Scholz Jana, Holland Gudrun, Laue Michael, Banhart Sebastian, Heuer Dagmar
Unit of Sexually Transmitted Bacterial Pathogens and HIV, Robert Koch Institute, Berlin, Germany.
Unit of Advanced Light and Electron Microscopy, Robert Koch Institute, Berlin, Germany.
Sci Rep. 2025 May 25;15(1):18241. doi: 10.1038/s41598-025-02077-w.
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 productive 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 specific 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.
细胞内病原体的排出受到高度调控且时间安排精确。对于人畜共患细菌鹦鹉热衣原体而言，主要的排出途径是形成含衣原体的球体（CCS），这是一种依赖钙的顺序机制，包括蛋白酶活性、包涵体膜去稳定化、细胞内钙增加以及质膜起泡。目前尚不清楚排出过程是如何被调控以确保其仅在鹦鹉热衣原体在细胞内进行有效发育之后才发生。在此，我们表明鹦鹉热衣原体在细胞内发育过程中会将细胞神经酰胺转运蛋白CERT募集到其包涵体上，但在排出前的后期时间点这种募集会减少。此外，由CERT-KO诱导的包涵体膜上CERT的早期缺失会通过CCS形成诱导过早排出。用不同的CERT-GFP变体对CERT-KO进行互补可防止过早排出，但用缺乏包涵体靶向PH结构域的变体进行互补除外，这表明CERT的特定定位对于CCS形成至关重要。CERT-KO诱导的过早CCS是通过针对成熟CCS描述的顺序过程形成的，但它们主要包含RB且大多无感染性。因此，我们的研究结果表明，通过CCS形成的鹦鹉热衣原体排出的时间是由CERT募集到包涵体上所调控的。我们提出，CERT在细胞内发育过程中通过形成内质网-包涵体膜接触位点来稳定衣原体包涵体，而CERT募集的丧失促进了包涵体膜去稳定化和CCS形成。
