Identification of ferredoxin PA1551 in the bacterial iron uptake pathway and exploration of its antibacterial synergistic as target for biofilm inhibitors against Pseudomonas aeruginosa infection

Identification of ferredoxin PA1551 in the bacterial iron uptake pathway and exploration of its antibacterial synergistic as target for biofilm inhibitors against Pseudomonas aeruginosa infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of ferredoxin PA1551 in the bacterial iron uptake pathway and exploration of its antibacterial synergistic as target for biofilm inhibitors against Pseudomonas aeruginosa infection Jun Liu, Anmin Ren, Zhiying Miao, Tian Zhou, Chenhui Zhang, Yiqun Chang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6842284/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2025 Read the published version in npj Biofilms and Microbiomes → Version 1 posted 9 You are reading this latest preprint version Abstract Addressing antibiotic-resistant bacterial biofilm infections without promoting drug resistance is a pressing challenge. Pseudomonas aeruginosa is well known for causing biofilm-associated drug-resistant infections that often lead to treatment failure. In this study, we identified a previously uncharacterized membrane protein ferredoxin encoded by PA1551 using photoaffinity-based biomimetic probes based on our previous dual-acting antibiofilm compound 2-(heptanamido)methyl 3-hydroxy-1,6-dimethylpyridin-4(1 H )-one ( 10d) . The precision-targeted ferredoxin PA1551 exhibited excellent effectiveness in various model systems, suppressing bacterial biofilm and virulence, and enhancing the antibacterial effects of tobramycin (Tob, by 200-fold) and ciprofloxacin (CIP, by 1000-fold) compared to single-dose antibiotic treatments in a mouse model of Pseudomonas aeruginosa infection. These results indicate that ferredoxin PA1551 can be used as target to design new antibiofilm drugs for the treatment of Pseudomonas aeruginosa infections, particularly challenging bacterial biofilms. Biological sciences/Microbiology/Antimicrobials Biological sciences/Microbiology/Biofilms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Antibiotics remain one of the most pivotal interventions in human medicine 1 – 4 . Effective and selective antibiotics have substantially prolonged the lifespan of humans, leading to a perception that bacterial infections have largely been controlled 5 , 6 . However, in recent years, the emergence of “superbugs” with broad-spectrum antibiotic resistance and the increasing prevalence of antibiotic-resistant biofilm infections have posed persistent global challenges 7 , 8 . Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen that can form biofilms, leading to the development of drug-resistant, hospital-acquired infections 9 , 10 . Biofilms formed by bacterial cells reduce the effectiveness of antimicrobials through various mechanisms, including reduced penetration of antimicrobials and persister cell formation 11 . The management of wound infections and lung infections is commonly complicated by the formation of biofilms 12 . It is estimated that 60–90% of delayed healing wounds and almost all lung infections are associated with biofilms 13 – 15 . Biofilms not only resist the host immune system and the killing effects of antibacterial drugs 16 , 17 but also exhibit high resistance to antibiotic therapy, extending healing durations. This leads to difficult-to-treat infections accompanied by elevated levels of inflammatory factors 18 , 19 . Given the complex biofilm formation process, which regulates virulence and antibiotic effectiveness, it has been identified as a potential therapeutic target for managing wound and lung infections. Nevertheless, important regulatory proteins in biofilm formation have not been fully characterized in P. aeruginosa , which is one of the reasons that no biofilm inhibitors have been marketed to date. Therefore, identifying precise targets that modulate biofilm formation is crucial for developing therapeutic strategies against drug-resistant bacteria, particularly for combating P. aeruginosa infections. Our previous study discovered a dual-acting antibiofilm strategy based on 2-substituted 3-hydroxy-1,6-dimethylpyridin-4-ones 20 , 21 . A hit compound 2-(heptanamido)methyl 3-hydroxy-1,6-dimethylpyridin-4(1 H )-one ( 10d ) exhibited promising antibiofilm activity for both the standard laboratory P. aeruginosa strain PAO1 (IC 50 = 6.6 µM) and several clinical multidrug-resistant P. aeruginosa strains 20 , which is able to interfere with iron uptake and the QS system of P. aeruginosa . Despite the unique biological activities of 10d , its exact target remains unknown. To identify its therapeutic targets for inhibiting biofilm, we used affinity-based proteome profiling (ABPP) combined with bioimaging for profiling drug-target interactions in situ 22 , 23 . ABPP is a powerful method for the in situ investigation of noncovalent ligand-receptor interactions, and this technique plays a crucial role in various fields, such as chemical biology and drug discovery 24 , 25 . As illustrated in Fig. 1 , we developed a series of chemical probes based on the scaffold of 2-(heptanamido)methyl 3-hydroxy-1,6-dimethylpyridin-4(1 H )-ones ( 10d) for the study of biofilm formation at the proteomic level to identify biological drug-resistant targets via ABPP. These probes possessed diazirine photoreactive moiety and alkyne analytical handles for mediating copper-mediated azide-alkyne coupling (CuAAC) reactions. We evaluated the biological activity of all these probes in P. aeruginosa and used the most optimal probe for the ABPP experiment. Using these biomimetic probes, we identified an iron metabolism protein (ferredoxin PA1551) as a drug target for biofilm inhibitors against difficult-to-treat bacterial biofilms. We purified the full-length membrane protein ferredoxin PA1551 and validated its interaction using biophysical techniques. Furthermore, a PA1551 -deficient mutant exhibited reduced virulence and altered biofilm phenotypic characteristics, supporting the antibacterial synergistic effect of targeting ferredoxin PA1551. In addition, in vivo assays confirmed that △ferredoxin ( PA1551 -deficient mutant) was effective in enhancing the antibacterial effects of tobramycin (Tob, by 200-fold) and ciprofloxacin (CIP, by 1000-fold) compared with single-dose antibiotic treatments in a mouse model of P. aeruginosa infection. In summary, our results demonstrate the suppression of biofilm formation in P. aeruginosa bacteria by inhibiting the ferredoxin PA1551 of iron homeostasis. This research provides new insights for inhibiting pathogenic disease. Results Compound 10d displays antibacterial synergistic efficacy on antibiotics against P. aeruginosa infection in vivo To discover the potential target for inhibiting P. aeruginosa biofilm, compound 10d stands out for its promising antibiofilm activity and could be used to explore the possible novel functional target. Considering that wound infection is one of the major infections caused by P. aeruginosa , we initially examined whether 10d can enhance the effectiveness of commonly used antibiotics, Tob and CIP 26 in an experimental open-wound mouse model of P. aeruginosa infection 27 . In this in vivo model, we created surface wounds in mice and inoculated the wound with 5×10 8 CFU of P. aeruginosa PAO1. Subsequently, the wounds were administered dropwise directly with physiological saline, Tob 28 (0.5 mg/mL), Tob (0.0025 mg/mL), or the combination of Tob (0.0025 mg/mL) with 10d over a 3-day period. We analyzed the bacterial load in each wound as a measure of the bacterial survival rate, and we monitored the status of mice for 9 days. The results indicated that 10d exhibited significant synergistic effects with Tob in controlling P. aeruginosa infection and improved wound healing in the open-wound murine model. In this model, lesions were significantly smaller in the 10d -Tob combination group than in the control group every day (Fig. 2 B, C). Nine days after infection, all the wounds treated with Tob (0.5 mg/mL) and Tob (0.0025 mg/mL) in combination with 10d were nearly closed, suggesting that the in vivo efficacy of combined Tob (0.0025 mg/mL) and 10d was comparable to that of high-dose Tob (0.5 mg/mL) treatment alone. After 3 days of continuous treatment (Fig. 2 A, D), the bacterial survival rate in the Tob (0.0025 mg/mL) + 10d combination group (2.28%) was significantly lower than that in the Tob (0.0025 mg/mL)-only group (16.78%) and the 10d -only group (99.93%). Moreover, the bacterial survival rate of the combination treatment group was comparable to that in the high-dose Tob (0.5 mg/mL)-only group (0%). This result indicated that 10d significantly improved the bactericidal effect of Tob by 200-fold. Tissue immunohistochemistry section images revealed no apparent toxicity to the organs following 3 days of treatment with the combination of Tob and 10d (Fig. 2 E). What’s more, 10d exhibited no obvious toxicity or effect on the body weight of the mice throughout the experiment ( Supplementary Fig. 1A ). We conducted additional experiments to investigate the synergistic effects of 10d with CIP, another commonly used antibiotic for P. aeruginosa treatment. The wound was treated with physiological saline, CIP 29 (1 mg/mL), CIP (0.01 mg/mL), CIP (0.001 mg/mL), the combination of CIP (0.01 mg/mL) with 10d , the combination of CIP (0.001 mg/mL) with 10d over a 3-day period. In addition, we monitored the status of mice for 9 days. Similar to the aforementioned findings, we observed that 10d enhanced the antibacterial effects of CIP by more than 200- to 1000-fold in an open-wound murine model of P. aeruginosa PAO1 infection (Fig. 3 A). Lesions caused by the infection were significantly smaller in the 10d -CIP combination group than in the control group on every day, except on day 1 (Fig. 3 B, C). Nine days after infection, all the wounds treated with CIP (1 mg/mL), the combination of CIP (0.01 mg/mL) with 10d , and the combination of CIP (0.001 mg/mL) with 10d were almost closed. This finding suggested that the in vivo efficacy of CIP (0.01 mg/mL)- 10d and CIP (0.001 mg/mL)- 10d combinations were comparable to that of the high-dose CIP (1 mg/mL) treatment alone. Furthermore, after 3 days of continuous treatment, the bacterial survival rate (0%) in the wound treated with the combination of CIP (0.01 mg/mL) and 10d was comparable to that in the wound treated with high-dose CIP (1 mg/mL) alone (0%) and was lower than that in the wound treated with low-dose CIP (0.01 mg/mL) alone (4.17%) (Fig. 3 D). In addition, the bacterial survival rate (2.35%) in the CIP (0.001 mg/mL)- 10d combination group was lower than that in the CIP (0.001 mg/mL) alone group (8.19%). These results indicated that 10d significantly improved the antibacterial effect of CIP. Histological sections of tissues revealed no apparent organ toxicity following 3 days of treatment with the combination of CIP and 10d (Fig. 3 E). Furthermore, 10d exhibited no obvious toxicity or effect on the body weight of the mice throughout the experiment ( Supplementary Fig. 1B ). In summary, using an open-wound murine model of P. aeruginosa infection, we found that the biofilm inhibitor 10d significantly enhanced the antibacterial effects of Tob (200-fold) and CIP (1000-fold) in vivo . These results suggest that 10d is a promising candidate as an antibiotic adjuvant for the treatment of P. aeruginosa infections. Design of chemical probes and evaluation of the antibiofilm effects of probes on P. aeruginosa. Our results revealed the promising antibacterial synergistic potential of biofilm inhibitor 10d for the treatment of P. aeruginosa infections. Next, we utilized affinity-based proteome profiling (ABPP) coupled with bioimaging for profiling drug-target interactions in situ . Over the past few years, Yao’s group has made several contributions to the field of ABPP by developing a set of minimalist diazirine photo-crosslinkers. These linkers, consisting of an alkyl diazirine, a reactive group, and a bioorthogonal handle, have not only substantially reduced the time and complexity involved in preparing photo-affinity probes but also considerably improved probe labeling efficiency. Diazirines, which remain stable under both acidic and basic conditions and are resistant to common nucleophiles and electrophiles, can be stored at room temperature, making them widely applicable in various labeling studies 30 , 31 . Inspired by the promising therapeutic effect of biofilm inhibitor 10d in P. aeruginosa infection, we further synthesized a series of probes based on the 10d scaffold by introducing a diazirine photoreactive moiety and alkyne handles for copper-mediated CuAAC reactions (complete synthetic details are provided in Supplementary Scheme 1–10 ) 20 . Among these probes, the commercially available minimalist photocrosslinker L1 (Fig. 4 A), which contains both diazirine and terminal alkyne, was selected for minimal modification of the affinity-based structural element, which is consistent with the fundamental principle that the ligand should closely resemble the natural ligand 25 , 32 . In the design of probe molecules, compound 10d-1 replaced the six-carbon chain of 10d , with the photocrosslinker L1 at the C-2 position to minimize ligand modification. In addition, previous studies examining structure-activity relationships have indicated that derivatives at the C-6 position of hydroxypyridinone exhibited minimal biofilm inhibitory effects 20 , 21 . This prompted us to focus on 2,6-disubstituted probe designs. Thus, we generated the affinity-based probe 10d-2 by introducing the photocrosslinker L1 at the C-6 position while preserving the six-carbon chain at the C-2 position of 10d . Initially, whether hydroxypyridinone derivatives could covalently modify their targets was unclear. Thus, we designed covalent probe molecules without a photoaffinity group. Among all three covalent probes, 10d-3 , 10d-4 , and 10d-5 corresponded to the parent drug 10d and the photoaffinity probe molecules 10d-1 and 10d-2 , respectively. Furthermore, three matching negative probes NP , NP-1 , and NP-2 were generated as controls in the following biological experiments to rule out any potential interference from the alkyne handle and photoaffinity group. We performed a crystal violet staining assay (Fig. 4 B) to examine the antibiofilm effects of these probes. Subsequently, we analyzed the capacity of these probes to inhibit the biofilms of P. aeruginosa PAO1. The antibiofilm effect of probe 10d-1 (IC 50 = 7.5 µM) on P. aeruginosa was comparable to that of 10d (IC 50 = 6.6 µM). Furthermore, to comprehensively evaluate the antibiofilm effects of probe 10d-1 , we examined its effect on the biofilm formation of P. aeruginosa PAO1 strains at concentrations of 1.25, 2.5, 5, 10, and 20 µM . 10d was concurrently tested as a reference compound. As expected, probe 10d-1 (Fig. 4 C, D), being the most suitable, significantly inhibited the biofilm formation of P. aeruginosa PAO1 in a dose-dependent manner. By contrast, the negative control probes ( NP ) displayed no biofilm inhibitory activity. Collectively, these results indicate that 10d-1 is a potent probe for labeling intact bacteria for the in situ profiling of antibacterial synergistic target-drug interactions. Affinity-based protein labeling and protein identification Next, we investigated whether probe 10d-1 can be used for simultaneous imaging and covalent labeling of target proteins in vivo . Probe 10d-1 contains a diazirine photoreactive moiety and alkyne analytical handles for CuAAC reactions. To evaluate the live-cell imaging capability of probe 10d-1 for P. aeruginosa PAO1 cells, we added this probe directly to P. aeruginosa cultures, both with and without 10× the amount of (+)- 10d as a competitor. Probe 10d-1 , 10× competitive (+)- 10d , and NP were added to P. aeruginosa cultures at a concentration of 10 µM. Subsequently, the CuAAC click reaction was performed with biotin azide under UV irradiation for the labeling of proteins. As shown in Fig. 5 A, photo labeling experiments with probe 10d-1 were highly successful (panel iii). Labeling bands were observed within the mass range of 25 to 55 kDa, and a strong band was visible in the mass range of 40 to 55 kDa. Weak fluorescence was detected in P. aeruginosa PAO1 cells treated with 10× competitive (+)- 10d (panel ii). These results indicated the efficiency of labeling with 10d-1 . When added at the same concentration as probe 10d-1 , NP (panel i) also exhibited weakly fluorescently labeled proteins in live bacteria, and most of the proteins obtained were similar to those obtained for probe 10d-1 . These “false positive” results might be attributed to structural similarities between NP and 10d-1 , because both are small molecules containing minimal terminal alkyne-containing diazirine photocrosslinkers. Thus, NP easily formed covalent bonds with proteins and could exist stably. However, in terms of fluorescence intensity, NP labeled the P. aeruginosa PAO1 proteome considerably less selectively than 10d-1 , resulting in several intense bands. The CuAAC click reaction of 10d-1 with biotin azide was performed under UV irradiation to label proteins. Subsequently, biotinylated proteins were captured using streptavidin agarose beads and identified LC − MS/MS. To minimize the “false hits” resulting from nonspecific protein binding, protein bands appearing in LC − MS/MS results that were obtained with 10× competitive (+)- 10d and NP were removed. The potential target proteins identified are summarized in Fig. 5 B and Supplementary Fig. 2A-D . Notably, most of the downregulated proteins were associated with iron homeostasis. Among the top identified proteins, PilJ is localized at cell poles and is essential for surface-associated twitching motility 33 . Twitching motility allows P. aeruginosa to respond to stimuli by extending and retracting its type IV pili. Q9I3G6/PA1551 was identified as an uncharacterized protein; it contains a domain belonging to the iron-sulfur family of cytoplasmic membrane proteins, possibly ferredoxin 34 . The PA4133 gene is predicted to encode the cytochrome c oxidase subunit (cbb3-type), which belongs to the heme-copper oxidase superfamily and facilitates the coupling of oxygen reduction to proton translocation across the membrane 35 . IscS, an L-cysteine desulfurase (pyridoxal phosphate-dependent), is a master enzyme responsible for delivering sulfur to various partners involved in Fe-S cluster assembly, tRNA modification, or cofactor biosynthesis. IscS plays a crucial role in sulfur delivery for Fe-S cluster synthesis onto IscU, a Fe-S scaffold assembly protein, and other sulfur acceptor proteins 36 . These results were consistent with the aforementioned results of antibiofilm experiments, indicating that 10d can disrupt iron homeostasis. Ferredoxin PA1551 may serve as a target to inhibit biofilm formation Based on the findings of affinity-based protein labeling experiments, we speculate that an uncharacterized protein ( PA1551 ) may play a key role in iron homeostasis ( Supplementary Fig. 2A-D ). The PA1551 gene is predicted to encode a ferredoxin cytoplasmic membrane protein, which we refer to as ferredoxin PA1551. The ferredoxin protein family comprises iron-sulfur (Fe-S) proteins that serve as electron carriers in various metabolic reactions and that connect biochemical pathways crucial for energy transduction, nutrient assimilation, and primary metabolism 37 . One of these subgroups is Fe-S ferredoxins, which are found in bacteria and often referred to as “bacterial-type” ferredoxins 37 – 39 . To directly investigate the labeling of ferredoxin PA1551 by the 10d-1 probe, we expressed and purified recombinant N-GST-PA1551 in the E. coli strain BL21 Star (DE3). The purified N-GST-PA1551 protein appeared as a single band with a molecular mass of 79.6 kDa ( Supplementary Fig. 3 ), consistent with the predicted molecular mass based on the DNA sequence. Next, we evaluated whether probe 10d-1 could be used for the simultaneous imaging and covalent labeling of the target protein. We incubated probe 10d-1 at concentrations of 1, 10, and 20 µM with P. aeruginosa cultures. Then, the CuAAC click reaction was performed with biotin azide under UV irradiation to efficiently label proteins. As shown in Fig. 5 C, the in-gel fluorescence scanning results for 10d-1 at concentrations of 1, 10, and 20 µM demonstrated highly specific labeling of the 80-kDa N-GST-PA1551 protein (panel i-iii) in a dose-dependent manner. Quantification of the fluorescent band intensity in in-gel fluorescence scanning also showed that ferredoxin PA1551 is the biological target of compound 10d . To evaluate the binding of 10d with ferredoxin PA1551, we carried out a microscale thermophoresis (MST) assay using the full-length membrane protein PA1551 fusion protein with 10d (Fig. 5 J), demonstrating that ferredoxin PA1551 directly interacts with 10d (K d = 8.49 ± 0.32 nM). To further validate the function of ferredoxin PA1551, we investigated the effects of the PA1551 -deficient mutant and PA1551 -overexpressing strain on iron homeostasis and biofilm formation in the presence of 10d . As shown in Fig. 5 D, E, 10d did not affect the biofilm formation of the PA1551 -deficient mutant or the PA1551 -overexpressing strain at various concentrations (1.25, 2.5, 5, 10, and 20 µM). However, the complementation of the PA1551 -deficient mutant with a plasmid carrying the PA1551 gene fully restored the biofilm inhibitory activity of 10d (Fig. 5 F). Furthermore, 10d still led to a significant reduction of biofilm in the absence of pilJ , PA4133 , and iscS genes (Fig. 5 G-I). These results suggest that 10d disrupted biofilm formation by targeting ferredoxin PA1551. To elucidate the mechanism of action between 10d and ferredoxin PA1551 at the molecular level, we built a computational model to identify the possible binding mode. The amino acid sequence of ferredoxin PA1551 was downloaded from Uniprot (Uniprot ID: Q9HGN8) 40 , and the protein structure was modeled using a local version of ColabFold 41 . ColabFold combines the fast homology search of MMseqs2 with AlphaFold2 to offer an accelerated prediction of unknown protein structures. The models were relaxed using amber force field after folding 42 . Despite the high-quality models, we still observed lower confidence (pLDDT < 50) in the loop region at the N terminus. Therefore, we conducted a molecular dynamics (MD) simulation to achieve the equilibrium of the N-terminal loop on a transmembrane model generated from the PPM server 43 . The entire structure reached equilibrium within 40 ns. To identify potential binding sites in the entire protein, a trajectory-based scan was conducted in the second half of the simulation ( Supplementary Fig. 4A-C ). We identified 43 site clusters from the equilibrium conformations (Fig. 6 A ). Among these, five clusters that appeared more than 75% of the time were inspected and filtered based on the size (> 75) and docking results ( Supplementary Table 1 ). Site 2, which was located around the luminal domain, could serve as a binding site, indicating that 10d is involved in Fe 3+ reduction mechanisms. (Fig. 6 B). Given that cystine plays a crucial role in Fe biological reduction 44 , this site, which is located along the channel rich in cysteine residues on the luminal domain, may be a candidate site for reduction reactions. On the ligand side, a conformational search was performed to account for the flexibility of 10d before docking. All possible conformations and protonated states were considered. The docking results provided promising affinity data both topologically and energetically. To verify the stability of the predicted binding mode, MD simulation was performed on the transmembrane complex model built using PPM 42 , As shown in Fig. 6 C, D, key interactions between 10d and THR282, ILE295, GLY296, and ALA298 were retained for most of the simulation. The MM/GBSA results based on the equilibrium trajectory supported the firm binding for the complex (ΔG = -64.96 Kcal/mol with STD 7.27). the site consisting of residues from Cys280 to Cys300 exhibited good compatibility for accommodating 10d -Fe 3+ , aligning with our speculation regarding the mechanism of ferredoxin PA1551. Analysis of PA1551-deficient mutants and -overexpressing strains Based on our exploration of the function of the previously uncharacterized cytoplasmic membrane protein ( PA1551 ), we hypothesized that its involvement in the reduction of ferric iron is a critical process in iron homeostasis in P. aeruginosa (Fig. 7 A). To determine whether ferredoxin PA1551 can reduce Fe 3+ to Fe 2+ and thus act as a reductase, we investigated the function of ferredoxin PA1551 in P. aeruginosa cells by performing validation experiments in vitro 45 . The reducing agent DTT can reduce ferric chloride to ferrous chloride. Ferrozine forms a magenta-red complex by chelating divalent iron ions, thus enabling the measurement of this reaction based on an increase in absorbance at 562 nm 46 . When ferric chloride was incubated with ferredoxin PA1551 and DTT, followed by the addition of ferrozine, we noted a gradual increase in absorbance at 562 nm over time. This result (Fig. 7 B) indicated that both the protein and DTT acted in the same manner for reducing trivalent iron ions to divalent ferrous iron ions, suggesting the essential role of ferredoxin PA1551 in the reduction of trivalent iron ions in P. aeruginosa . To investigate the role of ferredoxin PA1551 in the iron homeostasis of P. aeruginosa , we examined iron acquisition mechanisms in both the PA1551 -deficient mutant and PA1551 -overexpressing strains. P. aeruginosa has developed various systems for iron uptake, including those based on pyoverdine (Pvd) and pyochelin (Pch). To determine whether ferredoxin PA1551 interferes with iron homeostasis, we measured the levels of Pvd and Pch through fluorescence detection. These experiments were performed in ABTGC medium (a B-medium variant with 0.1% MgCl 2 , 0.1% CaCl 2 , and 0.1% FeCl 3 , supplemented with 10% A10, 0.2% glucose, and 0.2% casamino acids) and TSB medium, both containing 10 µM (iron-normal medium) and 0 µM (iron-deficient medium) of FeCl 3 . As depicted in Fig. 7 C-H, the PA1551 -deficient P. aeruginosa strain produced higher levels of Pvd and Pch to counteract iron deficiency in the ABTGC medium containing 10 µM FeCl 3 and in the TSB medium. However, the PA1551 -deficient P. aeruginosa strain exhibited negligible effects on the production of Pvd in the ABTGC medium containing 0 µM FeCl 3 . These results suggest that by interacting with high-affinity Pvd and Pch iron acquisition systems, ferredoxin PA1551 plays a role in iron homeostasis, particularly when a certain amount of iron is already present in the environment. Ferredoxin PA1551 may serve as an electron carrier in this process. The inhibition of electron transfer by disrupting the function of ferredoxin PA1551 can lead to bacterial iron limitation even under iron-rich conditions. To determine the role of ferredoxin PA1551 in the biofilm formation of P. aeruginosa , we examined the biofilm formation of the PA1551 -deficient mutant and PA1551 -overexpressing strains. As shown in Fig. 7 I, biofilm formation was significantly abolished in the PA1551 -deficient mutant. By contrast, biofilm formation was markedly increased in the PA1551 -overexpressing strain of P. aeruginosa . To investigate how ferredoxin PA1551 regulates the biofilm formation of P. aeruginosa , we measured the expression levels of pyocyanin and rhamnolipids and performed motility assays using the PA1551 -deficient mutant, PA1551 -overexpressing strain, and wild-type strain. The results revealed that the deletion of PA1551 significantly reduced the expression of pyocyanin and rhamnolipids. However, no significant differences were observed between the PA1551 -overexpressing strain and wild-type PAO1 (Fig. 7 J, K). The PA1551 -deficient mutant did not display swarming motility compared with the wild-type and PA1551 -overexpressing strain ( Supplementary Fig. 5 ). In addition, we investigated the effects of ferredoxin PA1551 on bacterial swimming and twitching motility ( Supplementary Fig. 5 ) and observed no change when compared with the wild-type strain. Because pyocyanin, rhamnolipids, and bacterial swarming motility are critical for the biofilm formation of P. aeruginosa , ferredoxin PA1551 may serve as an ideal target for developing novel antibiofilm therapeutics to combat P. aeruginosa biofilm-associated infections. Ferredoxin PA1551 mutant exhibits strong antibacterial synergistic effects in vivo Compared to the wild-type P. aeruginosa strain, the PA1551 -deficient mutant strain exhibited a significant reduction in the secretion of various virulence factors and biofilms. This finding indicates that ferredoxin PA1551 plays a crucial role in maintaining typical virulence and biofilm formation in P. aeruginosa . Thus, we used an open-wound infection murine model to determine whether ferredoxin PA1551 can serve as a target for inhibiting biofilm to combat P. aeruginosa infection. As shown in Fig. 8 A-D, wounds infected with the △ferredoxin mutant displayed a lower bacterial survival rate (21.44%). However, no therapeutic effects were observed for wounds infected with PAO1 and treated with saline (control), indicating a significant reduction in the virulence of the PA1551 -deficient mutant. Furthermore, the treatment of △ferredoxin-infected wounds with 0.0025 mg/mL Tob (3.95%) or 0.01 mg/mL (0%) and 0.001 mg/mL CIP (3.34%) resulted in the almost complete eradication of bacteria. These findings aligned with those for wounds infected with PAO1 and then treated with the combination of 10d and 0.0025 mg/mL Tob (2.28%) or the combination of 10d and 0.01/0.001 mg/mL CIP (0–2.35%), demonstrating that ferredoxin PA1551 plays a similar role as 10d in enhancing antibacterial effects. Similarly, compared with the control group (43.17%) and the wounds infected with △ferredoxin (74.67%), those infected with △ferredoxin and then treated with the combination of 10d and Tob or 10d and CIP exhibited high wound closure rates of 83.18–86.46%, emphasizing the importance of ferredoxin PA1551 in P. aeruginosa infection (Fig. 8 B, C). In addition, we observed no substantial organ toxicity (Fig. 8 E) or effects on the body weight of mice during the experiment ( Supplementary Fig. 1C ). These results demonstrated that ferredoxin PA1551 had a comparable effect to 10d in enhancing the antibacterial effects of Tob and CIP. These results raise the possibility that human infections could be treated by targeting ferredoxin PA1551 in difficult-to-treat bacterial biofilms. Discussion A major challenge in managing P. aeruginosa infections is the emergence of multidrug-resistant strains. This obstacle has prompted researchers to explore alternative, nonbiocidal methods to treat and eradicate bacterial infections 37 , 47 , 48 . The strategy of targeting biofilms is promising because it can reduce the use of antibiotics and curb the emergence and spread of antibiotic resistance. Using chemical proteomics, the PA1551 -deficient mutant, and the PA1551 -overexpressing strain, we identified that ferredoxin PA1551 can serve as a target to inhibit biofilm. We determined that the uncharacterized cytoplasmic membrane protein (ferredoxin PA1551) is involved in ferrite reduction, which plays an essential role in iron homeostasis in P. aeruginosa . In this study, we referred to ferredoxin encoded by PA1551 as ferredoxin PA1551. We propose that compound 10d 49 , 50 (Fig. 9 ) forms a complex with Fe 3+ outside the bacterial cell. This complex is then transported into the periplasm through the FpvA protein in the outer cell membrane of P. aeruginosa , where Fe 3+ is reduced to Fe 2+ by ferredoxin PA1551 in the inner membrane with the involvement of periplasmic proteins. Subsequently, the reduced Fe 2+ is chelated by periplasmic proteins, which can carry ferrous iron to the transporter proteins, allowing Fe 2+ to enter the cytoplasm. By inhibiting the action of ferredoxin PA1551, bacterial iron homeostasis, and quorum sensing can be disrupted, thus impeding biofilm formation. Our findings provide insights into the role of ferredoxin PA1551 in the antibiotic tolerance response, revealing a potential avenue to combat bacterial biofilm formation. In conclusion, by using our chemical probes designed on the basis of biofilm inhibitor 10d , we identified ferredoxin PA1551 as a new target for antibiofilm agents to combat P. aeruginosa infection. We revealed that under normal bacterial growth conditions where an iron source is available, iron homeostasis mediated by ferredoxin PA1551 is critical for the formation of P. aeruginosa biofilms and the secretion of virulence factors, such as pyocyanin, rhamnolipids, Pvd, and Pch (siderophores). Furthermore, we used an open-wound infection murine model to demonstrate that ferredoxin PA1551 can serve as an antibacterial synergistic target to inhibit biofilm in the treatment of P. aeruginosa infections. The ferredoxin PA1551 mutant exhibited the same efficacy as 10d in improving the antibacterial effect of Tob and CIP by more than 200- to 1000-fold. Our findings indicate the potential of ferredoxin PA1551 as a target for inhibiting biofilm formation to combat P. aeruginosa infections. Methods Strain and cell culture conditions Bacterial strains and plasmids used in this study are listed in Supplementary Table 2 . E. coli strains were grown at 37°C in either Luria-Bertani (LB) broth or agar. P. aeruginosa strains were grown at 37°C in either LB, tryptic soy broth (TSB), ABTGC medium (B-medium 51 (0.1% MgCl 2 , 0.1% CaCl 2 , 0.1% FeCl 3 ) supplemented with 10% A10, 0.2% glucose and 0.2% casamino acids). PA1551 mutant was grown in LB medium and on LB agar supplemented with 100 µg /mL Gentamicin. PA1551 -overexpressed was grown in LB medium and on LB agar supplemented with 100 µg /mL carbenicillin. Escherichia coli BL21 (DE3) carrying PA1551 cloned into the expression vector, pGEX-6P-1-1551 was grown in LB medium supplemented with 100 µg /mL ampicillin. Plasmid construction Primers 52 used in this study are listed in Supplementary Table 3 . To construct the knock-out plasmid for deletion, the PA1551 gene, the 1003-bp upstream fragment and the 1003-bp downstream fragment flanking PA1551 were amplified with primer pairs PA1551 up F/PA1551 up R and PA1551 low F/ PA1551 low R. The upstream and downstream PCR fragments were ligated by overlap PCR, and the resulting PCR products were inserted into the BamHI/HindIII sites of the suicide vector pK18. The gentamicin resistance cassette amplified from Gm was subsequently inserted into the same HindIII site to yield the knock-out plasmid pK18- PA1551. Expression plasmid constructs were generated by standard methods and verified by DNA sequencing. The plasmids were transformed into E. coli by heat shock and P. aeruginosa strain by electroporation unless otherwise stated. To generate the PA1551 -overexpressing construct p20- PA1551 , the encoding region of bswR was amplified with polymerase chain reaction (PCR) primers ( Supplementary Table 3 ), digested with EcoRI and HindIII inserted into the corresponding site of pUCP20. To construct pGEX-6P-1-PA1551, primer PA1551 F1/ PA1551 R1, PA1551 F2/ PA1551 R2, PA1551 F3/ PA1551 R3 were used to amplify the PA1551 gene. These PCR products of relevant genes were digested with BamHI/HindIII and inserted into the BamHI/HindIII sites of pGEX6P-1 resulting in plasmids pGEX6p-1-PA1551. The integrity of the insert in all constructs was confirmed by DNA sequencing. For constructing pET-28a-1551 (full-length), the primer pair 1551f and 1551r were used to amplify the PA1551 (full-length) fragment from the genomic DNA. Vector was digested with BamHⅠ and HindⅢ restriction enzymes and purified. The PCR products were digested and inserted into pET-28a to generate pET-28a-1551 (full-length) using the Seamless cloning kit. The resulting plasmid was transformed into E. coli DH5α, and the plasmid construct was verified by sequencing using primers that annealed to sites outside the multiple cloning site. N-GST-PA1551 expression A single bacterial colony of E. coli BL21 (DE3) carrying pGEX-6P-1-PA1551 strain was inoculated into 1 L of fresh LB medium flask supplemented with 100 ug/mL gentamycin. The culture was once again grown at 37°C with good aeration on a rotary shaker (200 rpm) until OD 600 reached 0.5-1. Protein expression was induced by the addition of IPTG to a final concentration of 1 mM. Following induction, the temperature was lowered to 16°C, and growth (with vigorous aeration, shaking at 200 rpm) was continued overnight. The bacterial pellet expressing recombinant protein was collected by sedimentation (4000 rpm, 15 min, 4°C). The supernatant was discarded and the cell pellet was washed with ice-cold 1×phosphate-buffered saline solution (PBS). Purification of N-GST-PA1551 protein using AKTA pureprotein purification layer system GST-tagged recombinant proteins were purified as described 53 . Fractions containing protein were pooled and analyzed by SDS-PAGE (4% stacking and 12% resolving gel). The purification protein samples were stored at -80°C. Protein expression and purification The plasmid pET-28a-1551 (full-length) was transformed into E. coli BL21 (DE3) , enabling the expression of PA1551 (full-length) membrane protein containing a 6-His tag. PA1551 (full-length) was induced with 0.5 mM IPTG overnight at 16°C. Cells were harvested and resuspended in lysis buffer composed of 30 mM HEPES pH 6.8, 150 mM NaCl, and 0.01% DDM (n-Dodecyl-β-D-Maltoside). Bacteria were lysed using probe sonication for a duration of 40 minutes, followed by the removal of cell debris through centrifugation at a speed of 15,000 rpm for 30 minutes. The supernatant was loaded onto a Ni-NTA column (L00250-50, GeneScript). The resin was washed with lysis buffer supplemented to 90 mM imidazole and subsequently eluted with lysis buffer supplemented to 300 mM imidazole. Where necessary, proteins were concentrated via filtration using centrifugation and a 10 kDa cut-off. Microscale thermophoresis (MST) assay The PA1551 (full-length)-His protein, at a 100 nmol/L concentration, was subjected to fluorescent tagging using a His-tag labeling kit (Nano Temper, MO-L008), adhering to the guidelines specified in the user manual. After the labeling was finalized, 10 µM of the fluorescently tagged ferredoxin PA1551 (full-length)-His protein was mixed with 10 M of PBST buffer, followed by the addition of 10d at a concentration of 1 mM. This mixture was then subject to serial dilution. The microscale thermophoresis assay was carried out on a NanoTemper Monolith NT.115 device, set to 20% LED power and medium MST power setting. Data collected from the assay were analyzed using the MO. Affinity Analysis software (X86). In situ protein labeling P. aeruginosa was inoculated in 5 mL LB and cultivated (200 rpm, 37°C) until the culture reached OD 600 to 0.05. Probe 10d-1 (10 µM), NP (10 µM), and 10d-1 (10 µM)- 10d (50 µM) as the competitor were incubated for 24 h at 37°C, with shaking (200 rpm). After 16 h incubation, the cultures were then centrifuged for 10 min at 4000 rpm and the cell pellets were washed with 2× PBS to remove excessive probe, followed by UV (365 nm) irradiation for 20 min on ice 25 . The cell pellets were resuspended in HEPES buffer (25 mM HEPES, 150 mM NaCl, 2 mM MgCl 2 ) with a 1% protease inhibitor cocktail (APExBIO, EDTA-Free in DMSO). The suspension was repeatedly frozen and thawed 3 times followed by sonication for 1 h (15s on and 15s off). The precipitate was removed by centrifugation and the supernatant was adjusted to 1 mg/mL 1.6 mL of a freshly premixed click chemistry reaction cocktail (50 mM TAMRA-biotin-N 3 from 50 mM stock solution in DMSO, 0.1 mM TBTA from 100 mM freshly prepared stock solution in DMSO, 1 mML TCEP from 1 M freshly prepared stock solution in deionized water, and 1 mM CuSO 4 from 1 M freshly prepared stock solution in deionized water) was added. The reaction was incubated for 2 h with gentle mixing at r.t before being terminated by the addition of prechilled Me 2 CO (incubation at 20°C for 1 h). Precipitated proteins were subsequently collected by centrifugation (10000 rpm, 10 min at 4°C). The supernatant was discarded and the pellet was washed twice with prechilled MeOH before redissolving in 2×loading buffer and heating for 10 min at 95°C. Proteins were separated by SDS-PAGE (10% gel) and then visualized by in-gel fluorescence scanning. Concentration-dependent N-GST-PA1551 labeling Purified N-GST-PA1551 protein samples were thawed, and the protein concentration was measured. Samples adjusted to contain 0.5 mg/mL of N-GST-PA1551 in PBS, were incubated in the presence of probe 10d-1 (1 µM, 10 µM, and 20 µM). Cu 2+ -mediated azide-alkyne coupling (CuAAC) reactions were above. Proteins were separated by SDS-PAGE (10% gel) and then visualized by in-gel fluorescence scanning. Large-scale Protein Identification by LC − MS/MS StageTips were prepared according to a modified protocoFl 54 with a C 18 solid-phase extraction disc (3 M Empore). Large-Scale Protein Identification by LC − MS/MS, for subsequent MS analysis, the samples were resuspended (with sonication in a water bath) in 10 mL of H 2 O containing 0.5% FA. Biofilm formation assay The measurement of compounds in biofilm formation was Crystal violet assay. P. aeruginosa PAO1 was grown overnight in LB medium at 37 o C for 24 h. Subsequently, the evaluation of biofilm formation using sterile 96-well flat-bottomed polystyrene microtiter plates (Corning/Costar, NY, USA) involved culture of 150 µL of bacterial (OD 600 = 0.05) in ABTGC medium at 37°C for 24 h. Subsequently, supernatant cells were removed and the biofilms were washed three times with sterile phosphate-buffered saline (PBS), then MeOH as a fixative. After 30 min, the MeOH was removed, and the microtiter plates were dried at RT. Crystal violet (0.1% in H 2 O) was then added to dye the biofilms which were then incubated for 30 min. Crystal violet was removed, microtiter plates were rinsed three times with sterile PBS, and add 150 µL of acetic acid (33%) to dissolve the biofilm. The absorbance values were measured at 570 nm by a microplate reader. All compounds were dissolved in DMSO to make 100 µM stock solutions before transferring to 96-well plates for the experiment. Rhamnolipid assay Rhamnolipid was quantified according to the method of Koch et al, with modifications. A subculture was conducted by directly diluting the overnight culture 1:100 into fresh Minimal Medium (49.3 mM Na 2 HPO 4 , 50 mM KH 2 PO 4 , 4.8 mM MgSO 4 , 7.6 mM (NH 4 ) 2 SO 4 , 0.6 mM CaCl 2 , 25 µM FeSO 4 , 0.162 µM (NH 4 ) 6 Mo 7 O 24 , 38 µM ZnSO 4 , 14 µM MnCl 2 , 1.6 µM CuSO 4 , 0.86 µM CoCl 2 , 1.9 µM boric acid, 5.5 µM NiCl 2 , 6.72 µM EDTA, 0.6% glycerol in 18 MΩ deionized H 2 O). The cultures were grown for 24 h at 37°C, with shaking (200 rpm). The final cell density was measured at 600 nm (OD 600 ) using a microplate reader (Bio-Tek). Supernatants were collected by ̈centrifuging at 10000 rpm for 10 min and extracted with Et 2 O (twice). Organic fractions were concentrated to yield a white solid. It was resuspended in deionized H 2 O and added with 0.19% (w/v) orcinol in 50% H 2 SO 4 . The resulting mixture was incubated at 80 o C for 30 min to give a yellow-orange solution. After cooling to rt, the absorbance was measured at 421 nm and results were normalized with final OD 600 values. Pyocyanin assay Overnight culture of P. aeruginosa PAO1, PA1551 deficient mutant, and PA1551 -overexpressing strain were standardized to OD 600 of 0.05 and diluted 100 times into 25 mL of LB Medium (Sangon Biotech, China) in 250 mL flask. The cultures were grown for 48 h at 37°C, with shaking (200 rpm). The cultures were then centrifuged for 10 min at 10000 rpm and 8 mL of the supernatants were transferred into new tubes. Briefly, 8 mL of culture supernatant conducted as described above was extracted with chloroform at a ratio of 4:1, followed by extraction with 1 mL of 0.2 M HCl. The absorbance of the upper red phase was measured using the OD 520 . The data was normalized by dividing the OD 520 reading with the final OD 600 values. Swarming motility assay To monitor swarming, petri dishes were filled with 20 mL of LB medium supplemented with 0.5% (w/v) bacto agar (Becton,Dickinson and Co.), 0.5% (w/v) casamino acids(Becton,Dickinson and Co.) and 0.5% (w/v) glucose (Solarbio, China) in the presence of P. aeruginosa PAO1, PA1551 deficient mutant, and PA1551 overexpressing strains. Petri dishes were dried in a single stack for 1 h at RT and then dropped 1 µL PAO1 (OD 600 = 1) onto the center of petri dishes and open the petri dishes to dry. The petri dishes were incubated for 16 h at 37°C. Swimming motility assay To monitor swimming, petri dishes contained 20 mL of LB medium supplemented with 0.3% (w/v) bacto agar, 0.5% (w/v) casamino acids and 0.5% (w/v) glucose in the presence of P. aeruginosa PAO1, PA1551 deficient mutant, and PA1551 overexpressing strains. Petri dishes were dried in a single stack for 1h at rt and then dropped 1 µL PAO1 (OD 600 = 1) into the bottom of the petri dishes and open the petri dishes to dry. The petri dishes were incubated for 16 h at 37°C. Twitching motility assay To monitor twitching, petri dishes were charged with 20 mL of LB medium supplemented with 1% (w/v) bacto agar, 0.5% (w/v) casamino acids and 0.5% (w/v) glucose in the presence of P. aeruginosa PAO1, PA1551 deficient mutant, and PA1551 overexpressing strains. Petri dishes were dried in a single stack for 1h at room temperature and then dropped 1 µL PAO1 (OD 600 = 1) into the bottom of the petri dishes and open the petri dishes to dry. The petri dishes were incubated for 16 h at 37°C. Pyoverdine and pyochelin assay An overnight culture of P. aeruginosa PAO1 (grown in LB medium at 37°C, 200 rpm) was diluted in ABTGC medium to a final optical density at 600 nm (OD 600 ) of 0.02 (2.5 × 10 8 CFU/mL). The microtiter plate was incubated at 37°C in a microplate reader (Bio-Tek) to measure the cell density (OD 600 ), Pyoverdine fluorescence (excitation at 400 nm, emission at 447 nm), pyochelin fluorescence (excitation at 350 nm, emission at 430 nm). The experiment assay for all test compounds and controls was done in triplicate. Reduction experiment of trivalent iron ions in vitro 20 µM FeCl 3 was incubated in 100 mM ammonium acetate buffer (pH = 6.5) in the presence of 100 mM DTT or protein, 200 µM ferrozine. Subsequently, the absorbance at 562 nm was monitored for 120 min. A 0 = absorbance at 0 min and A = absorbance at testing time. Validation of PA1551 protein for iron reduction in vitro After the constructed GST PGEX6P-1 strain induced a large amount of protein expression, the bacteria were lysed and the supernatant was extracted. To a solution of 20 µM Ferric chloride in 200 µL of 100 mM ammonium acetate buffer, 100 mM DTT or the supernatant of GST PGEX6P-1 strain was added at 37°C, then added separately 200 µM ferrozine in a 96-well plate. The 96-well plate was incubated at 37°C in a microplate reader (BioTek, Synergy H1) to measure the absorbance at 562 nm, every 5 min for 1.5 h. Computational methods The sequence of Ferredoxin PA1551 from Uniprot 40 was folded on a server with a local version of ColabFold 41 . Amber forcefield was used to relax the models. Top-ranked models were uploaded to PPM 2.0 server to predict the transmembrane region 43 . Apo protein with membrane system was prepared and constructed in Maestro protein preparation and Desmond system builder tools 55 , 56 . The orthorhombic simulation box was solvated of SPC water molecules, counterions, and an additional 0.15 M concentration of NaCl. A 100ns molecular dynamics was performed using Desmond to settle the loops, details are as in our previous work if not specified. SiteMap was utilized on every 10 frames of the second 50ns trajectory by a script 57 . All the sites identified were visually inspected. Top sites with preferable scores and sizes were all tested in Glide docking with the 10d conformations generated from LigPrep. The best 3 binding complexes were further validated in MD simulation, regular protein-ligand job settings were applied, and interaction and binding free energy analysis were performed to evaluate the affinity. Wound infection experiment in mice Ethical Statement. P. aeruginosa strain PAO1 was used for this animal experiment. Female 5-week-old BABL/C mice were purchased from SPF Biotechnology Co., Ltd with certificate number 20180302005. All animal experiments were performed under the animal care and use guidelines (IACUC approval number: Szwwbio-IACUC-20230930-01). During the whole experiment, the mice were kept at a constant temperature of 25 °C, with a 12 h light/night cycle, and provided with sufficient food and water without any animal cruelty. At the end of the experiment, the mice were all euthanized. Firstly, mice were randomly divided into different groups of 5/group and treated with 4% chloral hydrate, then the hair was shaved off the back of the mice and a circular wound of 4-5 mm was created on the back of each mouse. The wound was inoculated with 5×10 8 CFU of PAO1 (or △ferredoxin) to establish a wound infection model for 24 h. The mice in different groups were then treated with different drugs (saline, 0.5 mg/mL Tob, 0.0025 mg/mL Tob, 0.0025 mg/mL Tob+10 μM 10d , 10 μM 10d , 1 mg/mL CIP, 0.01 mg/mL CIP, 0.001 mg/mL CIP, 0.01 mg/mL CIP+10 μM 10d , 0.001 mg/mL CIP+10 μM 10d ). Mice were executed after 3 days of continuous dosing, wounded skin was cut off and homogenized, serially diluted and CFU counts were done on agar plates. In addition, histological sections were taken from the heart, liver, spleen, lung, and kidney of the mice. Declarations Data availability All data generated in this study are available upon request from the corresponding author. Acknowledgements This work was suppored by the National Natural Science Foundation of China (No. 82173651, and 82473788). Natural Science Foundation of Guangdong Province, China (2025A1515012144). Part of the images were created in BioRender.com with permission. Author contributions J. L. and A. R. contributed equally to this work. J. L. and A. R. wrote the manuscript and performed most of experiments. W.C. designed the project and supervised the chemical experiments as well as revised the manuscript; L.Y. supervised the experiments of target identification, and revised the manuscript; Z.M. performed chemical experiments. T.Z., C.Z., Y. C., S. Z., X.H., X. Z., T. J., Z. C., Z. L., J. L performed some chemical experiments or biological experiments. 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Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Published Journal Publication published 05 Dec, 2025 Read the published version in npj Biofilms and Microbiomes → Version 1 posted Editorial decision: Revision requested 23 Jul, 2025 Reviews received at journal 18 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 22 Jun, 2025 Reviewers agreed at journal 20 Jun, 2025 Reviewers invited by journal 16 Jun, 2025 Editor assigned by journal 12 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 07 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Jia","email":"","orcid":"","institution":"Southern University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tianyuan","middleName":"","lastName":"Jia","suffix":""},{"id":472079323,"identity":"65b0dcf9-8adb-4c96-9d36-6acb3fb1d662","order_by":10,"name":"Zhao Cai","email":"","orcid":"","institution":"Southern University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Cai","suffix":""},{"id":472079327,"identity":"8e71a204-f949-455d-934c-8fbb0ed99315","order_by":11,"name":"Zhengqiu Li","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Zhengqiu","middleName":"","lastName":"Li","suffix":""},{"id":472079328,"identity":"22cd6c17-14af-4b64-9371-d63e2c8be58d","order_by":12,"name":"Jing Lin","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Lin","suffix":""},{"id":472079329,"identity":"51708d29-3c62-4792-9a2f-a0290481e736","order_by":13,"name":"Liang Yang","email":"","orcid":"","institution":"Southern University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Yang","suffix":""},{"id":472079330,"identity":"959663ec-6078-4527-bf1a-be9733f5fa6a","order_by":14,"name":"Weimin Chen","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Weimin","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-06-07 10:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6842284/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6842284/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41522-025-00871-y","type":"published","date":"2025-12-05T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84922098,"identity":"885a6add-2300-4164-9604-ea0d06340a33","added_by":"auto","created_at":"2025-06-18 20:06:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ABPP methodology was used in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/0eb2c248b4e398510ceeed45.png"},{"id":84921808,"identity":"aaacca3f-dfcf-4342-b884-219bfd600ae2","added_by":"auto","created_at":"2025-06-18 19:58:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2600327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e antibacterial efficacy of 10d in combination with Tob. A\u003c/strong\u003e The results of CFU counting in the wound of mice on agar plate. \u003cstrong\u003eB\u003c/strong\u003eGraphical representation of the open-wound model of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 infection quantitative measurement of wound area within 9 days. \u003cstrong\u003eC\u003c/strong\u003e Visual observation of open-wound model of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 infection treated with control (saline), \u003cstrong\u003e10d\u003c/strong\u003e, Tob (0.5 mg/mL), Tob (0.0025 mg/mL), and the Tob(0.0025 mg/mL) - \u003cstrong\u003e10d\u003c/strong\u003e. \u003cstrong\u003eD\u003c/strong\u003e Related quantitative results of standard plate counting assay after different treatments with control (saline), \u003cstrong\u003e10d\u003c/strong\u003e, Tob (0.5 mg/mL), Tob (0.0025 mg/mL), and the Tob(0.0025 mg/mL) - \u003cstrong\u003e10d\u003c/strong\u003e in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 wound model. \u003cstrong\u003eE\u003c/strong\u003e Histological hematoxylin and eosin (H\u0026amp;E) staining images of heart, liver, spleen, lung, and kidney tissue sections at day 9 after different treatments indicated. All experiments are performed in triplicate, and data are presented as mean ± SD. *\u003cem\u003e p \u003c/em\u003e≤ 0.05; ** \u003cem\u003ep \u003c/em\u003e≤ 0.01; *** \u003cem\u003ep \u003c/em\u003e≤ 0.001; **** \u003cem\u003ep \u003c/em\u003e≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/d4b68b8f0f7c380e3ed9f917.png"},{"id":84921840,"identity":"675b707d-5e5c-4056-a094-7741d80a22a4","added_by":"auto","created_at":"2025-06-18 19:58:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2785140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eantibacterial efficacy of 10d in combination with CIP\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e The results of CFU counting in the wound of mice on agar plate. \u003cstrong\u003eB\u003c/strong\u003e Graphical representation of the open-wound model of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 infection quantitative measurement of wound area within 9 days. \u003cstrong\u003eC\u003c/strong\u003e Visual observation of open-wound model of \u003cem\u003eP. aeruginosa\u003c/em\u003ePAO1 infection treated with control (saline), CIP (1 mg/mL), CIP (0.01mg/mL), CIP (0.001mg/mL), CIP (0.01 mg/mL) - \u003cstrong\u003e10d\u003c/strong\u003e, and CIP (0.001 mg/mL) - \u003cstrong\u003e10d\u003c/strong\u003e. \u003cstrong\u003eD\u003c/strong\u003e Related quantitative results of standard plate counting assay after different treatments with control (saline), CIP (1mg/mL), CIP (0.01mg/mL), CIP (0.001mg/mL), CIP (0.01 mg/mL) - \u003cstrong\u003e10d\u003c/strong\u003e, and CIP (0.001 mg/mL) - \u003cstrong\u003e10d\u003c/strong\u003e in \u003cem\u003eP. aeruginosa \u003c/em\u003ePAO1 infected wound model.\u003cstrong\u003e E\u003c/strong\u003e Histological hematoxylin and eosin (H\u0026amp;E) staining images of heart, liver, spleen, lung, and kidney tissue sections at day 9 after different treatments indicated. All experiments are performed in triplicate, and data are presented as mean ± SD. * \u003cem\u003ep \u003c/em\u003e≤ 0.05; ** \u003cem\u003ep \u003c/em\u003e≤ 0.01; ***\u003cem\u003e p \u003c/em\u003e≤ 0.001; **** \u003cem\u003ep \u003c/em\u003e≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/cec1bec11571d6ce99bb9f79.png"},{"id":84921811,"identity":"048f2d9b-a351-4514-8a1c-894aa25fe43b","added_by":"auto","created_at":"2025-06-18 19:58:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":813567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of synthetic probes activity\u003c/strong\u003e. \u003cstrong\u003eA \u003c/strong\u003eThe structure of photoaffinity probes, covalent probes, and negative probes. \u003cstrong\u003eB\u003c/strong\u003e Activity of probes on biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1. \u003cstrong\u003eC\u003c/strong\u003eBiofilm formation inhibition rates of different concentrations of \u003cstrong\u003e10d \u003c/strong\u003e(1.25, 2.5, 5, 10, and 20 μM) for 24 h. \u003cstrong\u003eD\u003c/strong\u003e Biofilm formation inhibition rates of different concentrations of \u003cstrong\u003e10d-1 \u003c/strong\u003e(1.25, 2.5, 5, 10, and 20 μM) for 24 h. All experiments are performed in triplicate, and data are presented as mean ± SD. * \u003cem\u003ep \u003c/em\u003e≤ 0.05; ** \u003cem\u003ep \u003c/em\u003e≤ 0.01; ***\u003cem\u003e p \u003c/em\u003e≤ 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/919021674bd5ef92df483f40.png"},{"id":84922099,"identity":"6a8e49d4-8dbb-46b2-a0de-3bbfd195d71d","added_by":"auto","created_at":"2025-06-18 20:06:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1709431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLabeling of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Fluorescent scan of \u003cstrong\u003eNP\u003c/strong\u003e and \u003cstrong\u003e10d-1\u003c/strong\u003e labeled \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 proteins resolved by SDS-PAGE. \u003cstrong\u003eB\u003c/strong\u003e The most abundant protein hits (by score) were identified by ABPP experiments with probe \u003cstrong\u003e10d-1\u003c/strong\u003e. \u003cstrong\u003eC \u003c/strong\u003eFluorescent scan of \u003cstrong\u003e10d-1 \u003c/strong\u003e(1, 10, and 20 μM) labeled \u003cem\u003eP. aeruginosa\u003c/em\u003e N-GST-PA1551 proteins resolved by SDS-PAGE. \u003cstrong\u003eD\u003c/strong\u003e Effects of \u003cstrong\u003e10d-1\u003c/strong\u003e on biofilm formation in \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant strain, \u003cstrong\u003eE\u003c/strong\u003e \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain, \u003cstrong\u003eF\u003c/strong\u003e △PA1551 strain with a plasmid carrying the \u003cem\u003ePA1551\u003c/em\u003e gene. G \u003cem\u003epilJ\u003c/em\u003e-deficient mutant strain, \u003cstrong\u003eH\u003c/strong\u003e \u003cem\u003ePA4133\u003c/em\u003e-deficient mutant strain, and \u003cstrong\u003eI\u003c/strong\u003e \u003cem\u003eiscS\u003c/em\u003e-deficient mutant strain. \u003cstrong\u003eJ\u003c/strong\u003eMicroscale thermophoresis (MST) assay of membrane protein PA1551 with \u003cstrong\u003e10d\u003c/strong\u003e. All experiments are performed in triplicate, and data are presented as mean ± SD. * \u003cem\u003ep \u003c/em\u003e≤ 0.05; ** \u003cem\u003ep \u003c/em\u003e≤ 0.01; ***\u003cem\u003e p \u003c/em\u003e≤ 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/d176eff3748f717ca7122464.png"},{"id":84921841,"identity":"1edbedd3-1a2e-4685-871d-6213fcbd713d","added_by":"auto","created_at":"2025-06-18 19:58:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1019410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModeling of the binding of ferredoxin PA155 with 10d\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e Binding site cluster density based on trajectory frames. \u003cstrong\u003eB\u003c/strong\u003e Location of site2 in the optimized ferredoxin PA1551 model. \u003cstrong\u003eC\u003c/strong\u003e RMSD of protein Cα and \u003cstrong\u003e10d\u003c/strong\u003e. \u003cstrong\u003eD\u003c/strong\u003e \u003cstrong\u003e10d\u003c/strong\u003e binds in the bottom of cystine rich channel.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/71ef1a2a4a94e4aeb6a6ec5d.png"},{"id":84921813,"identity":"0af01475-23fd-43fe-a425-2303b8361f1b","added_by":"auto","created_at":"2025-06-18 19:58:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1017578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePA1551\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-deficient mutants and -overexpressing strains.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Iron uptake in \u003cem\u003eP. aeruginosa\u003c/em\u003e. \u003cstrong\u003eB\u003c/strong\u003e Reduction experiment of trivalent iron ions \u003cem\u003ein vitro\u003c/em\u003e. 20 μM FeCl\u003csub\u003e3\u003c/sub\u003e was incubated in 100 mM ammonium acetate buffer (pH=6.5) in the presence of 100 mM DTT or protein, 200 μM ferrozine. Experimental measurement of 562 nm absorbance. A0 = absorbance at 0 min and A = absorbance at time. \u003cstrong\u003eC-E\u003c/strong\u003e The production of iron-free pyoverdine was assessed in iron-supplemented ABTGC (0, 10 μM FeCl\u003csub\u003e3\u003c/sub\u003e), TSB medium. \u003cstrong\u003eF-H\u003c/strong\u003e The production of iron-free pyochelin was assessed in iron-supplemented ABTGC (0, 10 μM FeCl\u003csub\u003e3\u003c/sub\u003e), TSB medium. \u003cstrong\u003eI\u003c/strong\u003e Biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strains \u003cstrong\u003eJ \u003c/strong\u003ePyocyanin production of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain. \u003cstrong\u003eK\u003c/strong\u003e Rhamnolipid production of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain. All experiments are performed in triplicate, and data are presented as mean ± SD. * \u003cem\u003ep \u003c/em\u003e≤ 0.05; ** \u003cem\u003ep \u003c/em\u003e≤ 0.01; ***\u003cem\u003e p \u003c/em\u003e≤ 0.001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/b75e595133fb6b7703d52879.png"},{"id":84922100,"identity":"92fde212-ffc3-4faa-be07-ee45cd20634d","added_by":"auto","created_at":"2025-06-18 20:06:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2600666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe antibacterial synergistic effect of ferredoxin PA1551 in murine open-wound infection model.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The results of CFU counting in the wound of mice on agar plate. \u003cstrong\u003eB\u003c/strong\u003eGraphical representation of the open-wound model of \u003cem\u003eP. aeruginosa\u003c/em\u003e infection quantitative measurement of wound area within 9 days. \u003cstrong\u003eC\u003c/strong\u003e Visual observation of open-wound model of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (or △ferredoxin) infection treated with control (saline) (or 0.0025 mg/mL Tob, 0.01mg/mL, and 0.001mg/mL CIP). D Related quantitative results of standard plate counting assay after different treatments in PAO1 (or △ferredoxin)\u003cem\u003e \u003c/em\u003einfected wound model. \u003cstrong\u003eE\u003c/strong\u003e Histological hematoxylin and eosin (H\u0026amp;E) staining images of heart, liver, spleen, lung, and kidney tissue sections at day 9 after different treatments indicated. All experiments are performed in triplicate, and data are presented as mean ± SD. * \u003cem\u003ep \u003c/em\u003e≤ 0.05; ** \u003cem\u003ep \u003c/em\u003e≤ 0.01; ***\u003cem\u003e p \u003c/em\u003e≤ 0.001.**** \u003cem\u003ep \u003c/em\u003e≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/a683b2aa62dc3ea536ebd1bc.png"},{"id":84921822,"identity":"61cd14c2-80b1-442b-bb8e-e22aec147ab4","added_by":"auto","created_at":"2025-06-18 19:58:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1013915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA predictive model for the action of ferredoxin PA1551 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa.\u003c/strong\u003e\u003c/em\u003e Compound\u003cstrong\u003e 10d\u003c/strong\u003e (a synthetic siderophore) loads iron into the periplasm, Fe\u003csup\u003e3+\u003c/sup\u003e is reduced to Fe\u003csup\u003e2+ \u003c/sup\u003eby inner-membrane ferredoxin PA1551 in \u003cem\u003eP. aeruginosa.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/4890c48c73667005ffb9c08c.png"},{"id":97724033,"identity":"fd4b9aa0-1a11-45d7-ae06-386a17788396","added_by":"auto","created_at":"2025-12-08 16:11:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15699362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/0ced6634-f315-47ff-b3a6-c7f3155e82b6.pdf"},{"id":84921816,"identity":"c00019d3-a614-495d-a93b-83546843c37a","added_by":"auto","created_at":"2025-06-18 19:58:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4204319,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6842284/v1/fa2276eaeaf579feb2896345.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIdentification of ferredoxin PA1551 in the bacterial iron uptake pathway and exploration of its antibacterial synergistic as target for biofilm inhibitors against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e infection\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotics remain one of the most pivotal interventions in human medicine\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Effective and selective antibiotics have substantially prolonged the lifespan of humans, leading to a perception that bacterial infections have largely been controlled\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, in recent years, the emergence of \u0026ldquo;superbugs\u0026rdquo; with broad-spectrum antibiotic resistance and the increasing prevalence of antibiotic-resistant biofilm infections have posed persistent global challenges\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas aeruginosa (P. aeruginosa)\u003c/em\u003e is a common pathogen that can form biofilms, leading to the development of drug-resistant, hospital-acquired infections\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Biofilms formed by bacterial cells reduce the effectiveness of antimicrobials through various mechanisms, including reduced penetration of antimicrobials and persister cell formation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The management of wound infections and lung infections is commonly complicated by the formation of biofilms\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. It is estimated that 60\u0026ndash;90% of delayed healing wounds and almost all lung infections are associated with biofilms\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Biofilms not only resist the host immune system and the killing effects of antibacterial drugs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e but also exhibit high resistance to antibiotic therapy, extending healing durations. This leads to difficult-to-treat infections accompanied by elevated levels of inflammatory factors\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Given the complex biofilm formation process, which regulates virulence and antibiotic effectiveness, it has been identified as a potential therapeutic target for managing wound and lung infections. Nevertheless, important regulatory proteins in biofilm formation have not been fully characterized in \u003cem\u003eP. aeruginosa\u003c/em\u003e, which is one of the reasons that no biofilm inhibitors have been marketed to date. Therefore, identifying precise targets that modulate biofilm formation is crucial for developing therapeutic strategies against drug-resistant bacteria, particularly for combating \u003cem\u003eP. aeruginosa\u003c/em\u003e infections.\u003c/p\u003e \u003cp\u003eOur previous study discovered a dual-acting antibiofilm strategy based on 2-substituted 3-hydroxy-1,6-dimethylpyridin-4-ones\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. A hit compound 2-(heptanamido)methyl 3-hydroxy-1,6-dimethylpyridin-4(1\u003cem\u003eH\u003c/em\u003e)-one (\u003cb\u003e10d\u003c/b\u003e) exhibited promising antibiofilm activity for both the standard laboratory \u003cem\u003eP. aeruginosa\u003c/em\u003e strain PAO1 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.6 \u0026micro;M) and several clinical multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e strains\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which is able to interfere with iron uptake and the QS system of \u003cem\u003eP. aeruginosa\u003c/em\u003e. Despite the unique biological activities of \u003cb\u003e10d\u003c/b\u003e, its exact target remains unknown. To identify its therapeutic targets for inhibiting biofilm, we used affinity-based proteome profiling (ABPP) combined with bioimaging for profiling drug-target interactions \u003cem\u003ein situ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. ABPP is a powerful method for the \u003cem\u003ein situ\u003c/em\u003e investigation of noncovalent ligand-receptor interactions, and this technique plays a crucial role in various fields, such as chemical biology and drug discovery\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we developed a series of chemical probes based on the scaffold of 2-(heptanamido)methyl 3-hydroxy-1,6-dimethylpyridin-4(1\u003cem\u003eH\u003c/em\u003e)-ones (\u003cb\u003e10d)\u003c/b\u003e for the study of biofilm formation at the proteomic level to identify biological drug-resistant targets via ABPP. These probes possessed diazirine photoreactive moiety and alkyne analytical handles for mediating copper-mediated azide-alkyne coupling (CuAAC) reactions. We evaluated the biological activity of all these probes in \u003cem\u003eP. aeruginosa\u003c/em\u003e and used the most optimal probe for the ABPP experiment. Using these biomimetic probes, we identified an iron metabolism protein (ferredoxin PA1551) as a drug target for biofilm inhibitors against difficult-to-treat bacterial biofilms. We purified the full-length membrane protein ferredoxin PA1551 and validated its interaction using biophysical techniques. Furthermore, a \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant exhibited reduced virulence and altered biofilm phenotypic characteristics, supporting the antibacterial synergistic effect of targeting ferredoxin PA1551. In addition, \u003cem\u003ein vivo\u003c/em\u003e assays confirmed that △ferredoxin (\u003cem\u003ePA1551\u003c/em\u003e-deficient mutant) was effective in enhancing the antibacterial effects of tobramycin (Tob, by 200-fold) and ciprofloxacin (CIP, by 1000-fold) compared with single-dose antibiotic treatments in a mouse model of \u003cem\u003eP. aeruginosa\u003c/em\u003e infection. In summary, our results demonstrate the suppression of biofilm formation in \u003cem\u003eP. aeruginosa\u003c/em\u003e bacteria by inhibiting the ferredoxin PA1551 of iron homeostasis. This research provides new insights for inhibiting pathogenic disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCompound 10d displays antibacterial synergistic efficacy on antibiotics against\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo discover the potential target for inhibiting \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm, compound \u003cb\u003e10d\u003c/b\u003e stands out for its promising antibiofilm activity and could be used to explore the possible novel functional target. Considering that wound infection is one of the major infections caused by \u003cem\u003eP. aeruginosa\u003c/em\u003e, we initially examined whether \u003cb\u003e10d\u003c/b\u003e can enhance the effectiveness of commonly used antibiotics, Tob and CIP\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e in an experimental open-wound mouse model of \u003cem\u003eP. aeruginosa\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In this \u003cem\u003ein vivo\u003c/em\u003e model, we created surface wounds in mice and inoculated the wound with 5\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1. Subsequently, the wounds were administered dropwise directly with physiological saline, Tob\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (0.5 mg/mL), Tob (0.0025 mg/mL), or the combination of Tob (0.0025 mg/mL) with \u003cb\u003e10d\u003c/b\u003e over a 3-day period. We analyzed the bacterial load in each wound as a measure of the bacterial survival rate, and we monitored the status of mice for 9 days. The results indicated that \u003cb\u003e10d\u003c/b\u003e exhibited significant synergistic effects with Tob in controlling \u003cem\u003eP. aeruginosa\u003c/em\u003e infection and improved wound healing in the open-wound murine model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this model, lesions were significantly smaller in the \u003cb\u003e10d\u003c/b\u003e-Tob combination group than in the control group every day (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Nine days after infection, all the wounds treated with Tob (0.5 mg/mL) and Tob (0.0025 mg/mL) in combination with \u003cb\u003e10d\u003c/b\u003e were nearly closed, suggesting that the \u003cem\u003ein vivo\u003c/em\u003e efficacy of combined Tob (0.0025 mg/mL) and \u003cb\u003e10d\u003c/b\u003e was comparable to that of high-dose Tob (0.5 mg/mL) treatment alone. After 3 days of continuous treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, D), the bacterial survival rate in the Tob (0.0025 mg/mL)\u0026thinsp;+\u0026thinsp;\u003cb\u003e10d\u003c/b\u003e combination group (2.28%) was significantly lower than that in the Tob (0.0025 mg/mL)-only group (16.78%) and the \u003cb\u003e10d\u003c/b\u003e-only group (99.93%). Moreover, the bacterial survival rate of the combination treatment group was comparable to that in the high-dose Tob (0.5 mg/mL)-only group (0%). This result indicated that \u003cb\u003e10d\u003c/b\u003e significantly improved the bactericidal effect of Tob by 200-fold. Tissue immunohistochemistry section images revealed no apparent toxicity to the organs following 3 days of treatment with the combination of Tob and \u003cb\u003e10d\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). What\u0026rsquo;s more, \u003cb\u003e10d\u003c/b\u003e exhibited no obvious toxicity or effect on the body weight of the mice throughout the experiment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWe conducted additional experiments to investigate the synergistic effects of \u003cb\u003e10d\u003c/b\u003e with CIP, another commonly used antibiotic for \u003cem\u003eP. aeruginosa\u003c/em\u003e treatment. The wound was treated with physiological saline, CIP\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (1 mg/mL), CIP (0.01 mg/mL), CIP (0.001 mg/mL), the combination of CIP (0.01 mg/mL) with \u003cb\u003e10d\u003c/b\u003e, the combination of CIP (0.001 mg/mL) with \u003cb\u003e10d\u003c/b\u003e over a 3-day period. In addition, we monitored the status of mice for 9 days. Similar to the aforementioned findings, we observed that \u003cb\u003e10d\u003c/b\u003e enhanced the antibacterial effects of CIP by more than 200- to 1000-fold in an open-wound murine model of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Lesions caused by the infection were significantly smaller in the \u003cb\u003e10d\u003c/b\u003e-CIP combination group than in the control group on every day, except on day 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Nine days after infection, all the wounds treated with CIP (1 mg/mL), the combination of CIP (0.01 mg/mL) with \u003cb\u003e10d\u003c/b\u003e, and the combination of CIP (0.001 mg/mL) with \u003cb\u003e10d\u003c/b\u003e were almost closed. This finding suggested that the \u003cem\u003ein vivo\u003c/em\u003e efficacy of CIP (0.01 mg/mL)- \u003cb\u003e10d\u003c/b\u003e and CIP (0.001 mg/mL)- \u003cb\u003e10d\u003c/b\u003e combinations were comparable to that of the high-dose CIP (1 mg/mL) treatment alone. Furthermore, after 3 days of continuous treatment, the bacterial survival rate (0%) in the wound treated with the combination of CIP (0.01 mg/mL) and \u003cb\u003e10d\u003c/b\u003e was comparable to that in the wound treated with high-dose CIP (1 mg/mL) alone (0%) and was lower than that in the wound treated with low-dose CIP (0.01 mg/mL) alone (4.17%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, the bacterial survival rate (2.35%) in the CIP (0.001 mg/mL)- \u003cb\u003e10d\u003c/b\u003e combination group was lower than that in the CIP (0.001 mg/mL) alone group (8.19%). These results indicated that \u003cb\u003e10d\u003c/b\u003e significantly improved the antibacterial effect of CIP. Histological sections of tissues revealed no apparent organ toxicity following 3 days of treatment with the combination of CIP and \u003cb\u003e10d\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Furthermore, \u003cb\u003e10d\u003c/b\u003e exhibited no obvious toxicity or effect on the body weight of the mice throughout the experiment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, using an open-wound murine model of \u003cem\u003eP. aeruginosa\u003c/em\u003e infection, we found that the biofilm inhibitor \u003cb\u003e10d\u003c/b\u003e significantly enhanced the antibacterial effects of Tob (200-fold) and CIP (1000-fold) \u003cem\u003ein vivo\u003c/em\u003e. These results suggest that \u003cb\u003e10d\u003c/b\u003e is a promising candidate as an antibiotic adjuvant for the treatment of \u003cem\u003eP. aeruginosa\u003c/em\u003e infections.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDesign of chemical probes and evaluation of the antibiofilm effects of probes on\u003c/b\u003e \u003cb\u003eP. aeruginosa.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results revealed the promising antibacterial synergistic potential of biofilm inhibitor \u003cb\u003e10d\u003c/b\u003e for the treatment of \u003cem\u003eP. aeruginosa\u003c/em\u003e infections. Next, we utilized affinity-based proteome profiling (ABPP) coupled with bioimaging for profiling drug-target interactions \u003cem\u003ein situ\u003c/em\u003e. Over the past few years, Yao\u0026rsquo;s group has made several contributions to the field of ABPP by developing a set of minimalist diazirine photo-crosslinkers. These linkers, consisting of an alkyl diazirine, a reactive group, and a bioorthogonal handle, have not only substantially reduced the time and complexity involved in preparing photo-affinity probes but also considerably improved probe labeling efficiency. Diazirines, which remain stable under both acidic and basic conditions and are resistant to common nucleophiles and electrophiles, can be stored at room temperature, making them widely applicable in various labeling studies\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInspired by the promising therapeutic effect of biofilm inhibitor \u003cb\u003e10d\u003c/b\u003e in \u003cem\u003eP. aeruginosa\u003c/em\u003e infection, we further synthesized a series of probes based on the \u003cb\u003e10d\u003c/b\u003e scaffold by introducing a diazirine photoreactive moiety and alkyne handles for copper-mediated CuAAC reactions (complete synthetic details are provided in \u003cb\u003eSupplementary Scheme 1\u0026ndash;10\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Among these probes, the commercially available minimalist photocrosslinker \u003cb\u003eL1\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which contains both diazirine and terminal alkyne, was selected for minimal modification of the affinity-based structural element, which is consistent with the fundamental principle that the ligand should closely resemble the natural ligand\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In the design of probe molecules, compound \u003cb\u003e10d-1\u003c/b\u003e replaced the six-carbon chain of \u003cb\u003e10d\u003c/b\u003e, with the photocrosslinker \u003cb\u003eL1\u003c/b\u003e at the C-2 position to minimize ligand modification. In addition, previous studies examining structure-activity relationships have indicated that derivatives at the C-6 position of hydroxypyridinone exhibited minimal biofilm inhibitory effects\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This prompted us to focus on 2,6-disubstituted probe designs. Thus, we generated the affinity-based probe \u003cb\u003e10d-2\u003c/b\u003e by introducing the photocrosslinker \u003cb\u003eL1\u003c/b\u003e at the C-6 position while preserving the six-carbon chain at the C-2 position of \u003cb\u003e10d\u003c/b\u003e. Initially, whether hydroxypyridinone derivatives could covalently modify their targets was unclear. Thus, we designed covalent probe molecules without a photoaffinity group. Among all three covalent probes, \u003cb\u003e10d-3\u003c/b\u003e, \u003cb\u003e10d-4\u003c/b\u003e, and \u003cb\u003e10d-5\u003c/b\u003e corresponded to the parent drug \u003cb\u003e10d\u003c/b\u003e and the photoaffinity probe molecules \u003cb\u003e10d-1\u003c/b\u003e and \u003cb\u003e10d-2\u003c/b\u003e, respectively. Furthermore, three matching negative probes \u003cb\u003eNP\u003c/b\u003e, \u003cb\u003eNP-1\u003c/b\u003e, and \u003cb\u003eNP-2\u003c/b\u003e were generated as controls in the following biological experiments to rule out any potential interference from the alkyne handle and photoaffinity group.\u003c/p\u003e \u003cp\u003eWe performed a crystal violet staining assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) to examine the antibiofilm effects of these probes. Subsequently, we analyzed the capacity of these probes to inhibit the biofilms of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1. The antibiofilm effect of probe \u003cb\u003e10d-1\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.5 \u0026micro;M) on \u003cem\u003eP. aeruginosa\u003c/em\u003e was comparable to that of \u003cb\u003e10d\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.6 \u0026micro;M). Furthermore, to comprehensively evaluate the antibiofilm effects of probe \u003cb\u003e10d-1\u003c/b\u003e, we examined its effect on the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 strains at concentrations of 1.25, 2.5, 5, 10, and 20 \u003cb\u003e\u0026micro;M\u003c/b\u003e. \u003cb\u003e10d\u003c/b\u003e was concurrently tested as a reference compound. As expected, probe \u003cb\u003e10d-1\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D), being the most suitable, significantly inhibited the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 in a dose-dependent manner. By contrast, the negative control probes (\u003cb\u003eNP\u003c/b\u003e) displayed no biofilm inhibitory activity. Collectively, these results indicate that \u003cb\u003e10d-1\u003c/b\u003e is a potent probe for labeling intact bacteria for the \u003cem\u003ein situ\u003c/em\u003e profiling of antibacterial synergistic target-drug interactions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAffinity-based protein labeling and protein identification\u003c/h2\u003e \u003cp\u003eNext, we investigated whether probe \u003cb\u003e10d-1\u003c/b\u003e can be used for simultaneous imaging and covalent labeling of target proteins \u003cem\u003ein vivo\u003c/em\u003e. Probe \u003cb\u003e10d-1\u003c/b\u003e contains a diazirine photoreactive moiety and alkyne analytical handles for CuAAC reactions. To evaluate the live-cell imaging capability of probe \u003cb\u003e10d-1\u003c/b\u003e for \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 cells, we added this probe directly to \u003cem\u003eP. aeruginosa\u003c/em\u003e cultures, both with and without 10\u0026times; the amount of (+)-\u003cb\u003e10d\u003c/b\u003e as a competitor. Probe \u003cb\u003e10d-1\u003c/b\u003e, 10\u0026times; competitive (+)-\u003cb\u003e10d\u003c/b\u003e, and NP were added to \u003cem\u003eP. aeruginosa\u003c/em\u003e cultures at a concentration of 10 \u0026micro;M. Subsequently, the CuAAC click reaction was performed with biotin azide under UV irradiation for the labeling of proteins. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, photo labeling experiments with probe \u003cb\u003e10d-1\u003c/b\u003e were highly successful (panel iii). Labeling bands were observed within the mass range of 25 to 55 kDa, and a strong band was visible in the mass range of 40 to 55 kDa. Weak fluorescence was detected in \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 cells treated with 10\u0026times; competitive (+)-\u003cb\u003e10d\u003c/b\u003e (panel ii). These results indicated the efficiency of labeling with \u003cb\u003e10d-1\u003c/b\u003e. When added at the same concentration as probe \u003cb\u003e10d-1\u003c/b\u003e, NP (panel i) also exhibited weakly fluorescently labeled proteins in live bacteria, and most of the proteins obtained were similar to those obtained for probe \u003cb\u003e10d-1\u003c/b\u003e. These \u0026ldquo;false positive\u0026rdquo; results might be attributed to structural similarities between \u003cb\u003eNP\u003c/b\u003e and \u003cb\u003e10d-1\u003c/b\u003e, because both are small molecules containing minimal terminal alkyne-containing diazirine photocrosslinkers. Thus, \u003cb\u003eNP\u003c/b\u003e easily formed covalent bonds with proteins and could exist stably. However, in terms of fluorescence intensity, \u003cb\u003eNP\u003c/b\u003e labeled the \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 proteome considerably less selectively than \u003cb\u003e10d-1\u003c/b\u003e, resulting in several intense bands.\u003c/p\u003e \u003cp\u003eThe CuAAC click reaction of \u003cb\u003e10d-1\u003c/b\u003e with biotin azide was performed under UV irradiation to label proteins. Subsequently, biotinylated proteins were captured using streptavidin agarose beads and identified LC\u0026thinsp;\u0026minus;\u0026thinsp;MS/MS. To minimize the \u0026ldquo;false hits\u0026rdquo; resulting from nonspecific protein binding, protein bands appearing in LC\u0026thinsp;\u0026minus;\u0026thinsp;MS/MS results that were obtained with 10\u0026times; competitive (+)-\u003cb\u003e10d\u003c/b\u003e and NP were removed. The potential target proteins identified are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cb\u003eSupplementary Fig.\u0026nbsp;2A-D\u003c/b\u003e. Notably, most of the downregulated proteins were associated with iron homeostasis. Among the top identified proteins, PilJ is localized at cell poles and is essential for surface-associated twitching motility\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Twitching motility allows \u003cem\u003eP. aeruginosa\u003c/em\u003e to respond to stimuli by extending and retracting its type IV pili. Q9I3G6/PA1551 was identified as an uncharacterized protein; it contains a domain belonging to the iron-sulfur family of cytoplasmic membrane proteins, possibly ferredoxin\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ePA4133\u003c/em\u003e gene is predicted to encode the cytochrome c oxidase subunit (cbb3-type), which belongs to the heme-copper oxidase superfamily and facilitates the coupling of oxygen reduction to proton translocation across the membrane\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. IscS, an L-cysteine desulfurase (pyridoxal phosphate-dependent), is a master enzyme responsible for delivering sulfur to various partners involved in Fe-S cluster assembly, tRNA modification, or cofactor biosynthesis. IscS plays a crucial role in sulfur delivery for Fe-S cluster synthesis onto IscU, a Fe-S scaffold assembly protein, and other sulfur acceptor proteins\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. These results were consistent with the aforementioned results of antibiofilm experiments, indicating that \u003cb\u003e10d\u003c/b\u003e can disrupt iron homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFerredoxin PA1551 may serve as a target to inhibit biofilm formation\u003c/h3\u003e\n\u003cp\u003eBased on the findings of affinity-based protein labeling experiments, we speculate that an uncharacterized protein (\u003cem\u003ePA1551\u003c/em\u003e) may play a key role in iron homeostasis (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A-D\u003c/b\u003e). The \u003cem\u003ePA1551\u003c/em\u003e gene is predicted to encode a ferredoxin cytoplasmic membrane protein, which we refer to as ferredoxin PA1551. The ferredoxin protein family comprises iron-sulfur (Fe-S) proteins that serve as electron carriers in various metabolic reactions and that connect biochemical pathways crucial for energy transduction, nutrient assimilation, and primary metabolism\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. One of these subgroups is Fe-S ferredoxins, which are found in bacteria and often referred to as \u0026ldquo;bacterial-type\u0026rdquo; ferredoxins\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo directly investigate the labeling of ferredoxin PA1551 by the \u003cb\u003e10d-1\u003c/b\u003e probe, we expressed and purified recombinant N-GST-PA1551 in the \u003cem\u003eE. coli\u003c/em\u003e strain BL21 Star (DE3). The purified N-GST-PA1551 protein appeared as a single band with a molecular mass of 79.6 kDa (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e), consistent with the predicted molecular mass based on the DNA sequence. Next, we evaluated whether probe \u003cb\u003e10d-1\u003c/b\u003e could be used for the simultaneous imaging and covalent labeling of the target protein. We incubated probe \u003cb\u003e10d-1\u003c/b\u003e at concentrations of 1, 10, and 20 \u0026micro;M with \u003cem\u003eP. aeruginosa\u003c/em\u003e cultures. Then, the CuAAC click reaction was performed with biotin azide under UV irradiation to efficiently label proteins. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the in-gel fluorescence scanning results for \u003cb\u003e10d-1\u003c/b\u003e at concentrations of 1, 10, and 20 \u0026micro;M demonstrated highly specific labeling of the 80-kDa N-GST-PA1551 protein (panel i-iii) in a dose-dependent manner. Quantification of the fluorescent band intensity in in-gel fluorescence scanning also showed that ferredoxin PA1551 is the biological target of compound \u003cb\u003e10d\u003c/b\u003e. To evaluate the binding of \u003cb\u003e10d\u003c/b\u003e with ferredoxin PA1551, we carried out a microscale thermophoresis (MST) assay using the full-length membrane protein PA1551 fusion protein with \u003cb\u003e10d\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), demonstrating that ferredoxin PA1551 directly interacts with \u003cb\u003e10d\u003c/b\u003e (K\u003csub\u003ed\u003c/sub\u003e = 8.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 nM).\u003c/p\u003e \u003cp\u003eTo further validate the function of ferredoxin PA1551, we investigated the effects of the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain on iron homeostasis and biofilm formation in the presence of \u003cb\u003e10d\u003c/b\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E, 10d did not affect the biofilm formation of the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant or the \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain at various concentrations (1.25, 2.5, 5, 10, and 20 \u0026micro;M). However, the complementation of the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant with a plasmid carrying the \u003cem\u003ePA1551\u003c/em\u003e gene fully restored the biofilm inhibitory activity of \u003cb\u003e10d\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Furthermore, \u003cb\u003e10d\u003c/b\u003e still led to a significant reduction of biofilm in the absence of \u003cem\u003epilJ\u003c/em\u003e, \u003cem\u003ePA4133\u003c/em\u003e, and \u003cem\u003eiscS\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I). These results suggest that \u003cb\u003e10d\u003c/b\u003e disrupted biofilm formation by targeting ferredoxin PA1551.\u003c/p\u003e \u003cp\u003eTo elucidate the mechanism of action between \u003cb\u003e10d\u003c/b\u003e and ferredoxin PA1551 at the molecular level, we built a computational model to identify the possible binding mode. The amino acid sequence of ferredoxin PA1551 was downloaded from Uniprot (Uniprot ID: Q9HGN8)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and the protein structure was modeled using a local version of ColabFold\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. ColabFold combines the fast homology search of MMseqs2 with AlphaFold2 to offer an accelerated prediction of unknown protein structures. The models were relaxed using amber force field after folding\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Despite the high-quality models, we still observed lower confidence (pLDDT\u0026thinsp;\u0026lt;\u0026thinsp;50) in the loop region at the N terminus. Therefore, we conducted a molecular dynamics (MD) simulation to achieve the equilibrium of the N-terminal loop on a transmembrane model generated from the PPM server\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The entire structure reached equilibrium within 40 ns. To identify potential binding sites in the entire protein, a trajectory-based scan was conducted in the second half of the simulation (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A-C\u003c/b\u003e). We identified 43 site clusters from the equilibrium conformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e Among these, five clusters that appeared more than 75% of the time were inspected and filtered based on the size (\u0026gt;\u0026thinsp;75) and docking results (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). Site 2, which was located around the luminal domain, could serve as a binding site, indicating that \u003cb\u003e10d\u003c/b\u003e is involved in Fe\u003csup\u003e3+\u003c/sup\u003e reduction mechanisms. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Given that cystine plays a crucial role in Fe biological reduction\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, this site, which is located along the channel rich in cysteine residues on the luminal domain, may be a candidate site for reduction reactions. On the ligand side, a conformational search was performed to account for the flexibility of \u003cb\u003e10d\u003c/b\u003e before docking. All possible conformations and protonated states were considered. The docking results provided promising affinity data both topologically and energetically. To verify the stability of the predicted binding mode, MD simulation was performed on the transmembrane complex model built using PPM\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D, key interactions between \u003cb\u003e10d\u003c/b\u003e and THR282, ILE295, GLY296, and ALA298 were retained for most of the simulation. The MM/GBSA results based on the equilibrium trajectory supported the firm binding for the complex (ΔG = -64.96 Kcal/mol with STD 7.27). the site consisting of residues from Cys280 to Cys300 exhibited good compatibility for accommodating \u003cb\u003e10d\u003c/b\u003e-Fe\u003csup\u003e3+\u003c/sup\u003e, aligning with our speculation regarding the mechanism of ferredoxin PA1551.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAnalysis of PA1551-deficient mutants and -overexpressing strains\u003c/h3\u003e\n\u003cp\u003eBased on our exploration of the function of the previously uncharacterized cytoplasmic membrane protein (\u003cem\u003ePA1551\u003c/em\u003e), we hypothesized that its involvement in the reduction of ferric iron is a critical process in iron homeostasis in \u003cem\u003eP. aeruginosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). To determine whether ferredoxin PA1551 can reduce Fe\u003csup\u003e3+\u003c/sup\u003e to Fe\u003csup\u003e2+\u003c/sup\u003e and thus act as a reductase, we investigated the function of ferredoxin PA1551 in \u003cem\u003eP. aeruginosa\u003c/em\u003e cells by performing validation experiments \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. The reducing agent DTT can reduce ferric chloride to ferrous chloride. Ferrozine forms a magenta-red complex by chelating divalent iron ions, thus enabling the measurement of this reaction based on an increase in absorbance at 562 nm\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. When ferric chloride was incubated with ferredoxin PA1551 and DTT, followed by the addition of ferrozine, we noted a gradual increase in absorbance at 562 nm over time. This result (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) indicated that both the protein and DTT acted in the same manner for reducing trivalent iron ions to divalent ferrous iron ions, suggesting the essential role of ferredoxin PA1551 in the reduction of trivalent iron ions in \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of ferredoxin PA1551 in the iron homeostasis of \u003cem\u003eP. aeruginosa\u003c/em\u003e, we examined iron acquisition mechanisms in both the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strains. \u003cem\u003eP. aeruginosa\u003c/em\u003e has developed various systems for iron uptake, including those based on pyoverdine (Pvd) and pyochelin (Pch). To determine whether ferredoxin PA1551 interferes with iron homeostasis, we measured the levels of Pvd and Pch through fluorescence detection. These experiments were performed in ABTGC medium (a B-medium variant with 0.1% MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1% CaCl\u003csub\u003e2\u003c/sub\u003e, and 0.1% FeCl\u003csub\u003e3\u003c/sub\u003e, supplemented with 10% A10, 0.2% glucose, and 0.2% casamino acids) and TSB medium, both containing 10 \u0026micro;M (iron-normal medium) and 0 \u0026micro;M (iron-deficient medium) of FeCl\u003csub\u003e3\u003c/sub\u003e. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-H, the \u003cem\u003ePA1551\u003c/em\u003e-deficient \u003cem\u003eP. aeruginosa\u003c/em\u003e strain produced higher levels of Pvd and Pch to counteract iron deficiency in the ABTGC medium containing 10 \u0026micro;M FeCl\u003csub\u003e3\u003c/sub\u003e and in the TSB medium. However, the \u003cem\u003ePA1551\u003c/em\u003e-deficient \u003cem\u003eP. aeruginosa\u003c/em\u003e strain exhibited negligible effects on the production of Pvd in the ABTGC medium containing 0 \u0026micro;M FeCl\u003csub\u003e3\u003c/sub\u003e. These results suggest that by interacting with high-affinity Pvd and Pch iron acquisition systems, ferredoxin PA1551 plays a role in iron homeostasis, particularly when a certain amount of iron is already present in the environment. Ferredoxin PA1551 may serve as an electron carrier in this process. The inhibition of electron transfer by disrupting the function of ferredoxin PA1551 can lead to bacterial iron limitation even under iron-rich conditions.\u003c/p\u003e \u003cp\u003eTo determine the role of ferredoxin PA1551 in the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e, we examined the biofilm formation of the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strains. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI, biofilm formation was significantly abolished in the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant. By contrast, biofilm formation was markedly increased in the \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain of \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo investigate how ferredoxin PA1551 regulates the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e, we measured the expression levels of pyocyanin and rhamnolipids and performed motility assays using the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant, \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain, and wild-type strain. The results revealed that the deletion of \u003cem\u003ePA1551\u003c/em\u003e significantly reduced the expression of pyocyanin and rhamnolipids. However, no significant differences were observed between the \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain and wild-type PAO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K). The \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant did not display swarming motility compared with the wild-type and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). In addition, we investigated the effects of ferredoxin PA1551 on bacterial swimming and twitching motility (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e) and observed no change when compared with the wild-type strain. Because pyocyanin, rhamnolipids, and bacterial swarming motility are critical for the biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e, ferredoxin PA1551 may serve as an ideal target for developing novel antibiofilm therapeutics to combat \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm-associated infections.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFerredoxin PA1551 mutant exhibits strong antibacterial synergistic effects\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCompared to the wild-type \u003cem\u003eP. aeruginosa\u003c/em\u003e strain, the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant strain exhibited a significant reduction in the secretion of various virulence factors and biofilms. This finding indicates that ferredoxin PA1551 plays a crucial role in maintaining typical virulence and biofilm formation in \u003cem\u003eP. aeruginosa\u003c/em\u003e. Thus, we used an open-wound infection murine model to determine whether ferredoxin PA1551 can serve as a target for inhibiting biofilm to combat \u003cem\u003eP. aeruginosa\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D, wounds infected with the △ferredoxin mutant displayed a lower bacterial survival rate (21.44%). However, no therapeutic effects were observed for wounds infected with PAO1 and treated with saline (control), indicating a significant reduction in the virulence of the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant. Furthermore, the treatment of △ferredoxin-infected wounds with 0.0025 mg/mL Tob (3.95%) or 0.01 mg/mL (0%) and 0.001 mg/mL CIP (3.34%) resulted in the almost complete eradication of bacteria. These findings aligned with those for wounds infected with PAO1 and then treated with the combination of \u003cb\u003e10d\u003c/b\u003e and 0.0025 mg/mL Tob (2.28%) or the combination of \u003cb\u003e10d\u003c/b\u003e and 0.01/0.001 mg/mL CIP (0\u0026ndash;2.35%), demonstrating that ferredoxin PA1551 plays a similar role as \u003cb\u003e10d\u003c/b\u003e in enhancing antibacterial effects. Similarly, compared with the control group (43.17%) and the wounds infected with △ferredoxin (74.67%), those infected with △ferredoxin and then treated with the combination of \u003cb\u003e10d\u003c/b\u003e and Tob or \u003cb\u003e10d\u003c/b\u003e and CIP exhibited high wound closure rates of 83.18\u0026ndash;86.46%, emphasizing the importance of ferredoxin PA1551 in \u003cem\u003eP. aeruginosa\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, C). In addition, we observed no substantial organ toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) or effects on the body weight of mice during the experiment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u003c/b\u003e). These results demonstrated that ferredoxin PA1551 had a comparable effect to \u003cb\u003e10d\u003c/b\u003e in enhancing the antibacterial effects of Tob and CIP. These results raise the possibility that human infections could be treated by targeting ferredoxin PA1551 in difficult-to-treat bacterial biofilms.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA major challenge in managing \u003cem\u003eP. aeruginosa\u003c/em\u003e infections is the emergence of multidrug-resistant strains. This obstacle has prompted researchers to explore alternative, nonbiocidal methods to treat and eradicate bacterial infections\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The strategy of targeting biofilms is promising because it can reduce the use of antibiotics and curb the emergence and spread of antibiotic resistance.\u003c/p\u003e \u003cp\u003eUsing chemical proteomics, the \u003cem\u003ePA1551\u003c/em\u003e-deficient mutant, and the \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain, we identified that ferredoxin PA1551 can serve as a target to inhibit biofilm. We determined that the uncharacterized cytoplasmic membrane protein (ferredoxin PA1551) is involved in ferrite reduction, which plays an essential role in iron homeostasis in \u003cem\u003eP. aeruginosa\u003c/em\u003e. In this study, we referred to ferredoxin encoded by PA1551 as ferredoxin PA1551. We propose that compound \u003cb\u003e10d\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) forms a complex with Fe\u003csup\u003e3+\u003c/sup\u003e outside the bacterial cell. This complex is then transported into the periplasm through the FpvA protein in the outer cell membrane of \u003cem\u003eP. aeruginosa\u003c/em\u003e, where Fe\u003csup\u003e3+\u003c/sup\u003e is reduced to Fe\u003csup\u003e2+\u003c/sup\u003e by ferredoxin PA1551 in the inner membrane with the involvement of periplasmic proteins. Subsequently, the reduced Fe\u003csup\u003e2+\u003c/sup\u003e is chelated by periplasmic proteins, which can carry ferrous iron to the transporter proteins, allowing Fe\u003csup\u003e2+\u003c/sup\u003e to enter the cytoplasm. By inhibiting the action of ferredoxin PA1551, bacterial iron homeostasis, and quorum sensing can be disrupted, thus impeding biofilm formation. Our findings provide insights into the role of ferredoxin PA1551 in the antibiotic tolerance response, revealing a potential avenue to combat bacterial biofilm formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, by using our chemical probes designed on the basis of biofilm inhibitor \u003cb\u003e10d\u003c/b\u003e, we identified ferredoxin PA1551 as a new target for antibiofilm agents to combat \u003cem\u003eP. aeruginosa\u003c/em\u003e infection. We revealed that under normal bacterial growth conditions where an iron source is available, iron homeostasis mediated by ferredoxin PA1551 is critical for the formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms and the secretion of virulence factors, such as pyocyanin, rhamnolipids, Pvd, and Pch (siderophores). Furthermore, we used an open-wound infection murine model to demonstrate that ferredoxin PA1551 can serve as an antibacterial synergistic target to inhibit biofilm in the treatment of \u003cem\u003eP. aeruginosa\u003c/em\u003e infections. The ferredoxin PA1551 mutant exhibited the same efficacy as \u003cb\u003e10d\u003c/b\u003e in improving the antibacterial effect of Tob and CIP by more than 200- to 1000-fold. Our findings indicate the potential of ferredoxin PA1551 as a target for inhibiting biofilm formation to combat \u003cem\u003eP. aeruginosa\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStrain and cell culture conditions\u003c/h2\u003e \u003cp\u003eBacterial strains and plasmids used in this study are listed in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e. \u003cem\u003eE. coli\u003c/em\u003e strains were grown at 37\u0026deg;C in either Luria-Bertani (LB) broth or agar. \u003cem\u003eP. aeruginosa\u003c/em\u003e strains were grown at 37\u0026deg;C in either LB, tryptic soy broth (TSB), ABTGC medium (B-medium\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e (0.1% MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1% CaCl\u003csub\u003e2\u003c/sub\u003e, 0.1% FeCl\u003csub\u003e3\u003c/sub\u003e) supplemented with 10% A10, 0.2% glucose and 0.2% casamino acids). \u003cem\u003ePA1551\u003c/em\u003e mutant was grown in LB medium and on LB agar supplemented with 100 \u0026micro;g /mL Gentamicin. \u003cem\u003ePA1551\u003c/em\u003e-overexpressed was grown in LB medium and on LB agar supplemented with 100 \u0026micro;g /mL carbenicillin. \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) carrying \u003cem\u003ePA1551\u003c/em\u003e cloned into the expression vector, pGEX-6P-1-1551 was grown in LB medium supplemented with 100 \u0026micro;g /mL ampicillin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid construction\u003c/h3\u003e\n\u003cp\u003ePrimers\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e used in this study are listed in \u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e. To construct the knock-out plasmid for deletion, the \u003cem\u003ePA1551\u003c/em\u003e gene, the 1003-bp upstream fragment and the 1003-bp downstream fragment flanking PA1551 were amplified with primer pairs PA1551 up F/PA1551 up R and PA1551 low F/ PA1551 low R. The upstream and downstream PCR fragments were ligated by overlap PCR, and the resulting PCR products were inserted into the BamHI/HindIII sites of the suicide vector pK18. The gentamicin resistance cassette amplified from Gm was subsequently inserted into the same HindIII site to yield the knock-out plasmid pK18- PA1551.\u003c/p\u003e \u003cp\u003eExpression plasmid constructs were generated by standard methods and verified by DNA sequencing. The plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e by heat shock and \u003cem\u003eP. aeruginosa\u003c/em\u003e strain by electroporation unless otherwise stated. To generate the \u003cem\u003ePA1551\u003c/em\u003e -overexpressing construct p20- \u003cem\u003ePA1551\u003c/em\u003e, the encoding region of bswR was amplified with polymerase chain reaction (PCR) primers (\u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e), digested with EcoRI and HindIII inserted into the corresponding site of pUCP20.\u003c/p\u003e \u003cp\u003eTo construct pGEX-6P-1-PA1551, primer PA1551 F1/ PA1551 R1, PA1551 F2/ PA1551 R2, PA1551 F3/ PA1551 R3 were used to amplify the PA1551 gene. These PCR products of relevant genes were digested with BamHI/HindIII and inserted into the BamHI/HindIII sites of pGEX6P-1 resulting in plasmids pGEX6p-1-PA1551. The integrity of the insert in all constructs was confirmed by DNA sequencing.\u003c/p\u003e \u003cp\u003eFor constructing pET-28a-1551 (full-length), the primer pair 1551f and 1551r were used to amplify the PA1551 (full-length) fragment from the genomic DNA. Vector was digested with BamHⅠ and HindⅢ restriction enzymes and purified. The PCR products were digested and inserted into pET-28a to generate pET-28a-1551 (full-length) using the Seamless cloning kit. The resulting plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α, and the plasmid construct was verified by sequencing using primers that annealed to sites outside the multiple cloning site.\u003c/p\u003e\n\u003ch3\u003eN-GST-PA1551 expression\u003c/h3\u003e\n\u003cp\u003eA single bacterial colony of \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) carrying pGEX-6P-1-PA1551 strain was inoculated into 1 L of fresh LB medium flask supplemented with 100 ug/mL gentamycin. The culture was once again grown at 37\u0026deg;C with good aeration on a rotary shaker (200 rpm) until OD\u003csub\u003e600\u003c/sub\u003e reached 0.5-1. Protein expression was induced by the addition of IPTG to a final concentration of 1 mM. Following induction, the temperature was lowered to 16\u0026deg;C, and growth (with vigorous aeration, shaking at 200 rpm) was continued overnight. The bacterial pellet expressing recombinant protein was collected by sedimentation (4000 rpm, 15 min, 4\u0026deg;C). The supernatant was discarded and the cell pellet was washed with ice-cold 1\u0026times;phosphate-buffered saline solution (PBS).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePurification of N-GST-PA1551 protein using AKTA pureprotein purification layer system\u003c/h2\u003e \u003cp\u003eGST-tagged recombinant proteins were purified as described\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Fractions containing protein were pooled and analyzed by SDS-PAGE (4% stacking and 12% resolving gel). The purification protein samples were stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProtein expression and purification\u003c/strong\u003e \u003cp\u003eThe plasmid pET-28a-1551 (full-length) was transformed into \u003cem\u003eE. coli BL21 (DE3)\u003c/em\u003e, enabling the expression of PA1551 (full-length) membrane protein containing a 6-His tag. PA1551 (full-length) was induced with 0.5 mM IPTG overnight at 16\u0026deg;C. Cells were harvested and resuspended in lysis buffer composed of 30 mM HEPES pH 6.8, 150 mM NaCl, and 0.01% DDM (n-Dodecyl-β-D-Maltoside). Bacteria were lysed using probe sonication for a duration of 40 minutes, followed by the removal of cell debris through centrifugation at a speed of 15,000 rpm for 30 minutes. The supernatant was loaded onto a Ni-NTA column (L00250-50, GeneScript). The resin was washed with lysis buffer supplemented to 90 mM imidazole and subsequently eluted with lysis buffer supplemented to 300 mM imidazole. Where necessary, proteins were concentrated via filtration using centrifugation and a 10 kDa cut-off.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicroscale thermophoresis (MST) assay\u003c/h2\u003e \u003cp\u003e The PA1551 (full-length)-His protein, at a 100 nmol/L concentration, was subjected to fluorescent tagging using a His-tag labeling kit (Nano Temper, MO-L008), adhering to the guidelines specified in the user manual. After the labeling was finalized, 10 \u0026micro;M of the fluorescently tagged ferredoxin PA1551 (full-length)-His protein was mixed with 10 M of PBST buffer, followed by the addition of \u003cb\u003e10d\u003c/b\u003e at a concentration of 1 mM. This mixture was then subject to serial dilution. The microscale thermophoresis assay was carried out on a NanoTemper Monolith NT.115 device, set to 20% LED power and medium MST power setting. Data collected from the assay were analyzed using the MO. Affinity Analysis software (X86).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn situ protein labeling\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e was inoculated in 5 mL LB and cultivated (200 rpm, 37\u0026deg;C) until the culture reached OD\u003csub\u003e600\u003c/sub\u003e to 0.05. Probe \u003cb\u003e10d-1\u003c/b\u003e (10 \u0026micro;M), NP (10 \u0026micro;M), and \u003cb\u003e10d-1\u003c/b\u003e(10 \u0026micro;M)- \u003cb\u003e10d\u003c/b\u003e (50 \u0026micro;M) as the competitor were incubated for 24 h at 37\u0026deg;C, with shaking (200 rpm). After 16 h incubation, the cultures were then centrifuged for 10 min at 4000 rpm and the cell pellets were washed with 2\u0026times; PBS to remove excessive probe, followed by UV (365 nm) irradiation for 20 min on ice\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The cell pellets were resuspended in HEPES buffer (25 mM HEPES, 150 mM NaCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e) with a 1% protease inhibitor cocktail (APExBIO, EDTA-Free in DMSO). The suspension was repeatedly frozen and thawed 3 times followed by sonication for 1 h (15s on and 15s off). The precipitate was removed by centrifugation and the supernatant was adjusted to 1 mg/mL 1.6 mL of a freshly premixed click chemistry reaction cocktail (50 mM TAMRA-biotin-N\u003csub\u003e3\u003c/sub\u003e from 50 mM stock solution in DMSO, 0.1 mM TBTA from 100 mM freshly prepared stock solution in DMSO, 1 mML TCEP from 1 M freshly prepared stock solution in deionized water, and 1 mM CuSO\u003csub\u003e4\u003c/sub\u003e from 1 M freshly prepared stock solution in deionized water) was added. The reaction was incubated for 2 h with gentle mixing at r.t before being terminated by the addition of prechilled Me\u003csub\u003e2\u003c/sub\u003eCO (incubation at 20\u0026deg;C for 1 h). Precipitated proteins were subsequently collected by centrifugation (10000 rpm, 10 min at 4\u0026deg;C). The supernatant was discarded and the pellet was washed twice with prechilled MeOH before redissolving in 2\u0026times;loading buffer and heating for 10 min at 95\u0026deg;C. Proteins were separated by SDS-PAGE (10% gel) and then visualized by in-gel fluorescence scanning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConcentration-dependent N-GST-PA1551 labeling\u003c/h2\u003e \u003cp\u003ePurified N-GST-PA1551 protein samples were thawed, and the protein concentration was measured. Samples adjusted to contain 0.5 mg/mL of N-GST-PA1551 in PBS, were incubated in the presence of probe \u003cb\u003e10d-1\u003c/b\u003e (1 \u0026micro;M, 10 \u0026micro;M, and 20 \u0026micro;M). Cu\u003csup\u003e2+\u003c/sup\u003e-mediated azide-alkyne coupling (CuAAC) reactions were above. Proteins were separated by SDS-PAGE (10% gel) and then visualized by in-gel fluorescence scanning.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLarge-scale Protein Identification by LC\u0026thinsp;\u0026minus;\u0026thinsp;MS/MS\u003c/strong\u003e \u003cp\u003eStageTips were prepared according to a modified protocoFl\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e with a C\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e solid-phase extraction disc (3 M Empore). Large-Scale Protein Identification by LC\u0026thinsp;\u0026minus;\u0026thinsp;MS/MS, for subsequent MS analysis, the samples were resuspended (with sonication in a water bath) in 10 mL of H\u003csub\u003e2\u003c/sub\u003eO containing 0.5% FA.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation assay\u003c/h2\u003e \u003cp\u003eThe measurement of compounds in biofilm formation was Crystal violet assay. \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 was grown overnight in LB medium at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 h. Subsequently, the evaluation of biofilm formation using sterile 96-well flat-bottomed polystyrene microtiter plates (Corning/Costar, NY, USA) involved culture of 150 \u0026micro;L of bacterial (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05) in ABTGC medium at 37\u0026deg;C for 24 h. Subsequently, supernatant cells were removed and the biofilms were washed three times with sterile phosphate-buffered saline (PBS), then MeOH as a fixative. After 30 min, the MeOH was removed, and the microtiter plates were dried at RT. Crystal violet (0.1% in H\u003csub\u003e2\u003c/sub\u003eO) was then added to dye the biofilms which were then incubated for 30 min. Crystal violet was removed, microtiter plates were rinsed three times with sterile PBS, and add 150 \u0026micro;L of acetic acid (33%) to dissolve the biofilm. The absorbance values were measured at 570 nm by a microplate reader. All compounds were dissolved in DMSO to make 100 \u0026micro;M stock solutions before transferring to 96-well plates for the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRhamnolipid assay\u003c/h2\u003e \u003cp\u003eRhamnolipid was quantified according to the method of Koch et al, with modifications. A subculture was conducted by directly diluting the overnight culture 1:100 into fresh Minimal Medium (49.3 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 50 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 4.8 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 7.6 mM (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.6 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 25 \u0026micro;M FeSO\u003csub\u003e4\u003c/sub\u003e, 0.162 \u0026micro;M (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003eMo\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e24\u003c/sub\u003e, 38 \u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e, 14 \u0026micro;M MnCl\u003csub\u003e2\u003c/sub\u003e, 1.6 \u0026micro;M CuSO\u003csub\u003e4\u003c/sub\u003e, 0.86 \u0026micro;M CoCl\u003csub\u003e2\u003c/sub\u003e, 1.9 \u0026micro;M boric acid, 5.5 \u0026micro;M NiCl\u003csub\u003e2\u003c/sub\u003e, 6.72 \u0026micro;M EDTA, 0.6% glycerol in 18 MΩ deionized H\u003csub\u003e2\u003c/sub\u003eO). The cultures were grown for 24 h at 37\u0026deg;C, with shaking (200 rpm). The final cell density was measured at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) using a microplate reader (Bio-Tek). Supernatants were collected by ̈centrifuging at 10000 rpm for 10 min and extracted with Et\u003csub\u003e2\u003c/sub\u003eO (twice). Organic fractions were concentrated to yield a white solid. It was resuspended in deionized H\u003csub\u003e2\u003c/sub\u003eO and added with 0.19% (w/v) orcinol in 50% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The resulting mixture was incubated at 80 \u003csup\u003eo\u003c/sup\u003eC for 30 min to give a yellow-orange solution. After cooling to rt, the absorbance was measured at 421 nm and results were normalized with final OD\u003csub\u003e600\u003c/sub\u003e values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePyocyanin assay\u003c/h2\u003e \u003cp\u003eOvernight culture of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant, and \u003cem\u003ePA1551\u003c/em\u003e-overexpressing strain were standardized to OD\u003csub\u003e600\u003c/sub\u003e of 0.05 and diluted 100 times into 25 mL of LB Medium (Sangon Biotech, China) in 250 mL flask. The cultures were grown for 48 h at 37\u0026deg;C, with shaking (200 rpm). The cultures were then centrifuged for 10 min at 10000 rpm and 8 mL of the supernatants were transferred into new tubes. Briefly, 8 mL of culture supernatant conducted as described above was extracted with chloroform at a ratio of 4:1, followed by extraction with 1 mL of 0.2 M HCl. The absorbance of the upper red phase was measured using the OD\u003csub\u003e520\u003c/sub\u003e. The data was normalized by dividing the OD\u003csub\u003e520\u003c/sub\u003e reading with the final OD\u003csub\u003e600\u003c/sub\u003e values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSwarming motility assay\u003c/h2\u003e \u003cp\u003eTo monitor swarming, petri dishes were filled with 20 mL of LB medium supplemented with 0.5% (w/v) bacto agar (Becton,Dickinson and Co.), 0.5% (w/v) casamino acids(Becton,Dickinson and Co.) and 0.5% (w/v) glucose (Solarbio, China) in the presence of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant, and \u003cem\u003ePA1551\u003c/em\u003e overexpressing strains. Petri dishes were dried in a single stack for 1 h at RT and then dropped 1 \u0026micro;L PAO1 (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) onto the center of petri dishes and open the petri dishes to dry. The petri dishes were incubated for 16 h at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSwimming motility assay\u003c/h2\u003e \u003cp\u003eTo monitor swimming, petri dishes contained 20 mL of LB medium supplemented with 0.3% (w/v) bacto agar, 0.5% (w/v) casamino acids and 0.5% (w/v) glucose in the presence of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant, and \u003cem\u003ePA1551\u003c/em\u003e overexpressing strains. Petri dishes were dried in a single stack for 1h at rt and then dropped 1 \u0026micro;L PAO1 (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) into the bottom of the petri dishes and open the petri dishes to dry. The petri dishes were incubated for 16 h at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTwitching motility assay\u003c/h2\u003e \u003cp\u003eTo monitor twitching, petri dishes were charged with 20 mL of LB medium supplemented with 1% (w/v) bacto agar, 0.5% (w/v) casamino acids and 0.5% (w/v) glucose in the presence of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, \u003cem\u003ePA1551\u003c/em\u003e deficient mutant, and \u003cem\u003ePA1551\u003c/em\u003e overexpressing strains. Petri dishes were dried in a single stack for 1h at room temperature and then dropped 1 \u0026micro;L PAO1 (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) into the bottom of the petri dishes and open the petri dishes to dry. The petri dishes were incubated for 16 h at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePyoverdine and pyochelin assay\u003c/h2\u003e \u003cp\u003eAn overnight culture of \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 (grown in LB medium at 37\u0026deg;C, 200 rpm) was diluted in ABTGC medium to a final optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.02 (2.5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL). The microtiter plate was incubated at 37\u0026deg;C in a microplate reader (Bio-Tek) to measure the cell density (OD\u003csub\u003e600\u003c/sub\u003e), Pyoverdine fluorescence (excitation at 400 nm, emission at 447 nm), pyochelin fluorescence (excitation at 350 nm, emission at 430 nm). The experiment assay for all test compounds and controls was done in triplicate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReduction experiment of trivalent iron ions\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003e20 \u0026micro;M FeCl\u003csub\u003e3\u003c/sub\u003e was incubated in 100 mM ammonium acetate buffer (pH\u0026thinsp;=\u0026thinsp;6.5) in the presence of 100 mM DTT or protein, 200 \u0026micro;M ferrozine. Subsequently, the absorbance at 562 nm was monitored for 120 min. A\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;absorbance at 0 min and A\u0026thinsp;=\u0026thinsp;absorbance at testing time.\u003c/p\u003e \u003cp\u003e \u003cb\u003eValidation of PA1551 protein for iron reduction\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter the constructed GST PGEX6P-1 strain induced a large amount of protein expression, the bacteria were lysed and the supernatant was extracted. To a solution of 20 \u0026micro;M Ferric chloride in 200 \u0026micro;L of 100 mM ammonium acetate buffer, 100 mM DTT or the supernatant of GST PGEX6P-1 strain was added at 37\u0026deg;C, then added separately 200 \u0026micro;M ferrozine in a 96-well plate. The 96-well plate was incubated at 37\u0026deg;C in a microplate reader (BioTek, Synergy H1) to measure the absorbance at 562 nm, every 5 min for 1.5 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eComputational methods\u003c/h2\u003e \u003cp\u003eThe sequence of Ferredoxin PA1551 from Uniprot \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e was folded on a server with a local version of ColabFold\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Amber forcefield was used to relax the models. Top-ranked models were uploaded to PPM 2.0 server to predict the transmembrane region\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Apo protein with membrane system was prepared and constructed in Maestro protein preparation and Desmond system builder tools\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The orthorhombic simulation box was solvated of SPC water molecules, counterions, and an additional 0.15 M concentration of NaCl. A 100ns molecular dynamics was performed using Desmond to settle the loops, details are as in our previous work if not specified. SiteMap was utilized on every 10 frames of the second 50ns trajectory by a script\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. All the sites identified were visually inspected. Top sites with preferable scores and sizes were all tested in Glide docking with the \u003cb\u003e10d\u003c/b\u003e conformations generated from LigPrep. The best 3 binding complexes were further validated in MD simulation, regular protein-ligand job settings were applied, and interaction and binding free energy analysis were performed to evaluate the affinity.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eWound infection experiment in mice\u003c/h2\u003e \u003c/div\u003e \u003c/div\u003e\u003cp\u003eEthical Statement. \u003cem\u003eP. aeruginosa\u003c/em\u003e strain PAO1 was used for this animal experiment. Female 5-week-old BABL/C mice were purchased from SPF Biotechnology Co., Ltd with certificate number 20180302005. All animal experiments were performed under the animal care and use guidelines\u0026nbsp;(IACUC approval number: Szwwbio-IACUC-20230930-01). During the whole experiment, the mice were kept at a constant temperature of 25 \u0026deg;C, with a 12 h light/night cycle, and provided with sufficient food and water without any animal cruelty. At the end of the experiment, the mice were all euthanized.\u003c/p\u003e\n\u003cp\u003eFirstly, mice were randomly divided into different groups of 5/group and treated with 4% chloral hydrate, then the hair was shaved off the back of the mice and a circular wound of 4-5 mm was created on the back of each mouse. The wound was inoculated with 5\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU of PAO1 (or\u0026nbsp;△ferredoxin) to establish a wound infection model for 24 h. The mice in different groups were then treated with different drugs (saline, 0.5 mg/mL Tob, 0.0025 mg/mL Tob, 0.0025 mg/mL Tob+10 \u0026mu;M \u003cstrong\u003e10d\u003c/strong\u003e, 10 \u0026mu;M \u003cstrong\u003e10d\u003c/strong\u003e, 1 mg/mL CIP, 0.01 mg/mL CIP, 0.001 mg/mL CIP, 0.01 mg/mL CIP+10 \u0026mu;M \u003cstrong\u003e10d\u003c/strong\u003e, 0.001 mg/mL CIP+10 \u0026mu;M \u003cstrong\u003e10d\u003c/strong\u003e). Mice were executed after 3 days of continuous dosing, wounded skin was cut off and homogenized, serially diluted and CFU counts were done on agar plates. In addition, histological sections were taken from the heart, liver, spleen, lung, and kidney of the mice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated in this study are available upon request from the corresponding author.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was suppored by the National Natural Science Foundation of China (No. 82173651, and 82473788). Natural Science Foundation of Guangdong Province, China (2025A1515012144). Part of the images were created in BioRender.com with permission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ. L. and A. R. contributed equally to this work. J. L. and A. R. wrote the manuscript and performed most of experiments. W.C. designed the project and supervised the chemical experiments as well as revised the manuscript; L.Y. supervised the experiments of target identification, and revised the manuscript; Z.M. performed chemical experiments. T.Z., C.Z., Y. C., S. Z., X.H.,\u0026nbsp;X. Z., T. J., Z. C.,\u0026nbsp;Z. L.,\u0026nbsp;J. L \u0026nbsp;performed some chemical experiments or biological experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLewis, K. Platforms for antibiotic discovery. \u003cem\u003eNat. Rev. Drug Discovery\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 371-387 (2013).\u003c/li\u003e\n\u003cli\u003eBrown, E. D. \u0026amp; Wright, G. D. 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Shaw Research, New York, NY, 2023.\u003c/li\u003e\n\u003cli\u003eSchr\u0026ouml;dinger release 2023-1: sitemap, Schr\u0026ouml;dinger, LLC, New York, NY, 2023.\u003c/li\u003e\n\u003cli\u003eSchr\u0026ouml;dinger release 2023-1: glide, Schr\u0026ouml;dinger, LLC, New York, NY, 2023.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6842284/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6842284/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAddressing antibiotic-resistant bacterial biofilm infections without promoting drug resistance is a pressing challenge. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is well known for causing biofilm-associated drug-resistant infections that often lead to treatment failure. In this study, we identified a previously uncharacterized membrane protein ferredoxin encoded by \u003cem\u003ePA1551\u003c/em\u003e using photoaffinity-based biomimetic probes based on our previous dual-acting antibiofilm compound 2-(heptanamido)methyl 3-hydroxy-1,6-dimethylpyridin-4(1\u003cem\u003eH\u003c/em\u003e)-one (\u003cb\u003e10d)\u003c/b\u003e. The precision-targeted ferredoxin PA1551 exhibited excellent effectiveness in various model systems, suppressing bacterial biofilm and virulence, and enhancing the antibacterial effects of tobramycin (Tob, by 200-fold) and ciprofloxacin (CIP, by 1000-fold) compared to single-dose antibiotic treatments in a mouse model of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e infection. These results indicate that ferredoxin PA1551 can be used as target to design new antibiofilm drugs for the treatment of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e infections, particularly challenging bacterial biofilms.\u003c/p\u003e","manuscriptTitle":"Identification of ferredoxin PA1551 in the bacterial iron uptake pathway and exploration of its antibacterial synergistic as target for biofilm inhibitors against Pseudomonas aeruginosa infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 19:58:39","doi":"10.21203/rs.3.rs-6842284/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-23T12:32:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T07:25:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T10:25:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228451079963196354877102735747141161628","date":"2025-06-22T05:53:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243222981503732794615022504871866220548","date":"2025-06-20T12:26:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T15:17:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T13:05:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T12:59:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Biofilms and Microbiomes","date":"2025-06-07T10:23:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23cebbc7-a1e8-4881-b519-ae37a2330236","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50261469,"name":"Biological sciences/Microbiology/Antimicrobials"},{"id":50261470,"name":"Biological sciences/Microbiology/Biofilms"}],"tags":[],"updatedAt":"2025-12-08T16:05:20+00:00","versionOfRecord":{"articleIdentity":"rs-6842284","link":"https://doi.org/10.1038/s41522-025-00871-y","journal":{"identity":"npj-biofilms-and-microbiomes","isVorOnly":false,"title":"npj Biofilms and Microbiomes"},"publishedOn":"2025-12-05 15:57:35","publishedOnDateReadable":"December 5th, 2025"},"versionCreatedAt":"2025-06-18 19:58:39","video":"","vorDoi":"10.1038/s41522-025-00871-y","vorDoiUrl":"https://doi.org/10.1038/s41522-025-00871-y","workflowStages":[]},"version":"v1","identity":"rs-6842284","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6842284","identity":"rs-6842284","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}