Inclu sions are known to intercept the vesicular transport of the host cell and incorporate bodipy dyes. Infected HAEC were treated for 24 h with D3521 at 24 h, 48 h and 72 hpi in order to follow fluorescent ceramide incorpora sellekchem tion and distribution throughout the whole infection cycle. Colocalization of D3521 and C. pneumoniae was subsequently analyzed Infected HAEC harbouring inclusions stained with D3521 displayed ceramide labelling in all inclusions. Corresponding samples of spot like infected intact HAEC with a regular nuclear structure showed only low amounts of colocalized D3521 and C. pneumoniae signal in spots, indicating no or very much reduced metabolic activity of the chlamydial spots. In contrast, many spots of spot like infected aponecrotic HAEC colocalized with D3521 suggesting that these spots had resided in inclu sions.

C. pneumoniae spots of chloramphenicol treated infected cells colocalized with D3521 only in spots that still displayed Inhibitors,Modulators,Libraries DNA signal. DNA Inhibitors,Modulators,Libraries negative spots never colocalized with ceramide signal referring to metaboli cally inactive chlamydial particles. Taken together the results show that cHsp60 is produced exclusively by C. pneumoniae residing inside inclusions. Furthermore, 24 h after D3521supply, only ceramide pos itive inclusions occur, proving that metabolic activity is localised inside inclusions but not in spots. We never observed metabolically active aggregates alone, they were always accompanied by metabolically active spots, hence excluding the possibility of an aberrant inclusion forma tion.

These cHsp60 and D3521 positive bacteria are then released into the cytosol and cause cell death of the host. These results together with the LDH release suggest that metabolically active C. pneumoniae spots that derive from inclusions induce cell death in HAEC. Discussion In the present study we have demonstrated that Inhibitors,Modulators,Libraries metabol ically active C. pneumoniae stem from inclusions and lead to aponecrosis in human aortic endothelial cells. Healthy cells harbour metabolically active C. pneumoniae exclu sively in inclusions. Aponecrotic cells, in contrast, display an infection with scattered Inhibitors,Modulators,Libraries metabolically active chlamy dial spots or aggregates, indicating bacterial release from former inclusions. The release of Chlamydia into the Inhibitors,Modulators,Libraries cytosol, largely unno ticed during the past, seems to be a conserved mechanism between different Chlamydia species and is occurring under different experimental settings.

A remarkable study was recently published strongly supporting our data. It was shown that inclusions are lysed by proteases and that HeLa cells died shortly after release check this of Chlamydia trachom atis into the cytosol by a calcium dependent cell mem brane disruption. Our findings strongly suggest that also C. pneumoniae is released from inclusions leading to cell death of the host cell.