A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis

Abstract

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis and exhibit susceptibility to 'anti-PG' antibiotics, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the 'chlamydial anomaly'). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

Figure 1. Novel dipeptide PG labeling strategy

a, Biosynthesis of the terminal PG stem peptide of Gram negative bacteria. Two D-alanines are first ligated together by D-alanine-D-alanine ligase and the dipeptide is subsequently added to the stem tripeptide by MurF, resulting in a pentapeptide. The labeling strategy relies on the inherent tolerance of the PG machinery to accept DA-DA analogs. Subsequent cross-linking between neighboring peptide stems is carried out by a series of transpeptidases (penicillin binding proteins). Upon transpeptidation, a proximal m-DAP from a neighboring peptide stem attacks the subterminal D-alanine of the PG stem. The terminal D-alanine is thus cleaved from the stem peptide, which results in a tetrapeptide. Another pathway for the loss of terminal D-alanine is D,D-carboxypeptidation catalyzed by enzymes such as DacA. b, Chemical structures of D-Ala, DA-DA, and their derivatives carrying bioorthogonal handles used in this study. Abbreviations: MurNAc, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine; L-Ala, L-Alanine; D-Glu, D-Glutamic Acid; m-DAP, meso-diaminopimelic acid; D-Ala, D-Alanine; Alr, Alanine racemase; Ddl, D-alanine-D-alanine ligase; MurF, UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase; PBPs, penicillin-binding proteins; DA-DA, D-alanyl-D-Alanine; EDA, Ethynyl-D-alanine; ADA, Azido-D-alanine; EDA-DA, Ethynyl-D-alanyl-D-alanine; ADA-DA, Azido-D-alanyl-D-alanine.

Figure 2. Fluorescent labeling of intracellular C. trachomatis PG

Differential interference contrast (DIC) (a) and fluorescent (b–e) microscopy of L2 cells infected for 18 hours with C. trachomatis in the presence of the dipeptide probe EDA-DA (1 mM). Subsequent binding of the probe to an azide modified Alexa Fluor 488 (green) was achieved via click chemistry. Antibody to MOMP (red) was used to label chlamydial EBs and RBs. DAPI (blue) was used for nuclear staining. Panels B–E show a merge of all three fluorescent channels. Boxes indicate location of chlamydial inclusions, and magnification of the boxes is provided in panels c–e. Fluorescent images are maximum intensity projections of deconvoluted zstacks. Three dimensional renderings are provided in the supplemental materials (Videos S1 and S2).

Figure 3. Labeling in the presence of PG synthesis inhibitors

DIC and fluorescent microscopy of infected L2 cells 18 hours post infection in the presence of 1 mM EDA-DA and either (a) D-cycloserine (DCS) or (b) ampicillin (AMP). Labeling was conducted as described in the Figure 2 legend. A merge of all three fluorescent channels is presented in the right hand panels. Fluorescent images are maximum intensity projections of zstacks and three dimensional renderings are provided in the supplemental materials (Videos S3 and S4).

Extended Data Figure 1. Single D-amino acid probe EDA fails to label intracellular Chlamydia despite labeling intracellular Shigella flexneri

Phase contrast and epifluorescence microscopy of (a) Chlamydia-infected L2 cells 18 hours post infection, (b) Shigella flexneri strain 2457T two hour broth cultures, and (c) Shigella-infected L2 cells three hours post infection. All were grown in the presence of 1 mM EDA. Subsequent tethering of the probe to a modified Alexa Fluor 488 (green) was achieved via click chemistry. Antibody to chlamydial inclusion protein A (IncA, red) was used to visualize chlamydial inclusions. Experiments were conducted in technical duplicates and biological triplicates, with between 4–5 fields examined (with ~3–10 inclusions viewed per field) per technical replicate.

Extended Data Figure 2. D-enantiomer dipeptide probes do not affect growth in rich media, but differentially and specifically label PG of E. coli and B. subtilis

a, Growth of wild-type E. coli and B. subtilis in the presence of experimental concentrations of EDA-DA or DA-EDA. A representative growth curve from two biological replicates, each with three technical replicates, is shown. b, Phase contrast and epifluorescence microscopy of E. coli grown with 0.5 mM alkyne containing EDA-DA, DA-EDA or as a positive control with EDA at five minutes and 60 minutes. These samples together with unlabeled controls were ‘clicked’ to Alexa Fluor 488 azide and imaged. When the alkyne is on the C-terminus (DA-EDA), the labeling is not apparent. Signal from N-terminally tagged dipeptide (EDA-DA) is significantly higher, but still lower than EDA and the patterns of labeling at the earlier time points are different. This is probably due to periplasmic incorporation of D-amino acids (e.g. EDA) by E. coli L,D-transpeptidases, which result in more efficient peripheral labeling in addition to labeling due to lipid II-dependent PG synthesis. Therefore, in bacteria that have active L,D-transpeptidases, the cytoplasmic PG labeling through dipeptide probes provides a better measure of lipid II-dependent PG synthesis than single D-amino acids. The experiment was conducted twice and images are representative of a minimum of five fields viewed per condition/time point per replicate. c, Comparison of the labeling in E. coli grown with 0.5 mM alkyne containing EDA-DA or the L-enantiomer control Ethynyl-L-alanine-L-alanine (ELA-LA) for 45 min and clicked as above shows that the labeling is D-enantiomer specific. Images are representative of a minimum of four fields viewed per replicate and the experiment was conducted twice.

Extended Data Figure 3. Dipeptide probes differentially and specifically label PG of diverse gram-positive bacteria allowing live-cell experiments

Phase contrast and epifluorescence microscopy of B. subtilis (a–c), Streptococcus pneumoniae (d), and Streptomyces venezuelae (e). a, Five minute and 60 minute aliquots were taken from wild-type B. subtilis grown with 0.5 mM alkyne containing EDA-DA, DA-EDA or as a positive control with EDA. These aliquots together with unlabeled controls were ‘clicked’ to Alexa Fluor 488 azide and imaged. When the alkyne is on the N-terminus (EDA-DA), labeling is comparable to EDA. On the other hand, the labeling with C-terminal tag (DA-EDA) is much fainter. b, B. subtilis grown with 0.5 mM alkyne containing EDA-DA or the L-enantiomer control ELA-LA for 45 min and clicked as above indicates that the labeling is D-enantiomer specific. The partial lysis of the cells visible in phase contrast is caused by 70% EtOH fixation. c–d, When live B. subtilis and S. pneumoniae labeled with azide containing ADA-DA and DA-ADA (0.4 mM and 1.6 mM for c and 0.5 mM for d) were clicked to Alexa Fluor 488 DIBO alkyne using a non-toxic procedure, the signals from N-terminally tagged dipeptide ADA-DA were much higher than the signal from DA-ADA labeled cells. (c) Interestingly, the signal from DA-ADA can be elevated to the ADA-DA level, if the labeling is performed in a ΔdacA, D,D-carboxpeptidase-null mutant of B. subtilis. Since copper-free click-chemistry is not toxic to cells, a pulse-chase experiment was done, which shows the trapping of old PG at the poles of the cells (lower panel). e, When polarly growing S. venezuelae cells are grown with the blue fluorescent D-amino acid HADA (2 h, 0.5 mM) for several generations and briefly pulsed with EDA-DA (10 min, 0.5 mM) and clicked, the signal from EDA-DA complements the signal from HADA. This result shows that dipeptide probes label the cell wall at sites of new PG synthesis. Fluorescent images (a–d) were taken and processed in the same manner for comparison. In ‘Adjusted’ images, signal intensities were lowered for comparison of labeling patterns. All experiments were conducted in biological duplicates, and images are representative of 2–5 fields viewed per condition/time point/replicate.

Extended Data Figure 4. EDA-DA labeling is specific to the PG of bacteria

a, Alexa Fluor 488 Azide ‘clicked’ sacculi from B. subtilis and E. coli cells grown with 0.5 mM EDA-DA for several generations retained the alkyne label. The labeled cells were clicked before sacculi purification in the case of B. subtilis and after purification in the case of E. coli. Experiment was conducted in biological duplicates and images are representative of five fields viewed per replicate. b, The EDA-DA signal retained on the isolated PG can be released by PG-digesting enzymes (~ 10 mg/mL lysozyme + 200 µg/mL mutanolysin). The kinetics of signal disappearance from the lysozyme treated sacculi is much faster than the kinetics of the photo-bleaching during the time-course, indicating that the loss of signal is due to hydrolytic activity of lysozyme. Three experimental replicates were performed.

Extended Data Figure 5. Maximum intensity projections of confocal zstacks before and after deconvolution

Raw data used for generating Figure 2, showing merged (a–d) and green (e–h) channels are compared with the same maximum intensity projections from zstacks that have undergone deconvolution, (i–l) and (m–p), respectively.

Extended Data Figure 6. Fluorescence is specific to chlamydial infected cells in the presence of the dipeptide probe EDA-DA, and lysozyme treatment is capable of removing the label from fixed bacteria

Phase contrast and epifluorescence microscopy was conducted on (a) uninfected L2 cells grown in the presence of 1 mM EDA-DA, (b) 18 hour C. trachomatis-infected cells in the presence of 1 mM EDA-DA, and (c) 18 hour C. trachomatis-infected cells grown in the absence of probe. Subsequent binding of the probe to a modified Alexa Fluor 488 (green) was achieved via a click chemistry reaction. For lysozyme treatments, 18 hour C. trachomatis-infected cells (fixed and labeled as described above) were suspended in either (d) buffer (25 mM NaPO₄ pH 6.0, 0.5 mM MgCl₂) or (e) buffer and lysozyme (200 µg/mL) for two hours. Cells were subsequently washed, blocked, and counter labeled with anti-MOMP, as described previously. Images are representative of between 3–5 fields examined (with ~1–10 inclusions viewed per field) per technical replicate, each condition conducted in technical duplicates, and experiments represent a total of three biological replicates.

Extended Data Figure 7. D-cycloserine (DCS) and ampicillin (AMP) influence labeling of C. trachomatis PG by dipeptide probes EDA-DA and DA-EDA

Phase contrast and epifluorescence microscopy of L2 cells infected with C. trachomatis 18 hours post infection. Cells were grown in the presence of either EDA-DA or DA-EDA (1 mM) and were either untreated (a), or treated with 294 µM DCS (b) or 2.8 µM AMP (c). Subsequent binding of the probe to a modified Alexa Fluor 488 (green) was achieved via click chemistry. The image used for EDA-DA labeling in the absence of antibiotics is the same image from Extended Data Figure 6b and experiments were all conducted in parallel on the same day. Images showing labeling by EDA-DA and DA-EDA in the presence or absence of DCS are representative of the vast majority of over 100 inclusions measured 18 hours post-infection. Labeling by EDA-DA in the presence of ampicillin is representative of 97% (73/75) total aberrant bodies while labeling by DA-EDA in the presence of ampicillin is representative of 95% (73/77) total aberrant bodies, as viewed by epifluorescence microscopy. Experiments were conducted in technical duplicates and represent at least three biological replicates.

Extended Data Figure 8. Punctate labeling of aberrant bodies due to enlarged bacteria encompassing multiple focal planes

Phase contrast (a) and epifluorescence microscopy (b–i) of an 18 hour, EDA-DA labeled, ampicillin-induced aberrant body. Images were taken through sequential focal planes in order to show how the ring-like, PG structure is maintained in aberrant bodies and can appear punctate when viewed via an epifluorescence microscope. Images are representative of between 3–5 fields viewed per technical replicate, comprising over 20 independent biological replicates, and each experiment was conducted in technical duplicates.

Extended Data Figure 9. EDA-DA labeling of C. trachomatis is apparent as early as eight hours post infection

L2 cells infected with C. trachomatis (a) 6, (b) 8, and (c) 10 hours post infection grown in the presence of 1 mM EDA-DA. Time points examined covered 4, 6, 8, 10, 12, 18, 24, and 40 hour infected cells. Subsequent binding of the probe to a modified Alexa Fluor 488 (green) was achieved via a click chemistry reaction. Antibody to chlamydial MOMP (red) was used to label chlamydial EBs and RBs. Experiments were conducted in technical duplicates, and the time course was conducted three independent times, with between 3–5 fields viewed per time point per technical replicate.