Rhoptry-associated perforation of the host cell plasma membrane during Toxoplasma gondii invasion

The exocytosis and delivery of rhoptry proteins into the host cell is essential for Toxoplasma gondii invasion and virulence. The rhoptry secretory apparatus (RSA) has become increasingly well-defined and shows conservation among Apicomplexans. While we now know a great deal about both the RSA structure and the individual rhoptry effector proteins that are released, comparatively little is known about how those proteins are delivered into the host cell. Previous electrophysiology experiments revealed that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. We hypothesize that the conductance transient represents a perforation in the host cell plasma membrane through which rhoptry proteins are delivered into the host cell. To further investigate this event, we have developed a higher throughput assay that utilizes high-speed, multi-wavelength, fluorescence imaging to simultaneously visualize the host cell perforation and invasion of the parasite. Using this assay, we have interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking the rhoptry effector RON2 generate the perforation, suggesting that perforation is upstream of effector injection and moving junction formation. In contrast, parasites lacking components of the rhoptry exocytosis pathway (e.g., RASP2, Nd9) or proteins that regulate rhoptry secretion (e.g., CLAMP), are largely unable to induce the perforation. These data, which have been corroborated by electrophysiology studies, demonstrate that rhoptry exocytosis is required for formation of the perforation. Our results are consistent with a model in which rhoptry exocytosis results in transient perforation of the host cell membrane, enabling the subsequent delivery of rhoptry effector proteins into the host cell.