Live Cell Imaging of Chlamydia Inclusion Membrane Proteins Using Genetically Encoded Antibodies

Chlamydia subverts host cell pathways by secreting effector proteins into the membrane of its inclusion. These bacterial membrane proteins, called Incs, mediate interactions between the inclusion and the host cell. To study Inc protein dynamics in live cells, we have developed a methodology that relies on host cell expression of antibodies fused to fluorescent proteins, known as frankenbodies, which detect epitopes exposed to the cytosol. HeLa cells are first stably transfected with a plasmid encoding an mScarlet3-tagged anti-FLAG frankenbody. These cells are then infected with Chlamydia expressing a FLAG-tagged Inc protein, which is secreted into the inclusion membrane with the FLAG tag facing the cytosol. Recruitment of the fluorescent anti-FLAG frankenbody to the inclusion is monitored in real time using time-lapse confocal microscopy. This approach has been successfully employed to elucidate the dynamics of IncA on the inclusion membrane and can be expanded to simultaneously investigate multiple Incs, as well as Inc:Inc interactions, by using different tag-frankenbody combinations.