Fig. 6: Elementary bodies possess higher proportion of metabolized TFSMs than reticulate bodies.
a TFSM can be used to visualize chlamydial inclusion via 4xExM. HeLa cells were infected with C. trachomatis at MOI 1 in the presence of TFSM 1 for 24 h, fixed and stained with AlexaFluorTM (AF)546-azide (headgroup) and BODIPY-FL-DBCO (backbone). Chlamydia was stained with an anti-chlamydial heat-shock protein 60 (HSP60) anti-body and an AF405 secondary antibody. Samples were 4x expanded and imaged with CLSM. n = 3. Scale bars: 8 µm (with 4-fold expansion factor ~ 2 µm). b Periphery of inclusions rather possess non-metabolized TFSM compared to their center. The image depicted in (a) was zoomed, and the metabolic state of TFSM was calculated by determining the ratio of 3-times Förster resonance energy transfer (FRET) vs. donor (BODIPY-FL) signal. White arrows indicate intermediate chlamydial developmental forms. n = 3. Scale bars: 8 µm (with 4-fold expansion factor ~ 2 µm). c, d Inclusions possess lower FRET efficiency than host cell membranes. Chlamydia-infected HeLa cells stained with TFSM 1 (BODIPY-FL: Donor/AF546: Acceptor) were 4-fold expanded. FRET efficiency in regions containing an inclusion (white circles) or host cell membranes was determined by acceptor photobleaching. n = 3. Scale bars: 20 µm (with 4-fold expansion factor ~ 5 µm). e, f Sphingolipid composition of EBs and RBs differ. The area indicated by a white line in b.) was magnified, and the intensity profile was measured in Fiji. The resulting gray values were scaled to the highest and lowest values detected in individual channels. n = 3. Scale bar: 4 µm (with 4-fold expansion factor ~ 1 µm). g Identification of high metabolized TFSM levels in EBs by FLIM imaging of 4-fold expanded TFSM 1-stained chlamydial inclusion labeled with BODIPY-FL-DBCO (left) as well as BODIPY-FL-DBCO and AF546-azide (middle, right). Zoomed image (right) of BODIPY-FL-DBCO and AF546-azide labeled TFSM shows higher fluorescence lifetimes of EBs in comparison to RBs, indicating a higher proportion of metabolized TFSMs. n = 1. Scale bars: 10 µm (with 4x expansion factor ~ 2.5 µm). Statistics: Two-sided unpaired Student’s t test (d). Bars represent means ± SD. n corresponds to biological replicates. Source data and detailed statistics are provided as a Source Data file.
