AP2-L-Dependent Processes Promote PVM Maintenance and Plasmodium Liver-stage Development

Successful malaria infection requires transformation of Plasmodium sporozoites into replicative exo-erythrocytic forms (EEFs) within hepatocytes, a process requiring a functional parasitophorous vacuole membrane (PVM) and interactions with host cellular pathways. ApiAP2 transcription factors regulate developmental progression throughout the Plasmodium life cycle. AP2-L consists of an extended amino terminal region and two AP2 domains and is highly expressed in asexual stage trophozoites, sporozoites and early EEFs, but its role in these stages is poorly understood. We characterized the functional consequences of deleting the entire P. berghei ap2-l coding sequence on the erythrocytic cycle in vivo , sporozoite gene expression, liver-stage development, and hepatocyte-parasite interactions. AP2L-KO parasites maintained erythrocytic replication, but sporozoite inoculation failed to establish blood-stage infection in mice. AP2-L loss decreased transcripts encoding proteins involved in sporozoite gliding motility, host-cell entry, and rhoptry secretion. Motif discovery upstream of a subset of these genes suggests possible direct targets of AP2-L. Infected HepG2 cells showed reduced EEF numbers and sizes, impaired parasite DNA replication, and abnormal parasite positioning relative to the host nucleus. PVMs of AP2L-KO parasites exhibited aberrant localization of proteins, absence of tubular vacuolar network extensions, and sensitivity to detergent. These abnormalities were accompanied by increased accumulation of the host's autophagy-associated protein around parasites, suggesting efficient host-mediated elimination. Additionally, AP2L-KO parasites displayed reduced association with the host microtubule-organizing center, Golgi compartments, and COPB2-positive vesicular structures, suggesting an impairment in uptake of nutrients. Together, these findings demonstrate that AP2-L directs both vacuolar biogenesis and host cytoskeletal/vesicular capture in liver-stage parasites.

Before causing symptomatic malaria, Plasmodium parasites multiply silently inside liver cells within a specialized protective structure called the parasitophorous vacuole. Understanding how the parasite builds and defends this essential niche is critical for developing strategies to prevent malaria. In this study, we demonstrate that the parasite transcription factor AP2-L controls a gene expression program vital for liver-stage survival. By analyzing parasites with a complete gene deletion, we show that AP2-L, directly or indirectly, activates genes required for vacuole membrane formation and maintenance. Without AP2-L, parasites form compromised vacuoles, fail to evade host cell autophagic clearance, and cannot capture host cell organelles needed for growth, completely halting infection. These findings establish AP2-L as a key regulator connecting nuclear gene expression to vacuole integrity and host cell manipulation, processes that are fundamental to intracellular pathogen survival.