Chlamydia-containing spheres are the predominant egress structure of the zoonotic pathogen Chlamydia psittaci.

Egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci little is known about the mechanisms resulting in chlamydial egress from the infected epithelium. Using state-of-the-art imaging techniques including live cell imaging and electron microscopy, we describe and characterize Chlamydia-containing spheres (CCS), a novel type of egress structure of Chlamydia. The formation of CCS represents the predominant non-lytic egress pathway of C. psittaci. CCS are spherical, low phase contrast structures surrounded by a phosphatidylserine exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. CCS formation is a sequential process starting with proteolytic cleavage of a DEVD-containing substrate that can be detected inside of the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected cell. Subsequently, blebbing of the plasma membrane begins, the inclusion membrane destabilizes and the proteolytic cleavage of the DEVD-containing substrate increases rapidly within the whole infected cell. Finally, infected, blebbing cells detach and leave the monolayer thereby forming CCS. This sequence of events is unique for chlamydial CCS formation and fundamentally different from previously described Chlamydia egress pathways. Thus, CCS formation represents a new egress pathway for intracellular pathogens that could be linked to C. psittaci biology and might influence the outcome of the infection in organisms.