Plasmodium falciparum Liver Stage Antigen 3 plays a role in completing the final stages of merozoite invasion of red blood cells.

Understanding the red blood cell (RBC) invasion mechanism by Plasmodium falciparum, the causative agent of human malaria, is critical for developing blood-stage vaccines. Most research on the roles of parasite molecules during invasion into the RBC has focused on the pre-invasion and invasion phases, particularly on merozoite proteins in these early steps. Consequently, well-established blood-stage vaccine candidates, such as MSP1, AMA1, EBA175, Rh5, CyRPA, and Ripr, have been discovered by research targeting the early invasion phases. Recently, we identified that Liver Stage Antigen 3 (LSA3) is a merozoite dense granule protein and a novel blood-stage vaccine candidate, given that antigen-specific antibodies inhibit parasite growth by 24%. However, the precise role of LSA3 in RBC invasion remains unclear. In this study, we investigated the function of LSA3 during merozoite invasion of RBCs. In the late-invasion phase, LSA3 localized to the posterior end of invading wild-type merozoites accessible to anti-LSA3 antibodies in the culture medium. In vitro cultured lsa3 knockout parasites (ΔLSA3) exhibited slowed parasite growth. Time-lapse imaging revealed that knockout of lsa3 resulted in a 65% reduction in normal invasion completion relative to the wild-type. Furthermore, ΔLSA3 parasites displayed a high frequency of accolé-form: aberrant morphology, with a protruding convex-shaped ring-stage just beneath the RBC membrane, following successful internalization into RBCs. This study provides the first evidence that a merozoite dense granule protein potentially plays an important role in the late-invasion phase of the P. falciparum erythrocytic cycle.