L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.


Figure S11. Top:
MIC values for the antibiotics tested. Bottom: Flow cytometry analysis of E. faecium (M9) treated overnight with 100 µM TetraFl (blue bars) or PentaFl (orange bars) and increasing concentrations of meropenem, imipenem, ceftriaxone, or aztreonam. Data are represented as mean + SD (n = 3). S14 Figure S12. Above: chemical structure of TetraRh-3. Below: confocal microscopy image of E. faecium (WT) treated with 5-minute pulse of 500 µM TetraRh-3 (scale bar: 3 µm). S15 Figure S13. Structure of single D-amino acid derivative Diethyl-Amino-coumarin-D-Alanine (DADA). Figure S14. Above: chemical structure of PG monomers with DADA either on the 4 th or 5 th position within the stem peptide. Bottom: Compositional percentage of incorporation of DADA in E. faecium cells that were treated with 2 mM of DADA for 16 h. The sacculi was isolated and the composition of the monomeric strands was analyzed via LC -MS. The area of each individual peak was used to calculate the percentage of 4 th vs. 5 th position (100 % is total area of the peaks corresponding to the structures shown above). S17 Figure S15. Staining of M. smegmatis mc 2 155 for 3 h with 50 µM PentaRh or PentaRh-6. (Scale bar: 5 µm). Figure S16. Amidation of the stem peptide probes at D-glutamate is required for probe incorporation in M. tuberculosis. M. tuberculosis cells were labelled with 50 M of amidated probes (TetraFl and PentaFl) and non-amidated probe (TetraFl-4) for 30 min, 3 h and 24 h. Data are represented as a mean of the count of cells (n=300) of 3 independent replicas of the experiments. The student t test was used to detect statistical significance. * represents p values of < 0.0001. S19 Figure S17. Confocal microscopy image of M. tuberculosis H37Rv treated for 24 h with 50 µM TetraRh with 100 ug/mL meropenem. (Scale bar: 5 µm). Figure S18. Flow cytometry analysis of M. tuberculosis H37Rv treated overnight with 50 μM of tetrapeptide or pentapeptide with variations. The Rhodamine signal is indicated on the X-axis as detected by the PE-A channel (excitation/ emission maxima ~546/579). S21 Figure S19. Staining of M. tuberculosis for 24 h with 50 µM PentaRh or PentaRh-6. (Scale bar: 5 µm).

S22
Materials. All peptide related reagents (resin, coupling reagent, deprotection reagent, amino acids, and cleavage reagents) were purchased from ChemImpex. All other reagents were purchased from Sigma and were used without purification. Bacterial strains E. faecium D344S and M9 were grown in BHI for all experiments. D344S and M9 strains were kind gift from Dr. Arthur (Centre National de la Recherche Scientifique, Institut Pasteur). While D344S was characterized as a drug-sensitive strain, M9 was found to be highly drug-resistant. The main mode of drug-resistance related to M9 involves a D,D-carboxypeptidase step in the intracellular space that leads to both cross-resistance to glycopeptide and β-lactam antibiotics. E. faecium ATCC BAA-2317 (VanA) and E. faecium ATCC BAA-2127 (drug sensitive) were grown in Trypticase soy broth (TSB). All Mycobacterium smegmatis strains were grown in lysogeny broth (LB) containing 0.05% Tween-80 unless noted otherwise.
Confocal microscopy analysis of E. faecium labeled with TetraRh, PentaFl, and DADA. Brain heart infusion (BHI) broth containing 500 µM TetraRh, 500 µM PentaFl, and 5 mM DADA was prepared. E. faecium (D344S) from an overnight growth was added to the medium (1:10 dilution) and incubated at 37 o C with shaking at 250 rpm for 5 minutes. The bacteria were immediately harvested at 6,000g and washed three times with original culture volume of 1x PBS followed by fixation with 2% formaldehyde in 1x PBS for 30 min at ambient temperature. The cells were washed once more to remove formaldehyde and then analyzed using a Nikon C2 confocal microscope. For Mycobacterium smegmatis ATCC 14468 (WT), the previous procedure was repeated except using LB (0.05% tween) as the growth media and a 30 minute incubation.
In vivo labeling of E. faecium with TetraRh in live C. elegans. N2 Caenorhabditis elegans were maintained by standard protocol using nematode growth agar with bacterial lawns of E. coli OP50 (source) on a 60mm x 15mm cell culture dish. C. elegans were grown to contain primarily L4 larval stage nematodes by incubation at ambient temperature for ~48-52 h, washed off the plates with M9 buffer, and washed three times with M9 buffer. For washing steps, the C. elegans were pelleted at 1000g. The C. elegans were resuspended in 400 µL of M9 buffer containing 10% BHI broth and transferred to a sterile 24 multiwell plate. E. faecium (100 µL from overnight growth) was washed and added to the 400 µL suspension of C. elegans. The C. elegans were incubated at ambient temperature for 4 h, harvested at 1000g and washed three times with M9 buffer to remove bacteria on the outside of the C. elegans. The C. elegans were then resuspended in 500 µL of M9 buffer containing 10% BHI broth and 50 µM TetraRh, and incubated at ambient temperature for 2 h. The C. elegans were harvested at 1000g and washed three times with M9 buffer and resuspended in 10mM sodium azide in M9 buffer and analyzed by confocal microscopy.

Microscopy and Flow cytometry analysis of M. smegmatis mc 2 155 and M. tuberculosis
H37Rv labeled with TetraRh, PentaRh and PentaRh6 probes. Five militres cultures of M. smegmatis mc 2 155 and M. tuberculosis H37Rv were grown at 37 °C to an OD600nm of 0.8 and 1, respectively, in Middlebrook 7H9 broth. The 5 ml cultures of M. smegmatis and M. tuberculosis H37Rv were pelleted by centrifugation at 4000 xg for 5 min. The supernatant was discarded and the cells were resuspended in 2.5 ml of Middlebrook 7H9 broth. Four microliters of either the S23 TetraRh, PentaRh and PentaRh6 (5 mM) PG probe was added to 396 µl of M. smegmatis mc 2 155 and M. tuberculosis H37Rv cells (making a final concentration of 50 µM TetraRh, PentaRh or PentaRh6 probe). As controls, Meropenem-Clavulanate (100 µg/ml) treated -and heat killed -M. smegmatis mc 2 155 and M. tuberculosis H37Rv were also used for the labeling experiment. Heat killing was performed on a heating block set at 65 ºC and inserted into a BioSafety Cabinet for 24 h prior to addition of the PG probes. The M. smegmatis and M. tuberculosis labelling experiment was performed for 24 h, however, sampling for analysis of probe incorporation was done after 30 min, 3 h, 6 h, 9 h and 24 h of incubation at 37°C. Thereafter, the cells were washed in 1x PBS (500 µl) three times to remove unincorporated probe and the cells were then resuspended in 100 µl of PBS followed by fixation with 2.5% glutaraldehyde for 24 h. The cells were washed three times with 1x PBS (500 µl) to remove the glutaraldehyde and then resuspended in 500 µl 1x PBS. For microscopy, 5 µl of the cells was spotted on glass slides and viewed with the Zeiss Observer Z1 inverted fluorescence microscope under the DS-red channel (excitation/ emission maxima ~546/579) and the images were analyzed with the ZEN lite software (Zeiss). For flow cytometry analysis of incorporation of the different probes, 100 µl of the cells was transferred to a 96 well plate and the Cytoflex flow cytometer (Beckman Coulter) was us ed for analysis of probe incorporation. The PE-A channel (excitation/ emission maxima ~470/585) was used for detection of the Rhodamine signal (excitation/ emission maxima ~561/578). The gain for the PE-A channel was reduced from 370 to 120.
Peptidoglycan Isolation. BHI medium (200 mL) containing 500 µM TetraFl was prepared. E. faecium D344S bacteria were added to the BHI medium (1:100) and allowed to grow overnight at 37 o C with shaking at 250 rpm. The cells were harvested and washed with 1x phosphate buffer saline (PBS) (3 × 50 mL each). The cells were then resuspended in 1x PBS and boiled for 7 min and then centrifuged at 14,000g for 8 min at 4 °C. Cells were then placed in 25 mL of 5% (w/v) sodium dodecyl sulfate (SDS) and boiled for 25 min followed by centrifugation at 14,000g for 8 min at 4 °C. Following centrifugation, cells were boiled again in 25 mL of 4% (w/v) SDS for 15 min followed by centrifugation using same parameters as before. Cells were then washed 5 times with 60 °C DI water to remove all SDS. After washing, cells were resuspended in 6 mL of 20 mM Tris buffer (pH 8.0). The cells were treated with 800 g DNase for 24 hrs followed by Trypsin (800 ug) for another 24 hrs (37 O C shaking at 80 rpm). Cells were then boiled for 30 min. Lysozyme (250 ug/mL) was added and cells incubated for 24 hrs at 37 o C shaking at 250 rpm. Following lysozyme digestion, cells were frowzen and lyophilized, then dissolved in 0.375 M sodium borate buffer (pH 9.0) prepared in HPLC grade water. Sodium borohydride (10 mg) in 1 mL borate buffer was added for 30 min, then quenched with 125 uL phosphoric acid. The cell wall digest was filtered using a 0.45 um and 30K centrifuge filter respectively, freezed and lyophilized. The digest was then analyzed by LC-MS.
Mass Spectrometry Analysis of Stem Peptides incubated with E. faecium. BHI medium was prepared with 100 µM TetraFl or PentaFl. E. faecium D344S bacteria were added to the BHI medium (1:100) and allowed to grow overnight at 37 o C with shaking at 250 rpm. Samples of 100 µM TetraFl or PentaFl were also prepared in BHI medium and incubated overnight 37 o C with shaking at 250 rpm without the addition of bacteria (standards). The bacteria were harvested, supernatant collected and analyzed by Shimadzu LC 2020 with a Phenomenex Luna 5μ C18(2) 100Å (30 x 2.00 mm) column; gradient elution with H2O/CH3CN. PG Digestion/Lysozyme Treatment of Labeled Peptidoglycan. E. faecium D344S (1:100) were added to the BHI medium or BHI medium containing 100 M TetraFl and allowed to grow overnight at 37 o C with shaking. The bacteria were harvested at 6,000g and washed three times with original culture volume of 1x PBS followed by fixation with 2% formaldehyde in 1x PBS for 30 min at ambient temperature. Cells were washed once more to remove the formaldehyde. Cells were washed three times with 1x PBS. The bacteria were resuspended in 1x PBS containing 500 g/mL lysozyme (MP biomedicals) and incubated at 37°C. A portion of the cells were taken at 1, 5, 30, 60, and 120 min. At each time point, the collected bacteria were washed three times with 1x PBS and resuspended in a final solution of 1x PBS containing 4% formaldehyde to quench the lysozyme reaction. Cells were analyzed using a BDFacs Canto II flow cytometer using the previously stated parameters.
Confocal Imaging of Sacculi. Peptidoglycan Isolation. BHI medium (100 mL) containing 500 µM TetraFl-3 was prepared. E. faecium D344S bacteria were added to the BHI medium (1:100) and allowed to grow overnight at 37 o C with shaking at 250 rpm. The cells were harvested and washed with 1x phosphate buffer saline (PBS) (3 × 50 mL each). The cells were then resuspended in 1x PBS and boiled for 7 min and then centrifuged at 14,000g for 8 min at 4 °C. Cells were then placed in 25 mL of 5% (w/v) sodium dodecyl sulfate (SDS) and boiled for 25 min followed by centrifugation at 14,000g for 8 min at 4 °C. Following centrifugation, cells were boiled again in 25 mL of 4% (w/v) SDS for 15 min followed by centrifugation using same parameters as before. Cells were then washed 6 times with 60 °C DI water to remove all SDS. After washing, cells were resuspended in 6 mL of 20 mM Tris buffer (pH 8.0). Pellets were treated with 800 g DNase for 24 hrs followed by trypsin (800 g) for another 24 hrs (37 °C shaking at 80 rpm). Pellets were boiled for 25 min followed by centrifugation at 14,000g for 8 min at 4 °C, resuspended in 5 mL of 1X PBP, and imaged on a glass slide.
MIC Determination. MIC reported for the eight antibiotics were carried out using the microdilution method according to the Clinical and Laboratory Standards Institute recommendations with the change in medium (Brain-Heart Infusion).

Scheme S6. Synthesis of PentaFl-5
To a 25 mL peptide synthesis vessel charged with Fmoc-D-Alanine Wang resin (950 mg, 0.55 mmol). The Fmoc protecting group was removed with 6 M piperazine/100 mM HOBt in DMF (15 ml) for 30 min at ambient temperature, then washed with MeOH and DCM (3 x 15 mL each). Fmoc-D-alanine (3 eq, 513 mg, 1.65 mmol), HBTU (3 eq, 625 mg, 1.65 mmol), and DIEA (6 eq, 0.574 mL, 3.30 mmol) in DMF (15 mL) was added to the reaction flask and agitated for 2 h at ambient temperature. The Fmoc deprotection and coupling procedure was repeated as before using the same equivalencies with Fmoc-L-Lys(Mtt)-OH, Fmoc-D-glutamic acid α-amide and Fmoc-L-alanine. The Fmoc group of L-alanine was deprotected and resin coupled with 5(6)carboxyfluorescein (2 eq, 413 mg, 1.1 mmol), HBTU (2 eq, 416 mg, 1.1 mmol) and DIEA (6 eq, 0.574 mL, 3.30 mmol) in DMF (15 mL) shaking overnight. The resin was washed as before and added to a solution of 1% TFA / 5% TIPS in DCM and shaken for 10 min and washed. The step was repeated five times for removal of the Mtt group. Acetic anhydride (5 eq, 0.260 mL) and DIEA (10 eq, 0.956 mL) in DMF was added and resin shaken for 30 min at ambient temperature. The resin was washed and added to a solution of TFA/DCM (2:1, 20 mL) with agitation for 2 h at ambient temperature. The resin was filtered and resulting solution concentrated in vacuo. The residue was trituated with cold diethyl ether and purified using reverse phase HPLC using H2O/MeOH to yield TetraFl-3. The sample was analyzed for purity using a Shimadzu LC 2020 with a Phenomenex Luna 5µ C18(2) 100Å (30 x 2.00 mm) column; gradient elution with H2O/CH3CN.