dc.description.abstract
The genus Chlamydia comprises eleven pathogenic species, including the human specific, sexually transmitted pathogen C. trachomatis and the zoonotic pathogen C. psittaci, that causes lung infections. During their intracellular development, Chlamydia exist in two distinct forms, the infectious elementary bodies (EBs) and the metabolizing and replicating reticulate bodies (RBs). Intracellular development initiates with the invasion of EBs, which differentiate to RBs inside of a membrane-bound compartment, the chlamydial inclusion. RBs abuse host cellular metabolites to replicate. After redifferentiation to EBs, they egress from the host to start a new round of infection. Two chlamydial egress forms were described in the past: Lytic egress and Chlamydia-specific, non-lytic extrusion formation.
In the first part of this study, the intracellular development of C. psittaci in HeLa cells was investigated. It was shown that EB to RB differentiation was completed at 12 hours post infection (h pi) and RB replication initiates. At 24 h pi, RB redifferentiation to EB started and at 34 h pi, similar amounts of EBs, RBs and intermediate forms were present in the inclusion. Starting at 42 h pi, most of the bacteria were in EB form and the infectivity reached a maximum, suggesting to perform egress studies at this time point.
In the following part of this study, it was shown that C. psittaci uses a previously not characterized egress pathway, the formation of Chlamydia-containing spheres (CCS). CCS formation is a sequential process starting with proteolytic cleavage of an Aspartate-Glutamate-Valine-Aspartate (DEVD) tetrapeptide-containing substrate that can be detected inside of the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected epithelial 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. CCS contain infectious progeny and morphologically impaired cellular organelles and are surrounded by a phosphatidyl serine exposing membrane with specific barrier functions for TrypanRed or SYTOX Green. Interestingly, no extrusion formation was observed for C. psittaci infections, while CCS formation was the predominating egress type. In addition, CCS formation was also observed for C. trachomatis.
Next, investigations about the inclusion membrane stabilization during intracellular development mediated by the human ceramide transport protein CERT and about the inclusion membrane destabilization during CCS formation were realized. It was shown that CERT is recruited to the bacterial inclusion during intracellular development, while at late infection timepoints before CCS formation, this recruitment was not observed. An early lack of CERT at the inclusion membrane drove the formation of premature CCS containing non-infectious bacteria.
Finally, the lipidome of C. psittaci-infected HeLa cells during intracellular development and before host cell egress was investigated. It was shown that both, during intracellular development and during egress, increased concentration of phospholipid species with saturated, odd chain fatty acids were found, suggesting that they are a general characteristic of chlamydial infection. Before egress, the amount of specific phospholipids species comprising shorter fatty acids was increased, suggesting that they are a specific characteristic of late infections.
Taken together, this study shows that egress by CCS formation is a relevant part of the chlamydial developmental cycle. CCS formation depends on both, bacterial and host cellular factors, as a putative bacterial DEVD-cleaving protease and a CERT-recruiting factor or as the ceramide transporter CERT, calcium signaling and lipid metabolism, respectively. The zoonic pathogen C. psittaci and the human-specific pathogen C. trachomatis apply different egress strategies with CCS formation being the predominant egress pathway of C. psittaci. This suggests that the different biology of the different Chlamydia spp. including host tropism, protection from host cell defense mechanisms or bacterial pathogenicity could possibly be linked to different egress strategies.
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