A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma.

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

An obligate intracellular bacterial pathogen forms a direct, interkingdom membrane contact site.

Interorganelle communication regulates cellular homeostasis through the formation of tightly-associated membrane contact sites 1-3. Prior work has identified several ways that intracellular pathogens alter contacts between eukaryotic membranes 4-6, but there is no existing evidence for contact sites spanning eukaryotic and prokaryotic membranes. Here, using a combination of live-cell microscopy and transmission and focused-ion-beam scanning electron microscopy, we demonstrate that the intracellular bacterial pathogen Rickettsia parkeri forms a direct membrane contact site between its bacterial outer membrane and the rough endoplasmic reticulum (ER), with tethers that are approximately 55 nm apart. Depletion of the ER-specific tethers VAPA and VAPB reduced the frequency of rickettsia-ER contacts, suggesting these interactions mimic organelle-ER contacts. Overall, our findings illuminate a direct, interkingdom membrane contact site uniquely mediated by rickettsia that seems to mimic traditional host MCSs.