Chiral peptidoglycan mimics target bacterial wall biosynthesis for pathogen intervention

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
The global burden of microbial infections and antimicrobial resistance, coupled with the absence of
precise bacterial recognition modalities, demands innovative breakthroughs in antibacterial treatment.
Here, we report a chirality-specific biomimic—D-alanine-conjugated peptidoglycan mimics (D-PM)—
designed for bacterial recognition. D-PM exhibit broad-spectrum, effective recognition across ESKAPE
pathogens, antibiotic-resistant strains and clinical isolates, while displaying minimal interaction with
eukaryotic cells. We elucidate the bacterial recognition mechanism, wherein D-PM—act as biosynthetic
substrates—become incorporated into peptidoglycan biosynthesis. This reveals a mechanism by which
macromolecular mimetics are assimilated into bacterial biosynthesis, providing insights into bacterial
recognition. Beyond recognition, D-PM enable the construction of pathogen-specific imaging agents and
antibiotic-targeted delivery systems. In localized and systemic infection models, D-PM achieve efficient
pathogen localization, tissue penetration, and enhanced therapeutic outcomes. This work presents a
molecularly engineered strategy for bacterial recognition and intervention, offering a translational
approach to address the escalating threat of infectious diseases.

Introduction
Microbial infections continue to pose a formidable threat to human health, accounting for more than 7
million deaths annually1,2. This escalating crisis highlights an urgent need for effective approaches that
enable the precise diagnosis and targeted intervention of pathogenic infections3,4. Central to this
challenge is the accurate recognition of bacterial pathogens—an indispensable prerequisite for effective
treatment and resistance mitigation5,6. Despite significant advances in bacterial recognition,
differentiating pathogenic bacteria from host components within complex biological environments
remains profoundly difficult, posing a major obstacle to clinical antibacterial therapy7,8. As a result, the
development of robust strategies for bacterial recognition has emerged as a critical scientific imperative
in the global battle against infectious diseases.

The bacterial cell wall, built upon a peptidoglycan scaffold, constitutes one of the most fundamental
distinctions between prokaryotic pathogens and eukaryotic hosts9. Peptidoglycan—universally composed
of sugar moieties and chirality-specific amino acids—is highly conserved across bacterial taxa
(Supplementary Fig. 1)10-13, which presents unique molecular signatures for precise bacterial recognition14-
18. Although peptidoglycan-targeting antibiotics—such as vancomycin—have served as pivotal agents for
bacterial eradication and detection19-23, their efficacy is increasingly compromised by target mutations
and emerging resistance24-26. Concurrently, increasing efforts have been directed toward biosynthetic
labeling for bacterial recognition, drawing inspiration from the bacterial-specific sugars (e.g., N-
acetylglucosamine, N-acetylmuramic acid) and amino acids (e.g., D-alanine) essential for peptidoglycan
biosynthesis27-32. However, these small-molecule probes are intrinsically constrained by limited structural
tunability, suboptimal recognition efficiency, and restricted biological permeability33,34. These persistent
limitations necessitate bacterial recognition platforms that break through conventional intervention
paradigms and molecular designs.

Emerging evidence suggests that macromolecular analogues of bacterial peptidoglycan, endowed
with superior biomimetic properties, represent promising candidates for targeted bacterial intervention35.
For instance, chitin—a natural polysaccharide sharing partial structural features with peptidoglycan—
exhibits comparable recognition by host pattern recognition receptors36,37, while synthetic
glycoconjugates have been shown to elicit peptidoglycan-mimetic immune responses38,39. Inspired by
these findings, we rationally designed macromolecular mimics of peptidoglycan substrates that
recapitulate the chirality-specific amino acid motifs and glycan backbone architectures (Supplementary
Fig. 2). In this work, we delineate the design principles of peptidoglycan mimics, elucidate their structure–
function relationships and biological mechanisms of bacterial biosynthetic incorporation (Fig. 1a and
Supplementary Fig. 3), and demonstrate their capability to selectively distinguish pathogenic bacteria
from host cells. Furthermore, we engineered these peptidoglycan mimics into nanoparticulate probes and
antibiotic delivery platforms, enabling targeted intervention of bacterial infections. This multidisciplinary
study establishes a transformative solution for bacterial diagnostics and therapeutics, providing effective
strategies to tackle the global burden of resistant microbial infections.

Results
Chiral peptidoglycan mimics label bacteria
To enable the controlled synthesis of chiral peptidoglycan mimics (PM), protected chiral alanine (Ala) was
conjugated to glucosamine (GlcNH ) in chitosans (CS, (GlcN)ₙ analogous to the polysaccharide chain of
2
peptidoglycan) via amide bond formation under racemization-free conditions40,41. A series of chiral amino
acid-conjugated (GlcN) polymers was synthesized with varying degrees of L-Ala (7.5 ± 0.3%, 11.3 ± 0.4%,
n
17.7 ± 0.3%, and 23.5 ± 0.4%) or D-Ala (7.4 ± 0.6%, 11.6 ± 0.4%, 17.2 ± 0.3%, and 22.9 ± 0.8%) modification,
respectively (Supplementary Fig. 4). Following deprotection, L-PM of (GlcN–L-Ala) and D-PM of (GlcN–D-
n
Ala) were obtained with distinct Ala modification but identical fluorescein isothiocyanate (FITC) labeling.
n

The bacterial interactions of the chiral PM were quantified using Staphylococcus aureus (S. aureus
ATCC29213) as a model strain. L-PM exhibited consistently low binding efficiency with minimal
dependence on the modification ratio, yielding FITC-positive bacterial populations ranging from 2.9 ± 0.2%
to 14.1 ± 0.4% (Supplementary Fig. 5). In contrast, D-PM displayed a pronounced modification-ratio-
dependent recognition behavior, with the proportion of FITC-positive bacteria increasing from 27.3 ± 2.7%
to 94.1 ± 0.3%. Notably, maximal bacterial labeling was achieved at a D-Ala modification degree of 17.2%,
whereas further increasing the substitution to 22.9% resulted in diminished labeling efficiency, likely due
to steric congestion that impairs enzymatic incorporation during cell wall biosynthesis. On this basis, the
17.2% modification rate of D-PM was selected for all subsequent studies. 1H nuclear magnetic resonance
(NMR) spectra confirmed the successful conjugation of chiral amino acids onto (GlcN) , as evidenced by
n
the concurrent appearance of characteristic signals corresponding to sugar rings and amino acids
(Supplementary Fig. 6). Both L-PM and D-PM exhibited zeta potentials comparable to that of native CS
(approximately +23 mV) in aqueous solution (Supplementary Fig. 7a). Dynamic light scattering (DLS)
analysis indicated that CS could form aggregates around 350 nm in an aqueous solution, whereas both L-
PM and D-PM assembled into smaller nanostructures of 160 nm (Supplementary Fig. 7b-d). Transmission
electron microscopy (TEM) revealed well-defined nanostructures of both L-PM and D-PM, highlighting
their potential as functional nanomaterials. The circular dichroism spectra revealed distinct trends, with
L-PM showing a more pronounced attenuation of the negative band at 212 nm, but D-PM exhibiting a
greater increase in the positive band at 193 nm (Supplementary Fig. 8). These spectral variations are likely
attributed to the modifications induced by the chiral amino acids.

Chiral peptidoglycan mimics with abundant free amino groups facilitated effortless conjugation with
diverse fluorescent molecules, including FITC, 6-carboxytetramethylrhodamine (TAMRA), and sulfo-
cyanine5 (Cy5), facilitating sophisticated biological imaging (Supplementary Fig. 9). Moreover, both L-PM
and D-PM enhanced the stability of the fluorescent probes, with no detectable fluorescence decay over
10 days (Supplementary Fig. 10). To assess bacterial labeling performance, FITC-labeled CS, L-PM, and D-
PM were applied at identical concentrations and normalized to equivalent fluorescence intensities
(Supplementary Fig. 11). Strikingly, flow cytometric analysis revealed that D-PM displayed high-affinity
interactions across a broad panel of Gram-positive pathogenic bacteria42, including multiple
Staphylococcus aureus strains (S. aureus ATCC29213, S. aureus ATCC25904, and S. aureus RN4220), two
Enterococcus faecalis strains (E. faecalis ATCC29212 and E. faecalis ATCC19433), methicillin-resistant
Staphylococcus aureus (MRSA ATCC43300), as well as clinically isolated Staphylococcus aureus (Fig. 1b and
Supplementary Fig. 12). Meanwhile, D-PM also demonstrated significantly stronger recognition and
binding toward Gram-negative ESKAPE pathogens, including Klebsiella pneumoniae (K. pneumoniae
ATCC13883), Acinetobacter baumannii (A. baumannii ATCC19606), Pseudomonas aeruginosa (P.
aeruginosa ATCC27853), and Escherichia coli (E. coli ATCC25922), compared with CS and L-PM
(Supplementary Fig. 13). In addition, the relatively lower labeling efficiency observed for certain Gram-
negative species in the D-PM-treated group, including K. pneumoniae and E. coli, is likely attributable to
the presence of an outer membrane that partially restricts D-PM access to the peptidoglycan43. By
contrast, both CS and L-PM exhibited uniformly low binding efficacy toward both Gram-positive and
Gram-negative bacterial strains. More importantly, D-PM exhibited robust bacterial binding and
fluorescent labeling as visualized by confocal laser scanning microscopy (CLSM), clearly delineating the
coccoid morphology of S. aureus ATCC29213 (Fig. 1c and Supplementary Fig. 14). D-PM exhibited
pronounced dose-dependent binding to bacteria, indicative of its potential accumulation on the bacteria
(Supplementary Fig. 15). Taken together, D-PM exhibit chirality-dependent biological effects that enable
broad-spectrum and highly efficient recognition of pathogenic bacteria, including antibiotic-resistant
strains.

To investigate the bacterial recognition advantage conferred by chiral peptidoglycan mimic design,
we compared FITC-labeled D-PM with FITC-labeled small-molecule D-Ala probes at matched D-Ala molar
equivalents. Two control conditions were employed: (1) a D-Ala/FITC-labeled D-Ala mixture calibrated to
match the fluorescence intensity of FITC-labeled D-PM, and (2) fully FITC-labeled D-Ala with substantially
higher overall fluorescence than FITC-labeled D-PM (Supplementary Fig. 16). Under fluorescence-matched
conditions of probe, only 2.6 ± 0.2% of bacteria were FITC-positive following treatment with the D-
Ala/FITC-labeled D-Ala mixture, which was dramatically lower than the 85.7 ± 0.2% FITC-positive bacteria
observed in the FITC-labeled D-PM group. The mean fluorescence intensity (MFI) in the FITC-labeled D-
PM-treated bacteria was about 8-fold higher than that of the D-Ala/FITC-labeled D-Ala mixture. Notably,
even compared with fully FITC-labeled D-Ala with approximately 4-fold higher fluorescence than FITC-
labeled D-PM, both the proportion of FITC-positive bacteria and MFI remained higher in the FITC-labeled
D-PM-treated group. CLSM images clearly demonstrate the overwhelming performance of D-PM in
bacterial fluorescent labeling (Supplementary Fig. 17). These results underscore the superior bacterial
recognition efficiency afforded by D-chiral peptidoglycan mimics, highlighting the intrinsic advantages of
chirality-guided macromolecular design over small-molecule D-Ala–based probes for pathogenic bacteria.

D-PM incorporate biosynthetic downstream
To elucidate the recognition mechanisms, microscale thermophoresis (MST) was employed to generate
binding curves that characterized the interactions between chiral peptidoglycan mimics and S. aureus44.
The results revealed a significant interaction between D-PM and bacteria, with a dissociation constant (K )
d
of 5.4 ± 0.3 μM—an order of magnitude lower than that of both CS and L-PM (Fig. 2a). This finding
indicated that the D-chiral strategy enhanced the affinity between D-PM and pathogenic bacteria (S.
aureus ATCC29213). In parallel, flow cytometry analysis demonstrated that FITC-labeled D-PM rapidly
bound to bacteria over time, achieving near-complete binding efficiency (Fig. 2b and Supplementary Fig.
18). The maximal binding rates of fluorescent CS and L-PM with bacteria remained below 20%.
These striking differences suggested that D-PM may engage distinct mechanisms in binding to bacteria,
potentially involving biosynthetic processes.

Uridine 5’-diphosphate-N-acetylmuramic acid pentapeptide (UDP-MurNAc pentapeptide) is a well-
established biomarker of cell wall synthesis intervention, whose accumulation is notably induced by
vancomycin (Van)23,45. An ultra-high-performance liquid chromatography system coupled with a high-
resolution quadrupole–orbitrap mass spectrometer (UPLC-MS) revealed the presence of a characteristic
peak for UDP-MurNAc pentapeptide at 1,150.3583 in the bacterial wall of S. aureus following exposure to
D-PM, consistent with the signal observed after vancomycin treatment (Fig. 2c and Supplementary Fig.
19). In contrast, UDP-MurNAc pentapeptide was undetectable in both the CS and L-PM groups. Moreover,
flow cytometry results indicated that pretreatment with vancomycin significantly reduced D-PM binding
to S. aureus, likely due to competitive interference in cell wall synthesis (Supplementary Fig. 20).
Inspection of UPLC-MS results revealed that D-peptidoglycan mimics interfered with the biosynthesis
of the bacterial cell wall. The observed peak at m/z 322.1613 was assigned to the protonated molecular
ion [GlcN–D-Ala–D-Ala + H]⁺ (theoretical m/z 322.1609), which likely arises from the ligation of an
additional D-Ala to the D-PM substrate (GlcN–D-Ala) catalyzed by D-Ala–D-Ala ligase (Ddl, Fig. 2d,e)27. This
intermediate, GlcN-D-Ala-D-Ala, could subsequently be incorporated into the cell wall biosynthesis
pathway to form GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc through the biocatalyst of UDP-N-
acetylmuramoyl-tripeptide–D-alanyl-D-Ala ligase (MurF), with a characteristic ion peak at m/z 925.4353
(Fig. 2f,g). To further elucidate the structural basis of D-PM interference in cell wall synthesis, we used the
catalytic domain of Ddl bound to GlcN-D-Ala and determined the structure of the complex. Docking
analysis revealed that D-PM, as a substrate bind to Ddl in a pocket highly analogous to that of D-Ala within
the overall complex structure (Fig. 2h,i and Supplementary Fig. 21a,b). Furthermore, the strong binding
affinity of D-PM to Ddl was further supported by a lower docking score of -6.775 kcal mol-1, compared to
the D-Ala/Ddl complex, which had a docking score of -4.077 kcal mol-1. Similarly, the intermediate of GlcN-
D-Ala-D-Ala exhibited a better docking score of -8.088 kcal mol-1 as compared to natural D-Ala-D-Ala
(docking score of -6.042 kcal mol-1) when bound to the catalytic pocket of MurF (Supplementary Fig. 21c,d).
Subsequently, molecular dynamics simulations revealed that the GlcN–D-Ala/Ddl and GlcN–D-Ala–D-
Ala/MurF complexes exhibited sustained structural stability, with markedly stronger interactions than
those observed between conventional small-molecule substrates (D-Ala or D-Ala–D-Ala) and their
corresponding ligases (Supplementary Figs. 22 and 23). UPLC-MS evidence and molecular simulations
jointly confirmed that chirality-specific peptidoglycan mimics could efficiently integrate into the
metabolism downstream of bacterial cell wall biosynthesis, thereby facilitating the robust recognition and
biosynthetic labeling of pathogens.

In addition, key cell wall synthesis inhibitors, including D-cycloserine (D-Ala-D-Ala ligase inhibitor),
amoxicillin and meropenem (penicillin-binding protein transpeptidase inhibitor)43, were able to block the
incorporation of D-PM into downstream metabolic pathways (Fig. 2j). In contrast, a protein synthesis
inhibitor of tetracycline and a DNA synthesis inhibitor of ciprofloxacin showed no effect on the
incorporation of D-PM into S. aureus. In addition, none of the inhibitors significantly affected the binding
of CS and L-PM to the bacteria (Supplementary Fig. 24). The chiral-specific design of D-peptidoglycan
mimics enables their integration into bacterial cell wall synthesis metabolism, thereby facilitating efficient
bacterial recognition and targeting.

Encouraged by the molecular-level confirmation of downstream metabolism, we proceeded to
investigate the impact of D-PM on the morphology of pathogenic bacteria. FITC-labeled peptidoglycan
mimics were incubated with S. aureus over three passages in Luria–Bertani broth (Fig. 3a). As shown in
Fig. 3b, only a small proportion of fluorescent L-PM bound to S. aureus ATCC29213, always with around
10% of bacteria exhibiting fluorescence. This minimal binding of L-PM is attributable to weak interactions
governed by a dynamic adsorption-desorption equilibrium, as evidenced by the reduced bacterial surface
zeta potential following supramolecular electrostatic interactions between L-PM and the bacterial
envelope (Supplementary Fig. 25). Under the same conditions, continuous co-incubation with D-PM from
Passage 1 to Passage 3 led to a progressive increase in the percentage of fluorescent bacteria, rising from
16.1% to 44.3%. This outcome was due to the incorporation of D-PM into bacterial metabolism, enabling
the accumulation over successive generations during bacterial proliferation. To visualize this generational
integration, super-resolution three-dimensional structured illumination microscopy (3D-SIM) provided
direct evidence of D-PM incorporation into bacterial cell wall synthesis (Fig. 3c,d). In Passage 1, Cy5-
labeled D-PM well-delineated the bacterial cell wall (red fluorescence). In Passage 2, TAMRA-labeled D-
PM was incorporated into the biosynthesis of bacterial envelopes (purple fluorescence), while Cy5-labeled
D-PM remained prominently visible on the bacteria. The approximately half reduction in Cy5 fluorescence
intensity was attributed to fluorescence dilution during bacterial division in Passage 2. In Passage 3, co-
incubation with FITC-labeled D-PM (green fluorescence) demonstrated clear co-localization of red, purple,
and green fluorescence along the bacterial contours, indicating stable incorporation of D-PM into the
downstream metabolism of cell wall synthesis. In contrast, L-PM showed minimal co-localization with
bacteria, consistent with flow cytometry results, and failed to accumulate within the bacterial cell wall.

Scanning electron microscopy (SEM) images of bacteria over three passages in the presence of L-PM
or D-PM revealed distinct morphological differences (Fig. 3e). In the L-PM-treated groups, bacteria
retained well-defined surfaces, appearing consistent with both the control and CS-treated groups.
However, D-PM induced notable structural abnormalities, characterized by visible protrusions on the
bacterial surface. These deformities are likely attributed to the incorporation of D-PM into cell wall
synthesis, disrupting the formation of a cohesive peptidoglycan network. Additionally, propidium iodide
(PI) probe assays confirmed the compromised bacterial envelope integrity after three passages in the
presence of D-PM (Fig. 3f). The PI influx in the D-PM-treated group was significantly higher than that
observed in the control and L-PM-treated groups, indicating abnormal envelopes. In addition, colony-
forming unit (CFU) assays demonstrated that incorporation of D-PM into bacterial cell wall biosynthesis
elicited a modest bactericidal effect at Passage 1 (Supplementary Fig. 26). With successive passages up to
Passage 3, progressive accumulation of D-PM within the peptidoglycan increasingly compromised cell wall
integrity, leading to a stepwise enhancement of bactericidal activity. These findings confirmed that D-PM
could be efficiently incorporated into bacterial cell wall biosynthesis and progressively accumulates within
the peptidoglycan.

D-PM recognize bacteria over mammalian cells
D-PM achieved efficient integration into bacterial biosynthesis, exhibiting satisfactory bacterial
recognition. By leveraging the intrinsic differences in chiral amino acid metabolism between prokaryotic
pathogens and mammalian cells, D-PM demonstrated significant potential for selectively distinguishing
these entities, paving the way for advancements in the diagnosis and treatment of bacterial infections. As
expected, CS, L-PM, and D-PM exhibited minimal interactions with animal cell lines at concentrations
corresponding to those used for bacterial incubation, including mouse NIH 3T3 cells, human umbilical vein
endothelial cells, smooth muscle cells, and monkey Vero cells (Supplementary Fig. 27). Additionally, MST
measurements revealed no detectable specific binding of CS, L-PM, or D-PM to NIH 3T3 cells
(Supplementary Fig. 28), and UPLC-MS analysis targeting characteristic peptidoglycan-derived motifs
detected no corresponding signals in NIH 3T3 cells following incubation with CS, L-PM, or D-PM
(Supplementary Fig. 29). Co-culture with prokaryotic bacteria and eukaryotic cells in the presence of
fluorescent D-PM revealed a clear preference for bacterial recognition by flow cytometry analysis (Fig. 4a).
As shown in Fig. 4b, D-PM selectively associated with S. aureus, with approximately 30% of bacteria
exhibiting FITC-positive fluorescence, whereas binding to mammalian NIH 3T3 cells was much lower.
Additionally, D-PM exhibited robust specificity for identifying S. aureus over a range of the mammalian
cell lines (Supplementary Fig. 30). CLSM images directly showed that demonstrated that D-PM (green)
exhibited strong co-localization with S. aureus but poor co-localization with NIH 3T3 cells, confirming the
remarkable selectivity of D-PM in distinguishing bacteria from mammalian cells (Fig. 4c). In addition, flow
cytometry and CLSM images revealed that CS and L-PM exhibited minimal binding to S. aureus and NIH
3T3 cells under co-culture conditions (Supplementary Figs. 31 and 32). Similarly, D-PM effectively
distinguished multiple Gram-negative bacterial species from NIH 3T3 cells under co-culture conditions
(Supplementary Fig. 33). These findings highlight the potential of D-PM as a broad-spectrum recognition
tool for pathogenic bacteria.

Based on its strong bacterial recognition and self-aggregation into nanoparticles, D-PM holds great
potential as a vehicle for antibiotic loading and targeted delivery. Pyrene probes, serving as guest
molecules, were readily encapsulated into the internal pockets of nanoparticulate CS, L-PM, and D-PM,
demonstrating their potential as antibiotic carriers (Supplementary Fig. 34). Using hydrophobic
tetracycline (Tet) as a model drug, Tet was successfully encapsulated into the pockets of D-PM to form
Tet-loaded D-PM (T/D-PM)46, which exhibited a uniform size distribution of approximately 180 nm and
well-defined nanostructures (Fig. 4d and Supplementary Fig. 35). The drug-loaded T/D-PM delivery system
exhibited a loading capacity of ~ 6.6% and an encapsulation efficiency of ~ 32.9% (Supplementary Fig. 36).
T/D-PM significantly enhanced the antibacterial efficacy of antibiotics, reducing the minimum inhibitory
concentration (MIC) of tetracycline by 4-fold against S. aureus ATCC29213 (Supplementary Table 1).
Moreover, CFU assays further validated that, under the same Tet concentration conditions, T/D-PM
completely eradicated S. aureus, while free Tet, T/CS, and T/L-PM failed to eliminate the bacteria (Fig. 4e).
CLSM images revealed that D-PM improved the internalization of tetracycline through targeted delivery,
showing significantly stronger green fluorescence signals compared to free Tet, T/CS, and T/L-PM (Fig. 4f
and Supplementary Fig. 37a). Targeted antibiotic delivery by D-PM substantially promoted antibacterial
efficacy of antibiotics, as demonstrated by the highest proportion of PI-positive dead bacteria (red, Fig. 4g
and Supplementary Fig. 37b). Notably, T/D-PM potentiated the antibacterial efficacy of tetracycline
against MRSA, halving its MIC and markedly suppressing bacterial colony formation on agar plates
(Supplementary Table 2 and Supplementary Fig. 38).

As expected, tetracycline-containing formulations exhibited no detectable cytotoxicity toward
multiple mammalian cell lines even at concentrations exceeding 40 times the effective antimicrobial dose
(Supplementary Figs. 39 and 40). Additionally, treatment of erythrocytes collected from BALB/c mice with
these formulations at the high concentration revealed no hemolysis, demonstrating excellent
hemocompatibility (Supplementary Fig. 41). The combination of high-efficiency targeted delivery and
favorable biocompatibility underscores the potential of chiral-specific D-peptidoglycan mimics as
promising carriers for biomedical applications.

D-PM intervene wound bacterial infections
The robust incorporation into bacterial metabolism, high-efficiency bacterial recognition, and potential
for bacteria diagnosis and therapy of D-PM prompted further investigation of their efficacy in vivo. FITC-
labeled D-PM effectively marked nearly all S. aureus, which were used to infect wounds (~ 80 mm²) on
the dorsal skin of BALB/c mice with a bacterial load of 1.0 × 106 CFU ml⁻¹ per mouse (Fig. 5a). Following
bacterial infection, the wounds were treated with Cy5-labeled CS, L-PM, or D-PM, respectively. After an
additional 4 h, wound tissues were excised, cryosectioned, and visualized using CLSM. As illustrated in Fig.
5b, red fluorescence from D-PM exhibited prominent co-localization with the green fluorescence of S.
aureus, verifying their targeted association. In contrast, no significant red fluorescence was observed in
the CS- or L-PM-treated groups, and uninfected wounds also exhibited no fluorescence signal from D-PM.
Semi-quantitative analysis of co-localization rates demonstrated that D-PM selectively traced a
substantial number of pathogens, a capability absent in CS- and L-PM-treated groups (Supplementary Fig.
42). Moreover, their facile conjugation enables the incorporation of diverse imaging probes into D-PM,
potentially allowing for multimodal diagnostics.

To assess the targeted delivery capabilities of D-PM, a wound infection model was established by
inoculating BALB/c mice with a suspension of S. aureus (1.0 × 10⁶ CFU) for 4 h (Fig. 5c). Infected mice were
treated with PBS (control), free Tet, T/CS, T/L-PM, or T/D-PM, at a dosage of 10 mg Tet per kg of body
weight. Plate counting analysis demonstrated that T/D-PM exhibited the most effective antibacterial
performance in infected wounds, attributable to its targeted antibiotic delivery. As illustrated in Fig. 5d,
T/D-PM markedly enhanced the antibacterial efficacy of tetracycline, nearly eradicating all bacteria within
the wound area. In contrast, T/TCS and T/L-PM treatments failed to improve the antibacterial activity of
tetracycline (Fig. 5e). Gram staining of wound tissue sections further corroborated these findings, showing
that T/D-PM eliminated bacteria from the wound tissues, whereas abundant purple bacteria persisted in
the wounds of other treatment groups (Fig. 5f).

At the end of the observation period, T/D-PM exhibited the greatest efficacy in promoting infected
wound healing, as evidenced by the smallest wound area (Fig. 5g and Supplementary Fig. 43). Hematoxylin
and eosin (H&E) staining revealed that T/D-PM treatment promoted the formation of a thicker dermal
layer in the infected wounds, underscoring its superior antibacterial efficacy compared to free Tet, T/CS,
and T/L-PM (Supplementary Figs. 44 and 45). Masson staining further indicated enhanced collagen
deposition in the T/D-PM group, suggesting accelerated repair of the infected wounds (Fig. 5h,
Supplementary Figs. 46 and 47). Picrosirius red staining revealed that the proportion of type I collagen
relative to the total collagen was increased in the wound tissue of T/D-PM-treated wounds, suggesting
enhanced replacement of type III collagen by type I collagen to promote more effective wound healing
(Supplementary Figs. 48 and 49). Moreover, the infected wounds treated with T/D-PM exhibited elevated
CD31 expression relative to other groups, indicative of increased capillary formation with mature vascular
structures (Supplementary Figs. 50 and 51). The enhanced healing observed in infected wounds treated
with T/D-PM is primarily attributable to its potent antibacterial activity, whereas evaluation in non-
infected wound models further revealed that both D-PM and T/D-PM possessed intrinsic bioactivity that
promotes wound repair (Supplementary Fig. 52). Collectively, these findings highlight that T/D-PM not
only enhances the in vivo antibacterial potency of tetracycline but also facilitates wound healing through
promoting dermal regeneration, collagen deposition, and neovascularization.

D-PM manage systemic bacterial infection
Systemic bacterial infections, such as peritonitis, present a rigorous model to evaluate the targeted
bacterial recognition capability of D-PM under disseminated infection conditions. This model leverages
the highly specific integration of D-PM into bacterial metabolic pathways. To explore this, a bacterial
peritonitis model was established through the intraperitoneal injection of S. aureus ATCC29213 (2.5 × 10⁷
CFU) into the right lower abdomen of BALB/c mice (Fig. 6a). One hour post-infection, fluorescence CS, L-
PM, and D-PM were intraperitoneally administered into the left lower abdomen of the infected mice,
respectively. An in vivo imaging system provided a visual representation of the biomacromolecule
distribution within the body. As the observation time extended, fluorescence signals from CS and L-PM
gradually diminished, becoming almost undetectable by 4 h (Fig. 6b). In contrast, fluorescence signals
from D-PM remained stable even at 4 h post-administration and demonstrated site-specific accumulation
in the peritonitis mice. On the other hand, fluorescence signals of D-PM also decreased in uninfected mice
within 4 h, attributable to the absence of bacterial infections. Semi-quantitative analysis of whole-body
fluorescence, based on average radiant efficiency, demonstrated the progressive clearance of both CS and
L-PM from peritonitis mice, as reflected by the time-dependent attenuation of fluorescence signals (Fig.
6c). In contrast, D-PM demonstrated prolonged retention in peritonitis mice, with fluorescence intensities
approximately 3-fold higher than those of CS and L-PM. However, the signal decay of D-PM in uninfected
mice followed a similar pattern to that of CS and L-PM in bacterial peritonitis models. This infection-
dependent retention of D-PM is primarily due to the efficient incorporation into bacterial metabolic
pathways linked to cell wall biosynthesis, whereas no appreciable D-PM retention is observed in
uninfected mice.

Next, we sought to investigate the bacterial targeting ability of D-PM at the organ level, given that
peritonitis often leads to multiorgan infections. As shown in Fig. 6d, ex vivo imaging revealed pronounced
accumulations of D-PM in non-metabolic organs, specifically the spleens and lungs, distinguishing from
low fluorescence signals in the CS- and L-PM-treated groups. Plate counting revealed comparable bacterial
loads in the spleens and lungs of all peritonitis mice (Supplementary Fig. 53). However, D-PM exhibited
selective enrichment in these infected organs compared to the control, CS-, and L-PM-treated groups.
Notably, uninfected organs showed no obvious D-PM accumulation, highlighting the specificity of
bacterial tracing (Fig. 6e,f). Frozen sections of spleen and lung tissues were analyzed to investigate the
tissue distribution of D-PM within infected organs (Fig. 6g and Supplementary Figs. 54 and 55). A
significantly higher density of fluorescence signal spots was observed in the D-PM-treated group
compared to the L-PM group. The semi-quantitative analysis confirmed that the fluorescence intensity in
the D-PM-treated sections was substantially greater than that of the L-PM-treated group. These findings,
spanning from the systemic level to the organ level, consistently highlight the robust bacterial targeting
capabilities of D-PM, characterized by enhanced retention in infected mice and selective accumulation
within infected organs.

Beyond the ability to target and track bacteria, D-PM holds potential as a therapeutic carrier for
systemic bacterial infections. Following establishment of the S. aureus ATCC29213 peritonitis model,
antibacterial treatment was administered using the different antibiotic delivery systems (Fig. 6h). As
shown in Fig. 6i, the bacterial colony count in ascitic fluid was undetectable in the T/D-PM-treated group,
which was markedly lower than that observed in the control group (4.5 × 103 CFU ml⁻¹), the free Tet-
treated group (1.0 × 103 CFU ml⁻¹), the T/CS-treated group (2.2 × 102 CFU ml⁻¹), and the T/L-PM-treated
group (2.8 × 102 CFU ml⁻¹). Meanwhile, T/D-PM exhibited the highest antibacterial efficacy, effectively
eradicating bacteria in organs, including the spleens and lungs (Fig. 6j,k). Furthermore, T/D-PM exhibited
broad-spectrum and potent antibacterial activity in vivo, demonstrating robust therapeutic efficacy in
murine peritonitis models challenged with antibiotic-resistant S. aureus ATCC43300 (MRSA,
Supplementary Fig. 56) or the Gram-negative pathogen E. coli ATCC25922 (Supplementary Fig. 57). These
findings highlight the potential of D-PM as a drug delivery platform to enhance antibiotic efficacy and
mitigate bacterial resistance.

Last but most importantly, we conducted a comprehensive in vivo safety evaluation of D-PM, which
is critical for its future clinical translation. D-PM was administered intraperitoneally to female BALB/c mice
at doses of 200 or 500 mg per kg body weight twice on day 1, followed by a 7-day observation period
(Supplementary Fig. 58a). No significant changes in body weight were observed throughout the study
(Supplementary Fig. 58b). On day 7, complete blood counts in D-PM-treated mice remained within normal
ranges compared with control groups (Supplementary Fig. 59). Consistently, total plasma immunoglobulin
G (IgG) levels and plasma biochemistry indicators, including alanine aminotransferase (ALT), aspartate
aminotransferase (AST), and blood urea nitrogen (BUN), showed no abnormal alterations (Supplementary
Figs. 60 and 61). Moreover, H&E histological examination of major organs, including the heart, liver,
spleen, lungs, and kidneys, revealed no apparent pathological lesions (Supplementary Fig. 62). Collectively,
these results demonstrate that D-PM exhibits excellent in vivo biocompatibility, even at doses exceeding
those required for bacterial recognition and antibiotic-targeted delivery.

Methods
Bacterial strains and culture conditions
The bacterial strains utilized in this study are detailed in Supplementary Table 3. The strains were revived
from glycerol stocks and stored at -80 °C until further use. For liquid culture, bacterial strains were
cultured in tryptic soy broth (TSB) or Luria–Bertani broth (LB) at 37 °C with continuous shaking at 220 rpm.
For solid culture, bacteria were plated on LB agar or mannitol salt agar plates and incubated at 37 °C.

Synthesis and characterization of peptidoglycan mimics
Synthesis of chiral peptidoglycan mimics. Chiral peptidoglycan mimics (D-PM and L-PM) were synthesized
as follows: 200.0 mg of chitosans (CS, Mw ≈ 5,000 Da) and 94.6 mg of Boc-D-Ala, combined with 191.2 mg
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), 115.6 mg of N-
hydroxysuccinimide (NHS), and 384.0 μl of N,N-diisopropylethylamine (DIPEA), were dissolved in a 3:7
(v/v) mixture of dimethylformamide (DMF) and H O solution. The reaction was stirred for 48 h at room
2
temperature. Afterward, the solvent was removed, and the residue was purified by washing 3 times with
methanol and dichloromethane. This step yielded Boc-protected D-PM. To deprotect the Boc group of D-
Ala, trifluoroacetic acid (TFA, 1.0 ml) was added to the Boc-protected D-PM, and the mixture was stirred
for 6 h. The TFA was evaporated under reduced pressure, and the resultant residue was precipitated in
anhydrous diethyl ether. The D-PM product was obtained as a yellow solid (yield: 61.2%). The synthesis
of L-PM was performed under identical conditions, yielding a yellow solid (yield: 58.9%).

Characterizations of peptidoglycan mimics. To confirm the modification of chiral Ala, 1H NMR spectra of
Boc-protected L-PM or Boc-protected D-PM were recorded on a Bruker Avance III 400 MHz spectrometer
in D O, with a sample concentration of 5 mg ml-1. The modification rate of chiral Ala was calculated based
2
on the integral areas of the proton signals corresponding to the sugar rings (δ 3.94–3.10, 5H) and the
methyl group of Ala (-CH₃, δ 1.32–1.26, 3H). The modification rate of chiral Ala was also determined using
the amine titration method. Fluorescamine—a reagent for detecting primary amines—was employed to
assess the amine content in Boc-protected L-PM and Boc-protected D-PM. The fluorescamine solution
was prepared in DMSO at a concentration of 1 mg ml-1, containing 2% DIPEA. CS were used to construct a
standard curve with concentrations ranging from 0 to 200 μg ml-1 on a 96-well microplate. Fluorescence
measurements were taken using a plate reader (SpectraMax M2, Molecular Devices), with excitation at
402 nm and emission at 470 nm. The modification rate of Boc-protected L-PM and Boc-protected D-PM
was then calculated based on the standard curve.

Zeta potentials and nanostructures. The zeta potential and size distribution of CS, L-PM, and D-PM were
measured using a Zetasizer Nano (ZSE, Malvern Panalytical) at a concentration of 400 μg ml-1. The
nanostructures of CS, L-PM, and D-PM were observed using transmission electron microscopy (TEM, JEM-
2100Plus, JEOL) after staining with phosphotungstic acid.

Circular dichroism (CD) measurements. CD spectra were conducted for CS, L-PM, and D-PM at a
concentration of 0.5 mg ml-1 in aqueous solutions using a CD spectrometer (J-1500, Jasco). The
measurement parameters included a data pitch of 0.1 nm, a scanning rate of 100 nm min-1, a bandwidth
of 1.00 nm, and a wavelength range from 190 to 260 nm.

Fluorescent PM constructions. Peptidoglycan mimics possess abundant amino groups, making them
suitable for bioconjugation with fluorescent molecules such as FITC, TAMRA, and Cy5, which facilitates
the development of imaging probes. To construct fluorescent D-PM, 20.0 mg of D-PM was dissolved in 1
ml of water, and the pH was adjusted to 8.0 using 1 M NaOH. The fluorescent molecule solution was added
dropwise to the D-PM solution, and the reaction was stirred in the dark for 12 h. The resulting fluorescent
D-PM was purified by dialysis (MWCO 2,000) in water, followed by freeze-drying. The preparation of
fluorescent CS and fluorescent L-PM was carried out using the same procedure. To ensure uniform
fluorescence intensity across the experimental groups, unlabeled PM solutions of equivalent
concentration were used to adjust and standardize the fluorescence intensities. Fluorescent PM labeled
with FITC, TAMRA, and Cy5 were excited at wavelengths of 488 nm, 544 nm, and 637 nm, respectively.
Emission spectra were collected between 460 and 760 nm. The fluorescent stability of FITC-labeled CS,
FITC-labeled L-PM, and FITC-labeled D-PM was assessed at a concentration of 0.02 mg ml-1 in an aqueous
solution. The samples were incubated in the dark at room temperature for different time points.
Fluorescence intensity was measured using a plate reader, with an excitation wavelength of 488 nm and
an emission wavelength of 520 nm.

Bacterial recognition ability and mechanisms
Flow cytometry assays. Exponential-phase S. aureus ATCC29213 were harvested and resuspended in 10
mM PBS to achieve an optical density at 595 nm (OD ) of 0.3. The bacteria were incubated with FITC-
595
labeled CS, L-PM, or D-PM at specific concentrations for designated periods. Following incubation, the
bacteria were collected by centrifugation at ~9,000 g for 5 min at 4 °C, washed, and resuspended in 10
mM PBS. The treated bacteria were subsequently analyzed using flow cytometry (CytoFLEX, Beckman
Coulter). The bacterial recognition of chiral PM was assessed using the same treatment protocols for all
pathogenic Gram-positive and Gram-negative bacteria. All representative gating strategies are shown in
Supplementary Fig. 63.

CLSM observation. Exponential-phase S. aureus ATCC29213 were harvested and resuspended in 10 mM
PBS to achieve an OD of 0.3. The S. aureus was incubated with FITC-labeled CS, L-PM, or D-PM at a
595
concentration of 0.5 mg ml-1 for 1 h. After washing off unbound fluorescent PM, the bacterial nuclei were
stained with a 1 μg ml-1 Hoechst 33342 solution for 15 min. The fluorescence signals of the fluorescent
PM located with the bacteria were observed using CLSM (Stellaris 5, Leica).

MST assays. The binding affinity of CS, L-PM, and D-PM to S. aureus ATCC29213 was determined using
MST (Monolith NT.115, NanoTemper). S. aureus (OD = 1.0) was stained with 10 μg ml-1 FM4-64 dye in
595
10 mM PBS. Serial dilutions of CS, L-PM, and D-PM were prepared to create a concentration gradient and
incubated with S. aureus for 10 min at room temperature. The mixtures were loaded into capillaries, and
MST measurements were conducted and analyzed using Nanotemper Analysis software.

UPLC-MS assays. To investigate the effect of D-PM on cell wall biosynthesis, vancomycin was used as a
positive control based on the accumulation of UDP-MurNAc-pentapeptide in cultures. Exponential-phase
S. aureus ATCC29213 (OD = 0.3) grown in TSB was pretreated with 130 μg ml-1 chloramphenicol for 15
595
min at 37 °C. Subsequently, 1 mg ml-1 of CS, L-PM, and D-PM, along with 10 μg ml-1 vancomycin, were
added to the S. aureus cultures. After 1-h incubation, the S. aureus was harvested, and the pellets were
resuspended in 500 μl of aqueous solution. The cell suspension was boiled for 30 min and then centrifuged
at ~14,000 g for 15 min. The supernatant was analyzed using UPLC-MS (Orbitrap Exploris 120, Thermo),
and the data were analyzed by FreeStyle (Thermo Fisher Scientific, Xcalibur Version 4.2). By inputting the
chemical formula of UDP-MurNAc-pentapeptide into FreeStyle, the targeted molecular weight of
1,149.3516 was used to capture the molecular signal in UPLC-MS results.

To reveal the mechanism of D-PM intervention in synthesis, biosynthetic intermediates of GlcN-D-
Ala-D-Ala and GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc were tracked and studied. S. aureus (OD =
595
0.3) was treated with CS, L-PM, or D-PM. The supernatant was then analyzed by UPLC-MS to detect and
analyze the molecular signals of GlcN-D-Ala-D-Ala (Mw = 321.1536) and GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-
Ala-MurNAc (Mw = 924.4288).

Molecular docking and dynamics simulation. Molecular docking studies were conducted to predict the
binding interactions between peptidoglycan mimics and key proteins involved in peptidoglycan
biosynthesis, specifically Ddl and MurF. The three-dimensional structures of these proteins were obtained
using AlphaFold Protein Structure Database, and docking simulations were performed using AutoDock
Vina 1.2.3. The docking scores were computed using the built-in scoring functions of AutoDock Vina, which
evaluate binding affinities by considering intermolecular interactions. Following the docking procedure,
molecular dynamics (MD) simulations were carried out with GROMACS 2023.2, employing the
CHARMM36m force field and the TIP3P water model. The force field parameters for D-Ala, D-Ala-D-Ala,
GlcN-D-Ala, and GlcN-D-Ala-D-Ala were generated using CGenFF. Hydrogen bonds were constrained using
the LINCS algorithm, with an integration step of 2 fs, and nonbonded interactions were truncated at 12 Å.
The resulting data were visualized using PyMOL 3.0.4.

Biosynthetic inhibition. Exponential-phase S. aureus ATCC29213 (OD = 0.3) was resuspended in TSB
595
and pretreated with bacterial biosynthetic inhibitors, including Dcy (a D-Ala-D-Ala ligase inhibitor), Amo
(penicillin-binding protein transpeptidase inhibitor), Mer (penicillin-binding protein transpeptidase
inhibitor), Tet (a protein synthesis inhibitor), and Cip (a DNA synthesis inhibitor), all at a concentration of
100 μg ml-1 for 2.5 h. After the removal of the inhibitors, the pretreated S. aureus (OD = 0.3) was
595
incubated with 0.5 mg ml-1 FITC-labeled D-PM in 10 mM PBS for 1 h to analyze bacterial recognition using
flow cytometry. The un-pretreated group served as a positive control, allowing for a comparison of
bacterial recognition in the presence of each inhibitor.

D-PM accumulation in bacterial biosynthesis
Chirality-dependent bacterial accumulation. The accumulation of fluorescent peptidoglycan mimics in
bacteria was detected after multiple rounds of incubation with peptidoglycan mimics. Exponential-phase
S. aureus ATCC29213 (OD = 0.003) was collected and resuspended in an LB medium. After incubation
595
with FITC-labeled L-PM or FITC-labeled D-PM at a concentration of 0.5 mg ml-1 for 1 h, unbound L-PM and
D-PM were removed by washing, yielding Passage 1 (P1) S. aureus. P1 S. aureus was then subjected to a
second incubation with FITC-labeled L-PM or FITC-labeled D-PM for 1 h, followed by washing to remove
unbound PM, resulting in P2 S. aureus. This process was repeated to obtain P3 S. aureus. The accumulation
of chiral PM into each passage of S. aureus biosynthesis was quantified by flow cytometry. Exponential-
phase S. aureus ATCC29213 were harvested and diluted in PBS to an OD of 0.3. The bacterial
595
suspensions were then incubated with CS, L-PM, or D-PM at a concentration of 0.5 mg ml-1 for 1 h. After
incubation, the zeta potential of each sample was measured using a Zetasizer Nano instrument.

3D-SIM for PM hierarchical accumulation. Super-resolution 3D-SIM (N-SIM S, Nikon) was utilized to
visualize the hierarchical incorporation of D-PM into the bacterial cell wall biosynthesis. The bacterial
incubation and treatment protocols were consistent with those described in the flow cytometry assay for
studying chirality-dependent bacterial accumulation. To observe the hierarchical accumulation of PM into
the bacterial cell wall, P1 S. aureus ATCC29213 was derived from incubation with Cy5-labeled L-PM or
Cy5-labeled D-PM, and P2 S. aureus was observed following incubation with TAMRA-labeled L-PM or
TAMRA-labeled D-PM, and P3 S. aureus was examined after incubation with FITC-labeled L-PM or FITC-
labeled D-PM. Each passage of S. aureus (P1, P2, and P3) was observed using 3D-SIM to assess the
hierarchical accumulation of PM into the bacterial cell wall.

SEM for morphology observation. Exponential-phase S. aureus ATCC29213 was incubated with CS, L-PM,
or D-PM for three consecutive passages to assess morphological alterations. Following treatment, the
bacteria were thoroughly rinsed with PBS and sequentially dehydrated in a graded series of ethanol
solutions. After dehydration, the samples were air-dried and sputter-coated with gold to enhance
conductivity. The bacterial surface morphology was then observed using SEM (MIRA4 LMH, Tescan).

Propidium iodide (PI) influx assay. Exponential-phase S. aureus ATCC29213 (OD = 0.003) was harvested
595
and resuspended in LB medium. The bacterial suspension was incubated with CS, L-PM, or D-PM at
0.5 mg ml⁻¹ over three consecutive passages. Following treatment, bacteria were stained with 10 μg ml⁻¹
PI for 15 min in the dark, and fluorescence signals were quantified by flow cytometry.

CFU count assays. Exponential-phase S. aureus ATCC29213 was diluted in PBS to a final concentration of
5 × 10⁵ CFU ml⁻¹. The bacterial suspension was incubated with CS, L-PM, or D-PM at a concentration of 1
mg ml⁻¹ for 1 h to generate Passage 1 (P1). A 10 μl aliquot from each suspension was plated onto mannitol
salt agar. P1 bacteria were then subjected to a second round of incubation with the same materials under
identical conditions for 1 h to obtain Passage 2 (P2), followed by plating of a 10 μl aliquot. This incubation–
plating cycle was repeated once more to generate Passage 3 (P3). All agar plates were incubated at 37 °C
overnight, after which colony-forming units (CFUs) were enumerated.

Targeted delivery systems based on D-PM
Differentiation of bacteria and mammalian cells. NIH 3T3 fibroblasts and S. aureus ATCC29213 were co-
cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
FITC-labeled CS, L-PM, or D-PM (0.5 mg ml-1, equal fluorescence intensity) were added to the co-culture
system and incubated at 37 °C for 1 h. For flow cytometry, unbound fluorescent macromolecules were
removed by washing with PBS. S. aureus was collected by centrifugation, and NIH 3T3 cells were
enzymatically detached using 0.25% trypsin. For CLSM imaging, NIH 3T3 cells and S. aureus were stained
with Hoechst 33342 (1 μg ml-1) for 10 min after removing unbound macromolecules.

Preparation of Tet-loaded D-PM. Tet (20 mg ml-1 in 50 μl DMSO) was mixed with D-PM (20 mg ml-1 in 250
μl water) and sonicated in a water bath. The mixture was then added dropwise into 700 μl water under
ultrasonic conditions, obtaining nanoparticulate Tet-loaded D-PM (T/D-PM). Unencapsulated Tet and
residual organic solvents were removed by dialysis (MWCO: 2,000 Da) against distilled water for 24 h. And
the solution was freeze-dried to obtain the final T/D-PM formulation. Other formulations, including T/CS
and T/L-PM, were prepared using the same procedure. Encapsulation efficiency and drug loading content
were quantified by UV–Vis spectrophotometry (UV-1280, Shimadzu) at 360 nm, based on a standard curve
generated from known Tet concentrations. Pyrene was used as a fluorescent probe to evaluate the
loading capacity of D-PM for guest molecules. Particle size distribution and zeta potential were measured
by Zetasizer Nano. The nanostructure of T/D-PM was visualized using TEM.

Antibacterial activity of T/D-PM. The minimum inhibitory concentrations (MIC) of free Tet, T/CS, T/L-PM,
and T/D-PM were determined using the standard broth microdilution method following Clinical and
Laboratory Standards Institute guidelines. The exponential-phase bacteria were diluted in fresh TSB to a
final concentration of 5 × 10⁵ CFU ml-1 to prepare the working suspension. A 100 μl aliquot was added to
each well of a 96-well plate. Free Tet, T/CS, T/L-PM, or T/D-PM were then added to the first row at
designated initial concentrations (final volume: 200 μl), followed by a series of 2-fold serial dilutions across
the plate. After incubation at 37 °C for 24 h, the MIC was defined as the lowest antibiotic concentration
that completely inhibited visible bacterial growth. To further evaluate bactericidal activity, PI staining and
CFU assays were conducted. Exponential-phase S. aureus cultures were diluted to OD = 0.3 in PBS and
595
treated with each formulation containing Tet at a final concentration of 2 μg ml-1 for 1 h at 37 °C. Following
treatment, bacterial suspensions were incubated with PI (10 μg ml-1) for 15 min in the dark, followed by
CLSM to visualize PI-positive (dead) bacteria. For CFU assays, exponential-phase S. aureus ATCC29213 and
ATCC43300 were diluted in PBS to 5 × 10⁵ CFU ml⁻¹. S. aureus ATCC29213 was incubated with free Tet,
T/CS, T/L-PM, or T/D-PM (equivalent to 2 μg ml⁻¹ Tet) for 1 h, while S. aureus ATCC43300 was treated with
the same formulations at 0.25 μg ml⁻¹ Tet for 2 h. Each suspension (10 μl aliquot) was plated onto mannitol
salt agar, and CFUs were enumerated after overnight incubation at 37 °C.

Bacterial internalization of T/D-PM. Exponential-phase S. aureus ATCC29213 (OD = 0.3) was treated
595
with different formulations (equivalent to 2 μg ml-1 Tet) for 1 h. After treatment, S. aureus was stained
with Hoechst 33342 (1 μg ml-1, 15 min) to visualize nucleic acids. The intracellular localization of Tet was
examined by CLSM using excitation at 488 nm to detect the intrinsic fluorescence of Tet.

Animal study
All animal experiments were approved by the Laboratory Animal Ethics Committee of Hunan University
(SYXK(XIANG)2018-0006). Ethical guidelines related to animal experiments were strictly followed. Animals
were housed in a controlled environment with a temperature of 20–26 °C, humidity of 40–70%, and a 12
h light/dark cycle.

D-PM for local infection imaging and treatment. Following anesthesia, circular full-thickness wounds
(~80 mm²) were created on the dorsal skin of female BALB/c mice (6–8 weeks old), followed by inoculation
with FITC-D-PM-labeled S. aureus ATCC29213 (1 × 10⁶ CFU). The infected mice were subsequently treated
topically at the wound site with PBS or Cy5-labeled CS, L-PM, or D-PM (1 mg per kg body weight). After
4 h, wound tissues were collected, embedded, cryosectioned (~ 10 μm), and stained with Hoechst 33342
for CLSM analysis. Uninfected mice also received Cy5-labeled D-PM as a control. The Pearson’s correlation
coefficient (r) for the colocalization between bacteria (green) and peptidoglycan mimics (red) was
quantified using Fiji/ImageJ (version 1.54f).

For antibacterial efficacy evaluation, the same wound infection model was employed. After 4 h of
infection, the mice were treated with PBS, free Tet, T/CS, T/L-PM, and T/D-PM at a dosage of 10 mg Tet
per kg of body weight. Treatments were administered topically every 12 h for a total of 2 doses.
Throughout the treatment period, the wound area and the body weight of infected mice were closely
monitored. At designated time points, the infection wounds were swabbed under sterile conditions, and
the bacteria were suspended in PBS and plated on mannitol salt agar for CFU enumeration. Following
euthanasia, wound tissues were fixed in 4% polyformaldehyde, paraffin-embedded, and sectioned for
histological analysis. Gram staining assessed bacterial burden, while the stainings by H&E, Masson,
picrosirius red and CD31 immunofluorescence were performed to evaluate tissue regeneration and
neovascularization.

D-PM for systemic infection imaging and treatment. A mouse peritonitis model (female BALB/c) was
established by intraperitoneal injection of S. aureus ATCC29213 (2.5 × 10⁷ CFU in 100 μl PBS) into the right
side of the abdomen. After 1 h of infection, Cy5-labeled CS, L-PM, or D-PM (5 mg Tet per kg body weight)
were administered intraperitoneally into the left side of the abdomen. Control animals received the
treatment with Cy5-labeled D-PM in the absence of infection. In vivo imaging was conducted at 10 min, 1
h, 2 h, and 4 h post-administration using an in vivo imaging system (IVIS Spectrum CT, PerkinElmer). Major
organs were harvested and subjected to ex vivo fluorescence imaging. Fluorescence intensity was
quantified using Living Image software. Bacterial burdens in tissues were determined by plating tissue
homogenates on mannitol salt agar. These tissues were embedded, cryosectioned, and mounted as
described for wound samples, followed by CLSM imaging to assess the tissue distribution of Cy5-labeled
CS, L-PM, or D-PM.

For antibacterial efficacy assessment, female BALB/c mice were intraperitoneally inoculated with
100 μl of bacterial suspension containing S. aureus ATCC29213 (2.5 × 10⁷ CFU), S. aureus ATCC43300
(MRSA, 2.5 × 10⁷ CFU), or E. coli ATCC25922 (1.0 × 10⁷ CFU) in PBS. After 1 h, mice were treated with PBS,
free Tet, T/CS, T/L-PM, or T/D-PM. The administered dose was 10 mg Tet per kg body weight for S. aureus
ATCC29213 and MRSA ATCC43300 infections, and 20 mg Tet per kg body weight for E. coli ATCC25922
infection, delivered intraperitoneally every 12 h for a total of two doses. After 24 h of the first treatment,
mice were euthanized, and ascitic fluid, along with major organs, were aseptically collected. Samples were
homogenized, serially diluted, and plated on agar for CFU enumeration.

Safety evaluation of D-PM. D-PM suspended in PBS was administered intraperitoneally to female BALB/c
mice at 200 or 500 mg D-PM per kg body weight, with PBS alone serving as the control. Body weights were
recorded daily throughout the study. On day 7 post-administration, whole blood and plasma were
collected for routine hematological analysis and plasma biochemical profiling, including
immunoglobulin G (IgG), alanine transaminase (ALT), aspartate transaminase (AST), and blood urea
nitrogen (BUN). Mice were then euthanized, and major organs (heart, liver, spleen, lungs, and kidneys)
were excised, fixed overnight in 4% paraformaldehyde, paraffin-embedded, and sectioned for
hematoxylin and eosin (H&E) staining and histological examination.

Statistical information
Statistical significance between two groups was assessed using a two-tailed unpaired t-test. All
experiments were performed with at least three independent biological replicates. Data are presented as
mean ± s.d. Differences were considered statistically significant at P < 0.05 (*P < 0.05; **P < 0.01; ***P <
0.001; ****P < 0.0001); P > 0.05 was considered not significant (NS).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Deng et al presents an original article entitled “Chiral Peptidoglycan Mimics Target Bacterial Wall Biosynthesis for Pathogen Intervention” for publication in Nature communications.\nThis study describes the creation of a chiral peptidoglycan that mimics a bacterial cell wall component and allows its incorporation into the wall, to then be used either as a biomarker for pathogen detection or as a drug delivery system for antibiotics.\nThe study is rich, very comprehensive (including areas beyond my own expertise, such as the structural validation of the molecule), and very well written. The strategy described is innovative and very elegant.\nHowever, I have a few questions, comments, or discussion points.\n1. Targeting the bacterial cell wall is very interesting. However, even though peptidoglycan is highly conserved across species, several issues arise:\na. The possibility that the microorganism could change its peptidoglycan synthesis pathway, alter enzyme conformations, or use other mechanisms (as vancomycin-resistant strains do), meaning that D-PM can no longer be incorporated, becomes undetectable, or is unable to deliver the antibiotic.\nb. The bacterium must be metabolically active to incorporate D-PM — the authors should discuss this limitation.\nc. The need to cross the outer membrane in Gram-negative bacteria. In fact, the results shown in Supplementary Figure 12 are much less striking than in Gram-positive bacteria and should be discussed more thoroughly (particularly for E. coli). Moreover, in Supplementary Figure 27, the positive rate for E. coli is higher than in Supplementary Figure 12, which is not entirely consistent.\n2. More generally, many experiments are presented with only three replicates, which seems rather limited in some cases (e.g., Fig. 1d, Fig. 2j, Fig. 3f, Fig. 4b...).\n3. Supplementary Figure 3: some information is missing from the legend (e.g., what Ddl is).\n4. Fig. 1d: why not indicate the exact positive rates for D-PM (instead of stating between 80–100%)?\n5. For the comparison between FITC-labeled D-PM and FITC-labeled D-Ala (Supp. Fig. 14), while the difference is clearly significant, it remains relatively small. Wouldn’t it be simpler (and less expensive to synthesize) to just use D-Ala?\n6. D-PM seems to act like vancomycin (same UDP-MurNAC-pentapeptide peak). Moreover, dead bacteria (or at least damaged envelopes) are detected in the experiments shown in Figs. 3e and 3f. The authors should discuss this, report the MIC of D-PM, and include experiments with D-PM alone (without Tet loading) in addition to the other experimental conditions shown in Figs. 4, 5, and 6.\n7. Line 272: “almost no binding to mammalian cells.” This does not match what is observed. There is detectable binding, which could lead to false positives. What hypotheses do the authors have to explain this binding? Furthermore, in the experiment shown in Fig. 6, even though D-PMs are generally no longer detectable in mice after 6 h, they remain present in certain organs. This suggests nonspecific binding and the potential for false positives in pathogen detection. These limitations should be discussed.\n8. The results with S. aureus are promising in both in vivo models, but it would have been useful to test other bacteria, perhaps another Gram-positive or a Gram-negative species.\n9. Lines 356–357: the authors suggest that T/D-PM improves wound healing, but what is their hypothesis for this effect? Is it significant compared to a non-infected control? Could it simply be that clearing the infection is what enables wound healing?\nThis is a very well-designed and well-written study describing an innovative and elegant approach. Addressing the points above, particularly the mechanistic discussion of potential resistance, the clarification of Gram-negative results, the possible antibacterial effect of D-PM, and the issue of nonspecific binding, would significantly strengthen the manuscript and enhance its impact.", "reviewer_2": "In this work, macromolecular analogues of bacterial peptidoglycan was developed from chirality-specific biomimetics-D-alanine-conjugated chitosan (CS, (GlcN-D-Ala)n) to construct macromolecules for specific bacteria recognition. Chirality specific bacteria recognition of the D-PM was elucidated in contrast with the L-PM toward various strains. The D-PM was also shown to exclusively recognize bacteria and it was inactive toward various eukaryotic cells. Furthermore, the bacterial recognition by D-PM was demonstrated in vivo on the infected wound and peritonitis model in mice, whereby it showed specific accumulation toward the infecting bacteria. Overall, this finding is a highly noteworthy contribution in particular to the study of bacterial infection or treatment, and biomedical field in general. But there are comments below that need to be addressed:\n1. The experimental design lacks the characterization on the chemistry side. Can the authors measure the molecular weight of the CS, and the peptidoglycan mimics series after the syntheses (especially after treatment with TFA)?\n2. In general, EDC/NHS chemistry is rather inefficient and here the modification percentages are only around 17%. It means that the D-PM only has 17% of D-Ala moieties along the CS chain, which in theory does not differ much from, say, a mixture of CS and D-PM. Why and how could 17% modification result in stark difference of bacteria recognition?\n3. It is known that FITC can readily stain both the intact and compromised bacterial membranes, but here the authors claimed that FITC-labelled CS and L-PM were excluded or even cleared from bacterial surface. So just how big is the CS and L-PM molecules? Or is there any repulsive chemistry/force between them and the bacterial membrane?\n4. In Fig 1c, 3c, 3d, and 4c, I suggest the authors also show the zoom-out images (lower magnification), to show that this specific recognition by D-PM can be observed across all the bacteria in the much wider area.\n5. In Supplementary Fig 23, flow cytometry test showed negligible interaction of the peptidoglycan mimics (D-PM, L-PM and CS) on various eukaryotic cells. Can the authors test the binding characteristics/accumulation on those cells using MST measurement and take the LCMS peak too (similar to what the authors had shown on bacteria)? It is necessary because the whole idea emphasizes on selective bacteria recognition/targeting over eukaryotic cells.\n6. In Fig 2d-g, is it possible that the GlcN-D-Ala-D-Ala intermediate to GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc might be slightly off (by around 30 m/z maybe)? Can the authors do a confirmation whether this biosynthesis is via MurF ligase (i.e., not GlcN-D-Ala addition via MurC), for example using Edman degradation test (or any test) to confirm?\n7. In Supplementary Fig 37, it looks like the T/CS treated wound also showed relatively thick dermal layer. Can the authors show the images of all the biological replicates (not just 1 representative)? I also suggest to measure the dermal (and/or epidermal) thickness of the samples for quantitative data comparison.\n8. Similarly, for other in vivo images (Figs 5f, 5h, 6b, 6d, 6g, and Supplementary Figs 37, 38a, 39a) I suggest the authors also show all the biological replicates in the Supplementary Information, to visually gauge the extent of bacteria recognition by the D-PM.\n9. The wound model was not splinted, so we need a way to justify healing via re-epithelialization (inferably due to the infection relief) rather than via contraction. To support this, besides the information already provided in the trichrome Masson staining, more importantly the characterization of the collagen quality should be assessed to understand the type of new tissue that is being built. I suggest to do picrosirius staining to identify and quantify different types of collagen fibers in the tissue sections.\nMinor comments:\nIn the title, I suggest to change the word ‘intervention’ with something more meaningful or specific, maybe ‘recognition’?\nFor all the flow cytometry data/results, we need to see the gating strategy in each of the tests.\nIn Supplementary Fig 12, we also need to see the positive rate of CS and L-PM to the various bacteria.\nPage 6 line 113 mentioned E faecium, while Fig 1d mentioned E faecails.\nIn the captions of Supplementary Fig 6a, 29, 30, the replicates are not ‘biological’ replicates.\nIn the caption of Supplementary Fig 27 mentioned various mammalian cells, not just NIH 3T3.\nIn Supplementary Fig 38b, there is a typo ‘collangen’.", "reviewer_3": "This manuscript centres on a “chirality-specific peptidoglycan (PG) biomimetic macromolecule.” The authors graft D-alanine onto chitosan to construct D-PM, achieving efficient, broad-spectrum recognition of multiple pathogenic bacteria—including ESKAPE species, drug-resistant strains, and clinical isolates—while showing negligible binding to eukaryotic cells.\nMechanistically, D-PM acts as a non-native substrate for bacterial cell-wall biosynthesis and is incorporated into downstream metabolism (engaging Ddl and MurF and yielding characteristic intermediates), thereby enabling durable labeling and targeted delivery. In mouse models of localized wound infection and systemic peritonitis, D-PM supports imaging and targeted delivery of tetracycline, markedly improving antibacterial efficacy and tissue repair. The strengths of the work are: (1) proposing and validating a new concept and molecular design paradigm of “D-amino-acid-driven macromolecular substrate assimilation into the bacterial cell wall”; (2) assembling a mechanistic package spanning binding affinity, UPLC–MS identification of metabolic intermediates, inhibitor/competition experiments, and molecular docking/dynamics; and (3) demonstrating translational potential via an integrated diagnose-and-treat workflow from in vitro to in vivo. Overall, the study is innovative and practically relevant for pathogen-specific recognition and antibiotic delivery.\nHaving carefully read the manuscript and Supplementary Information, I find the overall conclusions plausible; however, to ensure reproducibility and strengthen the case for clinical translation, I recommend Major Revision. My main concerns and proposed revisions are:\n1.Insufficient evidence for broad in vivo effectiveness. The abstract and main text state that D-PM achieves broad recognition across ESKAPE/clinical isolates and is effective in both local and systemic infections in vivo. Yet both in vivo models (wound infection and peritonitis) are demonstrated only with S. aureus (Gram-positive), with no in vivo data for Gram-negative pathogens. To substantiate the claim of broad in vivo effectiveness, please add at least one Gram-negative animal infection model.\n2.Insufficient safety assessment. Safety evidence is largely limited to in vitro assays (multi-cell-line viability/Live–Dead showing minimal cytotoxicity) and hemocompatibility (low hemolysis), suggesting preliminary compatibility; in vivo observations mainly track body weight and histology within infection models, serving more as efficacy/localization readouts than as a conventional toxicology package. For a translational claim, key data are missing, including serum biochemistry and histopathology of major organs (heart, liver, spleen, lung, kidney), complement activation and cytokine panels, immunogenicity/hypersensitivity assessment, repeat-dose and accumulation toxicity, plasma/tissue PK/BD (including protein corona and blood stability), and potential impacts on commensal microbiota; dose/ exposure bridging to clinically comparable regimens and a defined safety window are also absent.\n3.Missing structure–activity/structure–performance relationships for the material. The current materials exploration uses a single chitosan molecular weight (Mw ≈ 5 kDa) and a single substitution level (~17% D-Ala), which is insufficient to establish the true dependence of “incorporation efficiency → bacterial enrichment → therapeutic benefit” on backbone Mw, degree of substitution, and choice of amino acid. Please, under otherwise matched conditions, systematically vary three axes: (i) several CS backbones of different Mw (e.g., low/medium/high) to assess length effects on incorporation and in vitro/in vivo enrichment; (ii) a substitution gradient at fixed Mw (e.g., ~5–10%, 15–20%, 30–40%) to build dose–response-like curves linking substitution to incorporation/accumulation/efficacy; and (iii) alternative D-amino-acid anchors at comparable Mw and substitution (e.g., D-Ala vs D-Glu or D-Lys) to clarify how the chiral substrate site governs acceptance by Ddl/MurF and downstream metabolic propagation."}, "author_response": "(Reviewer comments are shown in blue, and our responses are in black.)\nReviewer #1 (Remarks to the Author):\nDeng et al presents an original article entitled “Chiral Peptidoglycan Mimics Target Bacterial\nWall Biosynthesis for Pathogen Intervention” for publication in Nature Communications.\nThis study describes the creation of a chiral peptidoglycan that mimics a bacterial cell\nwall component and allows its incorporation into the wall, to then be used either as a bi-\nomarker for pathogen detection or as a drug delivery system for antibiotics.\nThe study is rich, very comprehensive (including areas beyond my own expertise, such as\nthe structural validation of the molecule), and very well written. The strategy described is in-\nnovative and very elegant.\nHowever, I have a few questions, comments, or discussion points.\nA1: We sincerely thank the reviewer for the highly positive and encouraging evaluation of our\nwork. We also appreciate the constructive comments and discussion points raised. In re-\nsponse, we have carefully addressed all concerns with additional clarifications, new control\nexperiments, and refined discussions where appropriate. All corresponding revisions have\nbeen incorporated into the revised manuscript and are detailed point-by-point below.\nQ1-1: Targeting the bacterial cell wall is very interesting. However, even though peptidoglycan\nis highly conserved across species, several issues arise:\na. The possibility that the microorganism could change its peptidoglycan synthesis path-\nway, alter enzyme conformations, or use other mechanisms (as vancomycin-resistant strains\ndo), meaning that D-PM can no longer be incorporated, becomes undetectable, or is unable\nto deliver the antibiotic.\nb. The bacterium must be metabolically active to incorporate D-PM, the authors should\ndiscuss this limitation.\nc. The need to cross the outer membrane in Gram-negative bacteria. In fact, the results\nshown in Supplementary Figure 12 are much less striking than in Gram-positive bacteria and\nshould be discussed more thoroughly (particularly for E. coli). Moreover, in Supplementary\nFigure 27, the positive rate for E. coli is higher than in Supplementary Figure 12, which is not\nentirely consistent.\nA1-1: We thank the reviewer for raising these important mechanistic considerations. We have\nnow expanded the discussion in the revised manuscript to clarify the following points:\n(a) We fully agree that alterations in peptidoglycan biosynthesis could, in principle, a(cid:431)ect D-\npeptidoglycan mimics (D-PM) incorporation. In designing the chiral peptidoglycan mimics,\nwe carefully evaluated the high conservation of the D-Ala–D-Ala terminus across clini-\ncally relevant pathogens listed by the WHO. As expected, D-PM e(cid:431)iciently recognizes\nmethicillin-resistant S. aureus (MRSA, ATCC 43300), as shown in Fig. 1b of the manuscript.\nWe also acknowledge that in strains with extensive remodeling of peptidoglycan precur-\nsors or markedly altered enzyme specificity, incorporation e(cid:431)iciency may decrease. We\nnow discuss this limitation in the revised manuscript and emphasize that D-PM is broadly\napplicable for bacterial detection but is not universal.\n…Looking forward, the broad-spectrum recognition of D-PM across diverse pathogenic bac-\nteria, including methicillin-resistant Staphylococcus aureus, supports future investigations of\nthe applicability to a wider range of multidrug-resistant pathogens…\n(b) The reviewer is correct that the incorporation of D-PM requires active peptidoglycan bio-\nsynthesis, which occurs only in metabolically active bacteria. Because D-PM functions as\na biosynthetic mimic, it may exhibit reduced incorporation e(cid:431)iciency in dormant or sta-\ntionary-phase bacteria. This limitation is now included in the revised discussion.\n…supports future investigations of the applicability to a wider range of multidrug-resistant\npathogens and to bacteria in distinct physiological states, such as dormant and stationary-\nphase populations…\n(c) We thank the reviewer for raising this important point. We agree that the Gram-negative\nouter membrane presents an intrinsic permeability barrier, which accounts for the lower\nincorporation and detection e(cid:431)iciency of D-PM in species such as Klebsiella pneumoniae\nand Escherichia coli compared with Gram-positive bacteria. We have clarified this mech-\nanistic consideration in the revised manuscript.\nWe also appreciate the reviewer’s careful comparison regarding the positive rate of\nE. coli. The apparent discrepancy arises from the distinct experimental conditions: Sup-\nplementary Fig. R1a (Supplementary Fig. 13d in the revised Supplementary Information)\nwas conducted in phosphate-bu(cid:431)ered saline to minimize background biosynthetic activ-\nity, whereas Fig. R1b (Supplementary Fig. 33d in the revised Supplementary Information)\nwas performed in DMEM to maintain mammalian cell viability during co-culture. This con-\ndition-dependent di(cid:431)erence was reproducibly observed in repeated experiments (Fig. R1\nc and d).\nFig. R1 a, Percentage of FITC-positive E. coli following co-incubation with FITC-labelled D-PM\nin phosphate-bu(cid:431)ered saline (PBS), used to minimize background biosynthetic activity. b, Per-\ncentage of FITC-positive E. coli following co-incubation with FITC-labelled D-PM in DMEM,\nemployed to preserve mammalian cell viability during co-culture. c, Independent repetition\nof the experiment shown in a, performed during the manuscript revision process. d, Inde-\npendent repetition of the experiment shown in b, performed during the manuscript revision\nprocess.\nThe discussion has been updated in the revised manuscript.\n…The relatively lower positive rates observed for certain Gram-negative bacteria (Klebsiella\npneumoniae and Enterobacter species), compared with Gram-positive bacteria under identi-\ncal D-PM treatment conditions, likely result from the presence of an outer membrane that par-\ntially impedes D-PM access to the peptidoglycan…\n…Supplementary Fig. 13 | Positive rate of FITC-labeled CS, L-PM, and D-PM binding to a, K.\npneumoniae ATCC13883, b, A. baumannii ATCC19606, c P. aeruginosa ATCC27853, and d E.\ncoli ATCC25922 after 1 h incubation at 0.5 mg ml-1 in PBS (n = 6 biological replicates per group;\ndata are presented as mean ± s.d.). Statistical analysis was performed using a two-tailed un-\npaired t-test (a-d); ****P < 0.0001…\n…Supplementary Fig. 33 | Flow cytometry analysis for co-cultures of a, K. pneumoniae\nATCC13883 and NIH 3T3 cells, b, A. baumannii ATCC19606 and NIH 3T3 cells, c, P. aeruginosa\nATCC27853 and NIH 3T3 cells, as well as d, E. coli ATCC25922 and NIH 3T3 cells after incu-\nbation with FITC-labeled D-PM at 0.5 mg ml-1 for 1 h in DMEM culture medium (n = 3 biological\nrepli-cates per group; data presented as mean ± s.d.). Statistical analysis was performed us-\ning a two-tailed unpaired t-test (a-d); ****P < 0.0001…\nQ1-2: More generally, many experiments are presented with only three replicates, which\nseems rather limited in some cases (e.g., Fig. 1d, Fig. 2j, Fig. 3f, Fig. 4b...).\nA1-2: We thank the reviewer for raising this important point regarding sample size. To directly\naddress this concern, all experiments explicitly highlighted by the reviewer—Fig. 1b (Fig. R2),\nFig. 2j (Fig. R3), Fig. 3f (Fig. R4), and Fig. 4b (Fig. R5)—as well as the corresponding in vivo\nstudies in Fig. 6i–k (Fig. R6), have now been independently repeated with increased biological\nreplicates (n = 6). In all cases, increasing the sample size did not alter the observed e(cid:431)ect\nsizes or statistical significance, and the results remained fully consistent with the original\nfindings, exhibiting low inter-sample variability. These data confirm the robustness and repro-\nducibility of our conclusions. The updated results, exact n values, and corresponding statisti-\ncal analyses have been incorporated into the revised manuscript and figure captions.\nFig. R2 The Relative positive rate of di(cid:431)erent pathogenic bacteria, including S. aureus\nATCC29213, S. aureus ATCC25904, S. aureus RN4220, E. faecalis ATCC29212, E. faecalis\nATCC19433, S. aureus ATCC43300 (MRSA), and a clinical isolate of S. aureus, incubated with\nFITC-labeled CS, L-PM, and D-PM at a concentration of 0.5 mg ml-1 for 1 h (n = 6 biological\nreplicates per group).\nFig. R3 E(cid:431)ect of di(cid:431)erent bacterial biosynthesis inhibitors on the incorporation of D-PM into\nS. aureus ATCC29213. All inhibitors were used at a concentration of 100 μg ml-1 (n = 6 biolog-\nical replicates per group; data presented as mean ± s.d.). Statistical analysis was performed\nusing a two-tailed unpaired t-test; ****P < 0.0001.\nFig. R4 Flow cytometry analysis of PI-positive rate in P3 bacteria (S. aureus ATCC29213) fol-\nlowing treatment with PBS, CS, L-PM, or D-PM (n = 6 biological replicates per group; data pre-\nsented as mean ± s.d.). Statistical analysis was performed using a two-tailed unpaired t-test;\n****P < 0.0001.\nFig. R5 Flow cytometry analysis for S. aureus ATCC29213 and NIH 3T3 cells co-cultures after\nincubation with FITC-labeled D-PM at 0.5 mg ml-1 for 1 h (n = 6 biological replicates per group;\ndata presented as mean ± s.d.). Statistical analysis was performed using a two-tailed un-\npaired t-test; ****P < 0.0001.\nFig. R6 a, Schematic diagram of antibacterial administrations in the S. aureus ATCC29213 in-\nfected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic fluid (b),\nspleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independent ani-\nmals; data presented as mean ± s.d.). Statistical analysis was performed using a two-tailed\nunpaired t-test (b-d); **P < 0.01, ****P < 0.0001.\nQ1-3: Supplementary Figure 3: some information is missing from the legend (e.g., what Ddl\nis).\nA1-3: We appreciate the reviewer pointing this out. The legend of Supplementary Fig. 3 in the\nrevised Supplementary Information has been revised to explicitly define Ddl as D-Ala–D-Ala\nligase, a key enzyme in peptidoglycan biosynthesis. In addition, we reviewed the revised man-\nuscript and Supplementary Information to ensure that all abbreviations (e.g., MurF, UDP-N-\nacetylmuramoyl-tripeptide–D-alanyl-D-Ala ligase) are clearly defined at first mention.\n…Key enzymes involved in bacterial cell wall biosynthesis include D-alanine–D-alanine ligase\n(Ddl) and UDP-N-acetylmuramoyl-tripeptide–D-alanine–D-alanine ligase (MurF)…\nThe observed peak at m/z 322.1613 was assigned to the protonated molecular ion [GlcN–D-\nAla–D-Ala + H]⁺ (theoretical m/z 322.1609), which likely arises from the ligation of an addi-\ntional D-Ala to the D-PM substrate (GlcN–D-Ala) catalyzed by D-Ala–D-Ala ligase (Ddl, Fig.\n2d,e)27…\n…This intermediate, GlcN-D-Ala-D-Ala, could subsequently be incorporated into the cell wall\nbiosynthesis pathway to form GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc through the bio-\ncatalyst of UDP-N-acetylmuramoyl-tripeptide–D-alanyl-D-Ala ligase (MurF), with a character-\nistic ion peak at m/z 925.4353 (Fig. 2f,g)…\nQ1-4: Fig. 1d: why not indicate the exact positive rates for D-PM (instead of stating between\n80-100%)?\nA1-4: We thank the reviewer for this important suggestion. In Fig. 1d, the values were inten-\ntionally normalized to facilitate direct cross-species comparison within a single heatmap, as\nthe absolute positive rates of D-PM vary substantially among di(cid:431)erent bacterial strains. This\nnormalization resulted in the visual range of 80–100% for D-PM labeling.\nWe fully agree that reporting the exact positive rates provides essential quantitative infor-\nmation. Therefore, in response to the reviewer’s comment, we have now included the com-\nplete unnormalized positive rates (Fig. R7) for each bacterial strain (mean ± s.d., with exact n\nvalues) in the revised Supplementary Information (Supplementary Fig. 12). These data con-\nfirm the consistently high labeling e(cid:431)iciency of D-PM while providing full quantitative trans-\nparency.\nFig. R7 Positive rate of FITC-labeled CS, L-PM, and D-PM binding to a, S. aureus ATCC29213,\nb, S. aureus ATCC25904, c, S. aureus RN4220, d, S. aureus ATCC43300 (MRSA), e, E. faecalis\nATCC29212, f, E. faecalis ATCC19433, and g, a clinical isolate of S. aureus after 1 h incubation\nat 0.5 mg ml-1 (n = 6 biological replicates per group; data are presented as mean ± s.d.). Sta-\ntistical analysis was performed using a two-tailed unpaired t-test (a-g); ****P < 0.0001.\nThe legend of the figure and the corresponding text have been updated to clearly direct\nreaders to these exact values in Supplementary Fig. 12.\n…b, Relative positive rate of di(cid:431)erent pathogenic bacteria, including S. aureus ATCC29213, S.\naureus ATCC25904, S. aureus RN4220, E. faecalis ATCC29212, E. faecalis ATCC19433, S. au-\nreus ATCC43300 (MRSA), and a clinical isolate of S. aureus, incubated with FITC-labeled CS,\nL-PM, and D-PM at a concentration of 0.5 mg ml-1 for 1 h (n = 6 biological replicates per group).\nAbsolute values are shown in Supplementary Fig. 12. c, Representative CLSM images of FITC-\nlabeled CS, L-PM, and D-PM incubated with S. aureus ATCC29213 at a concentration of 0.5\nmg ml-1 for 1 h. Scale bar represents 2 μm…\n…Supplementary Fig. 12 | Positive rate of FITC-labeled CS, L-PM, and D-PM binding to a, S.\naureus ATCC29213, b, S. aureus ATCC25904, c, S. aureus RN4220, d, S. aureus ATCC43300\n(MRSA), e, E. faecalis ATCC29212, f, E. faecalis ATCC19433, and g, a clinical isolate of S. au-\nreus after 1 h incubation at 0.5 mg ml-1 (n = 6 biological replicates per group; data are pre-\nsented as mean ± s.d.). Statistical analysis was performed using a two-tailed unpaired t-test\n(a-g); ****P < 0.0001…\nQ1-5: For the comparison between FITC-labeled D-PM and FITC-labeled D-Ala (Supp. Fig. 14),\nwhile the di(cid:431)erence is clearly significant, it remains relatively small. Wouldn’t it be simpler\n(and less expensive to synthesize) to just use D-Ala?\nA1-5: We thank the reviewer for this insightful question. To address this point, we included two\ncontrol groups based on D-Ala small-molecule probes (Fig. R8): (i) a D-Ala/FITC-labeled D-\nAla mixture under fluorescence-matched conditions, and (ii) fully FITC-labeled D-Ala with\nequimolar D-Ala content but substantially higher total fluorescence. Under fluorescence-\nmatched conditions, FITC-labeled D-PM produced an approximately 8-fold higher MFI than\nthe D-Ala mixture. Notably, even fully FITC-labeled D-Ala—despite exhibiting about 4-fold\nhigher overall fluorescence than FITC-labeled D-PM—resulted in both a lower percentage of\nFITC-positive bacteria and a reduced MFI. These results clearly demonstrate that D-PM exhib-\nits markedly superior bacterial recognition and labeling capability compared with small-mol-\necule probes. In addition, small-molecule probes such as D-Ala are intrinsically limited by\npoor structural tunability, reduced biosynthetic engagement, and suboptimal retention within\nbacterial cell walls (Nat. Chem. 2019, 11, 335-341).\nFig. R8 Chemical structure and fluorescence spectra for a, FITC-labeled D-PM (I), b, D-\nAla/FITC-labeled D-Ala mixture (II), and c, fully FITC-labeled D-Ala (III) with equivalent molar\nconcentrations of D-Ala in each sample. Representative flow cytometry plot for d, I, e, II, and\nf, III after incubation with S. aureus ATCC29213 in the presence of 40 μg ml-1 D-Ala for 1 h.\ng, Positive rate and h, MFI for I, II, and III after incubation with S. aureus ATCC29213 in the pres-\nence of 40 μg ml-1 D-Ala for 1 h (n = 3 biological replicates per group; data are presented as\nmean ± s.d.). Statistical analysis was performed using a two-tailed unpaired t-test (g, h); **P\n< 0.01, ****P < 0.0001.\nWe have clarified this design rationale in the revised manuscript and added a schematic\nsummary of D-PM over D-Ala (Supplementary Figs. 16 and 17).\n…To investigate the bacterial recognition advantage conferred by chiral peptidoglycan mimic\ndesign, we compared FITC-labeled D-PM with FITC-labeled small-molecule D-Ala probes at\nmatched D-Ala molar equivalents. Two control conditions were employed: (1) a D-Ala/FITC-\nlabeled D-Ala mixture calibrated to match the fluorescence intensity of FITC-labeled D-PM,\nand (2) fully FITC-labeled D-Ala with substantially higher overall fluorescence than FITC-la-\nbeled D-PM (Supplementary Fig. 16)…\n…Under fluorescence-matched conditions of probe, only 2.6 ± 0.2% of bacteria were FITC-\npositive following treatment with the D-Ala/FITC-labeled D-Ala mixture, which was dramati-\ncally lower than the 85.7 ± 0.2% FITC-positive bacteria observed in the FITC-labeled D-PM\ngroup. The mean fluorescence intensity (MFI) in the FITC-labeled D-PM-treated bacteria was\nabout 8-fold higher than that of the D-Ala/FITC-labeled D-Ala mixture. Notably, even com-\npared with fully FITC-labeled D-Ala with approximately 4-fold higher fluorescence than FITC-\nlabeled D-PM, both the proportion of FITC-positive bacteria and MFI remained higher in the\nFITC-labeled D-PM-treated group…\n…CLSM images clearly demonstrate the overwhelming performance of D-PM in bacterial flu-\norescent labeling (Supplementary Fig. 17). These results underscore the superior bacterial\nrecognition e(cid:431)iciency a(cid:431)orded by D-chiral peptidoglycan mimics, highlighting the intrinsic ad-\nvantages of chirality-guided macromolecular design over small-molecule D-Ala–based\nprobes for pathogenic bacteria…\n…Supplementary Fig. 16 | Chemical structure and fluorescence spectra for a, FITC-labeled D-\nPM (I), b, D-Ala/FITC-labeled D-Ala mixture (II), and c, fully FITC-labeled D-Ala (III) with equiv-\nalent molar concentrations of D-Ala in each sample. Representative flow cytometry plot for d,\nI, e, II, and f, III after incubation with S. aureus ATCC29213 in the presence of 40 μg ml-1 D-Ala\nfor 1 h. g, Positive rate and h, MFI for I, II, and III after incubation with S. aureus ATCC29213 in\nthe presence of 40 μg ml-1 D-Ala for 1 h (n = 3 biological replicates per group; data are pre-\nsented as mean ± s.d.). Statistical analysis was performed using a two-tailed unpaired t-test\n(g, h); **P < 0.01, ****P < 0.0001…\nQ1-6: D-PM seems to act like vancomycin (same UDP-MurNAC-pentapeptide peak). Moreover,\ndead bacteria (or at least damaged envelopes) are detected in the experiments shown in Figs.\n3e and 3f. The authors should discuss this, report the MIC of D-PM, and include experiments\nwith D-PM alone (without Tet loading) in addition to the other experimental conditions shown\nin Figs. 4, 5, and 6.\nA1-6: We appreciate the reviewer’s observations regarding the apparent similarity between D-\nPM and vancomycin, as well as the envelope impairment seen in the third-passage (P3)\nbacteria in Figs. 3e and 3f. First, although both D-PM and vancomycin lead to accumulation\nof UDP–MurNAc–pentapeptide, their mechanisms are distinct. Vancomycin binds the D-Ala–\nD-Ala terminus of lipid II and immediately blocks peptidoglycan polymerization. In contrast,\nD-PM is incorporated intracellularly as a D-Ala mimic during precursor synthesis; therefore,\ndefects emerge only after progressive intracellular accumulation. Second, as suggested, we\nmeasured the MIC of D-PM (Fig. R9). No detectable growth inhibition was observed even at\nhigh concentrations (up to 211 μg ml⁻¹). Third, to directly assess the antibacterial e(cid:431)ect of D-\nPM alone (without Tet loading), we performed CFU assays under increased cumulative co-\nincubation (Fig. R10). D-PM exhibited measurable antibacterial activity after three successive\npassages, whereas CS and L-PM showed no e(cid:431)ect. These results confirm that D-PM alone\nhas a delayed and modest bactericidal e(cid:431)ect under prolonged exposure, consistent with its\nproposed intracellular-incorporation mechanism.\nFig. R9 Inhibitory e(cid:431)ects of CS, L-PM, and D-PM against S. aureus ATCC29213 (n = 6 biological\nreplicates per group; data presented as mean ± s.d.).\nFig. R10 CFU count analysis of S. aureus ATCC29213 at a, Passage 1 (P1), b, Passage 2 (P2),\nand c, Passage 3 (P3) after multiple-round incubations with CS, L-PM, and D-PM at 0.5 mg ml-\n1 every 1 h (n = 6 biological replicates per group; data presented as mean ± s.d.). Statistical\nanalysis was performed using a two-tailed unpaired t-test; ***P < 0.001, ****P < 0.0001.\nThese new data, together with additional discussion, have been incorporated into the re-\nvised Supplementary Information (Supplementary Fig. 26), as detailed below.\n…In addition, colony-forming unit (CFU) assays demonstrated that incorporation of D-PM into\nbacterial cell wall biosynthesis elicited a modest bactericidal e(cid:431)ect at Passage 1 (Supplemen-\ntary Fig. 26). With successive passages up to Passage 3, progressive accumulation of D-PM\nwithin the peptidoglycan increasingly compromised cell wall integrity, leading to a stepwise\nenhancement of bactericidal activity. These findings confirmed that D-PM could be e(cid:431)iciently\nincorporated into bacterial cell wall biosynthesis and progressively accumulates within the\npeptidoglycan…\n…Supplementary Fig. 26 | CFU count analysis of S. aureus ATCC29213 at a, Passage 1 (P1), b,\nPassage 2 (P2), and c, Passage 3 (P3) after multiple-round incubations with CS, L-PM, and D-\nPM at 0.5 mg ml-1 every 1 h (n = 6 biological replicates per group; data presented as mean ±\ns.d.). Statistical analysis was performed using a two-tailed unpaired t-test; **P < 0.01, ****P <\n0.0001…\nQ1-7: Line 272: “almost no binding to mammalian cells.” This does not match what is ob-\nserved. There is detectable binding, which could lead to false positives. What hypotheses do\nthe authors have to explain this binding? Furthermore, in the experiment shown in Fig. 6, even\nthough D-PMs are generally no longer detectable in mice after 6 h, they remain present in cer-\ntain organs. This suggests nonspecific binding and the potential for false positives in pathogen\ndetection. These limitations should be discussed.\nA1-7: We thank the reviewer for raising these important points regarding mammalian-cell\nbinding and organ retention. The apparent inconsistency with the statement “almost no bind-\ning to mammalian cells” is mainly due to unclear cell boundaries and the large size di(cid:431)erence\nbetween mammalian cells and bacteria in the original merged images. To address this, we\nadded cell-outline masks derived from the corresponding bright-field images to the overlaid\nfluorescence panels. With these boundaries, FITC-labeled D-PM signals predominantly local-\nize to bacteria rather than to mammalian cells (Fig. R11). Flow cytometry further supports this\nconclusion: FITC-labeled D-PM incubation with mammalian cells alone, or in co-culture with\nbacteria, consistently yields very low FITC-positive mammalian cells. The figures and corre-\nsponding discussion have been updated in the revised manuscript.\nWe thank the reviewer for requesting clarification on nonspecific binding and the poten-\ntial for false positives. Cy5-labeled D-PM signals in uninfected mice decrease over time,\nfollowing a clearance pattern like Cy5-labeled CS or Cy5-labeled L-PM in bacteria-infected mice,\nindicating normal metabolic retention and minor nonspecific interactions. In contrast, in-\nfected mice show a characteristic infection-dependent increase in D-PM signal at the in-\nfected site. This divergence demonstrates that residual organ signals in uninfected animals\nprimarily reflect normal metabolic clearance rather than pathogen-specific binding.\nFig. R11 Representative CLSM images of S. aureus ATCC29213 and NIH 3T3 cells co-cultures\nfollowing incubation with FITC-labeled D-PM at 0.5 mg ml-1 for 1 h: a, wide-field images (Out-\nline the NIH 3T3 cells with white dashed lines) and b, magnified images. S. aureus and NIH\n3T3 cells were stained with Hoechst 33342 (blue). Scale bars represent 20 μm.\nWe have updated the discussion of these limitations and clarified nonspecific retention\nin the revised manuscript (Fig. 4c), with corresponding data in the Supplementary Infor-\nmation (Supplementary Fig. 32).\n…As shown in Fig. 4b, D-PM selectively associated with S. aureus, with approximately 30% of\nbacteria exhibiting FITC-positive fluorescence, whereas binding to mammalian NIH 3T3 cells\nwas much lower…\n…On the other hand, fluorescence signals of D-PM also decreased in uninfected mice within\n4 h, attributable to the absence of bacterial biosynthesis…\n…However, the signal decay of D-PM in uninfected mice followed a similar pattern to that of\nCS and L-PM in bacterial peritonitis models. This infection-dependent retention of D-PM is\nprimarily due to the e(cid:431)icient incorporation into bacterial metabolic pathways linked to cell\nwall biosynthesis, whereas no appreciable D-PM retention is observed in uninfected mice…\n…Outline the NIH 3T3 cells with white dashed lines based on the bright-field images…\nQ1-8: The results with S. aureus are promising in both in vivo models, but it would have been\nuseful to test other bacteria, perhaps another Gram-positive or a Gram-negative species.\nA1-8: We thank the reviewer for this valuable suggestion. To address this point, we performed\nadditional in vivo peritonitis studies using representative Gram-positive S. aureus (Fig. R12),\nGram-positive methicillin-resistant S. aureus (Fig. R13), and the Gram-negative bacterium E.\ncoli (Fig. R14). Across all three pathogens, T/D-PM consistently exhibited the highest antibac-\nterial e(cid:431)icacy, significantly reducing bacterial burdens in the ascitic fluid as well as in major\norgans, including the spleen and lungs. These results are consistent with our original findings\nand further demonstrate the broad-spectrum antibacterial therapeutic e(cid:431)icacy of T/D-PM.\nFig. R12 a, Schematic diagram of antibacterial administrations in the S. aureus ATCC29213\ninfected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic fluid (b),\nspleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independent ani-\nmals; data presented as mean ± s.d.). Statistical analysis was performed using a two-tailed\nunpaired t-test (b-d); **P < 0.01, ****P < 0.0001.\nFig. R13 a, Schematic diagram of antibacterial administrations in the MRSA ATCC43300 in-\nfected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic fluid (b),\nspleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independent ani-\nmals; data presented as mean ± s.d.). Statistical analysis was performed using a two-tailed\nunpaired t-test (b-d); ***P < 0.001, ****P < 0.0001.\nFig. R14 a, Schematic diagram of antibacterial administrations in the E. coli ATCC25922 in-\nfected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic fluid (b),\nspleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independent\nanimals; data presented as mean ± s.d.). Statistical analysis was performed using a two-tailed\nunpaired t-test (b-d); ****P < 0.0001.\nThe new data and corresponding discussion have been incorporated into the revised\nmanuscript and the Supplementary Information (Supplementary Figs. 56 and 57).\n…Following establishment of the S. aureus ATCC 29213 peritonitis model, antibacterial treat-\nment was administered using the di(cid:431)erent antibiotic delivery systems (Fig. 6h). As shown in\nFig. 6i, the bacterial colony count in ascitic fluid was undetectable in the T/D-PM-treated group,\nwhich was markedly lower than that observed in the control group (4.5 × 103 CFU ml⁻¹), the\nfree tetracycline-treated group (1.0 × 103 CFU ml⁻¹), the T/CS-treated group (2.2 × 102 CFU\nml⁻¹), and the T/L-PM-treated group (2.8 × 102 CFU ml⁻¹). Meanwhile, T/D-PM exhibited the\nhighest antibacterial e(cid:431)icacy, e(cid:431)ectively eradicating bacteria in organs, including the spleens\nand lungs (Fig. 6j,k)…\n…Furthermore, T/D-PM exhibited broad-spectrum and potent antibacterial activity in vivo,\ndemonstrating robust therapeutic e(cid:431)icacy in murine peritonitis models challenged with anti-\nbiotic-resistant S. aureus (MRSA ATCC 43300, Supplementary Fig. 56) or the Gram-negative\npathogen E. coli ATCC 25922 (Supplementary Fig. 57). These findings highlight the potential of\nD-PM as a drug delivery platform to enhance antibiotic e(cid:431)icacy and mitigate bacterial re-\nsistance…\nQ1-9: Lines 356–357: the authors suggest that T/D-PM improves wound healing, but what is\ntheir hypothesis for this e(cid:431)ect? Is it significant compared to a non-infected control? Could it\nsimply be that clearing the infection is what enables wound healing?\nA1-9: We thank the reviewer for raising this important point regarding T/D-PM–mediated\nwound healing. Our primary hypothesis is that T/D-PM promotes healing by clearing infection,\nthereby creating a favorable microenvironment for tissue regeneration. To test whether T/D-\nPM exerts additional pro-healing e(cid:431)ects beyond infection clearance, we performed experi-\nments in a non-infected wound model (Fig. R15).\nIn non-infected wounds, both D-PM and T/D-PM significantly accelerated wound closure\nrelative to PBS control. By day 8, the remaining wound areas were 17.7 ± 5.3% (D-PM) and 16.4\n± 3.3% (T/D-PM) of the original size, compared to 45.7 ± 7.9% in PBS-treated controls. These\nresults indicate that D-PM alone is su(cid:431)icient to promote wound healing.\nFig. R15 a, Schematic diagram of drug administrations in the non-infected wound model. b,\nChanges in wound area of mice after di(cid:431)erent administration from day 1 to day 8 (n = 5 bio-\nlogically independent animals; data presented as mean ± s.d.).\nThese new data, along with the corresponding discussion, have been incorporated into\nthe revised manuscript (Supplementary Fig. 52), providing insights into the observed\nwound-healing e(cid:431)ect of D-PM.\n…The enhanced healing observed in infected wounds treated with T/D-PM is primarily attribut-\nable to its potent antibacterial activity, whereas evaluation in non-infected wound models fur-\nther revealed that both D-PM and T/D-PM possessed intrinsic bioactivity that promotes wound\nrepair (Supplementary Fig. 52)…\nQ1-10: This is a very well-designed and well-written study describing an innovative and ele-\ngant approach. Addressing the points above, particularly the mechanistic discussion of po-\ntential resistance, the clarification of Gram-negative results, the possible antibacterial e(cid:431)ect\nof D-PM, and the issue of nonspecific binding, would significantly strengthen the manuscript\nand enhance its impact.\nA1-10: We sincerely thank the reviewer for the positive evaluation and constructive sugges-\ntions. We have addressed all points, including a mechanistic discussion of potential re-\nsistance, clarification of Gram-negative results, antibacterial activity of D-PM, and nonspe-\ncific binding. These revisions have been incorporated into the revised manuscript.\nReviewer #2 (Remarks to the Author):\nIn this work, macromolecular analogues of bacterial peptidoglycan was developed from chi-\nrality-specific biomimetics-D-alanine-conjugated chitosan (CS, (GlcN-D-Ala) ) to construct\nn\nmacromolecules for specific bacteria recognition. Chirality specific bacteria recognition of\nthe D-PM was elucidated in contrast with the L-PM toward various strains. The D-PM was also\nshown to exclusively recognize bacteria and it was inactive toward various eukaryotic cells.\nFurthermore, the bacterial recognition by D-PM was demonstrated in vivo on the infected\nwound and peritonitis model in mice, whereby it showed specific accumulation toward the\ninfecting bacteria. Overall, this finding is a highly noteworthy contribution in particular to the\nstudy of bacterial infection or treatment, and biomedical field in general. But there are com-\nments below that need to be addressed:\nA: We sincerely thank the reviewer for the positive evaluation of our work and for highlighting\nits significance. We have carefully addressed all specific comments, and all revisions and\nclarifications have been incorporated into the revised manuscript to strengthen the mecha-\nnistic discussion, experimental details, and interpretation of our findings.\nQ2-1: The experimental design lacks the characterization on the chemistry side. Can the au-\nthors measure the molecular weight of the CS, and the peptidoglycan mimics series after the\nsyntheses (especially after treatment with TFA)?\nA2-1: We thank the reviewer for this important comment regarding chemical characterization.\nThe molecular weights of both the chitosan backbone and the peptidoglycan mimics were\nmeasured by Gel permeation chromatography before and after TFA treatment (Fig. R16). The\nresults confirm that the polymeric backbone remains intact after synthesis and deprotection,\nwith only minor changes in dispersity.\nFig. R16 Gel permeation chromatography traces of CS, L-PM, and D-PM.\nQ2-2: In general, EDC/NHS chemistry is rather ine(cid:431)icient and here the modification percent-\nages are only around 17%. It means that the D-PM only has 17% of D-Ala moieties along the\nCS chain, which in theory does not di(cid:431)er much from, say, a mixture of CS and D-PM. Why and\nhow could 17% modification result in stark di(cid:431)erence of bacteria recognition?\nA2-2: We thank the reviewer for this insightful question. In our preliminary optimization stud-\nies, we synthesized a series of L-PM bearing L-Ala at modification levels of approximately 7.5\n± 0.3%, 11.3 ± 0.4%, 17.7 ± 0.3%, and 23.5 ± 0.4%, as well as D-PM bearing D-Ala at similar\nmodification levels (7.4 ± 0.6%, 11.6 ± 0.4%, 17.2 ± 0.3%, and 22.9 ± 0.8%) (Fig. R17). We\nquantified bacterial recognition and binding e(cid:431)iciency of L-PM and D-PM toward S. aureus. L-\nPM showed low binding e(cid:431)iciency with only minimal variation across all modification levels\n(positive-cell rates ranging from 2.9 ± 0.2% to 14.1 ± 0.4%), whereas D-PM exhibited a pro-\nnounced density-dependent e(cid:431)ect (positive-cell rates ranging from 27.3 ± 2.7% to 94.1 ±\n0.3%) (Fig. R18). Notably, D-PM achieved maximal recognition at a modification e(cid:431)iciency of\n~17%, reaching a positive rate of ~94%. A further increase to ~23% resulted in reduced label-\ning, likely due to steric hindrance impairing the enzymatic incorporation of densely grafted D-\nAla. Based on these findings, we selected the ~17% modification level for all subsequent stud-\nies.\nFig. R17 Quantification of the modification e(cid:431)iciency for protected L-PM and protected D-PM\nvia fluorescamine assays with di(cid:431)erent feeding ratios corresponding to the CS repeating units:\n10% (PM1), 20% (PM2), 40% (PM3), and 60% (PM4) (n = 3 independent samples per group;\ndata are presented as mean ± s.d.).\nFig. R18 a, Fluorescence spectra of FITC-labeled L-PM and D-PM at 0.02 mg ml-1 with di(cid:431)erent\nfeeding ratios corresponding to the CS repeating units. b, Positive rate of FITC-labeled L-PM\nand D-PM with di(cid:431)erent feeding ratios after incubation with S. aureus ATCC29213 at 0.5 mg\nml-1 for 1 h (n = 3 biological replicates per group; data are presented as mean ± s.d.).\nThe relevant data, including amino acid modification e(cid:431)iciencies and bacterial binding\ne(cid:431)iciencies, have been added to the Supplementary Information (Supplementary Figs. 4\nand 5), and the main text has been expanded to address this point, as detailed below.\n…A series of chiral amino acid-conjugated (GlcN) polymers was synthesized with varying de-\nn\ngrees of L-Ala (7.5 ± 0.3%, 11.3 ± 0.4%, 17.7 ± 0.3%, and 23.5 ± 0.4%) or D-Ala (7.4 ± 0.6%,\n11.6 ± 0.4%, 17.2 ± 0.3%, and 22.9 ± 0.8%) modification, respectively (Supplementary Fig. 4).\nFollowing deprotection, L-PM of (GlcN–L-Ala) and D-PM of (GlcN–D-Ala) were obtained with\nn n\ndistinct Ala modification but identical fluorescein isothiocyanate (FITC) labeling. The bacterial\ninteractions of the chiral PM were quantified using Staphylococcus aureus (S. aureus\nATCC29213) as a model strain. L-PM exhibited consistently low binding e(cid:431)iciency with mini-\nmal dependence on the modification ratio, yielding FITC-positive bacterial populations rang-\ning from 2.9 ± 0.2% to 14.1 ± 0.4% (Supplementary Fig. 5)…\n…In contrast, D-PM displayed a pronounced modification-ratio-dependent recognition be-\nhavior, with the proportion of FITC-positive bacteria increasing from 27.3 ± 2.7% to 94.1 ± 0.3%.\nNotably, maximal bacterial labeling was achieved at a D-Ala modification degree of 17.2%,\nwhereas further increasing the substitution to 22.9% resulted in diminished labeling e(cid:431)iciency,\nlikely due to steric congestion that impairs enzymatic incorporation during cell wall biosyn-\nthesis. On this basis, the 17.2% modification rate of D-PM was selected for all subsequent\nstudies…\nQ2-3: It is known that FITC can readily stain both the intact and compromised bacterial mem-\nbranes, but here the authors claimed that FITC-labelled CS and L-PM were excluded or even\ncleared from bacterial surface. So just how big is the CS and L-PM molecules? Or is there any\nrepulsive chemistry/force between them and the bacterial membrane?\nA2-3: We thank the reviewer for this important question. While free FITC can readily stain bac-\nterial membranes through nonspecific reactions with surface amines, this mechanism does\nnot apply in our experiments. In FITC-labeled CS and L-PM, FITC is covalently conjugated to a\nmacromolecular sca(cid:431)old, and bacterial fluorescence arises exclusively from the interaction\nof the macromolecules with the bacterial surface rather than direct dye staining. The chitosan\nused in this work has a number-average molecular weight of around 5,000 Da (supplier-spec-\nified), which undergoes only minor changes after chiral amino acid functionalization, as con-\nfirmed by Gel permeation chromatography. Thus, FITC-labeled CS and L-PM are macromo-\nlecular probes rather than small freely di(cid:431)usible dyes.\nBacterial surfaces carry a net negative zeta potential (-18.7 ± 0.5 mV), whereas CS, L-PM,\nand D-PM exhibit positive zeta potentials (24.2 ± 0.3 mV, 23.1 ± 0.6 mV, and 23.5 ± 1.3 mV,\nrespectively) (Fig. R19). Accordingly, their initial association with bacteria is primarily driven\nby electrostatic interactions, as further supported by the observed increase in bacterial sur-\nface zeta potential after incubation with these macromolecules (-0.4 ± 0.2 mV, -0.3 ± 0.2 mV,\nand 2.3 ± 0.6 mV, respectively). Importantly, for CS and L-PM, these interactions are non-co-\nvalent and reversible supramolecular associations. As a result, FITC-labeled CS and L-PM can\nbe excluded from or cleared o(cid:431) the bacterial surface over time. In contrast, D-PM acts as a\nnon-native substrate for peptidoglycan biosynthesis and becomes covalently incorporated\ninto the bacterial cell wall, rendering it resistant to surface clearance and enabling durable\nbacterial binding. Together, these findings explain why FITC-labeled CS and L-PM do not pro-\nduce persistent bacterial binding due to their dynamic supramolecular interactions, whereas\nD-PM displays stable bacterial binding through biosynthetic incorporation into the bacterial\ncell wall, despite comparable size and surface charge.\nFig. R19 a, Zeta potential of CS, L-PM, and D-PM (n = 3 independent samples per group; data\nare presented as mean ± s.d.). b, Zeta potential of S. aureus ATCC29213 before (Control) and\nafter treatment with CS, L-PM, and D-PM (n = 3 biological replicates per group; data are pre-\nsented as mean ± s.d.).\nThe corresponding discussion in the revised manuscript has been updated, and the re-\nsults have been incorporated into the Supplementary Information (Supplementary Figs.\n7 and 25), as detailed below.\n…As shown in Fig. 3b, only a small proportion of fluorescent L-PM bound to S. aureus\nATCC29213, always with around 10% of bacteria exhibiting fluorescence. This minimal bind-\ning of L-PM is attributable to weak interactions governed by a dynamic adsorption-desorption\nequilibrium, as evidenced by the reduced bacterial surface zeta potential following supramo-\nlecular electrostatic interactions between L-PM and the bacterial envelope (Supplementary\nFig. 25)…\nQ2-4: In Fig 1c, 3c, 3d, and 4c, I suggest the authors also show the zoom-out images (lower\nmagnification), to show that this specific recognition by D-PM can be observed across all the\nbacteria in the much wider area.\nA2-4: We thank the reviewer for this valuable suggestion. In response, we have added lower-\nmagnification (zoom-out) images for Figs. 1c, 3d, and 4c (Fig. R20, Fig. R21, and Fig. R22) in\nthe Supplementary Information. In these images, the regions shown in the original high-mag-\nnification panels are indicated by white boxes. The zoom-out views demonstrate that the spe-\ncific binding of D-PM is uniform and consistent across a much larger bacterial population,\nconfirming that the high-magnification images shown in the main text are representative.\nFig. R20 Representative CLSM wide-field images (top) and magnified images (bottom) of FITC-\nlabeled CS, L-PM, and D-PM following incubation with S. aureus ATCC29213 at a concentra-\ntion of 0.5 mg ml-1 for 1 h. Scale bars represent 2 μm.\nFig. R21 Representative 3D-SIM wide-field images (left) and magnified images (right) of S. au-\nreus ATCC29213 after incubation with Cy5-labeled D-PM (red) for P1, TAMRA-labeled D-PM\n(purple) for P2, and FITC-labeled D-PM (green) for P3. Scale bars represent 2 μm.\nFig. R22 Representative CLSM images of S. aureus ATCC29213 and NIH 3T3 cells co-cultures\nfollowing incubation with FITC-labeled D-PM at 0.5 mg ml-1 for 1 h: a, wide-field images (Out-\nline the NIH 3T3 cells with white dashed lines) and b, magnified images. S. aureus and NIH\n3T3 cells were stained with Hoechst 33342 (blue). Scale bars represent 20 μm.\nThe results have been incorporated into Supplementary Information (Supplementary Fig.\n14).\nQ2-5: In Supplementary Fig 23, flow cytometry test showed negligible interaction of the pep-\ntidoglycan mimics (D-PM, L-PM and CS) on various eukaryotic cells. Can the authors test the\nbinding characteristics/accumulation on those cells using MST measurement and take the\nLCMS peak too (similar to what the authors had shown on bacteria)? It is necessary because\nthe whole idea emphasizes on selective bacteria recognition/targeting over eukaryotic cells.\nA2-5: We thank the reviewer for this important suggestion. To further substantiate the selec-\ntive recognition of bacteria over eukaryotic cells, we performed microscale thermophoresis\n(MST) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analyses\nusing NIH 3T3 cells as a representative eukaryotic model. MST measurements showed no de-\ntectable specific binding of CS, L-PM, or D-PM to NIH 3T3 cells, with dissociation constants\nbeyond the measurable range, indicating only extremely weak and nonspecific interactions\n(Fig. R23). These results are fully consistent with the negligible association observed by flow\ncytometry. In parallel, UPLC-MS analysis targeting the characteristic peptidoglycan-derived\nmotif (GlcN-D-Ala-D-Ala and GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc) detected no\ncorresponding peaks in NIH 3T3 cell lysates (Fig. R24), confirming the absence of cellular ac-\ncumulation or metabolic incorporation of D-PM in eukaryotic cells.\nFig. R23 MST analysis of the binding a(cid:431)inities of CS, L-PM, and D-PM to NIH 3T3 cells (n = 3\nbiological replicates per group; data presented as mean ± s.d.).\nFig. R24 EIC of a, GlcN-D-Ala-D-Ala and b, GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc fol-\nlowing incubation of NIH 3T3 cells with CS, L-PM, and D-PM.\nThese new data have been included in the revised Supplementary Information (Supple-\nmentary Figs. 28 and 29), with the corresponding discussion provided below.\n…Additionally, MST measurements revealed no detectable specific binding of CS, L-PM, or D-\nPM to NIH 3T3 cells (Supplementary Fig. 28), and UPLC-MS analysis targeting characteristic\npeptidoglycan-derived motifs detected no corresponding signals in NIH 3T3 cells following\nincubation with CS, L-PM, or D-PM (Supplementary Fig. 29)…\nQ2-6: In Fig 2d-g, is it possible that the GlcN-D-Ala-D-Ala intermediate to GlcN-D-Ala-D-Ala-\nL-Lys-D-iGlu-L-Ala-MurNAc might be slightly o(cid:431) (by around 30 m/z maybe)? Can the authors\ndo a confirmation whether this biosynthesis is via MurF ligase (i.e., not GlcN-D-Ala addition\nvia MurC), for example using Edman degradation test (or any test) to confirm?\nA2-6: Thank you for raising these important points. We appreciate the reviewer’s insightful\nquestions regarding the possible mass deviation of the GlcN–D-Ala–D-Ala intermediate and\nthe suggestion to further validate the biosynthetic route. We have provided both theoretical\nand experimental values in the revised manuscript and have also expanded and standardized\nthe MS range. The observed peak at m/z 322.1613 was assigned to the protonated molecular\nion [GlcN–D-Ala–D-Ala + H]⁺ (theoretical m/z 322.1609), which likely arises from the ligation\nof an additional D-Ala to the D-PM substrate (GlcN–D-Ala) catalyzed by D-Ala–D-Ala ligase\n(Ddl). This intermediate, GlcN–D-Ala–D-Ala, can subsequently be incorporated into the cell\nwall biosynthesis pathway to form GlcN–D-Ala–D-Ala–L-Lys–D-iGlu–L-Ala–MurNAc via the bi-\nocatalyst UDP-N-acetylmuramoyl-tripeptide–D-alanyl–D-Ala ligase (MurF), with a character-\nistic ion peak at m/z 925.4353.\nFollowing the reviewer’s suggestion to use Edman degradation to confirm whether the\nbiosynthesis proceeds via MurF ligase rather than MurC-mediated addition of D-Ala to GlcN,\nwe attempted Edman degradation analysis. However, this approach failed because Edman\nsequencing requires relatively pure peptides, which are di(cid:431)icult to isolate from bacterial bio-\nsynthetic processes. As a complementary approach to test whether MurC could be involved,\nwe performed two experiments (Fig. R25). First, UPLC-MS analysis revealed no detectable\nsignal for the putative MurC-dependent intermediate in the relevant reaction mixtures,\nindicating that this intermediate is not formed in our system. Second, in a MurC inhibitor assay,\npre-incubation with a selective MurC inhibitor did not significantly a(cid:431)ect the incorporation of\nD-PM into bacteria, further supporting that the pathway does not rely on MurC activity for the\naddition of D-Ala to MurNAc.\nFig. R25 a, EIC of GlcN-D-Ala-MurNAc following incubation of S. aureus ATCC29213 with CS,\nL-PM, and D-PM. b, E(cid:431)ect of pretreatment of bacteria with the MurC inhibitor β-chloroalanine\n(Cla, 100 μg ml-1) on blocking D-PM binding (n = 3 biologically independent replicates; data\nare presented as mean ± s.d.).\nThank you again for these valuable comments, which have helped us strengthen the dis-\ncussion in the revised manuscript, as detailed below.\n…The observed peak at m/z 322.1613 was assigned to the protonated molecular ion [GlcN–D-\nAla–D-Ala + H]⁺ (theoretical m/z 322.1609), which likely arises from the ligation of an\nadditional D-Ala to the D-PM substrate (GlcN–D-Ala) catalyzed by D-Ala–D-Ala ligase (Ddl, Fig.\n2d,e)27. This intermediate, GlcN-D-Ala-D-Ala, could subsequently be incorporated into the\ncell wall biosynthesis pathway to form GlcN-D-Ala-D-Ala-L-Lys-D-iGlu-L-Ala-MurNAc through\nthe biocatalyst of UDP-N-acetylmuramoyl-tripeptide–D-alanyl-D-Ala ligase (MurF), with a\ncharacteristic ion peak at m/z 925.4353 (Fig. 2f,g)…\nQ2-7: In Supplementary Fig 37, it looks like the T/CS treated wound also showed relatively\nthick dermal layer. Can the authors show the images of all the biological replicates (not just 1\nrepresentative)? I also suggest to measure the dermal (and/or epidermal) thickness of the\nsamples for quantitative data comparison.\nA2-7: We thank the reviewer for this insightful comment. In response, we have included im-\nages from all biological replicates in the revised Supplementary Information (Fig. R26). In ad-\ndition, we performed quantitative measurements of epidermal thickness across all samples\n(Fig. R27). Statistical analysis revealed a significant increase in epidermal thickness in the\nT/D-PM treated group compared with control and other treatment groups, providing quantita-\ntive evidence of enhanced epidermal regeneration.\nFig. R26 H&E staining images of infected wounds after di(cid:431)erent administrations on day 8 (n =\n6 biologically independent replicates). The black dashed line indicates the epithelial\nboundary. Scale bars represent 200 μm and 50 μm, respectively.\nFig. R27 Epidermal thickness after di(cid:431)erent administrations on day 8 (n = 6 biologically inde-\npendent animals). Statistical analysis was performed using a two-tailed unpaired t-test; **P\n< 0.01.\nAll replicated images and the corresponding analysis have been added to the Supple-\nmentary Information (Supplementary Figs. 44 and 45).\nQ2-8: Similarly, for other in vivo images (Figs 5f, 5h, 6b, 6d, 6g, and Supplementary Figs 37,\n38a, 39a) I suggest the authors also show all the biological replicates in the Supplementary\nInformation, to visually gauge the extent of bacteria recognition by the D-PM.\nA2-8: We thank the reviewer for the suggestion to provide all biological replicate images. In\nresponse, we have included the complete set of replicates in the Supplementary Information.\nSpecifically, Masson and CD31 staining replicates are shown in Fig. R28–R29, in vivo imaging\nreplicates in Fig. R30, ex vivo spleen and lung images in Fig. R31–R32, and corresponding fro-\nzen tissue sections in Fig. R33–R34. These comprehensive data consistently demonstrate the\nrobust bacterial recognition capability of D-PM across all biological replicates. Figure legends\nand the manuscript text have been updated to guide readers to this supplementary data.\nFig. R28 Masson-stained images of infected wounds after di(cid:431)erent administrations on day 8\n(n = 6 biologically independent replicates). Scale bars represent 100 μm.\nFig. R29 CD31-stained images of infected wounds after di(cid:431)erent administrations on day 8 (n\n= 6 biological replicates per group). Scale bars represent 400 μm.\nFig. R30 In vivo imaging of Cy5 signals at di(cid:431)erent time points following di(cid:431)erent administra-\ntions (n = 3 biologically independent animals).\nFig. R31 Ex vivo imaging of spleens following di(cid:431)erent treatments over 6 h (n = 3 biologically\nindependent animals).\nFig. R32 Ex vivo imaging of lungs following di(cid:431)erent treatments over 6 h (n = 3 biologically in-\ndependent animals).\nFig. R33 Images of frozen tissue sections from spleens showing drug distribution (n = 3 bio-\nlogically independent animals).\nFig. R34 Images of frozen tissue sections from lungs showing drug distribution (n = 3 biologi-\ncally independent animals).\nQ2-9: The wound model was not splinted, so we need a way to justify healing via re-epitheli-\nalization (inferably due to the infection relief) rather than via contraction. To support this, be-\nsides the information already provided in the trichrome Masson staining, more importantly\nthe characterization of the collagen quality should be assessed to understand the type of new\ntissue that is being built. I suggest to do picrosirius staining to identify and quantify di(cid:431)erent\ntypes of collagen fibers in the tissue sections.\nA2-9: We thank the reviewer for highlighting this important point. To address it, we performed\npicrosirius red staining on wound sections to quantify collagen subtypes. T/D-PM-treated\nwounds showed a significant increase in type I collagen relative to type III, along with an over-\nall increase in total collagen (Fig. R35 and R36), indicating active tissue remodeling with ma-\nture, organized collagen deposition rather than mere wound contraction. These data support\nthat accelerated closure results from e(cid:431)ective infection control, facilitating proper re-epithe-\nlialization and stromal regeneration. The new results and discussion have been incorporated\ninto the revised manuscript and Supplementary Information.\nFig. R35 Picrosirius red staining of infected wounds following di(cid:431)erent administrations on day\n8 (observed under polarized light), showing type I collagen (yellow, orange, or red) and type III\ncollagen (green) (n = 6 biological replicates per group). Scale bar, 50 μm.\nFig. R36 Quantification of the ratio of type I collagen area to type III collagen area across dif-\nferent treatment groups (n = 6 biological replicates per group). Data are presented as mean ±\ns.d.\nThese results have been added to the Supplementary Information (Supplementary Figs.\n48 and 49), with the corresponding discussion provided below.\n…Picrosirius red staining revealed that the proportion of type I collagen relative to the total\ncollagen was increased in the wound tissue of T/D-PM-treated wounds, suggesting enhanced\nreplacement of type III collagen by type I collagen to promote more e(cid:431)ective wound healing\n(Supplementary Figs. 48 and 49)…\nQ2-10: Minor comments:\na) In the title, I suggest to change the word ‘intervention’ with something more meaningful or\nspecific, maybe ‘recognition’?\nb) For all the flow cytometry data/results, we need to see the gating strategy in each of the\ntests.\nc) In Supplementary Fig 12, we also need to see the positive rate of CS and L-PM to the vari-\nous bacteria.\nd) Page 6 line 113 mentioned E faecium, while Fig 1d mentioned E faecails.\ne) In the captions of Supplementary Fig 6a, 29, 30, the replicates are not ‘biological’ repli-\ncates.\nf) The caption of Supplementary Fig 27 mentioned various mammalian cells, not just NIH\n3T3.\ng) In Supplementary Fig 38b, there is a typo ‘collangen’.\nA2-10: We thank the reviewer for the thoughtful comments. Our responses are provided point\nby point below.\na) Title wording: We fully appreciate the reviewer’s point that “pathogen recognition” may be\nmore meaningful or specific. In our original wording, we used “pathogen intervention” to\nencompass both the initial pathogen recognition and the subsequent targeted antibiotic\ndelivery. We would greatly appreciate the reviewer’s guidance on whether it would be pref-\nerable to (i) replace “pathogen intervention” with “pathogen recognition” to emphasize\nspecificity, or (ii) explicitly incorporate both recognition and targeted delivery in the title to\nmore fully reflect the scope of this work. For now, we have retained “pathogen intervention”\nin the title, and we are very happy to revise it based on the reviewer’s preference.\nb) Flow cytometry gating strategy: In response, we have now included detailed gating strate-\ngies for all flow cytometry experiments. The sequential gating steps used to define the tar-\nget populations are provided in Fig. R37–R39, ensuring transparency and reproducibility\nof the analysis.\nFig. R37 Representative gating strategy for bacterial binding assays.\nFig. R38 Representative gating strategy for mammalian cell-binding assays.\nFig. R39 Representative gating strategy for a, bacteria, and b, mammalian cell co-culture as-\nsays.\nThese gating strategies have been added to the Methods section and the Supplementary\nInformation (Supplementary Fig. 63).\n…All representative gating strategies are shown in Supplementary Fig. 63…\nc) Positive rates of CS and L-PM in Supplementary Fig. 13: We agree with the reviewer and\nhave added the corresponding positive rates for CS and L-PM across the tested bacterial\nstrains. These data are now included in Fig. R40.\nFig. R40 Positive rate of FITC-labeled CS, L-PM, and D-PM binding to a, K. pneumoniae\nATCC13883, b, A. baumannii ATCC19606, c P. aeruginosa ATCC27853, and d E. coli\nATCC25922 after 1 h incubation at 0.5 mg ml-1 in PBS (n = 6 biological replicates per group;\ndata are presented as mean ± s.d.). Statistical analysis was performed using a two-tailed un-\npaired t-test (a-d); ****P < 0.0001.\nd) E. faecium vs. E. faecalis: We apologize for this inconsistency. The text has been corrected\nto E. faecalis to match Fig. 1b.\ne) Biological replicates: We thank the reviewer for pointing this out. The captions of Supple-\nmentary Figs. 6a, 29, and 30 have been corrected to accurately describe the replicates. We\nhave also carefully checked the entire manuscript to ensure correct usage of the terms\n“biological” and “technical” replicates throughout.\nf) Caption of Supplementary Fig. 27: We have revised the caption of Supplementary Fig. 27\nto accurately reflect that the experiments involved only NIH 3T3 cells, rather than multiple\nmammalian cell types. The caption has been corrected accordingly.\ng) Spelling error: The typo “collangen” in Supplementary Fig. 38b has been corrected to\n“collagen”. We have additionally performed thorough proofreading of the manuscript to\neliminate similar errors.\nReviewer #3 (Remarks to the Author):\nThis manuscript centres on a “chirality-specific peptidoglycan (PG) biomimetic macromole-\ncule.” The authors graft D-alanine onto chitosan to construct D-PM, achieving e(cid:431)icient,\nbroad-spectrum recognition of multiple pathogenic bacteria—including ESKAPE species,\ndrug-resistant strains, and clinical isolates—while showing negligible binding to eukaryotic\ncells. Mechanistically, D-PM acts as a non-native substrate for bacterial cell-wall biosynthesis\nand is incorporated into downstream metabolism (engaging Ddl and MurF and yielding char-\nacteristic intermediates), thereby enabling durable labeling and targeted delivery. In mouse\nmodels of localized wound infection and systemic peritonitis, D-PM supports imaging and tar-\ngeted delivery of tetracycline, markedly improving antibacterial e(cid:431)icacy and tissue repair. The\nstrengths of the work are: (1) proposing and validating a new concept and molecular design\nparadigm of “D-amino-acid-driven macromolecular substrate assimilation into the bacterial\ncell wall”; (2) assembling a mechanistic package spanning binding a(cid:431)inity, UPLC–MS identifi-\ncation of metabolic intermediates, inhibitor/competition experiments, and molecular dock-\ning/dynamics; and (3) demonstrating translational potential via an integrated diagnose-and-\ntreat workflow from in vitro to in vivo. Overall, the study is innovative and practically relevant\nfor pathogen-specific recognition and antibiotic delivery.\nHaving carefully read the manuscript and Supplementary Information, I find the overall\nconclusions plausible; however, to ensure reproducibility and strengthen the case for clinical\ntranslation, I recommend Major Revision. My main concerns and proposed revisions are:\nA3: We sincerely thank the reviewer for the positive evaluation of our work and for the con-\nstructive comments. We have carefully addressed all concerns raised, and all revisions and\nadditional data have been incorporated into the revised manuscript and Supplementary In-\nformation.\nQ3-1: Insu(cid:431)icient evidence for broad in vivo e(cid:431)ectiveness. The abstract and main text state\nthat D-PM achieves broad recognition across ESKAPE/clinical isolates and is e(cid:431)ective in both\nlocal and systemic infections in vivo. Yet both in vivo models (wound infection and peritonitis)\nare demonstrated only with S. aureus (Gram-positive), with no in vivo data for Gram-negative\npathogens. To substantiate the claim of broad in vivo e(cid:431)ectiveness, please add at least one\nGram-negative animal infection model.\nA3-1: We thank the reviewer for highlighting the need to demonstrate broad in vivo e(cid:431)ective-\nness. To address this point, we conducted additional murine peritonitis studies using E. coli\nas a representative Gram-negative pathogen (Fig. R41), alongside methicillin-sensitive S. au-\nreus (Fig. R42) and methicillin-resistant S. aureus (MRSA, Fig. R43). In all cases, T/D-PM treat-\nment markedly reduced bacterial loads in ascitic fluid, spleen, and lungs, demonstrating ro-\nbust antibacterial activity against both Gram-positive and Gram-negative infections.\nFig. R41 a, Schematic diagram of antibacterial administrations in the E. coli ATCC25922 in-\nfected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic fluid (b),\nspleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independent ani-\nmals; data presented as mean ± s.d.). Statistical analysis was performed using a two-tailed\nunpaired t-test (b-d); ****P < 0.0001.\nFig. R42 a, Schematic diagram of antibacterial administrations in the S. aureus ATCC29213\ninfected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic fluid (b),\nspleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independent ani-\nmals; data presented as mean ± s.d.). Statistical analysis was performed using a two-tailed\nunpaired t-test (b-d); **P < 0.01, ****P < 0.0001.\nFig. R43 a, Schematic diagram of antibacterial administrations in the S. aureus ATCC43300\n(MRSA) infected peritonitis model. b-d, Quantitative results of bacterial colonies for ascitic\nfluid (b), spleens (c), and lungs (d) following di(cid:431)erent treatments (n = 6 biologically independ-\nent animals; data presented as mean ± s.d.). Statistical analysis was performed using a two-\ntailed unpaired t-test (b-d); ***P < 0.001, ****P < 0.0001.\nThese results have been incorporated into the Supplementary Information (Supplemen-\ntary Figs. 56 and 57), and the corresponding discussion in the revised manuscript (Fig.\n6h-k) has been updated to reflect this expanded in vivo validation. The detailed revisions\nare summarized below:\n…Following establishment of the S. aureus ATCC29213 peritonitis model, antibacterial treat-\nment was administered using the di(cid:431)erent antibiotic delivery systems (Fig. 6h). As shown in\nFig. 6i, the bacterial colony count in ascitic fluid was undetectable in the T/D-PM-treated group,\nwhich was markedly lower than that observed in the control group (4.5 × 103 CFU ml⁻¹), the\nfree Tet-treated group (1.0 × 103 CFU ml⁻¹), the T/CS-treated group (2.2 × 102 CFU ml⁻¹), and\nthe T/L-PM-treated group (2.8 × 102 CFU ml⁻¹). Meanwhile, T/D-PM exhibited the highest anti-\nbacterial e(cid:431)icacy, e(cid:431)ectively eradicating bacteria in organs, including the spleens and lungs\n(Fig. 6j,k).\n…Furthermore, T/D-PM exhibited broad-spectrum and potent antibacterial activity in vivo,\ndemonstrating robust therapeutic e(cid:431)icacy in murine peritonitis models challenged with anti-\nbiotic-resistant S. aureus ATCC43300 (MRSA, Supplementary Fig. 56) or the Gram-negative\npathogen E. coli ATCC25922 (Supplementary Fig. 57). These findings highlight the potential of\nD-PM as a drug delivery platform to enhance antibiotic e(cid:431)icacy and mitigate bacterial re-\nsistance…\nQ3-2: Insu(cid:431)icient safety assessment. Safety evidence is largely limited to in vitro assays\n(multi-cell-line viability/Live–Dead showing minimal cytotoxicity) and hemocompatibility (low\nhemolysis), suggesting preliminary compatibility; in vivo observations mainly track body\nweight and histology within infection models, serving more as e(cid:431)icacy/localization readouts\nthan as a conventional toxicology package. For a translational claim, key data are missing,\nincluding serum biochemistry and histopathology of major organs (heart, liver, spleen, lung,\nkidney), complement activation and cytokine panels, immunogenicity/hypersensitivity as-\nsessment, repeat-dose and accumulation toxicity, plasma/tissue PK/BD (including protein\ncorona and blood stability), and potential impacts on commensal microbiota; dose/ exposure\nbridging to clinically comparable regimens and a defined safety window are also absent.\nA3-2: We thank the reviewer for highlighting the need for a comprehensive in vivo safety as-\nsessment. To address this point, we conducted a dedicated toxicity study in mice, in which D-\nPM was administered intraperitoneally at doses of 200 and 500 mg per kg body weight twice\non day 1, followed by a seven-day monitoring period. No significant changes in body weight\nwere observed (Fig. R44). On day 7, complete blood counts (Fig. R45), plasma total immuno-\nglobulin G (IgG) levels (Fig. R46), and plasma biochemistry parameters (Fig. R47), including\nalanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen\n(BUN), remained within normal ranges compared with control groups. Hematoxylin and eosin\n(H&E) histopathological analysis of the heart, liver, spleen, lungs, and kidneys revealed no ap-\nparent lesions (Fig. R48). Collectively, these results establish the primary in vivo safety profile\nof D-PM.\nFig. R44 a, Schematic diagram of the in vivo safety study conducted via intraperitoneal (i.p.)\ninjection. b, Changes in mouse body weight after treatment with PBS, D-PM at 200 mg kg-1 (D-\nPM(I)), and 500 mg kg-1 (D-PM(II)) (n = 6 biologically independent animals; data presented as\nmean ± s.d.).\nFig. R45 a, Blood routine analysis of a, red blood cells (RBC), b, white blood cells (WBC), and\nc, platelets (PLT) after treatment with PBS, D-PM(I), and D-PM(II) (n = 6 biologically independ-\nent animals; data presented as mean ± s.d.). Statistical analysis was performed using a two-\ntailed unpaired t-test; NS, not significant.\nFig. R46 Relative total IgG content in plasma after treatment with PBS, D-PM(I), and D-PM(II)\n(n = 6 biologically independent animals; data presented as mean ± s.d.). Statistical analysis\nwas performed using a two-tailed unpaired t-test; NS, not significant.\nFig. R47 Plasma biochemical analysis of a, ALT, b, AST, and c, BUN after treatment with PBS,\nD-PM(I), and D-PM(II) (n = 6 biologically independent animals; data presented as mean ± s.d.).\nStatistical analysis was performed using a two-tailed unpaired t-test; NS, not significant.\nFig. R48 Representative H&E-stained images of major organs after treatment with PBS, D-\nPM(I), and D-PM(II). Scale bar represents 50 μm.\nThe corresponding data and discussion have been incorporated into the revised manu-\nscript and the Supplementary Information (Supplementary Figs. 58-62). The specific de-\ntails are provided below:\n…Last but most importantly, we conducted a comprehensive in vivo safety evaluation of D-\nPM, which is critical for its future clinical translation. D-PM was administered intraperitoneally\nto female BALB/c mice at doses of 200 or 500 mg per kg body weight twice on day 1, followed\nby a 7-day observation period (Supplementary Fig. 58a). No significant changes in body weight\nwere observed throughout the study (Supplementary Fig. 58b). On day 7, complete blood\ncounts in D-PM-treated mice remained within normal ranges compared with control groups\n(Supplementary Fig. 59)…\n…Consistently, total plasma immunoglobulin G (IgG) levels and plasma biochemistry indica-\ntors, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood\nurea nitrogen (BUN), showed no abnormal alterations (Supplementary Figs. 60 and 61). More-\nover, H&E histological examination of major organs, including the heart, liver, spleen, lungs,\nand kidneys, revealed no apparent pathological lesions (Supplementary Fig. 62). Collectively,\nthese results demonstrate that D-PM exhibits excellent in vivo biocompatibility, even at doses\nexceeding those required for bacterial recognition and antibiotic-targeted delivery…\nQ3-3: Missing structure–activity/structure–performance relationships for the material. The\ncurrent materials exploration uses a single chitosan molecular weight (Mw ≈ 5 kDa) and a sin-\ngle substitution level (~17% D-Ala), which is insu(cid:431)icient to establish the true dependence of\n“incorporation e(cid:431)iciency → bacterial enrichment → therapeutic benefit” on backbone Mw, de-\ngree of substitution, and choice of amino acid. Please, under otherwise matched conditions,\nsystematically vary three axes: (i) several CS backbones of di(cid:431)erent Mw (e.g., low/me-\ndium/high) to assess length e(cid:431)ects on incorporation and in vitro/in vivo enrichment; (ii) a sub-\nstitution gradient at fixed Mw (e.g., ~5–10%, 15–20%, 30–40%) to build dose–response-like\ncurves linking substitution to incorporation/accumulation/e(cid:431)icacy; and (iii) alternative D-\namino-acid anchors at comparable Mw and substitution (e.g., D-Ala vs D-Glu or D-Lys) to clar-\nify how the chiral substrate site governs acceptance by Ddl/MurF and downstream metabolic\npropagation.\nA3-3: We thank the reviewer for the insightful suggestions. To elucidate structure-activity/per-\nformance relationships, we explored the three axes as recommended by the reviewer.\n(i) Chitosan backbone molecular weight (Mw): D-PM probes with backbones of 1,500, 3,000,\nand 5,000 Da (substitution ~17%) were evaluated (Fig. R49). FITC-labeled binding to S. aureus\nshowed a clear length dependence: 5,000 Da > 3,000 Da > 1,500 Da (92.2 ± 0.6%, 83.6 ± 0.9%,\n47.4 ± 3.2%, respectively), indicating that longer backbones enhance multivalent interactions\nand bacterial enrichment (Fig. R50).\n(ii) D-Ala modification rate (at fixed Mw 5,000 Da CS): In our preliminary optimization studies,\na series of L-PM and D-PM constructs with modification levels ranging from ~7% to ~23% were\nsynthesized (L-PM: 7.5 ± 0.3%, 11.3 ± 0.4%, 17.7 ± 0.3%, 23.5 ± 0.4%; D-PM: 7.4 ± 0.6%, 11.6\n± 0.4%, 17.2 ± 0.3%, 22.9 ± 0.8%) (Fig. R51). Binding assays against S. aureus revealed con-\nsistently low labeling for L-PM (<15%) across all modification levels (Fig. R52). In contrast, D-\nPM showed a pronounced density-dependent e(cid:431)ect, reaching maximal recognition at ~17%\nsubstitution (~94% positive cells). Further increasing the modification level (~23%) led to re-\nduced labeling e(cid:431)iciency, likely due to steric hindrance that impairs enzymatic incorporation\nof densely grafted D-Ala. Accordingly, a modification level of ~17% was selected for all sub-\nsequent studies.\n(iii) D-amino acid choice (fixed Mw 5,000 Da, ~17% substitution): GlcN-D-Ala exhibited high\nbinding to S. aureus (92.2 ± 0.6%) (Fig. R53), whereas GlcN-D-Lys showed lower binding at\nthe same modification level (72.8 ± 0.5%) (Fig. R54). Notably, GlcN-D-Lys still displayed par-\ntial bacterial association, consistent with previously reported chiral biological e(cid:431)ects. How-\never, the D-peptidoglycan mimic based on GlcN-D-Ala most closely resembles the native\npeptidoglycan structure, resulting in higher e(cid:431)iciency for bacterial recognition and biosyn-\nthetic incorporation (Nat. Chem. 2019, 11, 335-341).\nIn summary, our structure–activity relationship studies demonstrate that an optimal de-\nsign comprising a ~5,000 Da chitosan backbone, a ~17% D-Ala modification rate, and D-Ala\nfunctionalization in D-PM maximizes bacterial recognition. These findings elucidate key prin-\nciples of chiral material design and further strengthen the mechanistic foundation of our work.\nFig. R49 Quantification of the modification e(cid:431)iciency of protected D-PM via fluorescamine\nassays with di(cid:431)erent molecular weights of chitosan at the same feeding ratios: Mw ≈ 1,500 Da\n(PMI), Mw ≈ 3,000 Da (PMII), and Mw ≈ 5,000 Da (PMIII) (n = 3 independent samples per group;\ndata are presented as mean ± s.d.). Statistical analysis was performed using a two-tailed un-\npaired t-test; NS, not significant.\nFig. R50 a, Fluorescence spectra of FITC-labeled D-PM at 0.02 mg ml-1 with di(cid:431)erent molecu-\nlar weights of chitosan. b, Positive rate of FITC-labeled D-PM with di(cid:431)erent molecular weights\nof chitosan after incubation with S. aureus ATCC29213 at 0.5 mg ml-1 for 1 h (n = 3 biological\nreplicates per group; data are presented as mean ± s.d.). Statistical analysis was performed\nusing a two-tailed unpaired t-test (b); ***P < 0.001, ****P < 0.0001.\nFig. R51 Quantification of the modification e(cid:431)iciency for protected L-PM and protected D-PM\nvia fluorescamine assays with di(cid:431)erent feeding ratios corresponding to the CS repeating units:\n10% (PM1), 20% (PM2), 40% (PM3), and 60% (PM4) (n = 3 independent samples per group;\ndata are presented as mean ± s.d.).\nFig. R52 a, Fluorescence spectra of FITC-labeled L-PM and D-PM at 0.02 mg ml-1 with di(cid:431)erent\nfeeding ratios corresponding to the CS repeating units. b, Positive rate of FITC-labeled L-PM\nand D-PM with di(cid:431)erent feeding ratios after incubation with S. aureus ATCC29213 at 0.5 mg\nml-1 for 1 h (n = 3 biological replicates per group; data are presented as mean ± s.d.).\nFig. R53 Quantification of the modification e(cid:431)iciency of protected GlcN-D-Lys and protected\nGlcN-D-Ala via fluorescamine assays (n = 3 independent samples per group; data are pre-\nsented as mean ± s.d.). Statistical analysis was performed using a two-tailed unpaired t-test;\nNS, not significant.\nFig. R54 a, Fluorescence spectra of FITC-labeled GlcN-D-Lys and GlcN-D-Ala at 0.02 mg ml-\n1. b, Positive rate of FITC-labeled GlcN-D-Lys and protected GlcN-D-Ala after incubation with\nS. aureus ATCC29213 at 0.5 mg ml-1 for 1 h (n = 3 biological replicates per group; data are pre-\nsented as mean ± s.d.). Statistical analysis was performed using a two-tailed unpaired t-\ntest (b); ****P < 0.0001.\nNew data and the corresponding discussion have been incorporated into the revised\nmanuscript and the Supplementary Information (Supplementary Figs. 4 and 5). The de-\ntailed revisions are summarized below:\n…A series of chiral amino acid-conjugated (GlcN) polymers was synthesized with varying de-\nn\ngrees of L-Ala (7.5 ± 0.3%, 11.3 ± 0.4%, 17.7 ± 0.3%, and 23.5 ± 0.4%) or D-Ala (7.4 ± 0.6%,\n11.6 ± 0.4%, 17.2 ± 0.3%, and 22.9 ± 0.8%) modification, respectively (Supplementary Fig. 4).\nFollowing deprotection, L-PM of GlcN–L-Ala) and D-PM of (GlcN–D-Ala) were obtained with\nn n\ndistinct Ala modification but identical fluorescein isothiocyanate (FITC) labeling…\n…The bacterial interactions of the chiral PM were quantified using Staphylococcus aureus (S.\naureus ATCC29213) as a model strain. L-PM exhibited consistently low binding e(cid:431)iciency with\nminimal dependence on the modification ratio, yielding FITC-positive bacterial populations\nranging from 2.9 ± 0.2% to 14.1 ± 0.4% (Supplementary Fig. 5). In contrast, D-PM displayed a\npronounced modification-ratio-dependent recognition behavior, with the proportion of FITC-\npositive bacteria increasing from 27.3 ± 2.7% to 94.1 ± 0.3%...\n…Notably, maximal bacterial labeling was achieved at a D-Ala modification degree of 17.2%,\nwhereas further increasing the substitution to 22.9% resulted in diminished labeling e(cid:431)iciency,\nlikely due to steric congestion that impairs enzymatic incorporation during cell wall biosyn-\nthesis. On this basis, the 17.2% modification rate of D-PM was selected for all subsequent\nstudies…"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all the points raised in my initial review. I therefore recommend acceptance of this manuscript for publication.", "reviewer_2": "I thank and appreciate the revision efforts done by the authors. The additional data, information, and clarification provided in the revision have been sufficiently convincing. I recommend this work without further query.", "reviewer_3": "I have carefully reviewed the revised manuscript and the authors' point-by-point response to the reviewers' comments. I appreciate the substantial effort the authors have made to address my previous concerns with additional experiments and detailed data analysis.\nI have no further questions. I recommend the acceptance of this manuscript for publication in Nature Communications."}, "author_response": "(Reviewer comments are shown in blue, and our responses are in black.)\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all the points raised in my initial review. I therefore recommend\nacceptance of this manuscript for publication.\nA1: We appreciate the reviewer’s acknowledgment that all points raised in the initial review\nhave been addressed and thank you for recommending acceptance.\nReviewer #2 (Remarks to the Author):\nI thank and appreciate the revision e(cid:431)orts done by the authors. The additional data, infor-\nmation, and clarification provided in the revision have been su(cid:431)iciently convincing. I recom-\nmend this work without further query.\nA2: We are grateful that the additional data and clarifications provided have been convincing,\nand we appreciate the reviewer’s recommendation for publication.\nReviewer #3 (Remarks to the Author):\nI have carefully reviewed the revised manuscript and the authors' point-by-point response to\nthe reviewers' comments. I appreciate the substantial e(cid:431)ort the authors have made to ad-\ndress my previous concerns with additional experiments and detailed data analysis.\nI have no further questions. I recommend the acceptance of this manuscript for publication\nin Nature Communications.\nA3: We thank the reviewer for the careful review and recognition of the additional experiments\nand analyses. We are pleased that you have no further questions and recommend our manu-\nscript for publication.\nWe sincerely thank all reviewers for their time, constructive comments, and support."}]

Decision letter