Rhoptry neck protein 2 expressed in Plasmodium sporozoites plays a crucial role during invasion of mosquito salivary glands.

Malaria parasite transmission to humans is initiated by the inoculation of Plasmodium sporozoites into the skin by mosquitoes. Sporozoites develop within mosquito midgut oocysts, first invade the salivary glands of mosquitoes, and finally infect hepatocytes in mammals. The apical structure of sporozoites is conserved with the infective forms of other apicomplexan parasites that have secretory organelles, such as rhoptries and micronemes. Because some rhoptry proteins are crucial for Plasmodium merozoite infection of erythrocytes, we examined the roles of rhoptry proteins in sporozoites. Here, we demonstrate that rhoptry neck protein 2 (RON2) is also localized to rhoptries in sporozoites. To elucidate RON2 function in sporozoites, we applied a promoter swapping strategy to restrict ron2 transcription to the intraerythrocytic stage in the rodent malaria parasite, Plasmodium berghei. Ron2 knockdown sporozoites were severely impaired in their ability to invade salivary glands, via decreasing the attachment capacity to the substrate. This is the first rhoptry protein demonstrated to be involved in salivary gland invasion. In addition, ron2 knockdown sporozoites showed less infectivity to hepatocytes, possibly due to decreased attachment/gliding ability, indicating that parts of the parasite invasion machinery are conserved, but their contribution might differ among infective forms. Our sporozoite stage-specific knockdown system will help to facilitate understanding the comprehensive molecular mechanisms of parasite invasion of target cells.