Utilizing nanoinducers for precision degradation of bacterial protein to mitigate antibiotic resistance

Domain: chemistry
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
Antibiotic resistance stands as a formidable global challenge to public health. Herein, we present a bacterial nanoinducer (bacNID) designed for targeted protein degradation in treating bacterial infections. Specifically, bacNID is engineered by grafting targeting peptides of MurD and SspB onto gold nanoparticles (GNPs). MurD plays a pivotal role in peptidoglycan production for cell wall synthesis, while SspB recruits SsrA-tagged proteins for degradation by ClpXP protease. The effectiveness of bacNIDs in targeted MurD degradation via ClpXP is demonstrated across both Gram-positive and Gram-negative bacterial strains. Importantly, prolonged exposure to bacNIDs does not result in the acquisition of resistance in either Staphylococcus aureus (S. aureus) or Salmonella typhimurium (S. typhimurium), even after 25 successive treatment passages. This stands in stark contrast to the rapid emergence of robust resistance observed with norfloxacin, evidenced by a 243-fold reduction in antibacterial activity against S. aureus after just 15 passages, and a 7410-fold decrease in activity against S. typhimurium over 22 passages. Moreover, the antimicrobial potential of bacNIDs is evaluated in vivo using S. aureus-infected nonhealing skin and corneal wounds. In summary, this study unveils a potent nanotechnology-driven strategy for targeted bacterial protein degradation with promising implications for in vivo antimicrobial applications.

Introduction
Antibiotics are critical in protecting individuals from severe bacterial infections by disrupting essential metabolic pathways or cellular structures1,2. However, bacteria inherently acquire resistance to antibiotics over time, despite meticulous prescription and administration practices3,4. Mechanisms contributing to antibiotic resistance encompass modifications in membrane permeability, genetic alterations affecting drug targets, enzymatic inactivation of antibiotics, and active expulsion of antimicrobial agents3,5. Consequently, antibiotic resistance represents a pervasive global menace, compromising the efficacy of conventional antibiotics against bacterial pathogens6. Addressing this formidable challenge necessitates the continual exploration and development of antibacterial agents endowed with innovative mechanisms of action to evade resistance mechanisms. Nonetheless, the pharmaceutical industry still faces many hurdles, with a failure rate of approximately 95% in antibiotic development, coupled with high research and development expenses, making this pursuit economically unsustainable7.

One potentially revolutionary therapeutic approach involves the direct degradation of the protein of interest (POI), circumventing the occupancy-driven activity characteristic of traditional protein inhibitors8,9. This strategy diminishes reliance on high-affinity ligand binding, instead facilitating a catalytic-type mechanism for POI degradation10. Within eukaryotes, the 26S proteasome serves as a canonical organelle responsible for degrading ubiquitin-tagged proteins via a network of ubiquitin ligases11. Utilizing this process, a heterobifunctional molecule has been devised to harness the ubiquitin-proteasome system (UPS) for POI degradation, termed proteolytic targeting chimera (PROTAC)12,13. Subsequently, various other heterobifunctional molecules have been proposed to degrade POI via lysosomes or autophagy in eukaryotic cells14–16. In bacteria, protein degradation occurs in part through the transfer of messenger RNA (tmRNA) reaction, representing a well-studied pathway of regulated proteolysis17. Within this pathway, the SsrA tag attaches to incompletely synthesized polypeptides, subsequently recognized and degraded by the bacterial ClpXP protease18. While ClpXP is proficient in degrading SsrA-tagged proteins, the presence of the SspB adapter enhances the ability of ClpXP to recognize and degrade tagged proteins19,20. Previous research has illustrated the concept of fused protein degradation through direct targeting of the ClpCP protease21. In this study, our objective is to achieve targeted protein degradation in bacteria utilizing the SspB adapter.

UDP-N-acetylmuramoyl-L-alanine-D-glutamate ligase (MurD) is recognized as a prime target for combating antibiotic resistance in bacteria22. Working alongside other Mur ligases, MurD catalyzes the synthesis of peptidoglycan, the predominant structural component in bacterial cell walls, starting from UDP-N-acetylglucosamine (UDP-GlcNAc, Fig. 1a)23,24. Here, we develop a bacterial nanoinducer (bacNID) designed to specifically degrade MurD, effectively inhibiting the growth of both Gram-positive and Gram-negative bacteria. To achieve this goal, MurD-targeting peptides (pMurD) are modified on gold nanoparticles (GNPs), alongside the incorporation of a rapidly degradable SsrA peptide tag (tSsrA, Fig. 1b)25–27. GNPs serve not only as peptide delivery vehicles but also possess the capability to circumvent potential antibiotic resistance mechanisms, including membrane permeability issues and active efflux of antibiotics. By emulating SspB-mediated degradation of SsrA-tagged proteins by ClpXP, bacNIDs can specifically bind MurD, with the SspB adapter actively binding tSsrA on bacNIDs to tether the complex to ClpX, thereby enhancing MurD degradation by ClpXP (Fig. 1c). Treatment of both Gram-positive bacteria (Staphylococcus aureus, abbreviated as S. aureus) and Gram-negative bacteria (Salmonella typhimurium, abbreviated as S. typhimurium) with bacNIDs results in MurD degradation and cell wall damage. Remarkably, no resistance to bacNIDs is observed in either S. aureus or S. typhimurium after 25 continuous passages of treatment. In stark contrast, S. aureus rapidly develops robust resistance to norfloxacin, with a 243-fold reduction in antibacterial activity after just 15 continuous passages. The antibacterial activity of norfloxacin against S. typhimurium decreased 7410-fold over 22 continuous passages. Furthermore, the antibacterial activity of bacNIDs is validated in vivo using S. aureus-infected nonhealing skin wounds and corneal wound models. In summary, this study underscores the feasibility of inhibiting bacterial growth through targeted protein degradation, offering significant potential in mitigating the development of antimicrobial resistance.

Results
Preparation and characterization of bacNIDs
Due to the inherent limitations in membrane permeability of peptides, our initial step involved the synthesis of GNPs via reduction of HAuCl4 with glutathione, followed by modification with pMurD (CIPSHTPRC) and tSsrA (CAANDENYALAA) to form bacNIDs (Fig. 2a). Cysteine was introduced at the N-terminus of the peptides and subsequently modified onto the GNPs through gold-sulfur coordination (Fig. 2a). We commenced by simulating the binding of pMurD to MurD of S. typhimurium through molecular docking. Notably, pMurD formed 8 hydrogen bonds with specific residues of MurD of S. typhimurium, including Glu-158, Ser-117, Asn-139, Asp-186, and Asp-347 (Fig. 2b). Similarly, tSsrA formed nine hydrogen bonds with SspB of S. typhimurium at specific residues, such as Arg-47, Asn-54, Arg-58, and Arg-96 (Fig. 2c). Despite slight structural disparities between MurD or SspB of S. aureus and S. typhimurium, pMurD or tSsrA demonstrated binding affinity towards them, as illustrated in Supplementary Fig. 1. In this study, GNPs served as a versatile nanoplatform for tethering targeting peptides, deviating from the conventional single-chain structure. The ratio and density of pMurD or tSsrA on GNPs could be finely tuned by controlling the feeding ratio. Subsequently, we evaluated the antibacterial activity of bacNIDs at various feeding ratios. As shown in Supplementary Fig. 2, bacNIDs with a feeding ratio of 1:2:2 (GNPs: pMurD: tSsrA) exhibited the highest efficacy in inhibiting the growth of both S. aureus and S. typhimurium, thereby serving as the optimal formulation for subsequent experiments.

Next, we examined the morphology of both GNPs and bacNIDs using transmission electron microscopy (TEM). As shown in Fig. 2d, the TEM image and distribution analysis revealed that GNPs exhibited spherical nanostructures with an average diameter of 1.7 ± 0.2 nm. The conjugation efficiency of the targeting peptides was assessed via high-performance liquid chromatography (HPLC), indicating an efficiency of 90% or 95% for pMurD or tSsrA, respectively (Supplementary Fig. 3a). Notably, peptide modification did not induce significant alterations in the size of bacNIDs (Fig. 2e), as further confirmed by UV-vis absorption analysis. As shown in Supplementary Fig. 3b, the absorbance peak of GNPs is similar to that of bacNIDs. While the zeta potential of GNPs was approximately −19 mV, it increased to approximately −5 mV upon bacNID formation (Supplementary Fig. 3c). Additionally, we synthesized GNPs modified solely with pMurD or tSsrA (referred to as M-GNPs or S-GNPs, respectively) as control groups. TEM analysis revealed that M-GNPs and S-GNPs exhibited a distribution similar to that of bacNIDs, with most particles falling within the 1–2 nm range (Supplementary Fig. 3d). The zeta potential of M-GNPs was positive, while that of S-GNPs was negative (Supplementary Fig. 3c). To validate the membrane permeability of bacNIDs, we conducted confocal imaging after incubating S. aureus or S. typhimurium with fluorochrome-labeled tSsrA or bacNIDs. While free tSsrA exhibited limited cellular penetration, bacNID-treated bacteria displayed robust fluorescence, indicating effective intracellular delivery facilitated by GNPs (Fig. 2f). We also performed high-resolution TEM analyses of S. aureus and S. typhimurium treated with bacNIDs. As shown in Supplementary Fig. 4, bacNIDs were clearly observed on the bacterial surface, within the cell wall, and inside the cytoplasmic compartment of both S. aureus and S. typhimurium. These TEM observations provide direct confirmation of intracellular delivery. In combination with the ultrasmall dimensions of bacNIDs, these results support that these nanoparticles likely enter bacteria primarily through a non-specific diffusion mechanism rather than membrane damage or receptor-mediated uptake.

Specific degradation of MurD and antibacterial activity of bacNIDs
Subsequent to incubating bacNIDs with S. aureus and S. typhimurium for 12 h, we examined the specific degradation of MurD. As shown in Fig. 3a, immunoblot data showed a dose-dependent degradation of MurD induced by bacNIDs in both S. aureus and S. typhimurium. In contrast, GNP, M-GNP, or S-GNP treatment did not exhibit any significant effect on MurD levels in either strain (Fig. 3b). Moreover, immunoblot analyses showed that bacNIDs selectively reduced MurD levels without affecting other Mur ligases (MurA, MurE, MurG) in S. aureus and S. typhimurium (Supplementary Fig. 5). It is also worth noting that a single band was consistently observed in S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (P. aeruginosa), while two bands were detected in Staphylococcus epidermidis (S. epidermidis), S. typhimurium, and Escherichia coli (E. coli, Supplementary Fig. 6). These results suggest that the presence of two bands may be attributed to post-translational modifications or isoforms of MurD in certain bacterial species.

The antibacterial efficacy of bacNIDs was assessed, as shown in Fig. 3c. Turbidity and optical density assays conducted at 600 nm (OD600) clearly demonstrated a significant inhibition of S. aureus growth upon treatment with bacNIDs (Fig. 3c). No such effect was observed with GNPs, M-GNPs, or S-GNPs (Fig. 3c). Similarly, only bacNIDs treatment led to a reduction in the number of viable S. typhimurium following 12 h of incubation, with no discernible impact observed in the control groups (Fig. 3d). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are provided in Supplementary Table 1. BacNIDs with other feeding ratios (1:1:1 and 1:3:3) also inhibited the growth of both S. aureus and S. typhimurium, albeit less efficiently than the 1:2:2 formulation (Supplementary Fig. 7). Furthermore, we performed time-kill assays to directly evaluate bactericidal activity, using norfloxacin as a reference antibiotic. As shown in Supplementary Fig. 8, bacNIDs induced a time-dependent reduction in bacterial load in both S. aureus and S. typhimurium at a concentration of 2×MIC. Maximum bactericidal activity was achieved within 12 h, comparable to the effect observed with norfloxacin.

Moreover, we evaluated the antibacterial activity of bacNIDs under nutrient-limited conditions using a 10:90 medium (10% lysogeny broth: 90% PBS). As shown in Supplementary Fig. 9, bacNIDs retained potent antibacterial activity even under nutrient restriction. In addition, we investigated the activity of bacNIDs against bacterial persisters. Persister populations of S. aureus and S. typhimurium were isolated and subsequently treated with bacNIDs. As shown in Supplementary Fig. 10, bacNIDs induced significant inhibition of persister survival in both species. Immunoblot analyses further demonstrated a dose-dependent degradation of MurD in persister cells, consistent with the observed antibacterial effect (Supplementary Fig. 10). To broaden the scope of our study, we further investigated the antibacterial activity of bacNIDs against S. epidermidis and P. aeruginosa. As shown in Supplementary Fig. 11, measurements clearly demonstrated significant inhibition of bacterial growth in both species upon bacNID treatment. Immunoblot analyses further confirmed a dose-dependent degradation of MurD in S. epidermidis and P. aeruginosa, supporting that the observed antibacterial effect is mediated by selective MurD degradation (Supplementary Fig. 11).

To validate the ClpXP-mediated degradation of MurD, we conducted co-incubation experiments with bacNIDs and ClpP inhibitors, specifically (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one ((3R,4R)-A2-32-01) and hinokiflavone, in bacteria28. As shown in Fig. 3e, Supplementary Fig. 12, co-incubation with ClpP inhibitors effectively inhibited MurD degradation in bacNID-treated S. aureus. Consequently, treatment with ClpP inhibitors markedly attenuated the cytotoxicity of bacNIDs against S. aureus (Fig. 3f, Supplementary Fig. 12). To further validate that the antibacterial activity of bacNIDs arises from targeted MurD degradation, we introduced mutations in the key amino acid residues of tSsrA, thereby abolishing its ability to recognize MurD. Immunoblot analysis demonstrated that bacNIDs no longer induced MurD degradation in S. aureus and S. typhimurium after these mutations (Supplementary Fig. 13). Consistently, turbidity and OD assays revealed that mutated tSsrA abrogated the growth-inhibitory effect of bacNIDs on both species. These findings confirm the utilization of ClpXP by bacNIDs for the degradation of target proteins in bacteria. We also assessed the antibacterial ability of bacNIDs using the colony-forming unit (CFU) assay and survival rate analysis. As shown in Fig. 3g, the number of colonies formed in the groups treated with GNPs, M-GNPs, or S-GNPs resembled those in the PBS group, indicating that these treatments did not induce cytotoxicity against S. aureus or S. typhimurium. In contrast, significantly fewer colonies were observed in plates treated with bacNIDs, indicating effective antibacterial activity (Fig. 3g). Quantitative analysis further confirmed that bacNID treatment substantially inhibited colony formation of both S. aureus and S. typhimurium (~10%) compared to the other groups (more than 95%, Fig. 3h).

Next, we assessed the antibacterial activity by measuring the diameter of the inhibition zone. Figure 3i illustrates that only bacNID treatment exhibited antibacterial activity against S. aureus. The inhibitory diameter measurements revealed that bacNIDs inhibited the growth of S. aureus over 25 mm, whereas S. aureus proliferated extensively around the groups treated with GNPs, M-GNPs, or S-GNPs (Fig. 3i). Consistently, bacNIDs inhibited the growth of S. typhimurium within an area of 24 mm, which was significantly wider compared to the other groups (Fig. 3j). An essential requirement for the development of antibiotics is the ability to selectively kill or inhibit the growth of bacteria without causing harm to the host, known as selective toxicity. To confirm this, we conducted cytotoxicity examinations by incubating bacNIDs with selected NIH/3T3, human umbilical vein endothelial cells (HUVECs), or RAW264.7 cells. As shown in Supplementary Fig. 14a–c, treatment with varying concentrations of bacNIDs did not induce toxicity in any of the three different types of eukaryotic cells, affirming the excellent biocompatibility of bacNIDs. Additionally, sequence alignment revealed that the MurD protein of Bifidobacterium (a probiotic bacterium) shares only 27.86% sequence similarity with that of S. aureus. Consistent with this low level of homology, treatment of Bifidobacterium and Akkermansia muciniphila (A. muciniphila) with bacNIDs did not result in significant antibacterial activity (Supplementary Fig. 15). The stability of bacNIDs was assessed by incubating them in 10% fetal bovine serum (FBS), simulated body fluid, and simulated intestinal fluid for extended periods, followed by evaluation of their antibacterial activity. As shown in Supplementary Fig. 16, bacNIDs retained robust activity against S. aureus and S. typhimurium after incubation, demonstrating favorable stability under physiologically relevant conditions.

It is worth noting that other nanoparticles (NPs) can also serve as peptide conjugation platforms. To evaluate this, we synthesized Pt and Pd NPs and modified them with pMurD and tSsrA, generating Pt-bacNIDs and Pd-bacNIDs, respectively. As shown in Supplementary Figs. 17a, 18a, peptide modification did not alter the morphology of Pt or Pd NPs, and both types maintained a negative surface potential. CFU assay revealed that Pt NPs alone did not exhibit cytotoxicity against S. aureus, with colony numbers comparable to the PBS group (Supplementary Figs. 17b, 18b). In contrast, Pt-bacNIDs significantly inhibited bacterial proliferation, consistent with immunoblot data showing MurD degradation only in the Pt-bacNIDs group. Similar results were obtained in S. typhimurium, confirming the antibacterial activity of Pt-bacNIDs. Likewise, Pd-bacNIDs also effectively suppressed the growth of S. aureus and S. typhimurium by degrading MurD (Supplementary Figs. 17b, 18b). Collectively, these data demonstrate the successful development of bacNIDs for the specific degradation of MurD proteins in bacteria, underscoring their potential as a promising antibacterial therapeutic agent.

Antibacterial mechanism and drug resistance evaluation
After confirming the antibacterial efficacy of bacNIDs, we proceeded to investigate their antibacterial mechanism. Given the role of MurD in peptidoglycan synthesis for bacterial cell wall formation, we assessed the morphological changes in S. aureus and S. typhimurium using scanning electron microscopy (SEM). As shown in Fig. 4a, SEM images revealed the typical spherical structure of S. aureus in the PBS group, unaffected by treatment with GNPs, M-GNPs, or S-GNPs. Conversely, incubation with bacNIDs resulted in severe cell wall damage (Fig. 4a). Rod-like structures characteristic of S. typhimurium were observed in SEM images (Fig. 4b). While S. typhimurium treated with GNPs, M-GNPs, or S-GNPs retained intact rod-like structures, those treated with bacNIDs exhibited complete destruction (Fig. 4b). These SEM findings confirm that MurD degradation significantly impacts bacterial cell wall formation.

A compromised cell wall would facilitate the free passage of extracellular materials. Thus, we conducted a SYTO9-propidium iodide (PI) fluorescent staining assay to assess the membrane integrity of both S. aureus and S. typhimurium. SYTO9, capable of entering bacteria irrespective of membrane integrity, binds to DNA and emits green fluorescence. Conversely, PI cannot penetrate intact bacteria but fluoresces red upon binding to DNA upon entry into the cell. As shown in Fig. 4c, S. aureus treated with PBS, GNPs, M-GNPs, or S-GNPs exhibited strong green fluorescence from SYTO9, with negligible red fluorescence from PI, indicating intact membrane integrity. Conversely, bacNID-treated S. aureus displayed predominantly red fluorescence from PI, significantly stronger than other groups, confirming cell wall damage (Fig. 4c, Supplementary Fig. 19a). Similar trends were observed in staining of S. typhimurium, with predominant red fluorescence evident only in the bacNIDs group (Fig. 4d, Supplementary Fig. 19b). Compromised cell walls can result in the leakage of intracellular components29,30. To quantify this, we measured the DNA and ATP content in the culture supernatants of both S. aureus and S. typhimurium. Our results revealed a significantly higher DNA and ATP concentration in the supernatant of bacNID-treated S. aureus compared to other groups, indicating cell wall damage (Fig. 4e). The amount of DNA or ATP in the S. aureus culture supernatant was significantly higher than that of other groups, which may be due to damaged cell wall. Similarly, measurements in the supernatant of S. typhimurium cultures showed a significant increase in DNA and ATP content following bacNID treatment (Fig. 4f). To further validate this, we measured extracellular alkaline phosphatase (AKP) activity. As shown in Supplementary Fig. 20, AKP activity increased in a dose-dependent manner following bacNID treatment, further supporting cell wall impairment.

As mentioned above, diversifying antibacterial mechanisms is crucial to mitigate the development of resistance. To assess antibacterial resistance, we subjected S. aureus and S. typhimurium to repeated treatments with bacNIDs at a concentration of 0.75×MIC. For comparison, we employed the antibiotic norfloxacin, known for its potent antibacterial activity against both species (Supplementary Fig. 21). Following 16 continuous passages, the MIC of norfloxacin against S. aureus significantly increased, reaching 243 times the original MIC, resulting in the emergence of 5 generations of drug-resistant S. aureus (Fig. 4g, Supplementary Fig. 22). Similarly, for S. typhimurium, the MIC of norfloxacin increased to 7410 times the original MIC after 22 passages, leading to the identification of 8 generations of drug-resistant S. typhimurium (Fig. 4g, Supplementary Fig. 23). These findings underscore the propensity of bacteria to develop resistance over sustained treatment with conventional antibiotics. Interestingly, the MIC of bacNIDs remained unchanged over 25 continuous passages, indicating the inability of S. aureus and S. typhimurium to develop resistance mechanisms against bacNIDs (Fig. 4g). Subsequent treatment of the most drug-resistant strains, S. aureus-5 and S. typhimurium-8, with bacNIDs revealed continued inhibition of growth, akin to the inhibition observed with the original strains (Fig. 4h). Colony formation assays further supported these findings, with significantly fewer colonies observed in bacNID-treated plates compared to those treated with PBS or norfloxacin. In contrast, only a few colonies were observed in the bacNID-treated plate (Fig. 4i).

Moreover, we established resistant strains of S. epidermidis and P. aeruginosa by subjecting them to nine consecutive passages in the presence of norfloxacin. Following this treatment regimen, the MIC values for both bacterial species were markedly elevated (Supplementary Fig. 24a), confirming the successful induction of drug resistance. These resistant strains, designated S. epidermidis-9 and P. aeruginosa-9, were subsequently treated with bacNIDs. Remarkably, bacNIDs continued to effectively inhibit the growth of both resistant strains, showing inhibition comparable to that observed with their respective parental strains (Supplementary Fig. 24b, c). To strengthen this, we further used imipenem to treat S. aureus and S. typhimurium. As shown in Supplementary Fig. 25, serial passaging experiments revealed that the MIC of imipenem against S. aureus increased significantly after five passages. In S. typhimurium, prolonged exposure to imipenem led to a 128-fold elevation of the MIC after 16 passages, clearly demonstrating the acquisition of drug resistance. In sharp contrast, the MIC of bacNIDs remained unchanged after 16 successive passages in both S. aureus and S. typhimurium, further supporting the conclusion that bacNIDs circumvent conventional resistance mechanisms (Supplementary Fig. 25). Together, these results suggest that targeted protein degradation to combat bacteria may offer a promising approach to circumvent the challenge of drug resistance arising from prolonged antibiotic use.

In vivo anti-infectious efficacy of bacNIDs in S. aureus-infected skin wound
To assess the in vivo anti-infection efficacy of bacNIDs, we established a mouse wound model infected with S. aureus and administered various drug formulations, as shown in Fig. 5a. One day post-infection, we topically applied PBS, GNPs, M-GNPs, S-GNPs, or bacNIDs to the wound site. Wound images and counts were obtained every two days. As shown in Fig. 5b, c, the wound size in mice treated with GNPs, M-GNPs, or S-GNPs gradually decreased, albeit insignificantly compared to the PBS group. Notably, treatment with bacNIDs showed no change in wound size on days 1 and 3, but a significant reduction was observed on day 5, with near-complete healing by day 7.

Infection with S. aureus can induce severe inflammation31. Hence, we conducted immunohistochemistry to assess the levels of IL-6 and TNF-α in the wound tissue. As shown in Fig. 5d, tissues infected with S. aureus exhibited extensive IL-6 and TNF-α staining in the PBS, GNPs, M-GNPs, or S-GNPs groups. Importantly, treatment with bacNIDs markedly suppressed the infection of wound tissue (Fig. 5d). Quantitative analysis further confirmed that while GNPs, M-GNPs, or S-GNPs treatment had minimal effects on inflammation compared to PBS treatment, the bacNIDs group demonstrated profound inhibition of inflammation (Fig. 5e). Additionally, we examined the expression of CD31 and VEGF in the wound tissue, as these are crucial for tissue repair32. Immunoblot data revealed a substantial increase in the expression of CD31 and VEGF in wound tissue following bacNID treatment, suggesting enhanced vasculature and growth factors for wound healing (Fig. 5f). Similarly, immunohistochemical staining corroborated robust expression of CD31 and VEGF in bacNID-treated wound tissues, significantly higher than other groups (Fig. 5g, h). Importantly, we observed no significant changes in the body weight of mice during treatment, suggesting that bacNIDs may possess a favorable safety profile in mice (Supplementary Fig. 26). To investigate the biodistribution of bacNIDs, we labeled the nanoparticles with Cy5.5 and monitored their localization after topical application to the wound site using the IVIS animal imaging system. As shown in Supplementary Fig. 27a, b, bacNIDs predominantly accumulated at the wound site for up to 3 days, with fluorescence signals disappearing by day 5, likely due to eschar detachment. Inductively coupled plasma mass spectrometry (ICP-MS) analysis further confirmed that most bacNIDs remained at the wound site and gradually decreased over time (Supplementary Fig. 27c). Only minimal amounts entered systemic circulation, with renal clearance as the primary elimination pathway, likely due to the relatively small nanoparticle size (Supplementary Fig. 27c).

Next, we utilized diabetic mice to establish typical chronic non-healing skin wounds, representing a significant clinical challenge. Wounds were created on the dorsal surface of diabetic mice using a biopsy punch and subsequently infected with S. aureus. One day post-infection, bacNIDs and other formulations were topically applied at the wound site (Fig. 6a). As shown in Fig. 6b, c, treatment with bacNIDs completely eradicated S. aureus infection, leading to wound healing within 10 days, whereas other treatments resulted in incomplete recovery ranging from 34% to 45%. Immunohistochemical staining for IL-6 and TNF-α revealed that only bacNID treatment effectively suppressed wound inflammation, while GNPs, M-GNPs, or S-GNPs showed minimal inhibition of inflammation compared to the PBS group (Fig. 6d, e). Epithelial cell migration and proliferation are crucial for skin wound healing. Hematoxylin and eosin (H&E) staining demonstrated that post-injury re-epithelialization was hindered in the PBS, GNPs, M-GNPs, and S-GNPs groups, likely due to S. aureus infection (Fig. 6f). Conversely, treatment with bacNIDs expedited epithelialization of the wound site, with numerous blood vessels observed in bacNID-treated wounds but not in other groups (Fig. 6f). Masson trichrome (MT) staining was employed to analyze the healing effect in vivo by quantifying the amount of collagen tissue. As shown in Fig. 6g, Supplementary Fig. 28, only bacNID treatment exhibited intact collagen organization and the highest amount of collagen (stained in blue). In contrast, collagen tissue in the GNPs, M-GNPs, and S-GNPs groups was severely damaged, with significantly lower total amounts compared to the bacNIDs group (Fig. 6g, Supplementary Fig. 28). The body weight of diabetic mice remained relatively unchanged during treatment (Supplementary Fig. 29). Together, these findings from the wound healing model demonstrate that bacNIDs effectively inhibit the growth of S. aureus in skin tissue.

In vivo anti-infectious properties of bacNIDs against S. aureus-infected nonhealing keratitis model
After confirming their anti-infectious effects using wound models, we next explored the clinical translational potential of bacNIDs for the treatment of bacterial keratitis. A keratitis model of S. aureus infection was established in diabetic mice and treated with various drug formulations 24 h later (Fig. 7a). As shown in Fig. 7b, an oval-shaped focal abscess was seen on day 0, accompanied by gray-white corneal stromal infiltrate with sharp boundaries. Due to S. aureus infection, the stromal lesions in the PBS, GNPs, M-GNPs, or S-GNPs groups slowly shrunk on day 20, and corneal transparency slightly improved (Fig. 7b). In contrast, bacNID treatment minimized the stromal lesions, and corneal transparency became clear after day 15 (Fig. 7b). Clinical scoring further demonstrated the efficacy of bacNID treatment, evident by day 10 and consistently enhanced on days 15 and 20, with significantly lower scores compared to other groups (Fig. 7c).

S. aureus infection can disrupt fibronectin-binding proteins, collagen-binding adhesion, and other crucial components in the cornea33. As shown in Fig. 7d, H&E and MT staining revealed an irregular arrangement of corneal epithelial cells, stromal edema, and significant inflammatory cell infiltration in PBS-treated mice. Minimal improvement was observed with GNP, M-GNP, or S-GNP treatment (Fig. 7d). In contrast, only bacNID treatment restored the three-layer structure of the cornea to near-normal histological appearance, indicating accelerated lesion shrinkage and robust recovery of corneal function (Fig. 7d). Additionally, there were no significant changes in the body weight of treated mice (Supplementary Fig. 30a). H&E staining of major organs further confirmed that bacNID treatment did not induce notable histological damage (Supplementary Fig. 30b). Hematological examination also revealed no significant differences in bacNID-treated mice compared to the PBS group (Supplementary Fig. 31). These findings underscore the superior antibacterial efficacy and minimal side effects of bacNIDs.

In vivo anti-infectious efficacy of bacNIDs in S. typhimurium-induced colitis model
To directly assess the antibacterial efficacy of bacNIDs against S. typhimurium in vivo, we established a mouse infection model. After successful bacterial colonization, bacNIDs were orally administered to mice twice at 12-h intervals (100 mg/kg per dose), maintaining the same daily dosage throughout the treatment period. Ciprofloxacin treatment was included as a positive control. In the untreated infection group, no mortality was observed until day 5 post-infection, and by day 10 only 50% of the animals survived (Supplementary Fig. 32a). In contrast, all mice treated with bacNIDs or Ciprofloxacin survived throughout the 10-day observation period (Supplementary Fig. 32a). Body weight monitoring revealed that S. typhimurium infection led to a marked reduction in body weight, whereas bacNIDs or Ciprofloxacin treatment significantly restored weight loss (Supplementary Fig. 32b). Similarly, colon length-significantly shortened in the infection group compared with healthy PBS controls-was restored by bacNID or Ciprofloxacin treatment (Supplementary Fig. 32c, d).

Histopathological analyses further supported these protective effects. Infected mice exhibited submucosal edema, depletion of goblet cells, and severe damage to intestinal villi, whereas animals treated with bacNIDs or Ciprofloxacin displayed preserved villi architecture, normal goblet cell numbers, and only mild edema, comparable to healthy controls (Supplementary Fig. 32e). Consistently, cytokine measurements showed significantly lower levels of IL-6 and TNF-α in colon tissue from bacNID- or Ciprofloxacin-treated mice than in the untreated infection group (Supplementary Fig. 33). Hematological analysis revealed no significant differences between bacNID-treated and control groups (Supplementary Fig. 34a). Moreover, liver, kidney, and cardiac function markers remained within normal ranges, indicating that bacNIDs did not induce systemic toxicity (Supplementary Fig. 34b). Taken together, these results demonstrate that bacNIDs provide strong protection in an S. typhimurium infection model, comparable to Ciprofloxacin, thereby validating the in vivo antibacterial efficacy of our nanoplatform.

Methods
Materials
HAuCl4 was purchased from Sigma Co., Ltd. (Shanghai, China). L-GSH was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The (3R, 4R)-A2-32-01 was purchased from MCE Co., Ltd. (Beijing, China). Luria-Bertani (LB) medium and agar were purchased from Sigma Co., Ltd. (Shanghai, China). The Oxford cup was purchased from Biosharp Co., Ltd. (Beijing, China). PI and SYTO9 green fluorescent nucleic acid stain were purchased from Sigma Co., Ltd. (Shanghai, China). Skin puncher was purchased from Miltex Co., Ltd. (Beijing, China).

Synthesis of bacNIDs
Freshly prepared L-GSH solution (50 mM, 300 μl) and HAuCl4 solution (50 mM, 200 μl) were mixed in 4 ml ultrapure water at room temperature. The mixture was heated to 70°C under gentle stirring (300 rpm) for 24 h. The reaction mixture was cooled to room temperature and stopped by mixing with 5 ml isopropyl alcohol. After centrifugation at 18,000 × g for 10 min, GNPs were obtained by redispersing in 3 mL ultrapure water. pMurD and tSsrA were added to the above solution, and the mixture was heated to 37°C and stirred at 300 rpm for 12 h. M-GNPs and S-GNPs were obtained in the same way by adding relevant peptides. Peptides (95% purity) were supplied by the Guoping Pharmaceutical Co., Ltd.

Characterization of GNPs, M-GNPs, S-GNPs, and bacNIDs
For morphology characterization, samples were dropped on carbon-coated copper grids and imaged directly by TEM (Tecnai G2 F20 U-TWIN, FEI, USA). The hydrodynamic diameter and surface charge of the nanoparticles were determined by DLS (Nano ZS, Malvern, UK). LC-20AT HPLC was used to determine the binding efficiency of peptides (Shimadz, Japan).

Antibacterial activity evaluation
All utensils were autoclaved at 121°C for 20 min to ensure sterility. S. aureus (ATCC 6538) and S. typhimurium (ATCC 14028) were grown overnight in the LB liquid medium and harvested at the exponential growth phase via centrifugation. The supernatant was then discarded, and the cell pellet was resuspended in PBS. The bacteria concentration was monitored photometrically by measuring OD at wavelength of 600 nm. The OD600 values of bacteria stock solutions were adjusted to 0.1, corresponding to the concentration of 108 colony-forming units per milliliter (CFU/ml). For the plate count method, the bacteria with an approximate cell concentration of 104 CFU/ml were inoculated in PBS containing GNPs, M-GNPs, S-GNPs or bacNIDs at indicated concentrations. The mixture was applied uniformly on three LB agar plates per gradient solution. These plates were incubated at 37°C for 12 h. The resulting colonies were counted, and the percentage of live bacteria was determined.

For the bacterial Live/Dead staining assay, the bacteria were incubated with GNPs, M-GNPs, S-GNPs or bacNIDs in LB medium at 37°C for 12 h, respectively. Next, PI (75 μM) and SYTO9 (62.5 μM) were added to stain the bacteria for 30 min. After staining, the bacterial suspension was centrifuged to remove the materials and dyes in the medium and washed three times with PBS. Last, the fluorescence images were obtained on a Zeiss 760 confocal microscope. Excitation/emission wavelength of 515/635 nm and 488/500 nm were applied for PI and SYTO9, respectively.

A single colony of the strain was inoculated into LB liquid medium, and 100 μL of bacterial suspension grown to an OD of 1 at 600 nm was spread evenly on the LB solid medium in petri dishes. Oxford cups were then placed in petri dishes. Then GNPs, M-GNPs, S-GNPs or bacNIDs were added to the Oxford cups. The inhibitory effects of nanoparticles on the strain were assessed after 12 h by measuring the diameter of the bacteriostatic circle.

Time-kill curve
A single colony was selected and inoculated into LB broth for overnight culture at 37°C with shaking at 200 r/min. The overnight bacterial culture was diluted 1:1000 in fresh LB broth and adjusted to approximately 1 × 105 CFU/mL. Norfloxacin and bacNIDs were added to the diluted bacterial suspension. The cultures were then incubated at 37°C with shaking at 200 r/min. At 0 min, 2 h, 4 h, 8 h, and 12 h of incubation, 100 μL aliquots were collected, serially diluted 10-fold, and plated onto LB agar plates. After incubation at 37°C for 24 h, CFU were counted. A curve was plotted with time as the x-axis and the logarithm of bacterial count as the y-axis.

Morphological characterization of bacteria
S. aureus and S. typhimurium suspension was treated with GNPs, M-GNPs, S-GNPs or bacNIDs at 37°C for 12 h. Then, bacteria were harvested by centrifugation (1000 rpm) for 3 min and rinsed with PBS, followed by fixing in a centrifuge tube with diluted glutaraldehyde solution (2.5%) for 12 h. After that, the samples were washed three times (15 min each time) in PBS, dehydrating with the sequential treatment of ethanol-water mixtures (30%, 50%, 70%, 90%, 95%, and 100%) for 10 min in each mixing ratio at room temperature. Finally, dried bacteria were sputter-coated with gold. The morphology and elemental analysis of the bacteria were characterized on an SEM (Hitachi SU-8010, Japan) with EDS equipped.

Measurement of DNA and ATP
The levels of DNA and ATP in bacterial supernatants after different treatments were measured. The OD260 nm was measured with a Nano Drop UV–vis spectrophotometer to show the amount of released DNA. ATP levels were determined using an ATP assay kit (MAK190-1KT, Sigma-Aldrich) as instructed. Fluorescence intensity was assessed by a microplate reader.

Synthesis of Pd and Pt NPs
To synthesize Pt NPs, 2 mL of 50 mM chloroplatinic acid hexahydrate (H2PtCl6·6H2O) was dissolved in 18 mL of ethylene glycol. This solution was mixed with 200 mg of polyvinylpyrrolidone (PVP) in a round-bottom flask. The mixture was heated at 180°C in an oil bath for 10 min. After cooling, 40 mL of acetone was added to precipitate the nanoparticles. The precipitate was collected by centrifugation at 11,000 rpm for 5 min and washed with acetone and n-hexane to yield purified Pt NPs.

The synthesis of Pd NPs was adapted from a previously reported method34. The detailed procedure is as follows: Dissolve 105 mg of polyvinylpyrrolidone (PVP), 60 mg of L-ascorbic acid, and 300 mg of potassium bromide (KBr) in 8 mL of deionized water, then heat the solution in an oil bath at 80°C for 5 min. Next, dissolve 57 mg of sodium tetrachloropalladate(II) (Na2PdCl4) in 3 mL of deionized water and add it to the reaction mixture, followed by heating in an oil bath at 80°C for 3 h. After the reaction is complete, add 30 mL of acetone and centrifuge the mixture at 13,000 rpm for 5 min. Discard the supernatant and wash the precipitate with ethanol and acetone.

Cytotoxicity assay
RAW264.7, NIH/3T3 or HUVECs were incubated for 12 h in a 96-well plate, each well containing 1 × 104 cells. The medium of the 96-well plate was discarded, then washed with PBS. Subsequently, complete medium containing GNPs, M-GNPs, S-GNPs or bacNIDs at a serial concentration was added to a 96-well plate, followed by incubation for 24 h. CCK-8 assay was performed according to a standard protocol to determine cell viabilities.

Immunoblotting
Bacteria of S. aureus and S. typhimurium suspension were treated with GNPs, M-GNPs, S-GNPs or bacNIDs at 37°C for 12 h. The collected cells were washed with PBS three times and lysed in RIPA lysis buffer containing 1% (v/v) protease inhibitor (phenylmethylsulfonyl fluoride, PMSF). Total protein concentration was determined using a BCA kit (Servicebio, Wuhan, China), and 20 μg total protein was resolved by SDS-PAGE (10% acrylamide) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Merck, Germany). The membranes were analyzed by immunoblotting with primary antibodies as followings: MURD (orb822688, dilution 1:1000) and RNAP (ab191598, dilution 1:1000).

For in vivo, collection of skin from wounds in mice were lysed in RIPA lysis buffer containing 1% (v/v) PMSF. Treatment is as described above, membranes were analyzed by immunoblotting with primary antibodies as followings: VEGF (ab32152, dilution 1:1000), CD31 (ab182981, dilution 1:1000).

Evaluation of antibacterial resistance
S. aureus and S. typhimurium were inoculated and cultured in LB medium at 37°C for 12 h, and the bacteria were diluted to 1 × 104 CFU/mL in LB medium as the working suspension. After adding bacNIDs or norfloxacin at a concentration of 0.75× Minimum inhibitory concentration (MIC), respectively, into the bacterial working suspension, the mixtures were incubated at 37°C for 12 h. This cell suspension was diluted to 1 × 104 with LB medium in a new container for another cycle of bacteria-drug incubation. This cycle was repeated every 12 h. The concentration of the antibiotic and bacNIDs was adjusted accordingly to remain at 0.75× MIC. MIC tests were repeated at least twice at different times.

Determination of AKP activity
For qualitative and quantitative assessment of AKP activity in cells, the supernatant of bacNIDs bacterial culture treated with different concentrations was collected. The level of AKP was measured using a commercially available kit according to the manufacturer’s instructions, and the absorbance was detected at a wavelength of 450 nm.

Construction of mutant peptides
Molecular docking was performed between pSsrA and the receptor protein, and PyMOL was used to visualize the key residues involved in the binding of pSsrA to the receptor protein. These key residues were mutated to alanine to generate a mutant peptide of pSsrA, with the amino acid sequence CAAAAANAALAA.

In vivo treatment of S. aureus-infected skin wound
All animal experiments were performed in accordance with relevant national laws and regulations and followed institutional guidelines approved by the Institutional Animal Care and Use Committee of the National Center for Nanotechnology (NCNST21-202502-0008). BALB/c mice or db/db mice (female, 6 weeks old) were used for the antibacterial study. Wound was prepared on the backbone of each mouse with a surgical scalpel over the surface as a circle (diameter of 10 mm). The mice (n = 5) were randomly divided into four groups: PBS, GNPs, M-GNPs, S-GNPs or bacNIDs group. S. aureus suspension (1 × 108 CFU/mL, 50 μL) was used to infect the skin wound of all mice. After 24 h, the pus was observed on the infected wound, and PBS, GNPs, M-GNPs, S-GNPs or bacNIDs (75 μM, 50 μL) were smeared on the wounds. The changes in the size of wounds were monitored by a video camera throughout the treatment process. Wound diameter was measured by a vernier caliper every 2 days. After 7 (BALB/c mice) or 12 (db/db mice) days, the tissue samples were harvested for pathological histology and immunohistochemistry analysis.

In vivo treatment of S. aureus-infected keratitis model
Db/db mice (male, 6 week-age) were used for this experiment, and the whole corneal epithelium was scraped with a surgical scalpel under a stereomicroscope (Olympus SZ61, Japan). S. aureus suspension (1 × 108 CFU/mL, 20 μL) was used to infect the corneal wound of all mice. The mice (n = 5) were randomly divided into five groups as mentioned above. After 48 h of incubation, opacity and edema emerged in the eyeball, indicating the keratitis model had been successfully established. PBS, GNPs, M-GNPs, S-GNPs or bacNIDs (75 μM, 50 μL) were smeared on the cornea. Keratitis clinical scores were evaluated by three oculists following three criterions (opacity area, surface regularity, opacity density) every 5 days. After 20 days, the tissue samples were collected for pathological histology analysis.

Immunohistochemical staining of S. aureus-infected tissue
Tissues were fixed in 4% paraformaldehyde (10 min at room temperature), washed twice in PBS, blocked with sheep serum and 0.3% Triton X-100 in PBS for 30 min at room temperature, and incubated with IL-6 (ab290735, dilution 1:200), TNF-α (ab183218, dilution 1:200), VEGF (ab32152, dilution 1:200), CD31 (ab182981, dilution 1:200), antibodies at 4°C for 12 h. These sections were washed with washing buffer at least three times and covered with SignalStain® Boost IHC Detection Reagent HRP Rabbit (CST, Shanghai, China) for 1 h at room temperature. Afterwards, each section was incubated with 100 μl SignalStain® DAB (CST#8059) for 5 min and immersed in deionized water three times. The slides were examined using an inverted microscope.

In vivo treatment of S. typhimurium-infected colitis model
BALB/c mice were pretreated with streptomycin (20 mg per mouse) by oral gavage 24 h prior to bacterial infection, followed by a 12-h fasting period. The mice (n = 10 per group) were randomly divided into four groups: control, S. typhimurium, ciprofloxacin, and bacNIDs. Except for the control group, all other groups were orally inoculated with S. typhimurium at a dose of 5 × 108 CFU per mouse to induce colitis. 24 h post-infection, treatment was initiated via daily oral gavage for 10 consecutive days with the following: control group received PBS (100 μL); S. typhimurium group received PBS (100 μL); ciprofloxacin group received ciprofloxacin (16 mg/kg); and bacNIDs group received bacNIDs (100 mg/kg). Body weight was monitored daily throughout the treatment period. Upon completion of the treatment, colon length was measured, and liver and spleen samples were collected for further analysis.

Statistical analysis
All values are presented as the mean ± s.d. unless otherwise indicated in the figure legend. One-way analysis of variance was used for multiple comparisons when more than two groups were compared. Statistical calculation of two groups of experimental data was performed using a two-tailed Student’s t-test. Statistics were performed using GraphPad Prism 8 software. The threshold for statistical significance was < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, the authors designed a bacterial nanoinducer of bacNIP for targeted protein degradation in treating bacterial\ninfection. The bacNIP is developed by co-conjugating targeting peptides of MurD and SspB onto gold nanoparticles, which\nfacilitate the degradation of MurD by ClpXP in bacteria. Such BacNIPs showed a broad antibacterial spectrum including S.\naureus and S. typhimurium, without causing new resistance even after 25 passages, which is superior to antibitoics like\nnorfloxacin. In particular, the bacNIPs showed therapeutic effects in three in vivo infection models. The design is smart and\nthe antibacterial effects are significant for developing novel antibacterial candidate to overcome the challenge of bacterial\nresistance. I think the work merits the consideration of publication on Nature Communications. However, before considering\nthe acceptance, below concerns and questions need to be carefully addressed.\n1. Gold nanoparticles were used as the carrier for two peptides. Is there any special reason to use gold nanoparticles? It\nseems that any other nanoparticles can be used as long as they can be modified for peptide conjugation. In addition, what is\nthe destination of gold nanoparticles after infection treatment? How can they be degraded or metabolized in vivo?\n2. The MurD-targeting peptide sequence is CIPSHTPRC, which contains two cysteine. For the conjugation on gold\nnanoparticles, how to determine which cysteine interacts with gold surface? Does the direction of pMurD on gold\nnanoparticles affect the function of peptide to recognize MurD?\n3. MurD was selected as the targeted protein for degradation to kill bacteria. However, there are many types of Mur ligases in\nbacteria. The family of Mur ligases contains MurA-G (Molecules 2023, 28(24), 8076). Is it sufficient for bacterial killing if just\nMurD is degraded? Can pMurD bind to other type of Mur ligases in bacteria?\n4. Two bacteria, S. aureus and S. typhimurium, have been tested in the experiments. The type of bacteria is limited, making\nit hard to conclude that bacNIPs can kill both Gram-negative and Gram-positive bacteria. In particular, no antibiotic-resistant\nbacteria strains have been tested. More bacteria and those with antibiotic resistance are suggested to be tested with\nbacNIPs. In addition, if there is MurD mutant strain, it also needs to be tested.\n5. In Figure 2f, the authors claimed that effective intracellular delivery was facilitated by GNPs according to robust\nfluorescence. However, such evidence might be insufficient. High resolution TEM is suggested to determine the localization\nof GNPs in bacteria. Furthermore, the mechanism for GNPs entry into bacteria needs to be carefully elucidated, which is\nvery important for the whole design for antibacterial action. Currently the mechanism of how bacNIPs enter into bacteria\nremains unclear.\n6. Both peptides of pMurD and tSsrA are for proteins in S. typhimurium. How can they interact with proteins in other bacterial\nspecies? Are both proteins very conservative in all bacterial species? If yes, can they kill probiotic bacteria? In addition, the\ntest on in vivo model with S. typhimurium infection is also preferred, as the original design of bacNIPs was from this bacteria.", "reviewer_2": "This manuscript designed a bacterial nanoinducer of proximity (bacNIPs) that can specifically target and degrade MurD, an\nenzyme crucial for peptidoglycan synthesis in bacteria. Notably, bacNIPs demonstrate efficacy in treating S. aureus-infected\nskin and corneal wounds in vivo and show promise in combating bacterial infections without inducing resistance. The\nfindings of this study are interesting and highlight the potential of targeted protein degradation as an effective strategy to\ncombat bacterial infections, while there are still some critical issues that need to be addressed.\n1. Regarding the synthesis of bacNIPs, how to determine the specific feeding ratios of bacNIPs. In addition, why only\nbacNIPs with a feeding ratio of 1:2:2 (GNPs: pMurD: tSsrA) showed antibacterial activity.\n2. While the design of the nanocarriers is of interest, their high production costs may limit their clinical applications. What are\nthe advantages of targeted protein degradation nanodrugs over traditional antibiotic therapy. Meanwhile, the stability, safety\nand bioavailability of bacNIPs needs to be further studied and verified.\n3. MurD is involved in the synthesis of cell wall peptidoglycan, but Gram-negative bacteria have no intact cell wall, so why\ntargeting MurD is also effective against Gram-negative bacteria. In addition, the presence of outer membrane barrier in\nGram-negative bacteria may prevent the entry of bacNIPs, thereby constraining its activity.\n4. Fig. 3(a-b) shows that the MurD band of S. aureus is single, but S. typhimurium has two bands. Is this caused by the\nmodification of protein in the bacteria or other reasons? In the evaluation of the antibacterial activity of bacNIPs, the\nminimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) should be tested. Moreover, a time-\nkilling curve should be added to verify the bactericidal activity of bacNIPs, not only the inhibition of bacterial growth by OD\ndetection. Antibiotics should be used as the control.\n5. It is unclear whether the S. aureus and S. typhimurium used in this manuscript are standard or resistant strains. The\nstrains information should be provided in the supplementary materials. Does bacNIPs have similar antibacterial effects\nagainst drug-resistant strains?\n6. In the mechanism verification, the targeting and degradation of MurD by bacNIP can directly produce antimicrobial activity,\nwhether the knockout of MurD will cause direct bacterial death? Line 168, only a potential ClpP inhibitor was used to\nvalidate the ClpXP-mediated degradation of MurD. The construction of knockout and/or overexpressed strains of ClpXP\nwould strengthen these results.\n7. In the process of verifying bacNIPs specific targeting MurD, it should be considered whether the nano-preparation has a\ncomparable antibacterial effect under low nutrient conditions, as ClpP is an ATP-dependent protease. In addition, whether\nbacNIPs is also active against bacterial persisters.\n8. Given the role of MurD in peptidoglycan synthesis for bacterial cell wall formation, the direct effect of bacNIPs on cell wall\nsynthesis should be assessed, rather than focusing on the changes of membrane permeability. Why bacNIPs targeting MurD\ncan result in the membrane damage, which was different from cell wall damage. Line 227, the authors stated that the\ncompromised cell walls can result in the leakage of intracellular components, please add related references.\n9. Norfloxacin was used as an antibiotic control in this study. Why not use antibiotics targeting cell walls as a positive\ncontrol, such as carbapenems and cephalosporins, which are not easy to induce drug resistance. And Fig. 4(i) did not\nindicate the specific concentration used for norfloxacin or bacNIPs.\n10. The antibacterial effect of bacNIPs was verified in three animal models of S. aureus infection. What is the difference\nbetween the two models of skin infection and what does “nonhealing skin wound” mean? It could also be considered to\nsupplement the model of S. typhimurium infection in vivo while adding the antibiotic treatment group as the positive control.\n11. In the in vivo efficacy evaluation, how about the in vivo bioavailability and in vivo stability of bacNIPs. In addition, the\nnumber of animals used in each group in the animal studies is five, but the data presented is not consistent with it, for\nexample, Fig. 7(c)."}, "author_response": "Point-by-Point Responses to the Reviewers\nWe sincerely thank all the reviewers for their insightful and thoughtful comments. We\nhave revised the manuscript in accordance with their suggestions, which have\nsignificantly improved the clarity and quality of our work. Below is our point-by-point\nresponse to the reviewers’ comments along with the corresponding changes that we\nhave made to the paper. All substantive changes have been highlighted in the\nmanuscript text, also indicted in this letter.\nReviewer #1 (Remarks to the Author):\nIn this paper, the authors designed a bacterial nanoinducer of bacNIP for targeted\nprotein degradation in treating bacterial infection. The bacNIP is developed by co-\nconjugating targeting peptides of MurD and SspB onto gold nanoparticles, which\nfacilitate the degradation of MurD by ClpXP in bacteria. Such BacNIPs showed a broad\nantibacterial spectrum including S. aureus and S. typhimurium, without causing new\nresistance even after 25 passages, which is superior to antibitoics like norfloxacin.\nIn particular, the bacNIPs showed therapeutic effects in three in vivo infection models.\nThe design is smart and the antibacterial effects are significant for developing novel\nantibacterial candidate to overcome the challenge of bacterial resistance. I think the\nwork merits the consideration of publication on Nature Communications. However,\nbefore considering the acceptance, below concerns and questions need to be carefully\naddressed.\nResponse: We express our gratitude to the reviewer for the insightful and thoughtful\ncomments.\n1. Gold nanoparticles were used as the carrier for two peptides. Is there any special\nreason to use gold nanoparticles? It seems that any other nanoparticles can be used as\nlong as they can be modified for peptide conjugation. In addition, what is the destination\nof gold nanoparticles after infection treatment? How can they be degraded or\nmetabolized in vivo?\nResponse: We thank the reviewer for raising this important point. Gold nanoparticles\n(GNPs) are among the most extensively studied nanomaterials in biomedical research.\nCompared with other metal nanoparticles, GNPs are generally considered to be the least\ntoxic and, therefore, particularly suitable for biological and medical applications. In\naddition, their facile synthesis, excellent biocompatibility, and high stability allow\nefficient functionalization with a wide variety of ligands, making them a robust\nbiomedical platform. Based on these advantages, we employed GNPs as carriers for\nMurD-targeting peptides, together with SsrA peptide tag.\nNevertheless, we agree with the reviewer that other nanoparticles (NPs) can also\nserve as peptide conjugation platforms. To evaluate this, we synthesized Pt and Pd NPs\nand modified them with MurD-targeting peptides and SsrA peptide tag, generating Pt-\nbacNIPs and Pd-bacNIPs, respectively. As shown in new Supplementary Figure 21a\n1\nand 22a, peptide modification did not alter the morphology of Pt or Pd nanoparticles,\nand both types maintained a negative surface potential. Colony-forming unit (CFU)\nassay revealed that Pt NPs alone did not exhibit cytotoxicity against S. aureus, with\ncolony numbers comparable to the PBS group. In contrast, Pt-bacNIPs significantly\ninhibited bacterial proliferation, consistent with immunoblot data showing MurD\ndegradation only in the Pt-bacNIPs group. Similar results were obtained in S.\ntyphimurium, confirming the antibacterial activity of Pt-bacNIPs. Likewise, Pd-\nbacNIPs also effectively suppressed the growth of S. aureus and S. typhimurium by\ndegrading MurD (new Supplementary Figure 22b-c).\nTo investigate the biodistribution of bacNIPs, we labeled the nanoparticles with\nCy5.5 and monitored their localization after topical application to the wound site using\nIVIS animal imaging system. As shown in new Supplementary Figure 31, bacNIPs\npredominantly accumulated at the wound site for up to 3 days, with fluorescence signals\ndisappearing by day 5, likely due to eschar detachment. Inductively coupled plasma\nmass spectrometry (ICP-MS) analysis further confirmed that most bacNIPs remained\nat the wound site and gradually decreased over time. Only minimal amounts entered\nsystemic circulation, with renal clearance as the primary elimination pathway, likely\ndue to the relatively small nanoparticle size.\nThe text corresponding to these new results is now incorporated in lines 5-20 on\npage 10, and lines 8-17 on page 14 of the revised paper; the new results are now shown\nin Supplementary Figures 21, 22, and 31.\n2. The MurD-targeting peptide sequence is CIPSHTPRC, which contains two cysteine.\nFor the conjugation on gold nanoparticles, how to determine which cysteine interacts\nwith gold surface? Does the direction of pMurD on gold nanoparticles affect the\nfunction of peptide to recognize MurD?\nResponse: We appreciate the reviewer’s insightful question. Thiols form strong,\nspontaneous Au–S bonds on GNP surfaces; thus, for CIPSHTPRC (Cys–…–Cys), both\nsulfhydryls should be conjugated on GNPs, yielding a surface-anchored, loop-\nconstrained conformation. This is consistent with the MurD-recognition “loop”\nreported for CIPSHTPRC [Ref 1], in which the internal residues (IPSHTPR) constitute\nthe binding epitope while the terminal cysteines primarily serve as anchoring/constraint\nelements. Functionally, our data show preserved MurD engagement on GNPs (MurD\ndegradation and antibacterial efficacy), indicating that peptide orientation on gold does\nnot impair recognition. Even if, under certain local packing conditions, only one\ncysteine was to bind Au at a given site, the remaining thiol would still permit a\nconstrained presentation (via partial looping or transient surface interactions),\nmaintaining exposure of the IPSHTPR epitope. Taken together with Ref 1, these results\nsupport that the GNP-tethered, loop-like presentation of CIPSHTPRC preserves the\npeptide’s MurD-binding competence rather than disrupting it.\n3. MurD was selected as the targeted protein for degradation to kill bacteria. However,\nthere are many types of Mur ligases in bacteria. The family of Mur ligases contains\nMurA-G (Molecules 2023, 28(24), 8076). Is it sufficient for bacterial killing if just\nMurD is degraded? Can pMurD bind to other type of Mur ligases in bacteria?\nResponse: We thank the reviewer for this insightful question. Peptidoglycan\nbiosynthesis is indispensable for bacterial survival, and the ATP-dependent MurC–F\nligases mediate the cytoplasmic assembly of the stem peptide. MurD catalyzes the ATP-\ndriven addition of D-glutamic acid to UDP-MurNAc-L-Ala—an obligate step absent in\nmammalian cells—making MurD a well-established antibacterial target. In our study,\nbacNIPs that degrade MurD produced robust antibacterial effects in vitro and in vivo,\nindicating that selective depletion of this single, essential node is sufficient for bacterial\nkilling under our conditions.\nRegarding selectivity, pMurD was designed to recognize a MurD-specific epitope.\nConsistent with this, immunoblot analyses showed that bacNIPs selectively reduced\nMurD levels without affecting other Mur ligases for which commercial antibodies are\navailable (MurA, MurE, MurG) in S. aureus and S. typhimurium (new Supplementary\nFigure 9). Functionally, this biochemical specificity aligns with the phenotype:\nsignificant inhibition of bacterial proliferation and loss of viability occurred only when\nMurD was targeted and degraded. Together, these data support that (i) targeting MurD\nalone is sufficient for bactericidal activity in our models and (ii) pMurD does not\nmeasurably bind or degrade other Mur ligases under the tested conditions.\nThe text corresponding to our new results is now incorporated in lines 4-7 on page\n7 and the new results are shown in Supplementary Figure 9.\n4. Two bacteria, S. aureus and S. typhimurium, have been tested in the experiments.\nThe type of bacteria is limited, making it hard to conclude that bacNIPs can kill both\nGram-negative and Gram-positive bacteria. In particular, no antibiotic-resistant\nbacteria strains have been tested. More bacteria and those with antibiotic resistance are\nsuggested to be tested with bacNIPs. In addition, if there is MurD mutant strain, it also\nneeds to be tested.\nResponse: We thank the reviewer for this important suggestion. To broaden the scope\nof our study beyond S. aureus (Gram-positive) and S. typhimurium (Gram-negative),\nwe further investigated the antibacterial activity of bacNIPs against Staphylococcus\nepidermidis (S. epidermidis, Gram-positive) and Pseudomonas aeruginosa (P.\naeruginosa, Gram-negative). As shown in new Supplementary Figure 15, turbidity\nmeasurements and optical density (OD600) assays clearly demonstrated significant\ninhibition of bacterial growth in both species upon bacNIP treatment. Immunoblot\nanalyses further confirmed a dose-dependent degradation of MurD in S. epidermidis\nand P. aeruginosa, supporting that the observed antibacterial effect is mediated by\nselective MurD degradation.\nTo specifically address the reviewer’s concern regarding antibiotic resistance, we\nestablished resistant strains of S. epidermidis and P. aeruginosa by subjecting them to\nnine consecutive passages in the presence of norfloxacin at 0.75 × minimal inhibitory\nconcentration (MIC). Following this treatment regimen, the MIC values for both\nbacterial species were markedly elevated (new Supplementary Figure 28), confirming\nthe successful induction of drug resistance. These resistant strains, designated S.\nepidermidis-9 and P. aeruginosa-9, were subsequently treated with bacNIPs.\nRemarkably, bacNIPs continued to effectively inhibit the growth of both resistant\nstrains, showing inhibition comparable to that observed with their respective parental\nstrains (new Supplementary Figure 28). Together with drug-resistant S. aureus and S.\ntyphimurium, these results strongly indicate that bacNIPs remain effective even against\nbacteria that have acquired resistance to conventional antibiotics.\nRegarding Mur ligase specificity, we fully agree with the reviewer that testing\nagainst MurD mutant strains would further strengthen our conclusions. At present, we\ndo not have access to such strains; however, we recognize the importance of this\nexperiment and plan to incorporate it in future work. We anticipate that testing bacNIPs\non MurD mutants will provide additional mechanistic insights into the indispensability\nof MurD degradation for antibacterial activity. Together, these extended results\ndemonstrate that bacNIPs exhibit robust antibacterial effects not only against S. aureus\nand S. typhimurium, but also against additional Gram-positive and Gram-negative\nbacteria, including drug-resistant strains.\nThe text corresponding to these new data is now incorporated in the lines 9-16 on\npage 8, the first 8 lines of the second paragraph on page 12 of the revised paper, and the\nnew results are shown in Supplementary Figures 15 and 28.\n5. In Figure 2f, the authors claimed that effective intracellular delivery was facilitated\nby GNPs according to robust fluorescence. However, such evidence might be\ninsufficient. High resolution TEM is suggested to determine the localization of GNPs\nin bacteria. Furthermore, the mechanism for GNPs entry into bacteria needs to be\ncarefully elucidated, which is very important for the whole design for antibacterial\naction. Currently the mechanism of how bacNIPs enter into bacteria remains unclear.\nResponse: We thank the reviewer for this important comment. To provide direct\nevidence of intracellular localization, we performed additional high-resolution TEM\nanalyses of S. aureus and S. typhimurium treated with bacNIPs. As shown in new\nSupplementary Figure 8, and consistent with the fluorescence imaging in Figure 2f,\nbacNIPs were clearly observed on the bacterial surface, within the cell wall, and inside\nthe cytoplasmic compartment of both S. aureus and S. typhimurium. These TEM\nobservations provide direct confirmation of intracellular delivery.\nWith respect to the entry mechanism, we acknowledge that the internalization of\nnanoparticles by bacteria is not as well characterized as in eukaryotic systems. Current\nliterature suggests three possible routes for nanoparticle translocation across bacterial\ncell envelopes: (i) non-specific diffusion through the porous cell wall, (ii) non-specific\nmembrane damage associated with local perturbation or injury, and (iii) specific uptake\nmechanisms that accommodate larger molecules [Ref 2]. Among these, non-specific\ndiffusion is considered the predominant pathway, but is limited by the pore size of the\nbacterial cell wall and typically feasible only for nanoparticles below ~20 nm in\ndiameter [Ref 3]. Our bacNIPs are ultrasmall (~2 nm), which is well within the size\nrange previously reported to allow passive diffusion through bacterial cell wall pores.\nThe high-resolution TEM results, in combination with the ultrasmall dimensions of\nbacNIPs, strongly support that these nanoparticles likely enter bacteria primarily\nthrough a non-specific diffusion mechanism rather than membrane damage or receptor-\nmediated uptake. Together, these findings provide both structural and mechanistic\nevidence for effective intracellular delivery of bacNIPs, which is central to their design\nand antibacterial function.\nThe text corresponding to these new data is now incorporated in the last 9 lines of\nthe second paragraph on page 6 of the revised paper, and the new results are shown in\nSupplementary Figure 8.\n6. Both peptides of pMurD and tSsrA are for proteins in S. typhimurium. How can they\ninteract with proteins in other bacterial species? Are both proteins very conservative in\nall bacterial species? If yes, can they kill probiotic bacteria? In addition, the test on in\nvivo model with S. typhimurium infection is also preferred, as the original design of\nbacNIPs was from this bacteria.\nResponse: We thank the reviewer for this important question. Although MurD enzymes\nacross different bacteria share conserved residues that are critical for catalytic activity,\ntheir overall amino acid sequences vary substantially. Therefore, it is necessary to\nconfirm whether the pMurD sequence used in this study can effectively target MurD in\nthe specific bacterial species tested. For example, treatment of Bifidobacterium with\nbacNIPs did not result in significant antibacterial activity (new Supplementary Figure\n19). These findings suggest that bacNIPs selectively target pathogenic bacteria to S.\ntyphimurium or S. aureus MurD, while exhibiting minimal effects on probiotic bacteria\nsuch as Bifidobacterium. This specificity supports the safety potential of bacNIPs by\nreducing off-target antibacterial effects on beneficial microbiota.\nAs per the advice, to directly assess the antibacterial efficacy of bacNIPs against\nS. typhimurium in vivo, we established a mouse infection model. After successful\nbacterial colonization, bacNIPs were orally administered to mice twice at 12-hour\nintervals (100 mg/kg per dose), maintaining the same daily dosage throughout the\ntreatment period. Ciprofloxacin treatment was included as a positive control. In the\nuntreated infection group, no mortality was observed until day 5 post-infection, and by\nday 10 only 50% of the animals survived. In contrast, all mice treated with bacNIPs or\nCiprofloxacin survived throughout the 10-day observation period. Body weight\nmonitoring revealed that S. typhimurium infection led to a marked reduction in body\nweight, whereas bacNIP or Ciprofloxacin treatment significantly restored weight loss.\nSimilarly, colon length-significantly shortened in the infection group compared with\nhealthy PBS controls-was restored by bacNIPs or Ciprofloxacin treatment (new\nSupplementary Figure 36a-d).\nHistopathological analyses further supported these protective effects. Infected\nmice exhibited submucosal edema, depletion of goblet cells, and severe damage to\nintestinal villi, whereas animals treated with bacNIPs or Ciprofloxacin displayed\npreserved villi architecture, normal goblet cell numbers, and only mild edema,\ncomparable to healthy controls (new Supplementary Figure 36e). Consistently,\ncytokine measurements showed significantly lower levels of IL-6 and TNF-α in colon\ntissue from bacNIP- or Ciprofloxacin-treated mice than in the untreated infection group\n(new Supplementary Figure 37). Taken together, these results demonstrate that bacNIPs\nprovide strong protection in an S. typhimurium infection model, comparable to\nCiprofloxacin, thereby validating the in vivo antibacterial efficacy of our nanoplatform.\nThe text corresponding to these new data is now incorporated in the lines 14-18 of\nthe second paragraph on page 9, the last 16 lines on page 16, the first 7 lines of the\nsecond paragraph on page 17 of the revised paper, and the new results are shown in\nSupplementary Figures 19, 36 and 37.\nThis manuscript designed a bacterial nanoinducer of proximity (bacNIPs) that can\nspecifically target and degrade MurD, an enzyme crucial for peptidoglycan synthesis in\nbacteria. Notably, bacNIPs demonstrate efficacy in treating S. aureus-infected skin and\ncorneal wounds in vivo and show promise in combating bacterial infections without\ninducing resistance. The findings of this study are interesting and highlight the potential\nof targeted protein degradation as an effective strategy to combat bacterial infections,\nwhile there are still some critical issues that need to be addressed.\nResponse: We express our gratitude to the reviewer for their insightful and thoughtful\ncomments.\n1. Regarding the synthesis of bacNIPs, how to determine the specific feeding ratios of\nbacNIPs. In addition, why only bacNIPs with a feeding ratio of 1:2:2 (GNPs: pMurD:\ntSsrA) showed antibacterial activity.\nResponse: The specific feeding ratios of bacNIPs were determined by preparing a\nseries of formulations with varying GNPs:pMurD:tSsrA feeding ratios and\nsubsequently evaluating their antibacterial activity. Among these, the 1:2:2 ratio\nexhibited the highest antibacterial efficacy and was therefore selected for this study.\nAlthough other ratios, such as 1:1:1 and 1:3:3, also demonstrated antibacterial activity,\ntheir effects were comparatively weaker. As shown in new Supplementary Figure 11,\nboth 1:1:1 and 1:3:3 formulations were capable of inhibiting the proliferation of S.\naureus and S. typhimurium, albeit less efficiently than the 1:2:2 formulation. These data\nalso indicate that the number of targeting peptides can influence the protein-targeting\nefficiency.\nThe text corresponding to these new data is now incorporated in lines 9-11 of the\nsecond paragraph on page 7 of the revised paper. The new results are shown in\nSupplementary Figure 11.\n2. While the design of the nanocarriers is of interest, their high production costs may\nlimit their clinical applications. What are the advantages of targeted protein degradation\nnanodrugs over traditional antibiotic therapy. Meanwhile, the stability, safety and\nbioavailability of bacNIPs needs to be further studied and verified.\nResponse: We thank the reviewer for this insightful comment. In this study, gold\nnanoparticles (GNPs) were employed because they are generally regarded as minimally\ntoxic and particularly well-suited for biomedical applications owing to their facile\nsynthesis, excellent biocompatibility, high colloidal stability, and versatile surface\nchemistry that enables efficient ligand functionalization. While the use of GNPs may\nraise concerns about production costs, it is worth noting that silver nanoparticles have\nalready been widely incorporated into commercial products, and alternative\nnanomaterials can also serve as peptide-conjugation platforms. To evaluate this, we\nsynthesized Pt- and Pd-based bacNIPs by modifying Pt and Pd nanoparticles (NPs) with\nMurD-targeting peptides and the SsrA tag. As shown in Supplementary Figure 21 and\n22, peptide modification did not alter nanoparticle morphology, and both Pt- and Pd-\n7\nbased bacNIPs maintained a negative surface potential. CFU assays revealed that bare\nPt NPs displayed no intrinsic antibacterial activity against S. aureus, whereas Pt-\nbacNIPs markedly inhibited bacterial proliferation, consistent with immunoblot data\nconfirming MurD degradation. Comparable results were obtained with S. typhimurium,\nand Pd-bacNIPs likewise showed significant antibacterial efficacy.\nImportantly, prolonged exposure to bacNIPs did not induce resistance in S. aureus\nor S. typhimurium after 25 consecutive passages, in contrast to norfloxacin, which\nexhibited a 243-fold loss of activity against S. aureus after 15 passages and a 7,410-\nfold reduction against S. typhimurium after 22 passages. Given the global challenge of\nantibiotic resistance, this feature highlights the therapeutic potential of bacNIPs as\nnanodrugs with a novel mechanism of action that circumvents conventional resistance\npathways. To further validate this, we established resistant strains of S. epidermidis and\nP. aeruginosa by repeated exposure to sub-MIC norfloxacin. Both strains displayed\nmarkedly increased MIC values (new Supplementary Figure 28), confirming successful\ninduction of resistance. Remarkably, bacNIPs effectively inhibited these resistant\nstrains with potency comparable to that observed in the parental strains, underscoring\ntheir potential as broad-spectrum agents effective against multidrug-resistant bacteria.\nThe stability of bacNIPs was assessed by incubating them in 10% FBS, simulated\nbody fluid, and simulated intestinal fluid for extended periods, followed by evaluation\nof their antibacterial activity. As shown in new Supplementary Figure 20, bacNIPs\nretained robust activity against S. aureus and S. typhimurium after incubation,\ndemonstrating favorable stability under physiologically relevant conditions.\nFurthermore, as addressed in Question 10, orally administered bacNIPs demonstrated\nefficacy in the S. typhimurium infection model in vivo, confirming their stability in a\nphysiological context.\nBiodistribution was investigated by labeling bacNIPs with Cy5.5 and tracking\ntheir localization after topical application to wound sites using an IVIS imaging system.\nNew Supplementary Figure 31 shows that bacNIPs predominantly accumulated at the\nwound site for up to three days, with fluorescence signals disappearing by day 5, likely\ndue to eschar detachment. ICP-MS analysis confirmed that bacNIPs largely remained\nat the wound site, gradually declining over time, with minimal systemic distribution\nand predominant renal clearance, consistent with their relatively small size.\nIn addition, the biosafety of bacNIPs was evaluated both in vitro and in vivo. As\nshown in Supplementary Figure 18, bacNIPs did not induce toxicity in three different\ntypes of eukaryotic cells, including NIH/3T3 cells, human umbilical vein endothelial\ncells (HUVEC), and RAW 264.7 cells, demonstrating the excellent biocompatibility of\nbacNIPs. Importantly, it exhibited good biosafety in multiple in vivo therapeutic models,\nincluding the S. aureus-infected nonhealing keratitis model and the S. typhimurium-\ninduced colitis model. As shown in Supplementary Figure 35 and the new\nSupplementary Figure 38, hematological examinations of bacNIPs-treated mice\nshowed no significant differences compared to the PBS group. Moreover, liver, kidney,\nand cardiac function markers remained within normal ranges, indicating that bacNIPs\ndid not induce systemic toxicity. Taken together, these results demonstrate that bacNIPs\nnot only provide a robust antibacterial effect through targeted protein degradation but\n8\nalso possess favorable stability, biosafety, and biodistribution profiles, while offering a\nclear advantage over conventional antibiotics by minimizing the risk of resistance\ndevelopment.\nThe text corresponding to these new data is now incorporated in the last 2 lines on\npage 9, the first 20 lines on page 10, the first 8 lines of the second paragraph on page\n12, lines 8-16 on page 14, and lines 7-10 on page 17 of the revised paper. The new\nresults are shown in Supplementary Figures 20, 21, 22, 28, 31 and 38.\n3. MurD is involved in the synthesis of cell wall peptidoglycan, but Gram-negative\nbacteria have no intact cell wall, so why targeting MurD is also effective against Gram-\nnegative bacteria. In addition, the presence of outer membrane barrier in Gram-negative\nbacteria may prevent the entry of bacNIPs, thereby constraining its activity.\nResponse: MurD is a cytoplasmic ligase that catalyzes the ATP-dependent addition of\nD-glutamic acid to UDP-N-acetylmuramoyl-L-alanine, an essential step in\npeptidoglycan biosynthesis. Although Gram-negative bacteria possess an outer\nmembrane and a thinner peptidoglycan layer compared to Gram-positive bacteria, this\nlayer remains indispensable for maintaining cell morphology, osmotic stability, and\nviability. Inhibition of MurD therefore disrupts peptidoglycan synthesis and\ncompromises cell wall integrity in both Gram-positive and Gram-negative species,\nleading to growth arrest and bacterial death. Consistent with this, multiple previous\nstudies have demonstrated that selective inhibition of Mur ligases blocks peptidoglycan\nbiosynthesis and exerts antibacterial activity against both classes of bacteria [Ref 4-5].\nWe acknowledge the concern regarding the outer membrane barrier in Gram-\nnegative bacteria, which can hinder the entry of conventional drugs. To directly assess\nwhether bacNIPs can overcome this barrier, we performed confocal microscopy using\nfluorophore-labeled tSsrA or bacNIPs. Free tSsrA displayed minimal penetration into\nS. aureus or S. typhimurium, whereas bacNIP-treated bacteria exhibited strong\nintracellular fluorescence signals, confirming that the GNP-based platform enabled\neffective cytoplasmic delivery (Figure 2f). To corroborate these findings, high-\nresolution TEM was additional performed on bacNIP-treated bacteria. As shown in new\nSupplementary Figure 8, bacNIPs were clearly visualized at the bacterial surface,\nwithin the cell wall, and inside the cytoplasm of both bacNIP-treated S. aureus and S.\ntyphimurium. These results provide direct structural evidence that bacNIPs can traverse\nthe bacterial envelope and deliver functional payloads intracellularly, thereby\novercoming the permeability barrier of Gram-negative bacteria.\nThe text corresponding to these new data is now incorporated in the last 9 lines of\nthe second paragraph on page 6 of the revised paper, and the new results are shown in\nSupplementary Figure 8.\n4. Fig. 3(a-b) shows that the MurD band of S. aureus is single, but S. typhimurium has\ntwo bands. Is this caused by the modification of protein in the bacteria or other reasons?\nIn the evaluation of the antibacterial activity of bacNIPs, the minimum inhibitory\nconcentration (MIC) and minimum bactericidal concentration (MBC) should be tested.\nMoreover, a time-killing curve should be added to verify the bactericidal activity of\n9\nbacNIPs, not only the inhibition of bacterial growth by OD detection. Antibiotics should\nbe used as the control.\nResponse: We thank the reviewer for raising this important point. In our immunoblot\nanalysis, MurD from S. aureus appeared as a single band, whereas S. typhimurium\nexhibited two bands. To investigate whether this was strain-specific or related to post-\ntranslational modifications, we examined additional Gram-positive and Gram-negative\nbacteria. As shown in new Supplementary Figure 10, a single band was consistently\nobserved in S. aureus, MRSA, and P. aeruginosa, while two bands were detected in S.\nepidermidis, S. typhimurium, and E. coli. These results suggest that the presence of two\nbands may be attributed to post-translational modifications or isoforms of MurD in\ncertain bacterial species rather than an artifact of detection.\nIn line with the reviewer’s suggestion, we have now included MIC and MBC data\nin Supplementary Table 1. Furthermore, we performed time-kill assays to directly\nevaluate bactericidal activity, using norfloxacin as a reference antibiotic. As shown in\nnew Supplementary Figure 12, bacNIPs induced a time-dependent reduction in\nbacterial load in both S. aureus and S. typhimurium at a concentration of 2 × MIC.\nMaximum bactericidal activity was achieved within 12 hours, comparable to the effect\nobserved with norfloxacin. These results provide a more comprehensive evaluation of\nthe antibacterial efficacy of bacNIPs beyond OD-based growth inhibition assays.\nThe text corresponding to our new results is now incorporated in lines 7-11 and\nlast 6 lines on page 7 of the revised paper, and the new results are shown in\nSupplementary Figures 10 and 12.\n5. It is unclear whether the S. aureus and S. typhimurium used in this manuscript are\nstandard or resistant strains. The strains information should be provided in the\nsupplementary materials. Does bacNIPs have similar antibacterial effects against drug-\nresistant strains?\nResponse: In this study, standard laboratory strains of S. aureus and S. typhimurium\nwere used, and the corresponding strain numbers are provided in the Methods section.\nTo evaluate whether bacNIPs induce resistance, we subjected S. aureus and S.\ntyphimurium to 25 consecutive passages in the presence of 0.75 × MIC bacNIPs.\nNotably, prolonged exposure did not result in resistance development in either species.\nIn contrast, repeated exposure to norfloxacin led to rapid resistance, with a 243-fold\nincrease in MIC for S. aureus after 15 passages and a 7,410-fold increase for S.\ntyphimurium after 22 passages.\nTo further address the activity of bacNIPs against drug-resistant pathogens, we\nestablished resistant strains of S. epidermidis and P. aeruginosa by serially passaging\nthem nine times in sub-MIC norfloxacin. Both strains displayed markedly elevated\nMIC values, confirming successful resistance induction (new Supplementary Figure 28).\nRemarkably, bacNIPs remained highly effective against these resistant strains, showing\nantibacterial activity comparable to that observed in the parental strains. Together with\nthe results from resistant S. aureus and S. typhimurium, these findings strongly\ndemonstrate that bacNIPs maintain potent antibacterial efficacy even against strains\nthat have acquired resistance to conventional antibiotics.\nof the second paragraph on page 12 of the revised paper, and the new results are shown\nin Supplementary Figure 28.\n6. In the mechanism verification, the targeting and degradation of MurD by bacNIP can\ndirectly produce antimicrobial activity, whether the knockout of MurD will cause direct\nbacterial death? Line 168, only a potential ClpP inhibitor was used to validate the\nClpXP-mediated degradation of MurD. The construction of knockout and/or\noverexpressed strains of ClpXP would strengthen these results.\nResponse: As suggested, we attempted to construct a MurD knockout strain; however,\nthe bacteria were unable to proliferate following knockout, confirming that MurD is\nessential for survival. To further validate that the antibacterial activity of bacNIPs arises\nfrom targeted MurD degradation, we introduced mutations in the key amino acid\nresidues of pSsrA, thereby abolishing its ability to recognize MurD. Immunoblot\nanalysis demonstrated that bacNIPs no longer induced MurD degradation in S. aureus\nand S. typhimurium after these mutations (new Supplementary Figure 17). Consistently,\nturbidity and OD assays revealed that mutated pSsrA abrogated the growth-inhibitory\neffect of bacNIPs on both species (new Supplementary Figure 17).\nWith respect to ClpXP, we attempted to generate overexpression strains, but the\nexpression efficiency was low and no significant phenotypic changes were observed.\nTo further confirm ClpXP-mediated MurD degradation, we employed an alternative\nspecific ClpP inhibitor, hinokiflavone [Ref 6]. As shown in new Supplementary Figure\n16, treatment with hinokiflavone markedly attenuated bacNIP-mediated MurD\ndegradation, which was accompanied by a substantial reduction in antibacterial efficacy.\nTogether, these results demonstrate that bacNIPs exert antibacterial activity by inducing\nClpXP-dependent degradation of MurD, an essential enzyme for bacterial viability.\nThe text corresponding to our new results is now incorporated in the last 13 lines\non page 8 of the revised paper, and the new results are shown in Supplementary Figures\n16 and 17.\n7. In the process of verifying bacNIPs specific targeting MurD, it should be considered\nwhether the nano-preparation has a comparable antibacterial effect under low nutrient\nconditions, as ClpP is an ATP-dependent protease. In addition, whether bacNIPs is also\nactive against bacterial persisters.\nResponse: As per the advice, we evaluated the antibacterial activity of bacNIPs under\nnutrient-limited conditions using a 10:90 medium (10% lysogeny broth: 90% PBS) [Ref.\n7]. As shown in new Supplementary Figure 13, bacNIPs retained potent antibacterial\nactivity even under nutrient restriction, indicating that their efficacy is not significantly\ncompromised by the ATP dependence of ClpP.\nIn addition, we investigated the activity of bacNIPs against bacterial persisters.\nPersister populations of S. aureus and S. typhimurium were isolated and subsequently\ntreated with bacNIPs. As shown in new Supplementary Figure 14, bacNIPs induced\nsignificant inhibition of persister survival in both species. Immunoblot analyses further\ndemonstrated dose-dependent degradation of MurD in persister cells, consistent with\n11\nthe observed antibacterial effect (new Supplementary Figure 14). These findings\nconfirm that bacNIPs remain active under nutrient-limited conditions and are capable\nof\ncapable of eradicating bacterial persisters.\nThe text corresponding to our new results is now incorporated in the first 9 lines\non page 8 of the revised paper, and the new results are shown in Supplementary Figures\n13 and 14.\n8. Given the role of MurD in peptidoglycan synthesis for bacterial cell wall formation,\nthe direct effect of bacNIPs on cell wall synthesis should be assessed, rather than\nfocusing on the changes of membrane permeability. Why bacNIPs targeting MurD\ncan result in the membrane damage, which was different from cell wall damage. Line 227,\nthe authors stated that the compromised cell walls can result in the leakage of\nintracellular components, please add related references.\nResponse: To directly evaluate the effect of bacNIPs on bacterial cell wall synthesis,\nwe first performed SEM imaging of S. aureus and S. typhimurium, which revealed\nmarked structural alterations in the cell wall following bacNIP treatment (Figure 4a–b).\nConsistently, PI staining confirmed significant cell wall damage in both strains (Figure\n4c–d). In addition, we quantified DNA and ATP released into the culture supernatants,\nand bacNIP-treated S. aureus and S. typhimurium displayed markedly elevated\nextracellular DNA and ATP levels compared to controls, suggesting compromised cell\nwall integrity. As per the advice, to further validate this, we measured extracellular\nalkaline phosphatase (AKP) activity, as AKP resides between the cell wall and\nmembrane and is widely used as an indicator of cell wall disruption. As shown in new\nSupplementary Figure 24, AKP activity increased in a dose-dependent manner\nfollowing bacNIP treatment, further supporting cell wall impairment.\nMurD is an essential cytoplasmic ligase in the peptidoglycan biosynthetic pathway,\ncatalyzing the ATP-dependent addition of D-glutamic acid to UDP-MurNAc-L-Ala.\nInhibition or degradation of MurD disrupts stem peptide assembly and compromises\npeptidoglycan formation, thereby impairing bacterial cell wall integrity. Previous\nstudies have demonstrated that perturbations in peptidoglycan biosynthesis can\nsecondarily lead to membrane damage, resulting in leakage of intracellular components\n[Refs. 4 and 8]. Accordingly, we have now included the relevant references in the\nrevised manuscript to support this statement. Together, these findings confirm that\nbacNIPs disrupt peptidoglycan synthesis via MurD degradation, leading to cell wall\ndamage and subsequent membrane permeability defects.\nThe text corresponding to our new results is now incorporated in the last 3 lines of\nthe second paragraph on page 11 of the revised paper, and the new results are shown in\nSupplementary Figure 24.\n9. Norfloxacin was used as an antibiotic control in this study. Why not use antibiotics\ntargeting cell walls as a positive control, such as carbapenems and cephalosporins,\nwhich are not easy to induce drug resistance. And Fig. 4(i) did not indicate the specific\nconcentration used for norfloxacin or bacNIPs.\nResponse: We thank the reviewer for this important comment. In the initial version of\n12\nour study, norfloxacin was used as a representative antibiotic control because of its\nwell-documented tendency to induce resistance, allowing us to highlight the unique\nresistance-evading feature of bacNIPs. To strengthen the comparison, we have now\nincluded imipenem, a clinically used antibiotic that targets cell wall synthesis, making\nit effective against both Gram-positive and Gram-negative bacteria. As shown in new\nSupplementary Figure 29, serial passaging experiments revealed that the MIC of\nimipenem against S. aureus increased significantly after five passages, resulting in the\nemergence of resistant strains. In S. typhimurium, prolonged exposure to imipenem led\nto a 128-fold elevation of the MIC after 16 passages, clearly demonstrating the\nacquisition of drug resistance. These findings underscore that even highly effective cell\nwall–targeting antibiotics can eventually select for resistant populations under sustained\ntreatment pressure. In sharp contrast, the MIC of bacNIPs remained unchanged after 16\nsuccessive passages in both S. aureus and S. typhimurium, further supporting the\nconclusion that bacNIPs, by leveraging targeted MurD degradation, circumvent\nconventional resistance mechanisms. Together with the results obtained using\nnorfloxacin, these data emphasize that bacNIPs provide a fundamentally distinct\nantibacterial mechanism that is not prone to rapid resistance development, unlike\ntraditional antibiotics.\nAdditionally, to address the reviewer’s concern regarding clarity, we have now\nspecified the exact concentrations of both norfloxacin and bacNIPs used in Figure 4i in\nthe revised figure caption.\nThe text corresponding to our new results is now incorporated in the last 2 lines\non page 12 and the first 7 lines on page 13 of the revised paper, and the new results are\nshown in Supplementary Figure 29.\n10. The antibacterial effect of bacNIPs was verified in three animal models of S. aureus\ninfection. What is the difference between the two models of skin infection and what\ndoes “nonhealing skin wound” mean? It could also be considered to supplement the\nmodel of S. typhimurium infection in vivo while adding the antibiotic treatment group\nas the positive control.\nResponse: We thank the reviewer for this suggestion. To clarify, the two S. aureus skin\ninfection models were designed to mimic different clinical contexts. The first skin\nwound model was established in healthy, immunocompetent mice to represent acute\nbacterial skin infections in hosts with intact immune responses, reflecting typical\ninfection scenarios that usually resolve under normal healing conditions. The second\nskin wound model was established in diabetic mice to replicate the pathological\nmicroenvironment associated with diabetes, where impaired immune function, vascular\ncomplications, and altered metabolic states contribute to the formation of chronic,\nrefractory, and “nonhealing” wounds. Here, the term “nonhealing skin wound”\nspecifically refers to diabetic wounds that fail to progress through the normal healing\nphases (hemostasis, inflammation, proliferation, and remodeling), thereby remaining\nchronically infected and resistant to standard therapeutic approaches. This model is\nhighly relevant clinically, as diabetic wound infections are a major healthcare challenge\ndue to their high morbidity, chronicity, and risk of complications.\nS. typhimurium in vivo, we established a mouse infection model. After successful\nbacterial colonization, bacNIPs were orally administered to mice twice at 12-hour\nintervals (100 mg/kg per dose), maintaining the same daily dosage throughout the\ntreatment period. Ciprofloxacin treatment was included as a positive control. In the\nuntreated infection group, no mortality was observed until day 5 post-infection, and by\nday 10 only 50% of the animals survived. In contrast, all mice treated with bacNIPs or\nCiprofloxacin survived throughout the 10-day observation period. Body weight\nmonitoring revealed that S. typhimurium infection led to a marked reduction in body\nweight, whereas bacNIP or Ciprofloxacin treatment significantly restored weight loss.\nSimilarly, colon length-significantly shortened in the infection group compared with\nhealthy PBS controls-was restored by bacNIPs or Ciprofloxacin treatment (new\nSupplementary Figure 36a-d).\nHistopathological analyses further supported these protective effects. Infected\nmice exhibited submucosal edema, depletion of goblet cells, and severe damage to\nintestinal villi, whereas animals treated with bacNIPs or Ciprofloxacin displayed\npreserved villi architecture, normal goblet cell numbers, and only mild edema,\ncomparable to healthy controls (new Supplementary Figure 36e). Consistently,\ncytokine measurements showed significantly lower levels of IL-6 and TNF-α in colon\ntissue from bacNIP- or Ciprofloxacin-treated mice than in the untreated infection group\n(new Supplementary Figure 37). Taken together, these results demonstrate that bacNIPs\nprovide strong protection in an S. typhimurium infection model, comparable to\nCiprofloxacin, thereby validating the in vivo antibacterial efficacy of our nanoplatform.\nThe text corresponding to these new data is now incorporated in the last 16 lines\non page 16, the first 7 lines of the second paragraph on page 17 of the revised paper,\nand the new results are shown in Supplementary Figures 36 and 37.\n11. In the in vivo efficacy evaluation, how about the in vivo bioavailability and in vivo\nstability of bacNIPs. In addition, the number of animals used in each group in the animal\nstudies is five, but the data presented is not consistent with it, for example, Fig. 7(c).\nResponse: As mentioned above, the stability of bacNIPs was assessed by incubating\nthem in 10% FBS, simulated body fluid, and simulated intestinal fluid for extended\nperiods, followed by evaluation of their antibacterial activity. As shown in new\nSupplementary Figure 20, bacNIPs retained robust activity against S. aureus and S.\ntyphimurium after incubation, demonstrating favorable stability under physiologically\nrelevant conditions. As addressed in Question 10, orally administered bacNIPs\ndemonstrated efficacy in the S. typhimurium infection model in vivo, confirming their\nstability in a physiological context.\nBiodistribution was investigated by labeling bacNIPs with Cy5.5 and tracking\ntheir localization after topical application to wound sites using an IVIS imaging system.\nNew Supplementary Figure 31 shows that bacNIPs predominantly accumulated at the\nwound site for up to three days, with fluorescence signals disappearing by day 5, likely\ndue to eschar detachment. ICP-MS analysis confirmed that bacNIPs largely remained\nat the wound site, gradually declining over time, with minimal systemic distribution\n14\nand predominant renal clearance, consistent with their relatively small size. In addition,\nthe biosafety of bacNIPs was evaluated both in vitro and in vivo. As shown in\nSupplementary Figure 18, bacNIPs did not induce toxicity in three different types of\neukaryotic cells, including NIH/3T3 cells, human umbilical vein endothelial cells\n(HUVEC), and RAW 264.7 cells, demonstrating the excellent biocompatibility of\nbacNIPs. Importantly, it exhibited good biosafety in multiple in vivo therapeutic models,\nincluding the S. aureus-infected nonhealing keratitis model and the S. typhimurium-\ninduced colitis model. As shown in Supplementary Figure 35 and the new\nSupplementary Figure 38, hematological examinations of bacNIPs-treated mice\nshowed no significant differences compared to the PBS group. Moreover, liver, kidney,\nand cardiac function markers remained within normal ranges, indicating that bacNIPs\ndid not induce systemic toxicity.\nRegarding the number of animals per group, we have carefully re-examined the\noriginal experimental records. Each group indeed consisted of five animals, consistent\nwith the descriptions in the Methods section. However, due to overlapping or coincident\ndata points in the graphs (e.g., Fig. 7c), some data distributions may visually appear to\nrepresent fewer than five animals. To ensure transparency and reproducibility, we have\nprovided the complete raw data in Source Data file in the revised submission, allowing\nreaders to verify individual data points directly.\nThe text corresponding to these new data is now incorporated in the last 2 lines on\npage 9, the first 4 lines on page 10, lines 8-16 on page 14, and lines 7-10 on page 17 of\nthe revised paper. The new results are shown in Supplementary Figures 20, 31 and 38.\nReferences:\n1. Paradis-Bleau C, Beaumont M, Boudreault L, et al. Selection of peptide inhibitors against the\nPseudomonas aeruginosa MurD cell wall enzyme. Peptides, 2006, 27(7): 1693-1700.\n2. J. A. Kloepfer, R. E. Mielke, J. L. Nadeau. Uptake of CdSe and CdSe/ZnS quantum dots into\nbacteria via purine-dependent mechanisms. Appl. Environ. Microbiol. 2005, 71, 2548.\n3. K. S. Butler, D. J. Peeler, B. J. Casey, B. J. Dair, R. K. Elespuru, Silver nanoparticles: correlating\nnanoparticle size and cellular uptake with genotoxicity. Mutagenesis 2015, 30, 577.\n4. Xin Q, Liu Q, Geng L, et al. Chiral Nanoparticle as a New Efficient Antimicrobial Nanoagent.\nAdvanced Healthcare Materials, 2017, 6(23): 1601011.\n5. Humljan, J., Kotnik, M., Contreras-Martel, C., Blanot, D., Urleb, U., Dessen, A., Šolmajer, T.\n& Gobec, S. Novel naphthalene-N-sulfonyl-D-glutamic acid derivatives as inhibitors of MurD,\na key peptidoglycan biosynthesis enzyme. J. Med. Chem. 2008, 51, 7555–7564\n6. Kong X, et al. Hinokiflavone Attenuates the Virulence of Methicillin-Resistant Staphylococcus\naureus by Targeting Caseinolytic Protease P. Antimicrob Agents Chemother. 2022, 16,\ne0024022.\n7. Derek C K Chan, Katherine Dykema, Mahrukh Fatima, Hanjeong Harvey, Ikram Qaderi, Lori\nL Burrows. Nutrient Limitation Sensitizes Pseudomonas aeruginosa to Vancomycin. ACS Infect.\nDis. 2023, 9, 7, 1408–1423\n8. Andrew J Christofferson, et al. Conformationally tuned antibacterial oligomers target the\npeptidoglycan of Gram-positive bacteria. Journal of Colloid and Interface Science 2020, 15,\n15\n850-862\n16"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully addressed the concerns of the reviewers and significantly improved the manuscript. I thus recommend it to be accepted for publication.", "reviewer_2": "The additional data provided by the authors have partially addressed my initial concerns and improved the manuscript. However, several key points still remain unclear or not fully substantiated by the available evidence.\n1. The effect of the ClpP inhibitor was only tested on the reduction of MurD degradation in S. aureus. Given that the design of bacNIPs originated from S. typhimurium, it would be more comprehensive to also evaluate its effects on S. typhimurium. Moreover, given that mutated pSsrA and the ClpP inhibitor markedly suppressed bacNIPs-mediated MurD degradation, it remains unclear whether MurD overexpression would likewise attenuate the antibacterial effect of bacNIPs.\n2. The MIC of bacNIPs is reported to be 64 to 128 μM and the MBC is 512 μM, which is inconsistent with the potent antibacterial activity of bacNIPs even under 64 μM as shown in figures 3c and 3d and the Supplementary figures. Additionally, the cytotoxicity assays were conducted at concentrations below 100 μM, which may not be sufficient to reflect its safety profile. The tested concentrations of bacNIPs and norfloxacin in Figure S12 are also missing.\n3. While the authors have attempted to verify the specific antibacterial effect of bacNIPs against pathogens rather than probiotics, the evaluation of antibacterial activity in Bifidobacterium alone is insufficient to fully demonstrate its safety for beneficial microbiota. Moreover, it remains unclear whether this nanomaterial has the potential to impact gut microbiota homeostasis.\n4. In Supplementary Figure 17, the authors introduced mutations in the key amino acid residues of pSsrA, but the detailed methods and the specific mutant sites are not provided. This makes it difficult for other researchers to understand and reproduce this study.\n5. The graphs in the supplementary material appear to be too scattered. It is suggested that the authors merge and organize the graphs to make the results clearer and more coherent."}, "author_response": "Point-by-Point Responses to the Reviewers\nWe sincerely thank all the reviewers for their insightful and thoughtful comments. We\nhave revised the manuscript in accordance with their suggestions, which have\nsignificantly improved the clarity and quality of our work. Below is our point-by-point\nresponse to the reviewers’ comments along with the corresponding changes that we\nhave made to the paper. All substantive changes have been highlighted in the\nmanuscript text, also indicted in this letter.\nReviewer #1 (Remarks to the Author):\nThe authors have carefully addressed the concerns of the reviewers and significantly\nimproved the manuscript. I thus recommend it to be accepted for publication.\nResponse: We express our gratitude to the reviewer for the insightful and thoughtful\ncomments.\nReviewer #2 (Remarks to the Author):\nThe additional data provided by the authors have partially addressed my initial concerns\nand improved the manuscript. However, several key points still remain unclear or not\nfully substantiated by the available evidence.\nResponse: We express our gratitude to the reviewer for their insightful and thoughtful\ncomments.\n1. The effect of the ClpP inhibitor was only tested on the reduction of MurD degradation\nin S. aureus. Given that the design of bacNIPs originated from S. typhimurium, it would\nbe more comprehensive to also evaluate its effects on S. typhimurium. Moreover, given\nthat mutated pSsrA and the ClpP inhibitor markedly suppressed bacNIPs-mediated\nMurD degradation, it remains unclear whether MurD overexpression would likewise\nattenuate the antibacterial effect of bacNIPs.\nResponse: We agree with the reviewer’s suggestion and have now examined the effect\nof the ClpP inhibitor on the reduction of MurD degradation in S. typhimurium. As\nshown in the new Supplementary Figure 12b, treatment with (3R,4R)-A2-32-01\nmarkedly inhibited MurD degradation in S. typhimurium, thereby compromising the\nantibacterial efficacy of bacNIDs.\nRegarding MurD overexpression, in previously published studies employing\nPROTAC technology, inhibitors were typically used to confirm target protein\ndegradation via proteases, while overexpression experiments were not performed to\nfurther demonstrate the functional role of the target protein. The biological functions of\nMurD have been well established in prior studies, and we concur with the reviewer’s\nviewpoint that increased intracellular levels of MurD would, in principle, attenuate the\nantibacterial effect of bacNIDs.\n1\n2. The MIC of bacNIPs is reported to be 64 to 128 μM and the MBC is 512 μM, which\nis inconsistent with the potent antibacterial activity of bacNIPs even under 64 μM as\nshown in figures 3c and 3d and the Supplementary figures. Additionally, the\ncytotoxicity assays were conducted at concentrations below 100 μM, which may not be\nsufficient to reflect its safety profile. The tested concentrations of bacNIPs and\nnorfloxacin in Figure S12 are also missing.\nResponse: We agree that this requires clarification. The apparent discrepancy arises\nfrom the distinction between bacterial growth inhibition and complete bactericidal\nactivity, as well as methodological differences between MIC and MBC assays. Please\nallow us to explain as follows: The MIC reflects the lowest concentration of bacNIDs\nthat inhibits visible bacterial growth after incubation for a defined period, representing\nthe inhibitory effect on proliferation. In contrast, the MBC assesses the bactericidal\ncapacity of bacNIDs by subculturing samples from wells at or above the MIC\nconcentration onto drug-free agar plates, with the lowest concentration showing no\ncolony formation defined as the MBC. Figures 3c, 3d, and the corresponding\nSupplementary Figures plot the absolute OD600 values to visually demonstrate the\ndose-dependent inhibitory effect of bacNIDs on bacterial growth. At 64 μM, the OD600\nvalue remains low, indicating that bacNIDs effectively suppressed bacterial\nproliferation and prevented entry into the exponential growth phase. This demonstrates\npotent growth inhibition, but not necessarily complete bacterial eradication—hence the\ndistinction from MBC. Therefore, there is no inconsistency between the MIC/MBC data\nand the growth inhibition results presented.\nAs per the reviewer’s advice, we have also evaluated the cytotoxicity of bacNIDs\nat higher concentrations. As shown in the new Supplementary Figure 14, even at\nelevated doses, bacNID treatment did not induce significant toxicity in NIH/3T3,\nHUVEC, or RAW 264.7 cells.\n3. While the authors have attempted to verify the specific antibacterial effect of bacNIPs\nagainst pathogens rather than probiotics, the evaluation of antibacterial activity in\nBifidobacterium alone is insufficient to fully demonstrate its safety for beneficial\nmicrobiota. Moreover, it remains unclear whether this nanomaterial has the potential to\nimpact gut microbiota homeostasis.\nResponse: As per the reviewer’s suggestion, we further investigated the antibacterial\neffect of bacNIDs on Akkermansia muciniphila (A. muciniphila). As shown in the new\nSupplementary Figure 15, bacNID treatment induced slight toxicity to A. muciniphila\nat high concentrations, and the effect was much weaker than that observed in pathogenic\nbacteria. Overall, we believe that the antibacterial activity of bacNIDs is not determined\nby whether the bacteria are pathogenic or probiotic, but rather by the targeting\nefficiency of MurD- and SspB-binding peptides and the degree of bacterial dependence\non MurD function.\n4. In Supplementary Figure 17, the authors introduced mutations in the key amino acid\nresidues of pSsrA, but the detailed methods and the specific mutant sites are not\nprovided. This makes it difficult for other researchers to understand and reproduce this\n2\nstudy.\nResponse: As per the reviewer’s advice, we have added the detailed peptide sequence\nof the mutant targeting peptide, including the specific mutation sites, in the Methods\nsection to facilitate understanding and reproducibility of the study.\n5. The graphs in the supplementary material appear to be too scattered. It is suggested\nthat the authors merge and organize the graphs to make the results clearer and more\ncoherent.\nResponse: We agree with the reviewer’s suggestion. Accordingly, we have reorganized\nand merged several supplementary figures in this revision to present the results more\nclearly and coherently.\n3"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors has addressed my concerns."}, "author_response": ""}]

Decision letter