Fig 1.
Relevant steps of PG biosynthesis and D-amino acid dipeptide (DAAD) probes used in this study.
(a) DAADs are taken up by bacteria where they compete with endogenous D-Ala-D-Ala (DA—DA) for incorporation into PG. De novo synthesis of DA—DA is inhibited by D-cyloserine. The pentapeptide PG subunit is then flipped across the inner membrane into the periplasm where it is transglycosylated to form glycan polymers (nascent PG) and crosslinked by penicillin binding proteins (PBPs). Transpeptidation causes cleavage of the terminal D-Ala at position 5. Because the N-terminally labeled portion of DAAD becomes the amino acid at position 4 of the pentapeptide, the label is resistant to this processing and remains on the stem peptide. (b) PG-labeling reagents used in this study. Clickable DAADs EDA—DA and ADA—DA. Star represents the clickable amino acid.
Fig 2.
Structured illumination microscopy of DAAD PG labeling in pathogenic Chlamydia.
Structured illumination microscopy (SIM) was conducted on four pathogenic Chlamydia species. EDA-DA was added 2 hours post infection (hpi) and coverslips were fixed at 18 hpi. PG labeling (represented by EDA—DA) is shown in green, the major C. trachomatis outer membrane protein (MOMP) is shown in red, and cell nuclei are in blue (this labeling scheme is maintained in all subsequent figures, unless otherwise stated). MOMP staining is not shown for the three other species. Images are representative of ~20 inclusions viewed per strain. Scale bar = 1 μm.
Fig 3.
Chlamydial EBs do not retain PG labeling.
(a) SIM of EDA—DA labeled C. trachomatis inclusions at 22 hpi. Arrowheads indicate locations of EBs. Tissue culture cells were infected with C. trachomatis and placed on a rocker for 2 hours, ensuring asynchronous infection of the cell monolayer. Bacteria were then incubated with 4 mM EDA-DA for 22 hours. (b) Confocal maximum intensity projections of a mature, Chlamydia inclusion (40 hpi) in which bacteria were grown in the presence of 4 mM EDA—DA for the entire developmental cycle. The arrowheads point to EBs (distinguished by their MOMP labeling and smaller size). (c) Confocal maximum intensity projections of Chlamydia-infected cells 4 hpi. EBs used for infection were harvested from cells that had been incubated with EDA—DA for 18 hours. Exogenous EDA—DA (4 mM) was present throughout the EB harvest as well as the subsequent reinfection. MOMP and PG labeling is the same as in Fig 2. Images are all representative of at least three separate experiments. Scale bar = 1 μm.
Fig 4.
3D SIM visualization of labeled chlamydial PG.
(a-e) Maximum intensity projection (7 μm thick Z stack) of C. trachomatis incubated with 4 mM EDA-DA at 2 hpi and fixed at 18 hpi. Panels (b-e) are maximum intensity projections of 0.5 μm thick planes of interest selected from panel a. Arrowheads indicate areas of punctate PG staining (as opposed to PG rings). (f) The PG ring dimensions: diameter x, width y, and thickness z. (g-k) 3D-SIM of cases of asymmetric cell division are outlined and marked with arrowheads for clarity. Panels (h) and (i) are magnifications of the image in panel (g). Images in panels (j) and (k) are independent cases. MOMP and PG labeling is the same as in Fig 2. Image is representative of ~ 20 inclusions analyzed. Scale bar = 1 μm.
Fig 5.
C. trachomatis incorporates DAADs rapidly during exponential growth.
(a) SIM of chlamydial inclusions (18 hpi) incubated for 10 min with 4 mM EDA—DA. Z-stacks from chlamydial inclusions (18 hpi) incubated with EDA—DA were collected, maximum intensity projections constructed, and average pixel intensities calculated for entire inclusions for both MOMP (b) and EDA—DA (c) channels. ~200 inclusions were assigned average pixel intensities per time point measured and the entire experiment was carried out in biological duplicates. MOMP and PG labeling is as presented in Fig 2. Scale bar = 1 μm.
Fig 6.
Chlamydial MreB is patchy, co-localizes to PG rings, and is required for chlamydial PG synthesis.
(a) SIM of chlamydial PG localization in relation to polymerized MreB in inclusions 18 hpi incubated with EDA—DA for one hour. Right-most panels are magnifications of single imaging planes from a. (b) Chlamydial inclusions (18 hpi) grown in the presence of 4 mM EDA—DA and MreB inhibitors A22 or MP265 (added 12 hpi). MOMP and PG labeling is the same as in Fig 2. (c) SIM of EDA-DA labeled PG (green) and MOMP (red) within chlamydial inclusions (18 hpi) incubated with 4 mM EDA-DA for 5 minutes. Scale bars = 1 μm.
Fig 7.
Pulse-chase experiments establish preliminary kinetics of PG disassembly in chlamydial RBs.
Change in mean fluorescence (a) and integrated fluorescence (b) per inclusion over time from chlamydial inclusions pulsed 1 h with EDA—DA after which the medium was removed and replaced with probe-free medium. Subsequent measurements were taken at indicated time points (i.e. chase). (c) Representative epifluorescence (EPI) maximum intensity projections of labeled chlamydial inclusions after the initial pulse and successive chases are shown. Scale bar = 1 μm.
Fig 8.
(a) SIM of chlamydial inclusions in which cells were pulsed for two hours with 4 mM EDA—DA, after which the MreB polymerization inhibitor A22 (75 μM) was added to the medium. In the two far-right panels, medium containing A22 was removed after one hour. Cells were then washed, fresh medium without new EDA—DA was added, and cells were allowed to grow an additional 30 and 60 minutes, respectively. At indicated time points, cells were fixed, permeabilized, and fluorescently labeled via click chemistry and anti-CTMOMP. Scale bars = 1 μm. (b) Quantitative analysis of the mean fluorescence intensity of chlamydial inclusions initially pulsed with EDA-DA and subsequently chased under the times and conditions indicated. Fluorescence values were collected for ~250 inclusions per group, statistics were obtained utilizing an unpaired t test with Welch's correction and error bars represent standard deviations of the mean. Analysis was conducted in triplicate and is representative of two biological replicate experiments. (c) Graphical model representing the experimental approach and results from (b).
Fig 9.
Proposed model for PG biosynthesis and maintenance in pathogenic Chlamydia.
(a) Upon invading a host cell, MreB facilitates mid-cell PG (green) ring formation 8 hpi and the first cell division occurs. Inhibition of MreB (red) prior to EB-RB transition prevents this first division and results in accumulation of intracellular ‘EB-like’ particles in the host cytosol. Once RBs form, PG is constantly synthesized and incorporated into the ring non-uniformly by dynamic MreB patches. If MreB polymerization is inhibited, the PG ring dissociates, resulting in slightly enlarged, static, PG-less aberrant bodies. Inhibition of PG crosslinking by β-lactams does not affect transglycosylase activity and MreB localization, but instead results in unchecked enlargement of the RB. Bottom, upon cell division, the new division planes form immediately and perpendicular to the previous division plane. During transition to non-replicative EBs, MreB and PG biosynthesis enzymes are downregulated [58], resulting in the complete disassembly of the PG ring. (b) Chlamydial PG biosynthesis and degradation mechanisms overlap and are active throughout the cell cycle. MreB (red) moves along the ring plane (black arrows), initiating the non-uniform synthesis of new PG (green) and influencing the action of an unknown degradation mechanism that continuously removes older material (white arrows). PG degradation enzymes (amidases and lytic transglycosylases) are visualized as blue pacmen.