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Upon invasion into the host cell, a subset of bacterial pathogens resides exclusively in the cytosol. While previous research revealed how they reshape the plasma membrane during invasion, subvert the immune response, and hijack cytoskeletal dynamics to promote their motility, it was unclear if these pathogens also interacted with the organelles in this crowded intracellular space. Here, we examined if the obligate intracellular pathogen Rickettsia parkeri interacts with the endoplasmic reticulum (ER), a large and dynamic organelle spread throughout the cell. Using live-cell microscopy and transmission and focused-ion-beam scanning electron microscopy, we show that R. parkeri forms extensive contacts with the rough ER that are ∼55 nm apart and cover more than half the bacterial surface. Depletion of the ER-specific tethers VAPA and VAPB reduced rickettsia-ER contacts, and VAPA and VAPB were localized around intracellular rickettsiae. Overall, our findings illuminate an interkingdom ER contact uniquely mediated by rickettsiae that mimics some characteristics of traditional host membrane contact sites., (© 2025 Acevedo-Sánchez et al.)

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., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)