Identification of a novel rhoptry protein expressed predominantly in Plasmodium sporozoites

The infective stages of apicomplexan protozoans, such as the malaria parasite <i>Plasmodium</i>, possess apical organelles rhoptries and micronemes, which contain secretory proteins required for host cell invasion. The mechanisms mediating invasion are largely conserved among apicomplexan parasites; for example, rhoptry proteins are secreted to form a tight junction prior to invasion, which facilitates parasite entry into target cells. Stage-specific invasion mechanisms have also been described; such as those differentially mediating <i>Plasmodium</i> merozoite infection of erythrocytes versus sporozoite stage invasion of mosquito salivary glands and mammalian hepatocytes. Sporozoites are the transmission stage present within the salivary glands of infected mosquitoes, and can efficiently infect the mammalian liver after being deposited in the skin during a blood meal. While some sporozoite rhoptry proteins have been demonstrated to be critical for invasion of mosquito salivary glands and mammalian hepatocytes, their comprehensive molecular mechanisms have not been elucidated due to the limited availability of material. To screen for <i>Plasmodium</i> sporozoite-specific rhoptry proteins in the rodent malaria parasite, <i>Plasmodium berghei</i>, a proximity-dependent biotin identification method was employed combined with a genome editing strategy. Rhoptry neck protein 12 (RON12) was identified as a rhoptry molecule with the highest transcript levels in sporozoites; and was selected for use as a bait following tagging with UltraID. In RON12::ultraID expressing transgenic sporozoites, several secretory proteins were successfully biotinylated during parasite maturation in mosquitoes, including known rhoptry proteins. A novel rhoptry molecule was identified, PBANKA_1363400, which was localized to sporozoite rhoptries and was predominantly expressed in sporozoites rather than merozoites. This study demonstrates that the UltraID strategy enables highly sensitive and comprehensive protein identification in a species- or stage-specific manner in <i>Plasmodium</i> sporozoites.