
==== Front
J Am Chem Soc
J Am Chem Soc
ja
jacsat
Journal of the American Chemical Society
0002-7863
1520-5126
American Chemical Society

39197836
10.1021/jacs.4c05138
Article
The Dual Mode of Antibacterial Action of the Synthetic Small Molecule DCAP Involves Lipid II Binding
https://orcid.org/0000-0003-3534-0729
Ludwig Kevin C. †‡○
https://orcid.org/0000-0002-8130-7375
Puls Jan-Samuel †○
https://orcid.org/0000-0002-0098-2783
Matos de Opitz Cruz L. §
Innocenti Paolo ∥
Daniel Jan-Martin †‡
Bornikoel Jan §
https://orcid.org/0000-0003-0344-6107
Arts Melina †
Krannich Sebastian †
Straetener Jan §
Brajtenbach Dominik ⊥
Henrichfreise Beate †
https://orcid.org/0000-0002-4477-2382
Sass Peter §#
Mueller Anna †
https://orcid.org/0000-0001-8246-3006
Martin Nathaniel I. ∥
https://orcid.org/0000-0001-9364-1832
Brötz-Oesterhelt Heike §#∇
Kubitscheck Ulrich ⊥
Grein Fabian *†‡
https://orcid.org/0000-0001-7269-4716
Schneider Tanja *†‡
† Institute for Pharmaceutical Microbiology, University of Bonn, University Hospital Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
‡ German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, 53115 Bonn, Germany
§ Department of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology & Infection Medicine, University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
∥ Biological Chemistry Group, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, the Netherlands
⊥ Clausius-Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstraße 12, 53115 Bonn, Germany
# German Center for Infection Research (DZIF), Partner Site Tübingen, 72076 Tübingen, Germany
∇ Cluster of Excellence “Controlling Microbes to Fight Infections”, University of Tübingen, 72076 Tübingen, Germany
* Email: grein@uni-bonn.de.
* Email: tschneider@uni-bonn.de.
28 08 2024
11 09 2024
146 36 2485524862
16 04 2024
06 08 2024
05 08 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322–11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466–471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

Deutsches Zentrum fÃ¼r Infektionsforschung 10.13039/100009139 NA Deutsche Forschungsgemeinschaft 10.13039/501100001659 TRR261 398967434 Deutsche Forschungsgemeinschaft 10.13039/501100001659 EXC2124 390838134 Deutsche Forschungsgemeinschaft 10.13039/501100001659 53223775 document-id-old-9ja4c05138
document-id-new-14ja4c05138
ccc-price
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pmcIntroduction

The occurrence of multidrug resistance among most bacterial pathogens is increasing rapidly, endangering the efficacy and lifespan of currently used antibiotics.1,2 These days, 1.27 million people die per year due to infections attributable to antibiotic-resistant bacteria and it was estimated that, if the trend continues, this number will dramatically increase in the future.3,4 Thus, there is an urgent need to develop new therapeutic drugs with new modes of action that circumvent existing resistance mechanisms and limit resistance development.

A previous in vitro high-throughput screening for novel cell division inhibitors identified the synthetic small molecule 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP).5 The compound was active against Gram-positive and Gram-negative bacteria and was found to efficiently eradicate bacterial biofilms.5 Furthermore, DCAP reduced uropathogenic Escherichia coli infections in vitro by modulating autophagy.6 The results of the original study pointed toward a multifaceted membrane-targeting mechanism of action, inducing the collapse of transmembrane potential, an increase of membrane permeability, and mislocalization of essential membrane-associated proteins.5 Therefore, it was hypothesized, that DCAP may combine membrane effects with specific target binding.5 It has been speculated that the compound interacts with lipids or proteins in the bacterial membrane, however, no molecular target had been identified so far. Therefore, we set out to characterize the cellular consequences of DCAP treatment that lead to cell death and to ultimately identify a molecular target.

Results and Discussion

Previous studies had already tested a variety of important bacterial pathogens and model organisms for their susceptibility to DCAP.5,7 We expanded this list and observed moderate antibacterial activity against a variety of Gram-positive pathogens including multidrug-resistant Staphylococcus aureus (MRSA), methicillin-resistant S. epidermidis (MRSE), and vancomycin-resistant enterococci (VRE). A slightly lower activity was observed against mycobacteria and Gram-negative species (Table S1). We next assessed the killing kinetics of DCAP and found it to be rapidly bactericidal for S. aureus (Figure 1a). In line with the previously described bacteriolytic effect on Caulobacter crescentus,5 we found that DCAP is also bacteriolytic against S. aureus. DCAP caused lysis already at low concentrations and lysis was largely mediated by the major S. aureus autolysin AtlA (Figure 1b). These observations supported the idea of a more complex mode of action that goes beyond membrane targeting and dissipation of the transmembrane potential.5

Figure 1 DCAP rapidly kills S. aureus. (a) Killing kinetics of early exponential phase-grown S. aureus in the presence of DCAP with a detection limit of 100 colony forming units (CFU) mL–1. (b) DCAP treatment resulted in AtlA-mediated lysis as demonstrated with an ΔatlA mutant of S. aureus exhibiting markedly reduced lysis. Survival of exponential phase-grown S. aureus cells after 24 h of DCAP-treatment. Exemplary images of cultures are shown below the respective columns. Data of both graphs are mean values from 3 independent experiments. Error bars represent the standard deviation (SD).

To gain deeper insight into the membrane effects induced by DCAP, we used a live-cell microscopy-based method that allows the simultaneous spatiotemporal visualization of membrane effects and analysis of impacts on the integrity of FtsZ-rings as a measure of the transmembrane potential in Bacillus subtilis.8,9 FtsZ is the key scaffolding protein essential for divisome formation and organization in bacteria. FtsZ-rings are formed by condensation of treadmilling FtsZ-filaments in a GTP-dependent process.10−12 Loss of transmembrane potential leads to FtsZ-ring disassembly due to loss of GTPase activity.11,13 As expected, the membrane was uniformly stained by the membrane dye FM4–64 in the course of the experiment and FtsZ was found to form FtsZ-rings at the division sites in untreated cells (Figure S1a and Video S1). In contrast, DCAP treatment led to a specific accumulation of the dye at the septum, that increased over time and subsequent disassembly of the FtsZ-ring (Figure 2 and Video S2). Of note, such an effect was not observed for cells treated with the transmembrane potential dissipating agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP), where the disassembly of FtsZ-rings was not accompanied by an accumulation of membrane dye at the septum (Figure S1b and Video S3). We therefore hypothesized that DCAP may preferably bind to the septum area in a target-mediated manner.

Figure 2 DCAP leads to localized membrane alteration at the septum and subsequent delocalization of FtsZ. (a) Time-lapse fluorescence microscopy of exponentially growing B. subtilis 2020 cells treated with DCAP at 0.5× MIC. Images show channel overlays of phase contrast with either FM4–64-labeled cell membrane (upper panel, FM4–64/membrane in red) or GFP-tagged FtsZ (lower panel, in yellow). Micrographs show that DCAP treatment induces localized perturbation of the cell membrane at the septum area, which precedes the delocalization of FtsZ from midcell. Scale bars = 5 μm. Images are representative of at least three biological replicate cultures of B. subtilis 2020 with >700 septa/FtsZ-rings monitored over time. Time-lapse video is provided by Supplementary movie 2. (b) Time-resolved fold change of the septal FM4–64 fluorescence signal during DCAP treatment. In contrast to the untreated or CCCP-treated controls, DCAP leads to a significant increase in fluorescence intensity of the lipophilic dye FM 4–64 at the septum, which is indicative for membrane perturbation in this area. Data are represented as mean values of three independent experiments analyzing >700 septa over time. Error bars represent the SD. Two-way ANOVA, ****p < 0.0001. (c) Time-resolved fold change of the number of disintegrated FtsZ-rings during DCAP exposure. Data are represented as mean values of three independent experiments analyzing >700 FtsZ-rings over time. Error bars represent the SD. Two-way ANOVA, ****p < 0.0001.

We thus set out to acquire a better understanding of the distinct cellular effects of DCAP on S. aureus. DCAP-treated cells displayed aberrant septa including multiple septation, failed cell separation and a unique bent septum phenotype (Figures 3a and S2). CCCP had no significant effect on septum morphology, as virtually all cells with a distinct septal signal showed a single, centrally positioned, straight septum (Figure 3a), equally to cells in the untreated control. As septum formation is closely linked to cell division and cell wall biosynthesis, we examined the effect of DCAP on FtsZ and the essential peptidoglycan (PGN) biosynthesis protein FtsW. FtsW localization is closely associated with FtsZ, and both proteins localize to the cell division site.14 Upon DCAP treatment, we found FtsZ and FtsW to localize at misplaced or aberrant septa in almost 40% of the cells (Figure 3b,c), an effect characteristic for antibiotics that inhibit cell wall biosynthesis.15−17 CCCP in contrast did not alter septum morphology significantly, but in line with its activity on the transmembrane potential induced disintegration of the FtsZ-ring,13 which led to delocalization of FtsZ and FtsW into the cytoplasm. DCAP-treated cells showed less pronounced FtsZ/FtsW delocalization than CCCP-treated cells. These observations further corroborated the hypothesis that DCAP exhibited a complex mechanism of action, that not exclusively relied on membrane potential dissipation, but involves interference with PGN biosynthesis.

Figure 3 DCAP disrupts S. aureus cell division and cell wall biosynthesis. (a) DCAP (1× MIC) induces aberrant septum morphology, including multiple septation, impaired cell separation and a unique bent septum phenotype (black bars, white arrows). Cell morphology was visualized via vancomycin-FL cell wall staining. Scale bar = 1 μm. (b) and (c) FtsZ and FtsW localize at aberrant septa after 1 h of DCAP treatment. Higher DCAP concentrations lead to additional Z-ring disassembly with subsequent effects on FtsW localization. In contrast, CCCP does not alter septum morphology significantly, but affects Z-ring disintegration more effectively compared to DCAP. Representative images are shown for FtsZ-SNAP stained with SNAP-TMR-Star (red) and FtsW-GFP (green). Scale bar = 1 μm. (d) DCAP (2× MIC) causes diffuse distribution of HADA signal, indicating inhibition of septal transpeptidase activity, which occurs across the leading edge of the developing septum. CCCP (2× MIC) does not alter HADA fluorescence distribution. Representative images (individually adjusted) and density rendering of HADA fluorescence maxima analysis. Scale bar = 1 μm. Density scale bar = 0.1 μm for x and y, respectively. All data quantified from n ≥ 300 cells from 3 independent biological experiments (n ≥ 100 per replicate).

To further investigate a potential effect of DCAP on PGN biosynthesis, we studied the effect of DCAP on the transpeptidase activities of S. aureus and B. subtilis through the incorporation of the fluorescent amino acid HCC-amino-d-alanine (HADA) into nascent PGN.18 While CCCP and DCAP both reduced HADA fluorescence intensity (Figures S3 and S4), DCAP remarkably caused complete dislocation of the signal resulting in homogeneous fluorescence distribution across the cell (Figure 3d). CCCP instead had considerably less effect on the spatial organization of the transpeptidase activity with most of the HADA fluorescence remaining septal (Figures 3d and S4). From these experiments we concluded that DCAP may directly impact the cell wall biosynthesis machinery. Corroborating, treatment with DCAP resulted in characteristic membrane blebbing as observed for other cell wall-targeting antibiotics, whereas compounds exclusively affecting proton motive force or membrane potential, such as the protonophore CCCP and the ionophore valinomycin, did not (Figures 4a and S5). In line with the notion that DCAP impairs cell wall integrity,19 we observed the specific induction of cell wall stress in pathway-selective bioreporter strains, while other pathways (DNA, RNA and protein) remained unaffected (Figures 4a and S6).

Figure 4 DCAP targets bacterial cell wall biosynthesis. (a) Left: DCAP activated theB. subtilisypuA promotor, which signals cell wall stress, as observed by a blue halo at the edge of the inhibition zone. Right: Phase-contrast microscopy of B. subtilis revealed that DCAP causes impairment of cell wall integrity as severe cell-shape deformations and characteristic membrane blebs were observed. Scale bar = 2 μm. (b) Cytoplasmic accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid pentapeptide (UDP-MurNAc-pp, indicated by arrow) in DCAP-treated S. aureus. (c) Antagonization of DCAP-induced cell wall stress response of B. subtilis PliaI-lux by lipid II. Full antagonization of the PliaI-lux signal is achieved at a 2:1 molar ratio (lipid II:DCAP). Error bars represent the SD of three independent experiments (d) Impact of DCAP on individual cell wall biosynthesis reactions in vitro using purified substrates and S. aureus enzymes. DCAP inhibits all reactions with C55PP-containing lipid substrates or C55PP in a dose-dependent manner and almost completely inhibits enzymatic activity when added in 4-fold molar excess. Amount of reaction product formed in the absence of DCAP was taken 100%. DCAP was added at molar ratios of 0.5 to 10:1 with respect to the lipid substrates as indicated. Data represent mean values from three independent experiments and error bars represent the SD. pp, pentapeptide; tp, tetrapeptide.

Treating cells with compounds that interfere with late-stage PGN biosynthesis steps usually leads to intracellular accumulation of the last soluble PGN precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pp). Indeed, we found a strong and dose-dependent accumulation of UDP-MurNAc-pp upon DCAP treatment, comparable to that of vancomycin (Figure 4b). The glycopeptide antibiotic binds to the cell wall precursor lipid II, which inhibits the final PGN biosynthesis reactions. This also disrupts the localization of S. aureus penicillin binding protein 2 (PBP2), whose septal recruitment during cell division and septal localization is dependent on binding to its transpeptidation substrate, i.e., the availability of lipid II for PGN biosynthesis at the septum.14,17,20 Similar to vancomycin, DCAP significantly decreased the fraction of cells with PBP2 present at the septum and reduced the ratio of septal to peripheral fluorescence in those cells (Figure S7).

From these results we hypothesized that DCAP may bind to lipid II and/or interfere with biosynthesis of the peptidoglycan precursor. To test this, we monitored the induction of the B. subtilis LiaRS two-component system, which is known to respond to lipid II cycle-interfering antibiotics.21 Indeed, DCAP induced the LiaRS-mediated cell envelope stress response. Moreover, preincubation with purified lipid II was found to antagonize this response in a dose-dependent manner, indicating a direct and specific interaction of DCAP with lipid II (Figure 4c). Having identified lipid II as a molecular target of DCAP, we next determined the minimal binding motif. To this end, we tested different undecaprenyl pyrophosphate (C55PP)-containing PGN precursors for their ability to antagonize LiaRS activation. Lipid I, lipid II, the wall teichoic acid (WTA) precursor lipid IIIWTA (C55PP-GlcNAc), and the capsule biosynthesis precursor lipid IIcap (C55PP-glucose-galactose) all antagonized the DCAP-induced LiaRS response at molar ratios of 2:1 (lipid:DCAP), suggesting that DCAP is likely to complex with lipids containing a C55PP motif (Figures 4c and S8). In contrast, undecaprenyl phosphate (C55P), the soluble precursor UDP-MurNAc-pp and UDP-N-acetylglucosamine (UDP-GlcNAc) did not antagonize the LiaRS response even at elevated concentrations (10:1 molar ratio). Importantly, a lipid II variant found in vancomycin-resistant enterococci and vancomycin-resistant S. aureus carrying a modified stem peptide ending in d-Ala-d-Lac antagonized the response as efficiently as the variant ending in d-Ala-d-Ala. These findings strongly pointed toward the lipid-bound pyrophosphate moiety as the minimal molecular binding motif of DCAP, as also observed for structurally related tetrahydrocarbazoles (THCz).22 To further confirm this, we tested the impact of DCAP on a cascade of individual S. aureus cell wall biosynthesis reactions in vitro.

The initial membrane-associated step of PGN synthesis is catalyzed by the glycosyltransferase MraY that links UDP-MurNAc-pp to the lipid carrier C55P, yielding lipid I (Figure 4d).23 This reaction was not affected, in accordance with the inability of the substrate C55P to antagonize DCAP activity (Table S2). In contrast, the MurG-catalyzed synthesis of lipid II through addition of UDP-GlcNAc to lipid I was inhibited in a dose-dependent manner when DCAP was added (Figure 4d).24 Likewise, the MurT-GatD-catalyzed amidation of lipid II25 as well as the PBP2-mediated transglycosylation and PBP4-mediated carboxypeptidation of the PGN precursor were blocked.26 Furthermore, DCAP inhibited dephosphorylation of C55PP to C55P (Figure 4d), an essential recycling step conducted by C55PP phosphatases such as the tested YbjG.27 Also, synthesis of the WTA precursor lipid IVWTA catalyzed by the glycosyltransferase TarA was inhibited (Figure 4d).28 In summary, all reactions consuming a lipid substrate containing a pyrophosphate group were inhibited, strongly suggesting this moiety to be the molecular binding motif of DCAP. In line with that, only those lipids antagonized the bactericidal effect of DCAP in vivo, which contained this pyrophosphate moiety while the length of the prenyl chain appeared to be not important (Table S2).

In agreement, isothermal calorimetry (ITC) studies using large unilamellar vesicles (LUVs) comprised of DOPC doped with lipid II titrated into a DCAP solution revealed binding of DCAP to the lipid II target (Figures S9–S11). The collected data, after subtraction of the weaker, background heat generated by the association of DCAP to DOPC, revealed a dissociation constant (KD) of 1.56 μM (Figure S9) for the DCAP/lipid II interaction. This value is in the same range as observed for other small molecule lipid II binders, i.e., the synthetic BAS00127538 (1.71 μM),29 and the natural product tridecaptin A1 (4 μM).30 Additionally, we performed docking studies using AutoDock Vina 1.2.531 to visualize interaction and to support a molecular model of DCAP binding to the pyrophosphates of lipid II and C55PP (Figure S12). For the best fit model of DCAP/lipid II interaction, we calculated a Gibbs free energy (ΔG) of ΔG = −4.5 kcal/mol, which is in the same range of the binding we predicted for BAS00127538 and lipid II, that matches previous models (Table S3).29 For the best fit model of DCAP binding to the pyrophosphate moiety of C55PP, we calculated a ΔG = −3.2 kcal/mol (Figure S12, Table S3). Interestingly, our model indicates hydrogen-bond interaction of the DCAP central amino-linker with the pyrophosphates of lipid II and C55PP, respectively. In contrast, no consistent binding model was calculated for C55P and DCAP after three replica runs, further supporting binding to the pyrophosphate moiety.

Combining in vivo and in vitro results we concluded that DCAP interacts with all C55PP-containing lipids of PGN, capsule, and WTA synthesis, including lipid II. This implies a complex dual mode of action (Figure S13) that exceeds the previously described activity of DCAP. In addition to membrane impairment, DCAP disrupts the organization of cell division and of the cell wall biosynthesis machinery via cell wall synthesis inhibition. It may be hypothesized that binding to lipid precursors could promote the observed membrane effects and thus link both activities. The complexity and variety of precursors involved reduces proneness to resistance development. Accordingly, we were unable to isolate resistant mutants of S. aureus, when serially passaged in the presence of sub-MIC levels of DCAP over a period of 25 days (Figure S14).

Finally, we expanded previous toxicity studies which demonstrated that DCAP is moderately toxic to rabbit red blood cells at 50 μM (20.7 μg mL–1).5 We assessed hemolysis in human red blood cells and found DCAP but not CCCP to be inert up to 128 μg mL–1 (Figure S15a). Time- and dose-dependent cytotoxic effects were observed in both human epithelial (HEp-2) and murine McCoy B cells upon treatment with DCAP (Figure S15b–e). Importantly, toxicity did not arise from major membrane lesions as no effect of DCAP on the membrane integrity of eukaryotic cells was found even at 4× IC50 (Figure S16a). The fact that membrane integrity of B. subtilis cells was severely impaired at the 4× MIC (Figure S16b) again substantiates the relevance of the specific interaction with a molecular target that is present in bacterial cells but absent in human plasma membranes. However, eukaryotic polyprenyl-pyrophosphate analogs, such as nona (C45PP)- or decaprenyl-pyrophosphate (C50PP) and dolichyl-pyrophosphate (C95PP) are present in mitochondrial membranes and involved in protein N-glycosylation in the endoplasmic reticulum (ER), respectively.32−34 In line, we found DCAP to depolarize mitochondria within HEp-2 cells at 4× IC50 (Figure S16c) which is well in accordance with previous observations reporting depolarization of mitochondria within human epithelial kidney (HEK) cells.5 Corroborating and comparable to observations in HEK cells5 the viability of HEp-2 cells was almost unaffected at short treatment time (Figure S16a), while longer incubations decreased the cell viability (Figure S15b–d).

Conclusions

The results obtained in this study illustrate that the mode of action of small synthetic molecules like DCAP can be multifaceted and complex, despite the straightforward first impression of their antibiotic activity and their small size. Furthermore, the synthetic character of these molecules facilitates compound optimization and allows for studying the individual structural features required for the distinct aspects of their molecular action. As such, Hurley et al. improved the antibacterial activity of the DCAP scaffold by increasing the hydrophobicity of the tail-region and could prove essentiality of the carbazole moiety for activity.7 Based on our results and the predicted model, this moiety in concert with the central amino-linker is responsible for interaction of DCAP with the lipid pyrophosphate. Structurally related THCz22 share similar features of spatially close amino groups, while lacking the distinctive chlorine substitutions of DCAP, indicating an important role of the amines and a possible explanation for the difference in binding between one and two phosphates (meaning more negative charges to interact with the positively polarized amino groups). Further improvement of this molecular interaction combined with increased hydrophobicity as shown by Hurley et al. could maximize the bactericidal action of the DCAP scaffold. In order to improve the therapeutic index for bacterial cells, conjugating DCAP to other antibiotic compounds (e.g., glycopeptides) may increase specificity for target bacterial cells and may further limit entry into eukaryotic cells and interaction with the intracellular target analogs, thus reducing cytotoxicity. The small molecule inhibitor DCAP joins the class of lipid II-binding cell wall inhibitors and represents a promising scaffold for antibiotic development.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c05138.Experimental details, materials, and methods; including fluorescence microscopy, isothermal calorimetry analysis and binding thermograms, bioreporter strain analysis, toxicity and resistance studies, molecular docking, schematic model for the mechanism of action; and including minimal inhibitory concentrations, antagonization of antimicrobial activity, and docking energy calculations (PDF)

Supplementary Material

ja4c05138_si_001.mp4

ja4c05138_si_002.mp4

ja4c05138_si_003.mp4

ja4c05138_si_004.pdf

Author Contributions

○ K.C.L. and J.-S.P. contributed equally.

Funding was provided by the German Research Foundation (DFG), TRR261, project ID 398967434, EXC2124, project ID 390838134 and project ID 53223775, and the German Center for Infection Research (DZIF).

The authors declare no competing financial interest.

Acknowledgments

We thank Vanessa Becker for preparation and purification of streptococcal lipid IIcap and Thorsten Mascher for providing strain TMB1617 B. subtilis W168 sacA::pCHlux101 (PliaI-lux).

Abbreviations

CCCP carbonyl cyanide m-chlorophenyl hydrazone

CFU colony forming unit

PBP penicillin-binding protein

PGN peptidoglycan

THCz tetrahydrocarbazoles

WTA wall teichoic acid
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