-containing spheres are a novel and predominant form of egress by the pathogen .

The 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 little is known about the mechanisms resulting in bacterial egress from the infected epithelium. Here, we describe and characterize -containing spheres (CCSs), a novel and predominant type of non-lytic egress utilized by spp. CCSs 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 the proteolytic cleavage of a DEVD tetrapeptide-containing substrate that can be detected inside 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 a 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 egress pathways. Thus, CCS formation represents a major, previously uncharacterized egress pathway for intracellular pathogens that could be linked to biology in general and might influence the infection outcome .IMPORTANCEHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host transmission. Here, we characterize -containing sphere (CCS) formation as a novel and predominant non-lytic egress pathway of the intracellular pathogens and . CCS formation is fundamentally different from extrusion formation, the previously described non-lytic egress pathway of . CCS formation is a unique sequential process, including proteolytic activity, followed by an increase in intracellular calcium concentration, inclusion membrane destabilization, plasma membrane blebbing, and the final detachment of a whole phosphatidylserine-exposing former host cell. Thus, CCS formation represents an important and previously uncharacterized egress pathway for intracellular pathogens that could possibly be linked to biology, including host tropism, protection from host cell defense mechanisms, or bacterial pathogenicity.