Abstract Egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This egress 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. Here, we describe and characterize a novel non-lytic egress pathway of C. psittaci by formation of Chlamydia -containing spheres (CCS). 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. The formation of CCS shares characteristics of apoptotic cell death including a proteolytic cleavage of the peptide DEVD albeit independent of active caspase-3, an increase in the intracellular calcium concentration of infected cells, followed by blebbing of the plasma membrane and rupture of the inclusion membrane. Finally, infected blebbing cells detach and leave the monolayer thereby forming CCS. These results support that Chlamydia psittaci egresses the epithelial cell by a novel non-lytic egress pathway, a process beneficial for the bacterium, which might influence the outcome of the infection in organisms. Importance Host cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host-transmission. Here, we describe CCS formation as a novel egress pathway for the intracellular, zoonotic bacterial pathogen C. psittaci . This non-lytic egress pathway is fundamentally different from previously described Chlamydia egress pathways. Interestingly, CCS formation shares several characteristics of apoptotic cell death. However, the sequence of proteolytic activity, followed by plasma membrane blebbing and the final detachment of a whole phosphatidylserine exposing former host cell is unique for C. psittaci . Thus, CCS formation represents a new egress pathway for intracellular pathogens that could possibly be linked to C. psittaci biology including host tropism, protection from host cell defense mechanisms or bacterial pathogenicity.

The formation ofChlamydia-containing spheres, a novel non-lytic egress pathway of the zoonotic pathogenChlamydia psittaci