and sporozoites, together with a role in the development of parasites in the hepatocytes. Consistent with its probable role in hepatocyte invasion, the RON6 is localized to the sporozoite membrane, with its C-terminal domain being extracellular. Our observations reveal that RON6 maintains the virulence of the parasite, and mutants lacking RON6 enhance host survival and induce hyper-reactive malarial splenomegaly.