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

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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 pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis2,3,4,5,6,7 and exhibit susceptibility to ‘anti-PG’ antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). 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.

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Figure 1: Novel dipeptide PG labelling strategy.
Figure 2: Fluorescent labelling of intracellular C. trachomatis PG.
Figure 3: Labelling in the presence of PG synthesis inhibitors.

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Acknowledgements

This work was supported by NIH grants to A.T.M. (AI044033) and Y.V.B. (GM51986). We would like to thank D. McDaniel and M. Murgai for their help with image acquisition and presentation, P. Foster and D. Kearns for help in early stages of the project, R. Calvo for help with strain construction, and M. Winkler for providing strains and advice.

Author information

Affiliations

Authors

Contributions

G.W.L. conducted all labelling, imaging, intracellular growth-rescue, and probe characterization studies in C. trachomatis. E.K. developed the dipeptide labelling strategy, conducted all labelling, imaging and probe characterization studies for E. coli, B. subtilis, S. pneumoniae and S. venezuelae, purification and imaging of labelled bacterial PG, and E. coli and B. subtilis growth rescue experiments. E.H. and A.K. were involved in the synthesis of probes. M.V.N., Y.V.B. and A.T.M. were all involved in study design. G.W.L. and E.K. designed the study, analysed the data and wrote the manuscript. G.W.L. and E.K. contributed equally to this work. All authors discussed the results and commented on the manuscript. The opinions or assertions contained herein are ours and are not to be construed as official or as reflecting the views of the Department of Defense or the Uniformed Services University.

Corresponding authors

Correspondence to M. VanNieuwenhze or A. T. Maurelli.

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The authors declare no competing financial interests.

Extended data figures and tables

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

ac, Phase contrast and epifluorescence microscopy of Chlamydia-infected L2 cells 18 h post infection (a), Shigella flexneri strain 2457T two hour broth cultures (b) and Shigella-infected L2 cells three hours post infection (c). 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 bacterial growth in rich media, but differentially and specifically label PG of E. coli.

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 min. These samples together with unlabelled controls were ‘clicked’ to Alexa Fluor 488 azide and imaged. When the alkyne is on the C terminus (DA-EDA), the labelling is not apparent. Signal from N-terminally tagged dipeptide (EDA-DA) is significantly higher, but still lower than EDA and the patterns of labelling at the earlier time points are different. This is probably due to periplasmic incorporation of d-amino acids (for example, EDA) by E. coli l,d-transpeptidases, which result in more efficient peripheral labelling in addition to labelling due to lipid II-dependent PG synthesis. Therefore, in bacteria that have active l,d-transpeptidases, the cytoplasmic PG labelling 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 labelling 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 labelling 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.

ae, Phase contrast and epifluorescence microscopy of B. subtilis (ac), Streptococcus pneumoniae (d), and Streptomyces venezuelae (e). a, Five minute and 60 min 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 unlabelled controls were ‘clicked’ to Alexa Fluor 488 azide and imaged. When the alkyne is on the N terminus (EDA-DA), labelling is comparable to EDA. On the other hand, the labelling with carboxy-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 labelling is d-enantiomer specific. The partial lysis of the cells visible in phase contrast is caused by 70% ethanol fixation. c, d, When live B. subtilis and S. pneumoniae labelled 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 labelled cells. c, Interestingly, the signal from DA-ADA can be elevated to the ADA-DA level, if the labelling is performed in a ΔdacA, d,d-carboxypeptidase-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)19 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 ad were taken and processed in the same manner for comparison. In ‘Adjusted’ images, signal intensities were lowered for comparison of labelling 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 labelling 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 labelled 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−1 lysozyme + 200 µg ml−1 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 Fluorescent labelling of intracellular C. trachomatis PG: maximum intensity projections of confocal z-stacks before and after deconvolution.

ap, Raw data used for generating Fig. 2, showing merged (ad) and green (eh) channels compared with the same maximum intensity projections from z-stacks that have undergone deconvolution, (il) and (mp), 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.

ac, Phase contrast and epifluorescence microscopy was conducted on uninfected L2 cells grown in the presence of 1 mM EDA-DA (a), 18 h C. trachomatis-infected cells in the presence of 1 mM EDA-DA (b), and 18 h C. trachomatis-infected cells grown in the absence of probe (c). Subsequent binding of the probe to a modified Alexa Fluor 488 (green) was achieved via a click chemistry reaction. d, e, For lysozyme treatments, 18 h C. trachomatis-infected cells (fixed and labelled as described above) were suspended in either buffer (25 mM NaPO4 pH 6.0, 0.5 mM MgCl2) (d) or buffer and lysozyme (200 µg ml−1) (e) for two hours. Cells were subsequently washed, blocked and counter-labelled 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 labelling 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 h post infection. ac, 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 labelling in the absence of antibiotics is the same image from Extended Data Fig. 6b and experiments were all conducted in parallel on the same day. Images showing labelling 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 h post-infection. Labelling by EDA-DA in the presence of ampicillin is representative of 97% (73/75) total aberrant bodies and labelling 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 labelling of aberrant bodies due to enlarged bacteria encompassing multiple focal planes.

ai, Phase contrast (a) and epifluorescence microscopy (bi) of an 18 h, EDA-DA labelled, 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 labelling of C. trachomatis is apparent as early as eight hours post infection.

ac, L2 cells infected with C. trachomatis 6 (a), 8 (b) and 10 h (c) post infection grown in the presence of 1 mM EDA-DA. Time points examined covered 4, 6, 8, 10, 12, 18, 24 and 40 h 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.

Extended Data Table 1 Exogenous dipeptide analogues rescue growth of cells that have been depleted of DA-DA

Supplementary information

Supplementary Information

This file contains the Supplementary Methods. (PDF 403 kb)

3D projections of fluorescent labeling of intracellular C.trachomatis PG

Three dimensional renderings were generated from confocal zstacks (seen in Figure 2) of chlamydial inclusions 18 hours post infection. Video S1 displays both the EDA-DA labeled chlamydial PG (green) and the labeled chlamydial major outer membrane protein (red), while in video S2 only the green channel is shown to emphasize the ring-like chlamydial PG structure. DAPI stain is indicated in blue. Renderings are rotated 180 degrees about the y axis. (MOV 2454 kb)

3D projections of fluorescent labeling of intracellular C.trachomatis PG

Three dimensional renderings were generated from confocal zstacks (seen in Figure 2) of chlamydial inclusions 18 hours post infection. Video S1 displays both the EDA-DA labeled chlamydial PG (green) and the labeled chlamydial major outer membrane protein (red), while in video S2 only the green channel is shown to emphasize the ring-like chlamydial PG structure. DAPI stain is indicated in blue. Renderings are rotated 180 degrees about the y axis. (MOV 2362 kb)

3D projections of fluorescent PG labeling in D-cycloserine and ampicillin treated, intracellular C.trachomatis

Three dimensional renderings were generated from confocal zstacks (seen in Figure 3a and 3b) of chlamydial inclusions 18 hours post infection. Video S3 displays zstack of Figure 3a and Video S4 displays the zstack of Figure 3b. EDA-DA labeled chlamydial PG is shown in green and the labeled chlamydial major outer membrane protein in red, with DAPI stain indicated in blue. Renderings are rotated 180 degrees about the y axis. (MOV 2112 kb)

3D projections of fluorescent PG labeling in D-cycloserine and ampicillin treated, intracellular C.trachomatis

Three dimensional renderings were generated from confocal zstacks (seen in Figure 3a and 3b) of chlamydial inclusions 18 hours post infection. Video S3 displays zstack of Figure 3a and Video S4 displays the zstack of Figure 3b. EDA-DA labeled chlamydial PG is shown in green and the labeled chlamydial major outer membrane protein in red, with DAPI stain indicated in blue. Renderings are rotated 180 degrees about the y axis. (MOV 2170 kb)

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Liechti, G., Kuru, E., Hall, E. et al. A new metabolic cell-wall labelling method reveals peptidoglycan in Chlamydia trachomatis . Nature 506, 507–510 (2014). https://doi.org/10.1038/nature12892

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