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 constructs 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. 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.