Fig. 4
Premature, CERT-KO-induced CCS contain mainly non-infectious reticulate bodies. (A) CCS in the supernatant of C. psittaci-infected CERT-KO and KO control cells (MOI 2) were separated from free bacteria by centrifugation (300 x g, 5 min, RT) at 24 h pi. Infectious progeny was titrated after glass bead lysis and numbers were normalized to genome copy numbers determined by qRT-PCR. Data show mean ± SEM; n = 3. (B) Transmission electron microscopy (TEM) of a thin section through the chemically fixed supernatant of C. psittaci-infected HeLa CERT-KO cells (MOI 2, 24 h pi). Numerous Chlamydia, mostly in RB stage, were found; n = 3. The image shows a group of RBs (*) which are associated with cellular debris, which is the main component of the supernatant. (C) Representative fluorescence images of an early live CCS isolated of the supernatant of C. psittaci-infected HeLa CERT-KO cells (MOI 2, 24 h pi). The surrounding membrane was visualized using the membrane marker FM 4–64 and DNA was counterstained by Hoechst; n = 3. (D) Representative fluorescence images of a PFA-fixed early CCS isolated from C. psittaci-infected HeLa CERT-KO cells (MOI 2, 24 h pi). Bacteria inside the CCS were detected using a chlamydial Hsp60 (Cy3) antibody and the DNA was counterstained using DAPI; n > 3.
