Heuer, D. | bioRxiv Science

bioRxiv2024

Recruitment of the cellular lipid transport protein CERT to C. psittaci inclusions regulates the timing of bacterial egress

Egress of intracellular pathogens is highly regulated and carefully timed. For the zoonotic bacterium C. psittaci, the predominant egress pathway is Chlamydia-containing sphere (CCS) formation, a calcium-dependent sequential mechanism including protease activity, inclusion membrane destabilization, intracellular calcium increase, and plasma membrane blebbing. How egress is regulated to ensure that it takes place only after C. psittaci intracellular development is thus far unknown. Here, we show that C. psittaci recruits the cellular ceramide transporter CERT to its inclusion during intracellular development, but this recruitment is reduced at late time points prior to egress. In addition, an early loss of CERT at the inclusion membrane induced by CERT-KO induces premature egress by CCS formation. Complementation of the CERT-KO with different CERT-GFP variants prevents premature egress, except of complementation with a variant lacking the inclusion targeting PH domain, showing that the localization of CERT is critical for CCS formation. The CERT-KO induced premature CCS are formed by the sequential process described for mature CCS, but they contain mostly RBs and are predominantly non-infectious. Thus, our findings suggest that the timing of C. psittaci egress by CCS formation is regulated by the recruitment of CERT to the inclusion. We propose that CERT stabilizes the chlamydial inclusion by the formation of ER-inclusion membrane contact sites during intracellular development, and the loss of CERT recruitment facilitates inclusion membrane destabilization and CCS formation.

microbiology↗

bioRxiv2023

The formation of Chlamydia-containing spheres, a novel non-lytic egress pathway 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 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. ImportanceHost 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.

microbiology↗