Golgi Fragmentation and Sphingomyelin Transport to Chlamydia trachomatis during Penicillin-Induced Persistence Do Not Depend on the Cytosolic Presence of the Chlamydial Protease CPAF

Abstract

Chlamydia grows inside a cytosolic vacuole (the inclusion) that is supplied with nutrients by the host through vesicular and non-vesicular transport. It is unclear in many respects how Chlamydia organizes this transport. One model posits that the Chlamydia-induced fragmentation of the Golgi-apparatus is required for normal transport processes to the inclusion and for chlamydial development, and the chlamydial protease CPAF has been controversially implicated in Golgi-fragmentation. We here use a model of penicillin-induced persistence of infection with Chlamydia trachomatis to test this link. Under penicillin-treatment the inclusion grew in size for the first 24 h but after that growth was severely reduced. Penicillin did not reduce the number of infected cells with fragmented Golgi-apparatus, and normal Golgi-fragmentation was found in a CPAF-deficient mutant. Surprisingly, sphingomyelin transport into the inclusion and into the bacteria, as measured by fluorescence accumulation upon addition of labelled ceramide, was not reduced during penicillin-treatment. Thus, both Golgi-fragmentation and transport of sphingomyelin to C. trachomatis still occurred in this model of persistence. The portion of cells in which CPAF was detected in the cytosol, either by immunofluorescence or by immune-electron microscopy, was drastically reduced in cells cultured in the presence of penicillin. These data argue against an essential role of cytosolic CPAF for Golgi-fragmentation or for sphingomyelin transport in chlamydial infection.

In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

“…C. trachomatis expresses CPAF, which is likely to translocate in part from the inclusion to the cytosol (16,49,50), although it is difficult to be absolutely certain of this for technical reasons and although the bulk of it is probably released only if the inclusion ruptures toward the end of the developmental cycle (27). Most of the reported massive degradation events due to CPAF activity are probably extraction artifacts (26), although small amounts of degradation occurring in intact cells cannot be excluded at present (see discussion in reference 35).…”

“…HeLa cells and the stable HeLa cell line YFP-Golgi-HeLa (16) were maintained in Dulbecco modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS; tetracycline negative; PAA Laboratories) and cultured at 37°C and 5% CO 2 .…”

“…Cells Ceramide transport and live-cell imaging. The experimental setup for ceramide transport has been described before (16). Briefly, HeLa cells were seeded in glass-bottom dishes (MatTek, Ashland, MA, USA) and infected with W. chondrophila or C. trachomatis L2 for 24 h. The medium was replaced with fresh medium containing 100 nM BODIPY (borondipyrromethene) FL C 5 ceramide (Invitrogen no.…”

“…For Western blot analysis, 200,000 HeLa cells were seeded in 6-well plates and infected as described above. Extracts using radioimmunoprecipitation assay (RIPA) buffer or 8 M urea dissolved in water were prepared as described before (16). Ten micrograms of protein was loaded onto 10% SDS gels and transferred to nitrocellulose membranes.…”

Characterization of CPAF Critical Residues and Secretion during Chlamydia trachomatis Infection

“…A chlamydial mutant deficient in type II secretion also significantly restricted CPAF secretion (10), suggesting that CPAF may get out of the outer membrane via the type II secretion apparatus, which, however, only sends CPAF into the lumen of the inclusion. Due to lack of knowledge about how CPAF reaches the host cell cytoplasm, some have questioned the validity of the detection of CPAF in the host cell cytoplasm (11) despite the fact that CPAF has been localized to the host cell cytosol consistently (3,(12)(13)(14)(15)(16)(17). Interestingly, the cytosolically localized CPAF is not colocalized with the secreted Pgp3 protein (18,19), which may suggest that CPAF and Pgp3, although both are secreted into the host cell cytoplasm, may have different host targets.…”