Inhibition of Growth and Biofilm Formation in Staphylococcus aureus by LLY-507

Inhibition of Growth and Biofilm Formation in Staphylococcus aureus by LLY-507 | 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 Research Article Inhibition of Growth and Biofilm Formation in Staphylococcus aureus by LLY-507 Yuanyuan Tang, Zhichao Xu, Yingying Lai, Jialang Zhang, Jinxin Zheng, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5353061/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The emergence of methicillin-resistant Staphylococcus aureus (MRSA) as a prevalent antibiotic-resistant pathogen underscores the urgent need for novel antibacterial agents. This study investigates the potential of LLY-507 against gram-positive bacteria, particularly S. aureus , focusing on its antibacterial and anti-biofilm properties. Here, our data exhibited favorable antibacterial activity of LLY-507 with MIC50 and MIC90 values of 25 µM against S. aureus . Additionally, LLY-507 at sub-MIC concentrations effectively reduced the planktonic growth and biofilm formation of S. aureus . Proteomic analysis of S. aureus treated with LLY-507 revealed the classification of the functional proteins with significant expression level alterations in bacterial metabolism, particularly amino acid biosynthesis. Furthermore, we demonstrated the disruption of S. aureus cell integrity by LLY-507 through membrane permeability assays and direct binding experiments between LLY-507 and cardiolipin. Lastly, the effectiveness of LLY-507 was proven in vivo using the G. mellonella infection model. Overall, these findings highlight the promising antibacterial and anti-biofilm activities of LLY-507 against S. aureus and provide insights into its mechanism of action, implicating its potential as a lead compound for developing novel antibacterial agents targeting Gram-positive bacteria. LLY-507 Staphylococcus aureus Antimicrobial activity Biofilm Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Gram-positive bacteria, including Staphylococcus aureus ( S. aureus ), Enterococcus faecalis ( E. faecalis ), Enterococcus faecium ( E. faecium ) et al., are commonly found species as clinical pathogens causing infections in both hospital and community settings [ 1 ]. With an increasing number of patients undergoing prolonged hospitalization and the widespread use of broad-spectrum antibiotics, reports of gram-positive bacteria developing resistance to first-line antibiotics have gradually risen in recent years, posing significant challenges to clinical anti-infective therapy [ 2 , 3 ]. In the United States and the European Union, drug-resistant bacteria are responsible for over 35,000 deaths annually [ 4 , 5 ]. Some multidrug-resistant gram-positive bacteria strains, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococci (VRE), are disseminated and increasingly detected worldwide, holding a high-priority position on the World Health Organization's list of "critical priority pathogens" for antibiotic resistance [ 6 ]. MRSA and VRE, known for their relatively high virulence and remarkable adaptability, can thrive in various environmental conditions and have evolved resistance mechanisms against nearly all antimicrobial agents used in therapy [ 7 ]. Some bacteria form biofilms when freely floating planktonic cells attach to available surfaces and initiate colonization. Biofilms are structured and functional communities formed by the mutual adhesion of bacteria through extracellular matrix secretion. Approximately 80% of bacterial infections are closely associated with biofilm formation, in some cases, which even initiates the occurrence and development of bacterial infectious diseases [ 8 ]. Biofilm mass formation facilitates the bacterial cells to easily resist the various stress conditions and the host immune system [ 9 ]. Most gram-positive bacteria forming biofilms in clinical settings are prone to be explained as one of the crucial reasons for the poor clinical outcome of gram-positive bacterial infections [ 10 ]. For instance, biofilm formation in MRSA infections renders it difficult to remove by antibiotics and the host immune system, sometimes even leading to chronic infectious diseases [ 11 ]. Biofilm-embedded bacterial cells can detach from the biofilm mass into surrounding tissues and blood, resulting in recurrence and prolonged infection especially when antibiotic concentrations decrease [ 12 ]. Therefore, developing novel anti-infective drugs that can inhibit bacterial growth and suppress biofilm formation has become one of the current research hotspots [ 13 ]. LLY-507 is a cell-active and selective inhibitor of lysine methyltransferase SMYD2, obtained through chemical synthesis [ 14 ]. Overexpression of SMYD2 protein in the cancer cells often serves as a significant adverse prognostic factor in cancer. It is closely associated with tumorigenesis factors such as tumor infiltration, tumor proliferation, lymph node metastasis, lymphatic invasion, et al. [ 15 ]. However, the mechanism by which SMYD2 promotes tumorigenesis remains unclear. LLY-507 selectively targets a subset of histone molecules, binding to and inhibiting lysine methyltransferase SMYD2's methylation of p53 peptide and H4 with high specificity, which is employed to elucidate SMYD2's function in cancer and other biological processes [ 16 ]. During the screening of clinical compound libraries for antibacterial activity, LLY-507 was found to exhibit broad-spectrum inhibitory activity against gram-positive bacteria. Investigation of the activity of LLY-507 in inhibiting the bacterial growth and biofilm formation of gram-positive bacteria against S. aureus was demonstrated by examining LLY-507's effects on planktonic bacterial growth curves, killing curves, and biofilm activity. The action mechanism of LLY-507 was further evaluated by proteomics analysis, cell membrane permeability, and biolayer interferometry assays. These findings shed light on the potential of LLY-507 and its derivatives based on its lead structure as the development candidate for antibacterial drugs. 2. Materials and Methods 2.1. Bacterial isolates and growth conditions and antibiotics Strains of Staphylococcus aureus, comprising methicillin-resistant S. aureus (MRSA, n = 15) and methicillin-sensitive S. aureus (MSSA, n = 21), were collected from various clinical specimens of individual patients at Shenzhen Nanshan People's Hospital between 2015 and 2019. Identification of all clinical strains was performed using the Phoenix™100 automated microbial identification system, followed by re-identification through matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS, Bruker, Germany). Quality control strains, S. aureus YUSA145 and SA113, were sourced from the American Type Culture Collection. The S. aureus strains were cultured in tryptic soy broth (TSB, Oxoid Ltd., Basingstoke, United Kingdom), either with or without the addition of 0.5% glucose (TSBG). Cultivation conditions included incubation at 37°C with shaking at 220 rpm. The identified strains were preserved at -80°C in TSB supplemented with 15% glycerol for subsequent analyses. Antibiotics LLY-507, Vancomycin (Van), and Linezolid (LZD) were procured from MCE (Princeton, United States). The cell membrane phospholipids, such as cardiolipin (CL, C0563, Sigma-Aldrich), phosphatidylcholine (840051P, Sigma-Aldrich), phosphatidylethanolamine (Y0001953, Sigma-Aldrich), and phosphatidylglycerol (841138P, Sigma-Aldrich), were dissolved in methanol for further use. 2.2. Determination of antibacterial activities of LLY-507 2.2.1. MIC and MBC assays The minimum inhibitory concentrations (MICs) of antimicrobial agents were determined using the broth microdilution method in cation-adjusted Mueller–Hinton broth (CAMHB; Oxoid Ltd), following the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI M100, 29th ed.) as previously described. In brief, two-fold serial dilutions of the compounds were prepared in 96-well microtiter plates (Falcon), achieving final concentrations ranging from 200 to 0.39 µM in 100 µL of CAMHB medium. Bacterial cells were cultured overnight with shaking, then adjusted to match the density of a 0.5 McFarland standard (approximately 1×10 8 CFU/mL), and further diluted 1:200 into the CAMHB medium. The mixtures in the 96-well plates were thoroughly mixed and incubated at 37°C with shaking at 220 rpm for 16–20 hours. The MIC was defined as the lowest concentration of the compound that exhibited no visible growth in the wells. For the minimum bactericidal concentration (MBC) assay, samples from the wells showing MIC were spread onto TSB agar plates and incubated overnight at 37°C. The MBC was defined as the concentration that resulted in a 99.9% reduction of the original inoculum. All experiments were conducted in duplicate or more. 2.2.2. Bacterial dynamic growth curve assay Strains of Staphylococcus aureus were cultivated to stationary phase (16 hours) and subsequently diluted 1:200 in Tryptic Soy Broth (TSB). The diluted bacterial cultures were then incubated at 37°C with the specified concentrations of antibiotics while shaking at 220 rpm. Bacterial growth was monitored by measuring the optical density at 600 nm (OD 600 ) at 1-hour intervals over a 24-hour period using an automatic bacterial growth analyzer (Bioscreen C, Turku, Finland). All experiments were conducted in biological triplicates. 2.2.3. Dynamic time-kill curve assay For the bactericidal experiments involving planktonic cells, overnight-cultured Staphylococcus aureus were diluted to approximately 1×10 7 CFU in Tryptic Soy Broth (TSB). Antibiotics were then added to achieve final concentrations of 2 × MIC during the logarithmic growth phase and again at 2 × MIC during the stationary phase (16 hours). Colony counts were conducted at multiple time points: 0, 2, 4, 6, and 24 hours during the logarithmic phase, and at 24, 48, 72, 96, and 120 hours during the stationary phase. Cells were plated for colony-forming unit (CFU) counts and challenged with Linezolid (LZD) and Vancomycin (Van) antibiotics, respectively. Data presented are representative of three independent experiments, with bacteria treated with 0.1% DMSO serving as a control. Results are reported as the mean ± standard deviation (SD) from triplicate measurements. 2.3. Detection of Anti-biofilm Activity of the LLY-507 2.3.1. Microtiter plate assays of biofilm formation A detailed protocol for the inhibition of biofilm formation assay has been previously reported. Isolates of Staphylococcus aureus were cultured overnight in Tryptic Soy Broth (TSB) at 37°C with shaking at 220 rpm. The overnight cultures were then diluted 1:200 and inoculated into a polystyrene 96-well plate containing 200 µL of TSBG (TSB supplemented with 2% glucose) at concentrations of 1/8 ×, 1/4 ×, or 1/2 × MIC of LLY-507. For the biofilm eradication assay, plates were incubated at 37°C for 24 hours to allow the formation of mature biofilms. Following incubation, the medium was carefully removed, and the wells were washed gently three times with phosphate-buffered saline (PBS). Fresh medium containing various concentrations of LLY-507 was then added to each well and incubated for an additional 24 hours. Biofilms were stained with crystal violet and assessed by measuring the optical density at 570 nm (OD 570 ). Data were obtained from at least two independent experiments, yielding consistent results. 2.3.2. Adhesion cell counts in bacterial biofilm Biofilm inhibitory effects and mature biofilm removal effects were determined. Briefly, 1 mL of TSB medium containing 2% glucose with a dilution of 500 times was added to each well of a 24-well plate. For biofilm inhibitory effect assay, LLY-507 at different concentrations (1 × MIC, 1/2 × MIC, 1/4 × MIC, and 1/8 × MIC) were cultured overnight and added to the bacterial solution and incubated at 37℃ for 24 hours. After removing the culture solution carefully, wells were washed twice with sterile saline before being suspended, and then incubated in TSB plate and cells were counted. The 0.1% DMSO was treated as the negative control group. 2.3.3. Determination of cell viability in mature biofilms by CLSM The effect of LLY-507 on cell viability in mature biofilms (24 hours) was assessed using the Live/Dead Bacterial Viability method (Live/Dead BacLight, Molecular Probes, USA), employing SYTO 9 and propidium iodide (PI) dyes to stain live and dead cells within the biofilms. Staphylococcus aureus YUSA145 was cultured in cell-culture dishes (Fluorodish, FD35-100) using TSBG medium and incubated at 37°C for 24 hours. After incubation, planktonic bacteria were removed and discarded, and fresh TSBG containing LLY-507 (at 1/2 × MIC concentrations) was added. The cultures were then incubated at 37°C for an additional 24 hours. Following staining, the mature biofilms were visualized using a confocal laser scanning microscope (CLSM, Leica) with an oil-immersion objective. Image editing and analysis were performed using IMARIS 7.0.0 software (Bitplane). Green fluorescence indicated viable cells, while red fluorescence represented dead cells. The proportion of dead cells within the mature biofilm was quantified as the PI/total percentage using ImageJ software (Rawak Software Inc., Stuttgart, Germany). This assay was conducted in triplicate, yielding consistent results. 2.4. Proteomics analysis Proteomics analysis was performed following a previously reported method with modifications. Staphylococcus aureus YUSA145 in the exponential growth phase was treated with 1/2 × MIC of LLY-507 or DMSO at 37°C for 2 hours on a shaker set to 200 rpm, with each treatment group consisting of three biological replicates. Bacteria were harvested by centrifugation at 5,000 g for 10 minutes at 4°C and washed three times with 1 × PBS. The cell pellets were suspended in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime) supplemented with a protease inhibitor cocktail (P1065, Beyotime). Zirconia/Silica beads (0.1 mm diameter, catalog number: 11079101z) were added, and the samples were homogenized at 4°C using a Cell Disruption Device (JXFSTPRP-24L, Shanghai Jingxin Industrial Development Co., Ltd.) with a lysis protocol of 70 Hz for 1 minute, followed by 10-second pauses over four cycles. The samples were centrifuged at 4°C and 12,000 rpm for 10 minutes, and the supernatant was collected. Protein concentrations were determined using the BCA assay and SDS-PAGE. For protein reduction, 50 mg of total protein was transferred into 10 KD ultrafilter tubes and treated with 200 µL of 8 M urea solution containing 10 mM DTT for 2 hours at 37°C. The urea solution was removed by centrifugation (12,000 rpm at 20°C for 10 minutes), and then 200 µL of 8 M urea solution with 50 mM iodoacetamide (IAA) was added to the pellet and incubated in the dark for 15 minutes at room temperature. Samples were desalted using Amicon ultracentrifugal filters (10 KD, Millipore, Billerica, MA, USA), centrifuged at 12,000 g for 10 minutes at room temperature, and washed three times with 200 µL of 8 M urea solution containing 25 mM ammonium bicarbonate. Subsequently, 100 µL of 25 mM ammonium bicarbonate with 1 µg of trypsin (protein ratio of 50:1) was added to each filter tube and incubated at 37°C for 12 hours. The filter tubes were washed twice with 100 µL of 25 mM ammonium bicarbonate, and the resulting flow-through peptide fractions were collected and lyophilized. The lyophilized peptide fractions were re-suspended in 40 µL of ddH2O containing 0.1% formic acid, with 30 µL placed in the inner cannula of the autosampler vial and a 4 µL aliquot loaded into a C18 trap column (PepMap Neo Trap Cartridge, 164946, Thermo Scientific). Online chromatographic separation was performed on an Ultimate 3000 RSLCnano (Thermo Scientific) using 20 µL of 100% solvent A (0.1% formic acid) for trapping and desalting. An elution gradient of 5–38% solvent B (80% acetonitrile, 0.1% formic acid) over 60 minutes was applied to an analytical column (Acclaim PepMap RSLC, 75 mm × 25 cm C18-2 µm, 164941, Thermo Scientific). Data-dependent acquisition (DDA) mass spectrometry techniques were utilized to obtain tandem mass spectrometry (MS) data on a Thermo Fisher Q Exactive Plus mass spectrometer equipped with a Nano Flex ion source. Data were collected using an ion spray voltage of 1.9 kV and an interface heater temperature of 320°C. Protein identification and quantitative analysis of MS/MS data were conducted using Thermo Proteome Discoverer. After searching the target database, the local false discovery rate for peptides was set at 1.0%, allowing a maximum of two missed cleavages. In addition to a p-value threshold of < 0.05, a two-fold cutoff value was employed to identify upregulated and downregulated proteins. Differentially expressed proteins were uploaded to the OMICSBEAN database ( http://www.omicsbean.com ) for volcano plot generation, heatmap visualization, gene ontology (GO) annotation (encompassing biological processes, cellular components, and molecular functions), KEGG pathway analysis, and protein-protein interaction (PPI) network analysis. 2.5. Membrane permeability and depolarization assay For the membrane permeability assay, mid-log phase cells of Staphylococcus aureus YUSA145 were washed three times with sterile PBS and adjusted to a turbidity of 0.5 McFarland. The cell suspension was then diluted 1:10. Following this, the cells were incubated with 2 µM propidium iodide (PI) at 37°C for 10 minutes. Subsequently, the cell suspension was treated with varying concentrations of candesartan cilexetil, 0.1% Triton X-100, or 0.1% DMSO. The treated cell suspensions were placed in a 96-well plate with a black border and transparent bottom. Fluorescence intensity was dynamically monitored at an excitation wavelength of 493 nm and an emission wavelength of 534 nm. This assay allowed for the assessment of alterations in cell membrane integrity based on the fluorescence intensity of PI-stained cells in response to different treatments. For the determination of membrane potential, mid-log phase cells of S. aureus YUSA145 were similarly washed three times with PBS, adjusted to a 0.5 McFarland turbidity, and further diluted 1:10. The cells were then incubated with 1 µM DiBaC4(3) at 37°C for 10 minutes. Following incubation, the cell suspension was treated with different concentrations of candesartan cilexetil (12.5–100 µM) or 0.1% DMSO as a control. The treated cell suspensions were again placed in a 96-well plate with a black border and transparent bottom. Fluorescence intensity was dynamically monitored at an excitation wavelength of 492 nm and an emission wavelength of 515 nm. This assay facilitated the evaluation of changes in cell membrane potential based on the fluorescence intensity of DiBaC4(3)-stained cells in response to various treatments. 2.6. Antibacterial activity of LLY-507 with phospholipids and fatty acids Various phospholipids and fatty acids, including phosphatidylglycerol (PG, Aladdin, China), phosphatidylethanolamine (Y0001953, Sigma-Aldrich, USA), cardiolipin (CL, Sigma-Aldrich, C0563, USA), and arachidonic acid (A5837, Sigma-Aldrich, USA), were dissolved in methanol. The effects of these phospholipids (concentrations ranging from 8 to 128 µg/mL) and fatty acids (concentrations ranging from 7.8 to 500 µM) on the minimum inhibitory concentration (MIC) values of LLY-507 in TSB medium were evaluated using the checkerboard method, as previously described [ 17 ]. 2.7. In vitro induction of LLY-507-tolerant isolates of S. aureus Staphylococcus aureus YUSA145 was serially subcultured in TSB containing LLY-507, with initial inducing concentrations ranging from 6.25 µM to 200 µM. Each strain was cultured for 10 days at each concentration before being exposed to the subsequent concentration. On day 60, three separate isolated clones were selected for culture on TSB agar plates, and the biofilm formation of these isolated clones was assessed. Wild-type (WT) strains of S. aureus YUSA145 were utilized as controls. The isolated clones were preserved at -80°C in glycerol containing 50% TSB for future studies. 2.8. Whole-genome sequencing detection of mutations in LLY-507-tolerant clones Chromosomal DNA extracted from LLY-507-induced resistant strains of Staphylococcus aureus YUSA145 was prepared for whole-genome sequencing. Nextera libraries were constructed, and sequencing was performed on the Illumina HiSeq platform by Novogene Co. Ltd. (Beijing, China). The resulting sequences were aligned to the reference genome of S. aureus YUSA145 using bwa-mem software (version 0.7.5a) with standard parameters. Single nucleotide polymorphisms and indels in the resistant strains YUSA145 were identified using MUMmer (version 3.23). The data have been deposited in the National Center for Biotechnology Information (NCBI) under accession number PRJNA755754 (NCBI: http://www.ncbi.nlm.nih.gov/bioproject/1166673 ). 2.9. Biolayer interferometry assay (BLI) Biotinylated cardiolipin (L-C16B, Echelon Biosciences, USA) was utilized to assess the binding affinities between LLY-507 and these lipids using a biolayer interferometry (BLI) assay employing Gatorprime instrumentation (Gator Bio, San Francisco, USA). Streptavidin biosensor tips were first conditioned with kinetic buffer (PBS, 0.05% bovine serum albumin, 0.01% Tween 20) and then immobilized with biotin-labeled cardiolipin. Various concentrations of LLY-507 were subsequently applied to the streptavidin biosensors. Duplicate sets of biosensors were employed as background controls, incubated in buffer without proteins to account for nonspecific binding and signal variability. The assays were conducted in 96-well black plates at 30°C with a total volume of 300 µL per well, following standardized protocols. Data analysis utilized Gatorprime software, employing a double reference subtraction method for accurate determination of binding kinetics. 2.10. In vivo efficacy in Galleria mellonella model To evaluate the potential toxic effects of LLY-507, G. mellonella larvae were injected with LLY-507 at concentrations of 25, 50, 100 and 200 µM. Post-injection, the larvae were maintained at 37°C in the dark, and their phenotypic changes, including melanization and mobility, were observed every 24 hours for a total of 96 hours. Control larvae were injected with 1×PBS. To assess the in vivo efficacy of LLY-507 against S. aureus LLY-507 infection, groups of G. mellonella larvae were infected with a predetermined lethal dose of S. aureus SA113. Thirty minutes post-infection, LLY-507 was administered at a concentration of 50 and 100 µM and 12 µM vancomycin. PBS-injected larvae served as negative controls. After injection, all larvae were placed in clean petri dishes and incubated at 37°C for 3 days. Larvae viability was monitored and recorded at 6 to 12-hour intervals. 2.11. Quantification and statistical analysis Statistical analysis was performed using GraphPad Prism software (version 8.0), employing analysis of variance (ANOVA) or Tukey's test for multiple comparisons. The Log-rank (Mantel-Cox) test was utilized to assess survival rates. Data are presented as mean ± SD, with a p-value of < 0.05 deemed statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). Most experiments were conducted at least twice, yielding similar results. 3. Results 3.1. Antimicrobial activity of LLY-507 in vitro Our data indicated that the MIC50/MIC90 values of LLY-507 against MRSA and MSSA strains were 25/50 µM and 25/25 µM (14.37 µg/mL), respectively (Table 1), Whereas no antibacterial activities of this agent towards gram-negative bacteria, including E. coli , K. peneumoniae and A. baumanii were found in this study. In addition, the planktonic growth curve analysis using an automatic bacterial growth analyzer (Bioscreen C) showed that LLY-507 could significantly inhibit the growth of S. aureus planktonic cells at 1/2 × MIC (Fig. 1 ). To evaluate the bactericidal effect of LLY-507 on S. aureus , time-killing curve studies were carried out. As shown in the logarithmic growth phase (Fig. 1 ), LLY-507 could completely kill the S. aureus cells at 2 × MIC value and the CFUs were rapidly decreased to be undetectable within 2 h, suggesting its bactericidal activity was significantly stronger than vancomycin and linezolid at 2 × MIC. Moreover, LLY-507 also displayed a strong bactericidal effect on S. aureus isolates at 2 × MIC value in the stationary phase, further demonstrating the potent bactericidal activity of LLY-507 might significantly surpass that of clinically traditional antibiotics such as linezolid and vancomycin. The specific MIC values for various bacteria could be found in the supplementary materials (Table S1 ). 3.2. Antibiofilm activity of LLY-507 LLY-507 with 1/4 × MIC can cause a significant decrease in the biofilm formation of tested S. aureus isolates (Fig. 2 A). Moreover, LLY-507 decreased at least 80% biofilm formation of all MSSA isolates tested at the concentration of 1/2 × MIC, which indicated that LLY-507 significantly inhibited the biofilm formation of S. aureus . Moreover, adhesion viable cell counts can be significantly decreased by sub-MIC concentration of LLY-507 through confocal laser scanning microscopy (CLSM) using live/dead staining, further suggesting a noticeable S. aureus biofilm reduction by LLY-507 with the concentration of 1/2 × MIC (Fig. 2 B and 2 C). Table 1. MICs values of LLY-507 against various bacterial species 3.3. Determination of genetic mutation in LLY-507-induced S. aureus by whole-genome sequencing YUSA145 was induced with the continuous passage under LLY-507 pressure for 60 generations and then, the LLY-507-induced S. aureus , which was renamed with YUSA145N60, and its MIC was elevated to 200 µM increased from YUSA145 with MIC of 25 µM, were selected (Fig. 3 ). Whole genome sequencing of YuSA145N60 was performed and the whole genome comparison indicated 6 SNPs, including 5 non-synonymous mutations and 1 synonymous mutation, were found (Table 2 ). Of particular note is the non-synonymous mutation in the fatty acid kinase binding subunit FakB1. Fatty acid kinase generates acyl-phosphate, which is utilized in synthesizing membrane phospholipids in Gram-positive bacterial pathogens, suggesting the possible interaction between LLY-507 and the cell membrane and phospholipids of S. aureus . Phosphatidate cytidylyltransferase catalyzes the synthesis of the phospholipid intermediate cytidine diphosphate (CDP)-diacylglycerol, which serves as a precursor for the production of phosphatidylglycerol (PG) and cardiolipin (CL). This further suggests that the resistance gene mutations induced by long-term exposure to LLY-507 may be associated with alterations in the cell membrane. 3.4. Proteomic response of S. aureus to LLY-507 Quantitative label-free proteomic analysis was performed to understand the impact of LLY-507 on the proteomic level of the S. aureus YUSA145 strain. The proteomic response of S. aureus treated with 1/2 × MIC LLY-507 or DMSO alone was analyzed by mass spectrometry. Overall, 1382 proteins were confidently identified (matched peptides ≥ 1, and FDR < 0.01). Among the 1382 proteins quantified, 51 proteins showed significantly different expression levels (≥|1.5|-fold change, P ≤ 0.05) compared with that of the control, containing 22 upregulated and 29 downregulated proteins in the LLY-507 treatment (Fig. 4 A and 4 B). The proteins that are significantly upregulated or downregulated are listed in Table S2 . Gene ontology (GO) annotations of the differential proteins according to biological processes, molecular functions, and cellular components were done using the Omicsbean online database (Fig. 4 C). Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. EGG analysis revealed that the differential proteins were mainly enriched in the degradation of xylene, epithelial cell invasion, ribosomal component proteins, biosynthesis of peptidoglycan, sulfur metabolism, degradation of chloroalkanes, ascorbate and aldarate metabolism, β-ketone metabolism, selenium compound metabolism, and interconversion between pentose and glucuronate (Fig. 4 D). Table 2 Determination of genetic mutations inYUSA145N60 by whole-genome sequencing. Ref_gene_ID Mutation Type NC mutations AA mutations Subject Description CYUSA145_GM000364 nonsense T439A K147X YkyA family protein CYUSA145_GM000551 nonsyn C765A D255E ATPase/histidine kinase/DNA gyrase B/HSP90 domain protein CYUSA145_GM001001 nonsyn G686A S229L phosphatidate cytidylyltransferase CYUSA145_GM001209 nonsyn T285A S95R fatty acid kinase binding subunit FakB1 CYUSA145_GM001478 nonsyn C944T G315D Na(+)/H(+) antiporter subunit A1 CYUSA145_GM001937 nonsyn A669T E223D pur operon repressor Using the KEGG database, a protein-protein interaction network of differentially expressed proteins was constructed (Fig. 4 E). The results indicated that interactions among downregulated proteins primarily centred around SAOUHSC_00139, SAOUHSC_02654, FemA protein, FemX protein, and iron-sulfur lipoyl synthase LipM. These proteins are mainly involved in the metabolic pathways of xylene degradation, sulfur metabolism, biosynthesis of peptidoglycan, and selenium compound metabolism. Upregulated proteins were enriched in ribosomal component proteins such as rpsE, rpsG, rpsQ, and rpsK, fibronectin-binding protein FnbA, and aldehyde dehydrogenase (SAOUHSC_02363). These proteins primarily participate in metabolic pathways associated with protein biosynthesis, epithelial cell invasion, and interconversion between pentose and glucuronate. 3.5. Antibacterial mechanism of LLY-507 against S. aureus Through proteomic analysis, we observed downregulation of femA expression after treatment with LLY-507. Previous studies have linked femA to cell wall or membrane biosynthesis. Additionally, whole-genome sequencing revealed mutations in genes associated with membrane phospholipid synthesis in MRSA strains, suggesting that LLY-507 may exert its antibacterial activity by disrupting cell surface integrity. Propidium iodide (PI) fluorescent dye is used to detect membrane permeability. When it enters bacteria and binds to nucleic acids through damaged cell membranes, the fluorescence intensity increases. We found that treatment of S. aureus with LLY-507 resulted in a significant increase in PI fluorescence intensity (Fig. 5 A). After stabilization of LLY-507 treatment (56 min later), the membrane permeability of the 2×MIC and 1×MIC groups attained 70% and 60% of the positive control group, respectively (Fig. 5 B). DiBaC4(3) fluorescent dye reflects changes in membrane potential. A significant increase in DiBaC4(3) fluorescence intensity was observed in S. aureus treated with LLY-507, indicating membrane depolarization (Fig. 5 C). These results suggest that LLY-507 induces membrane damage in S. aureus . We further validated the impact of LLY-507 on the S. aureus cell membrane. As shown in Fig. 5 D, when different concentrations of various types of membrane phospholipids, especially cardiolipin, were added, the MIC values of LLY-507 against S. aureus YUSA145 increased multiplicatively, up to 16 × MIC. The result of BLI kinetic analysis between LLY-507 and cardiolipin further confirmed our speculation that there exists binding affinity between LLY-507 and CL (Fig. 5 E). Phospholipids are major constituents of cell membranes, and their addition neutralized the antibacterial activity of LLY-507. This further suggests that cardiolipin in the cell membrane may be the primary target of LLY-507's antibacterial activity. 3.6. Effects of exogenous fatty acids on the antibacterial activity of LLY-507 Previous results indicate that LLY-507 may exert its antibacterial impacts by disrupting the cell membrane permeability of S. aureus . To further explore the potential antibacterial mechanism of LLY-507, we investigated the effect of the additional mixture of exogenous fatty acid components with various concentrations in the bacterial culture medium on the antibacterial activity of LLY-507. Fatty acids are crucial components in phospholipid synthesis. By observing the impact of fatty acids on the antibacterial activity of LLY-507, we aim to further elucidate LLY's antibacterial mechanism. Checkerboard assay results (Fig. 6 ) showed that the unsaturated long-chain fatty acids palmitoleic acid, oleic acid, linoleic acid, α-linoleic acid, and arachidonic acid could decrease the antibacterial activity of LLY-507 against S. aureus , with a significant rise in MIC values with the concentration-dependent manner. Therefore, we propose that LLY-507 might exert its antibacterial activity by affecting the fatty acid-related pathways in S. aureus . 3.7. In vivo efficacy studies of LLY-507 in G. mellonella infection model Antimicrobial medication toxicity has been extensively tested in vivo using Galleria mellonella as a reliable model. We studied the toxicity of LLY-507 against larvae of G. mellonella and their capacity to shield the larvae from a fatal dosage of S. aureus SA113 infection. A pre-experiment was first conducted to determine that LLY-507 showed no toxic effect in G. mellonella at either 2 × MIC or 4 × MIC concentrations. The SA113 infective dose was determined as being 1 × 10 5 CFU/larvae. In Fig. 7 , larvae infected with SA113 and no treatment (PBS as negative control) rapidly died in 24h and the death rate reached 100% at 32h. However, infected larvae treated with 4 × MIC LLY-507 (100 µM) or 4 × MIC vancomycin (12 µM) showed 50% and 20% survival after 32 h. According to these findings, LLY-507 demonstrated an anti-infection protective action against this clinically significant drug-resistant pathogen in the in vivo model and markedly increased the survival of G. mellonella larvae infected with a lethal dosage of S. aureus SA113 bacteria. 4. Discussion LLY-507, a cell-active and selective inhibitor of SMYD2, serves as a valuable chemical probe for elucidating the role of SMYD2 in cancer and other biological processes. Our results demonstrate that LLY-507 exhibits significant antimicrobial activity against clinically isolated strains of S. aureus and E.faecalis, with MIC50/MIC90 values of 25 µM. The bactericidal activity of antimicrobial agents is a crucial determinant of their clinical efficacy [ 18 ]. Notably, LLY-507 exhibits more pronounced bactericidal activity than vancomycin and linezolid, particularly against MRSA. Furthermore, the formation of biofilms by S. aureus is a critical virulence factor contributing to chronic infections [ 19 ]. Currently, clinically available antibiotics have limited efficacy against S. aureus biofilms. LLY-507 effectively inhibits the formation of S. aureus biofilms and can penetrate mature biofilms, killing S. aureus persisters within them. These findings suggest the potential clinical application of LLY-507 in the treatment of biofilm-associated Gram-positive bacterial infections. Elucidating the mechanism of action of antimicrobial compounds is beneficial for the modification of promising derivatives for improved efficacy and provides new targets for the development of novel antimicrobial agents. To clarify the bactericidal mechanism of LLY-507 against planktonic S. aureus , we employed proteomics analysis to identify antimicrobial targets. Proteomics analysis has emerged as a commonly used research method in recent years and has shown promising prospects in elucidating the mechanism of action of novel antimicrobial drugs [ 20 – 21 ]. In this study, we utilized proteomics technology to preliminarily find that LLY-507 affects multiple metabolic pathways in S. aureus , as evidenced by the inhibition of the expression of various oxidative stress-related proteins, including proteins involved in vanillin degradation (SAOUHSC_00139), iron-sulfur sulfur transferase LipM, and selenium compound metabolism-related protein SAOUHSC_02654. Vanillin is a natural substance known for its anti-inflammatory, antioxidant, and antimicrobial properties [ 22 ]. Iron-sulfur sulfur transferase LipM is an essential cofactor for mitochondrial oxidative metabolism, involved in the catalysis and regulation of various enzyme complexes [ 23 ]. Inorganic selenium is passively absorbed in metabolism and primarily participates in redox processes. The inhibition of the expression of these proteins suggests that LLY-507-induced stress leads to a decrease in bacterial reducing power and an imbalance in redox processes. Furthermore, the upregulation of the pathway involved in the conversion between pentoses and glucuronic acid indicates that LLY-507-induced stress promotes normal carbohydrate and energy metabolism in S. aureus , thereby reducing damage caused by stress [ 24 ]. Additionally, proteins related to protein synthesis and sorting systems are upregulated by LLY-507 activation, possibly due to the inhibition of bacterial primary metabolism. In response to the inhibited protein synthesis, bacteria accelerate the renewal and turnover of ribosomes to ensure the synthesis of essential proteins for maintaining basic life activities. The protein product of the FemA gene is a 48kDa protein closely associated with methicillin resistance in S. aureus and is intimately involved in the biosynthesis of the cell wall or membrane of S. aureus [ 25 ]. FemX, a member of the FemA family, is an essential component of the cell wall synthesized only in a subset of Gram-positive bacteria and serves as a target for pathogen-specific antibiotics [ 26 ]. Proteomic analysis results showed a decrease in the expression of FemA and FemX, indicating that the antimicrobial activity of LLY-507 may be related to the disruption of cell surface integrity. Using fluorescent dyes, we observed that LLY-507 could depolarize the cell membrane, increasing its permeability, and demonstrating its ability to damage the cell membrane of S. aureus . The cell membrane of S. aureus consists of a phospholipid bilayer, which can be divided into phosphatidylglycerol and sphingomyelin according to the glycerol backbone. In this experiment, mutations were observed in the FakB1 gene associated with membrane phospholipid synthesis in the methicillin-resistant S. aureus strain YuSA145N60, which may be a target for the antimicrobial activity of LLY-507. Our results showed that the addition of cardiolipin increased the MIC value of LLY-507 against S. aureus , a phenomenon commonly found in drugs that need to penetrate the bacterial cell membrane to exert their effects. Phospholipids in the cell membrane of S. aureus might, at least partly, neutralize the antibacterial activity of LLY-507 by blocking its penetration from entering the S. aureus cell. Moreover, phospholipids are also important components of the bacteria membrane, and studies have shown that some antimicrobial peptides can penetrate the membrane, affect the activity of enzymes inside the bacteria, disrupt substance metabolism, and ultimately lead to bacterial death [ 27 ]. Therefore, LLY-507 may disrupt the membrane of bacteria, affecting the activity of various enzymes, and thereby exerting antimicrobial activity, but further experimental evidence is still needed. It is worth noting that our results showed a gene mutation in the DNA gyrase B subunit in the MRSA strain YuSA145N60. DNA gyrase is a member of bacterial type IIA topoisomerases [ 28 ], which controls the topological structure of DNA by introducing transient breaks in both DNA strands during transcription, replication, and recombination processes [ 29 – 30 ]. The DNA gyrase B subunit in bacteria is a promising target for the discovery and development of novel antibiotics [ 31 ]. According to our research results, another potential target for the antimicrobial activity of LLY-507 is DNA gyrase B, but further experimental validation is still needed. We evaluated the in vivo efficacy of LLY-507 and its interaction with exogenous fatty acids to investigate its possible antibacterial mechanism. The production of phospholipids, which are essential to bacterial cell membranes, depends on fatty acids. The MIC values of LLY-507 against S. aureus were dramatically enhanced in a concentration-dependent manner upon the addition of unsaturated long-chain fatty acids, including arachidonic acid, oleic acid, palmitoleic acid, linoleic acid, and α-linolenic acid. According to this, LLY-507 may interact with pathways linked to fatty acids, possibly causing disruptions in the synthesis of membrane phospholipids and jeopardizing the integrity of bacterial cell membranes. Fatty acids play a well-documented function in the formation of membrane phospholipids, and numerous antibacterial substances work to suppress bacterial growth by targeting fatty acid synthesis. In the in vivo efficacy studies using the G. mellonella infection model, LLY-507 exhibited a significant protective effect against a lethal dosage of S. aureus SA113. Larvae treated with 4 × MIC of LLY-507 demonstrated a 50% survival rate after 32 hours, compared to 20% for those treated with vancomycin, highlighting LLY-507's potential as an effective antimicrobial agent. The G. mellonella model is a reliable and widely-used in vivo system for assessing antimicrobial toxicity and efficacy, further validating our findings. However, the survival rate of the larvae indicates that while LLY-507 is effective, there is still a need for improvement. Future studies should focus on optimizing the dosing regimen and exploring the pharmacokinetics and pharmacodynamics of LLY-507 in more complex mammalian models. Despite LLY-507's promising activity, the observed MIC values remain relatively high, indicating that there is room for optimization. Future research should focus on modifying the structure of LLY-507 to improve its potency and reduce the effective concentration required to inhibit bacterial growth. Exploring combination treatments with other antimicrobial agents that target different pathways may also enhance the overall antibacterial efficacy of LLY-507. 5. Conclusions The present study demonstrates that LLY-507 exhibits antimicrobial activity against Gram-positive bacteria, significantly inhibiting biofilm formation and effectively killing bacteria within mature biofilms. Furthermore, quantitative proteomic analysis identified differentially expressed proteins and metabolic pathways closely associated with the inhibition of S. aureus growth by LLY-507. Fluorescent staining confirmed LLY-507's ability to disrupt bacterial cell membranes. These results suggest the potential of LLY-507 for treating the infectious diseases caused by gram-positive bacteria. However, the relatively high MIC of LLY-507 compared to clinically used antibiotics may limit its clinical application. Further exploration to identify the key pharmacophores of LLY-507, based on its molecular structure, is necessary to obtain derivatives with improved antimicrobial activity. And in vivo tests utilizing the G. mellonella infection model demonstrated the efficacy of LLY-507. Therefore, further elucidating the direct targets of LLY-507 against S. aureus is crucial for advancing the development of antimicrobial drugs based on LLY-507. Declarations Author Contributions : Conceptualization, T.H., P.L., Y.T., S.H., and Z.Y.; methodology, Y.T., P.L., Z.X., and Y.L.; validation, Y.T., T.H., P.L.; formal analysis, Y.T., P.L., Z.X., Y.L. and J.Z.; investigation, Y.T., Z.X., Y.L., J.Z., Z.W., and Z.C.; resources, Y.T., Z.X., and Y.L.; data curation, Y.T., P.L.; writing—original draft preparation, Y.T.; writing—review and editing, T.H, P.L, Y.T., and S.H.; visualization, Y.T., P.L; supervision, T.H., P.L., and S.H.; project administration, T.H., P.L., Y.T., and S.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (82172283); Guangdong Basic and Applied Basic Research Foundation (2023A1515220082, 2022A1515110096, ); Provincial medical funds of Guangdong (A2023468, A2022497); Shenzhen Key Medical Discipline Construction Fund (SZXK06162); Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20230807115814030, JCYJ20220530142006015) and the Shenzhen Nanshan District Scientific Research Program of the People's Republic of China (NS2024007, NSZD2024023, NSZD2023035, NS2022046, NSZD2023019, NS2023049, NS2023027, NS2023097; The Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTD 2219). Data Availability Statement: The whole-genome sequencing data were deposited in the National Center for Biotechnology Information (NCBI) with accession number PRJNA1166673 (NCBI: http://www.ncbi.nlm.nih.gov/bioproject/1166673). Ethics approval and consent to participate All methods were carried out in accordance with relevant guidelines and regulations and were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Bacterial strains were collected as part of the routine clinical management of patients, according to the national guidelines in China (Clinical trial number: not applicable). Therefore, informed consent was not sought, and informed consent waiver was approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Consent for publication Not applicable. Acknowledgements The authors thank Weiguang Pan (Department of Laboratory Medicine, Shenzhen Nanshan People’s Hospital and the 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen 518052, China) for helping identify and preserve the bacterial strains. Competing interests The authors declare that they have no competing interests. Disclaimer/Publisher’s Note: All methods were carried out in accordance with relevant guidelines and regulations and were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Bacterial strains were collected as part of the routine clinical management of patients, according to the national guidelines in China. 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Targeting Staphylococcal cell–wall biosynthesis protein FemX through steered molecular dynamics and drug-repurposing approach. ACS Omega 2023 , 8 , 29292-29301. Zhang, Q. Y.; Yan, Z. B.; Meng, Y. M.; Hong, X. Y.; Shao, G.; Ma, J. J.; Fu, C. Y. Antimicrobial peptides: mechanism of action, activity and clinical potential. Mil. Med. Res . 2021 , 8 , 1-25. Hirsch, J.; Klostermeier, D. What makes a type IIA topoisomerase a gyrase or a Topo IV?. Nucleic Acids Res . 2021 , 49 , 6027-6042. Tomašić, T.; Peterlin Masic, L. Prospects for developing new antibacterials targeting bacterial type IIA topoisomerases. Curr. Top. Med. Chem . 2014 , 14 , 130-151. Buzun, K.; Bielawska, A.; Bielawski, K.; Gornowicz, A. DNA topoisomerases as molecular targets for anticancer drugs. J. Enzyme Inhib. Med. Chem . 2020 , 35 , 1781-1799. Collin, F.; Karkare, S.; Maxwell, A. Exploiting bacterial DNA gyrase as a drug target: current state and perspectives. Appl. Microbiol. Biotechnol . 2011 , 92 , 479-497. Barbee, L. A.; Soge, O. O.; Holmes, K. K.; Golden, M. R. In vitro synergy testing of novel antimicrobial combination therapies against Neisseria gonorrhoeae. J. Antimicrob. Chemother . 2014 , 69 , 1572-1578. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TableS1.SpecificMICsvaluesofLLY507againstvariousbacterialspecies.docx TableS2.ProteinquantificationofsignificantlyreducedorincreasedexpressionofYUSA145aftercinacalcettreatment.RelatedtoFigure4..xlsx Table1.1.png Table 1. MICs values of LLY-507 against various bacterial species Table1.2.png Table 1. MICs values of LLY-507 against various bacterial species Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5353061","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374707901,"identity":"0a573a94-7ae0-41db-ac87-810983f3df93","order_by":0,"name":"Yuanyuan Tang","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Tang","suffix":""},{"id":374707902,"identity":"30fe6bf3-f1ba-4d0f-86ce-1fbd2e75184f","order_by":1,"name":"Zhichao Xu","email":"","orcid":"","institution":"Nanjing Tech 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School","correspondingAuthor":false,"prefix":"","firstName":"Tieying","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2024-10-29 09:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5353061/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5353061/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68922417,"identity":"9628e777-fbca-47d2-a35e-73098bc15d92","added_by":"auto","created_at":"2024-11-13 13:54:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195933,"visible":true,"origin":"","legend":"\u003cp\u003eThe planktonic cells of MSSA SA113 (A) and MRSA YUSA145 (B) were treated with 1/16 ×, 1/8 ×, 1/4 ×, 1/2 ×, 1 × MIC of LLY-507. The LLY-507 MIC for these \u003cem\u003eS. aureus\u003c/em\u003e clinical isolates was 25 μM. The OD600 of the bacterial cells was then measured by Bioscreen C (Turku, Finland) at 1-h intervals for 24 h. TSB without antimicrobials was used as an untreated control. Data are shown as Mean ± SD, n=3. Time-killing curve of LLY-507 against clinical MRSA (YUSA145) and impact of LLY-507 on the growth curves of \u003cem\u003eS. aureus\u003c/em\u003e planktonic cells. Bacterial cultures in the logarithmic growth phase (OD600=0.6-0.8) were treated with 2× MIC of LLY-507, Vancomycin (Van), and Linezolid (LZD) separately (C). Bacterial cultures in the stationary phase (OD600 \u0026gt; 3) were treated with 2× MIC of LLY-507, Vancomycin, and Linezolid, followed by overnight incubation (D). Bactericidal curves were plotted accordingly.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/d3caa33c24d57112f29f14e3.png"},{"id":68922418,"identity":"391a52f8-7ddb-4156-8bbc-9bf97b24690b","added_by":"auto","created_at":"2024-11-13 13:54:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5689164,"visible":true,"origin":"","legend":"\u003cp\u003eAntibiofilm activity of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e. (A) Eight clinical \u003cem\u003eS. aureus\u003c/em\u003eisolates were tested for the inhibitory effect of LLY-507 on the biofilm formation under different concentrations. (B) The reduction in CFU count of \u003cem\u003eS. aureus\u003c/em\u003e cells in biofilms at various concentrations of LLY-507 was compared to the corresponding untreated biofilms. (C) YUSA145 treated with 1/2×MIC of LLY-507 for 24 hours were stained with SYTO9/PI nucleic acid dye and observed under CLSM to visualize biofilm cells. Data are represented as means ± SD, n=3. *p \u0026lt; 0.05; **p \u0026lt; 0.01 (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/5aea104e2d7d79cf8231a8b8.png"},{"id":68922681,"identity":"5f0bbef6-f233-4e3a-bfb8-b02cf7a82ea7","added_by":"auto","created_at":"2024-11-13 14:02:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103133,"visible":true,"origin":"","legend":"\u003cp\u003eThe dynamics of LLY-507 MIC values in LLY-507-induced \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/e297de5e62bf05c8fbc4d8c4.png"},{"id":68922409,"identity":"79152583-d11a-4f4b-b231-0088cdd9eb81","added_by":"auto","created_at":"2024-11-13 13:54:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":398835,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic response of \u003cem\u003eS. aureus\u003c/em\u003e to LLY-507. (A-B) Differential protein volcano plot and statistical analysis after treatment with a sub-inhibitory concentration of LLY-507. The x-axis represents the fold change of differentially expressed proteins between the LLY-507 treated group and the untreated group of\u003cem\u003e S. aureus\u003c/em\u003e, and the y-axis represents the significance of the difference (p-value). Red dots represent upregulated proteins after treatment, while blue dots represent downregulated proteins. (C) Gene Ontology (GO) biological process analysis of differentially expressed proteins. (D) KEGG analysis of\u003cem\u003e S. aureus\u003c/em\u003eafter treatment with LLY-507. (E) Protein-protein interaction network analysis of differentially expressed proteins between the LLY-507 treatment group and the DMSO group in\u003cem\u003e S. aureus\u003c/em\u003e. The Protein-Protein Interaction Networks (PPI) analysis was based on the KEGG database. Rectangles represent GO/KEGG pathways, with colors indicating the significance of the p-values. Circles represent proteins or genes, with colors representing fold changes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/5edbf2f02ac3f741c37f51a0.png"},{"id":68922680,"identity":"ec787b99-4a3a-44f1-b979-6cfa0c8a7c77","added_by":"auto","created_at":"2024-11-13 14:02:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3076329,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of LLY-507 on \u003cem\u003eS. aureus\u003c/em\u003e cell membrane. (A and B) Disruption and (C) depolarization of \u003cem\u003eS. aureus\u003c/em\u003e bacterial membrane function by LLY-507. Triton X-100 at 0.1% concentration serves as a positive control, while DMSO serves as a negative control. (D) Changes in the MIC values of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003eYUSA145 after the addition of different concentrations of phospholipids. The x-axis represents different concentrations of phospholipids, and the y-axis represents the fold change in MIC values of LLY-507. PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; CL: cardiolipin. (E) Kinetic analysis by BLI of the binding of LLY-507 to cardiolipin.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/86fa48c75d060a0481f5d21a.png"},{"id":68922415,"identity":"0ec6ee2d-f8a5-4b5c-8679-b876794cd17b","added_by":"auto","created_at":"2024-11-13 13:54:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3911117,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exogenous fatty acids on the antibacterial activity of LLY-507. (A-G) Twelve types of medium- and long-chain fatty acids. The strain used was all SA113, and the MIC of LLY-507 against it was 25 μM.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/e7e976a364f1dea7527fe5d7.png"},{"id":68922419,"identity":"bccaeb74-8736-4b42-a6cb-c964e2dbff9d","added_by":"auto","created_at":"2024-11-13 13:54:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3003741,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo\u0026nbsp;efficacy assessment in the\u0026nbsp;G. \u003cem\u003emellonella\u003c/em\u003e larvae-\u003cem\u003eS. aureus\u003c/em\u003e\u0026nbsp;SA113 infection model. (A) Survival curves of\u0026nbsp;G. \u003cem\u003emellonella\u003c/em\u003e\u0026nbsp;infected with 1×10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;CFU/larvae of\u0026nbsp;\u003cem\u003eS. aureus\u003c/em\u003e\u0026nbsp;SA113 and treated with LLY-507 and vancomycin. *,\u0026nbsp;p\u0026nbsp;\u0026lt;0.05; **,\u0026nbsp;p\u0026nbsp;\u0026lt;0.01 (Student's t-test).\u0026nbsp; (B) Representative images of toxicity assay of (i) PBS, (ii) vancomycin (12 µM), (iii) LLY-507 (50 µM), and (iv) LLY-507 (100 µM) in\u0026nbsp;G. \u003cem\u003emellonella\u003c/em\u003e\u0026nbsp;32 h post-treatment.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/5f2d5c3bf54c85ea5153bce1.png"},{"id":93565942,"identity":"240159c1-3529-4e65-ba53-7fae3449c1fd","added_by":"auto","created_at":"2025-10-15 08:25:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17715824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/49b066d1-6c7f-45a6-a543-834b2946922c.pdf"},{"id":68922408,"identity":"df7512f5-d6ac-4013-9713-38ce9171d2e8","added_by":"auto","created_at":"2024-11-13 13:54:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17769,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.SpecificMICsvaluesofLLY507againstvariousbacterialspecies.docx","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/b2ea48c4433fffd664ceea5e.docx"},{"id":68922411,"identity":"ab29d727-79e8-40ea-9a26-304987707926","added_by":"auto","created_at":"2024-11-13 13:54:27","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17337,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.ProteinquantificationofsignificantlyreducedorincreasedexpressionofYUSA145aftercinacalcettreatment.RelatedtoFigure4..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/fa7f1703e3607ad98affc3cd.xlsx"},{"id":68922414,"identity":"4385bf66-7194-4e73-a0a6-1c4f8223351f","added_by":"auto","created_at":"2024-11-13 13:54:27","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":76777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e MICs values of LLY-507 against various bacterial species\u003c/p\u003e","description":"","filename":"Table1.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/3ff6a2bc5a9c2971276e42ca.png"},{"id":68922412,"identity":"2f1e7059-6aff-4116-a425-b2957c15e275","added_by":"auto","created_at":"2024-11-13 13:54:27","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e MICs values of LLY-507 against various bacterial species\u003c/p\u003e","description":"","filename":"Table1.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5353061/v1/d9d38c67f45b9e27ed4fe61e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of Growth and Biofilm Formation in Staphylococcus aureus by LLY-507","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGram-positive bacteria, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e), \u003cem\u003eEnterococcus faecium\u003c/em\u003e (\u003cem\u003eE. faecium\u003c/em\u003e) et al., are commonly found species as clinical pathogens causing infections in both hospital and community settings [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With an increasing number of patients undergoing prolonged hospitalization and the widespread use of broad-spectrum antibiotics, reports of gram-positive bacteria developing resistance to first-line antibiotics have gradually risen in recent years, posing significant challenges to clinical anti-infective therapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the United States and the European Union, drug-resistant bacteria are responsible for over 35,000 deaths annually [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Some multidrug-resistant gram-positive bacteria strains, such as methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) and vancomycin-resistant Enterococci (VRE), are disseminated and increasingly detected worldwide, holding a high-priority position on the World Health Organization's list of \"critical priority pathogens\" for antibiotic resistance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. MRSA and VRE, known for their relatively high virulence and remarkable adaptability, can thrive in various environmental conditions and have evolved resistance mechanisms against nearly all antimicrobial agents used in therapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome bacteria form biofilms when freely floating planktonic cells attach to available surfaces and initiate colonization. Biofilms are structured and functional communities formed by the mutual adhesion of bacteria through extracellular matrix secretion. Approximately 80% of bacterial infections are closely associated with biofilm formation, in some cases, which even initiates the occurrence and development of bacterial infectious diseases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Biofilm mass formation facilitates the bacterial cells to easily resist the various stress conditions and the host immune system [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Most gram-positive bacteria forming biofilms in clinical settings are prone to be explained as one of the crucial reasons for the poor clinical outcome of gram-positive bacterial infections [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For instance, biofilm formation in MRSA infections renders it difficult to remove by antibiotics and the host immune system, sometimes even leading to chronic infectious diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Biofilm-embedded bacterial cells can detach from the biofilm mass into surrounding tissues and blood, resulting in recurrence and prolonged infection especially when antibiotic concentrations decrease [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, developing novel anti-infective drugs that can inhibit bacterial growth and suppress biofilm formation has become one of the current research hotspots [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLLY-507 is a cell-active and selective inhibitor of lysine methyltransferase SMYD2, obtained through chemical synthesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Overexpression of SMYD2 protein in the cancer cells often serves as a significant adverse prognostic factor in cancer. It is closely associated with tumorigenesis factors such as tumor infiltration, tumor proliferation, lymph node metastasis, lymphatic invasion, et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the mechanism by which SMYD2 promotes tumorigenesis remains unclear. LLY-507 selectively targets a subset of histone molecules, binding to and inhibiting lysine methyltransferase SMYD2's methylation of p53 peptide and H4 with high specificity, which is employed to elucidate SMYD2's function in cancer and other biological processes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring the screening of clinical compound libraries for antibacterial activity, LLY-507 was found to exhibit broad-spectrum inhibitory activity against gram-positive bacteria. Investigation of the activity of LLY-507 in inhibiting the bacterial growth and biofilm formation of gram-positive bacteria against \u003cem\u003eS. aureus\u003c/em\u003e was demonstrated by examining LLY-507's effects on planktonic bacterial growth curves, killing curves, and biofilm activity. The action mechanism of LLY-507 was further evaluated by proteomics analysis, cell membrane permeability, and biolayer interferometry assays. These findings shed light on the potential of LLY-507 and its derivatives based on its lead structure as the development candidate for antibacterial drugs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Bacterial isolates and growth conditions and antibiotics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStrains of Staphylococcus aureus, comprising methicillin-resistant S. \u003cem\u003eaureus\u003c/em\u003e (MRSA, n\u0026thinsp;=\u0026thinsp;15) and methicillin-sensitive S. \u003cem\u003eaureus\u003c/em\u003e (MSSA, n\u0026thinsp;=\u0026thinsp;21), were collected from various clinical specimens of individual patients at Shenzhen Nanshan People's Hospital between 2015 and 2019. Identification of all clinical strains was performed using the Phoenix\u0026trade;100 automated microbial identification system, followed by re-identification through matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS, Bruker, Germany). Quality control strains, S. \u003cem\u003eaureus\u003c/em\u003e YUSA145 and SA113, were sourced from the American Type Culture Collection. The S. \u003cem\u003eaureus\u003c/em\u003e strains were cultured in tryptic soy broth (TSB, Oxoid Ltd., Basingstoke, United Kingdom), either with or without the addition of 0.5% glucose (TSBG). Cultivation conditions included incubation at 37\u0026deg;C with shaking at 220 rpm. The identified strains were preserved at -80\u0026deg;C in TSB supplemented with 15% glycerol for subsequent analyses. Antibiotics LLY-507, Vancomycin (Van), and Linezolid (LZD) were procured from MCE (Princeton, United States). The cell membrane phospholipids, such as cardiolipin (CL, C0563, Sigma-Aldrich), phosphatidylcholine (840051P, Sigma-Aldrich), phosphatidylethanolamine (Y0001953, Sigma-Aldrich), and phosphatidylglycerol (841138P, Sigma-Aldrich), were dissolved in methanol for further use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Determination of antibacterial activities of LLY-507\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. MIC and MBC assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe minimum inhibitory concentrations (MICs) of antimicrobial agents were determined using the broth microdilution method in cation-adjusted Mueller\u0026ndash;Hinton broth (CAMHB; Oxoid Ltd), following the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI M100, 29th ed.) as previously described. In brief, two-fold serial dilutions of the compounds were prepared in 96-well microtiter plates (Falcon), achieving final concentrations ranging from 200 to 0.39 \u0026micro;M in 100 \u0026micro;L of CAMHB medium. Bacterial cells were cultured overnight with shaking, then adjusted to match the density of a 0.5 McFarland standard (approximately 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL), and further diluted 1:200 into the CAMHB medium. The mixtures in the 96-well plates were thoroughly mixed and incubated at 37\u0026deg;C with shaking at 220 rpm for 16\u0026ndash;20 hours. The MIC was defined as the lowest concentration of the compound that exhibited no visible growth in the wells. For the minimum bactericidal concentration (MBC) assay, samples from the wells showing MIC were spread onto TSB agar plates and incubated overnight at 37\u0026deg;C. The MBC was defined as the concentration that resulted in a 99.9% reduction of the original inoculum. All experiments were conducted in duplicate or more.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Bacterial dynamic growth curve assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStrains of Staphylococcus aureus were cultivated to stationary phase (16 hours) and subsequently diluted 1:200 in Tryptic Soy Broth (TSB). The diluted bacterial cultures were then incubated at 37\u0026deg;C with the specified concentrations of antibiotics while shaking at 220 rpm. Bacterial growth was monitored by measuring the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) at 1-hour intervals over a 24-hour period using an automatic bacterial growth analyzer (Bioscreen C, Turku, Finland). All experiments were conducted in biological triplicates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Dynamic time-kill curve assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the bactericidal experiments involving planktonic cells, overnight-cultured Staphylococcus aureus were diluted to approximately 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU in Tryptic Soy Broth (TSB). Antibiotics were then added to achieve final concentrations of 2 \u0026times; MIC during the logarithmic growth phase and again at 2 \u0026times; MIC during the stationary phase (16 hours). Colony counts were conducted at multiple time points: 0, 2, 4, 6, and 24 hours during the logarithmic phase, and at 24, 48, 72, 96, and 120 hours during the stationary phase. Cells were plated for colony-forming unit (CFU) counts and challenged with Linezolid (LZD) and Vancomycin (Van) antibiotics, respectively. Data presented are representative of three independent experiments, with bacteria treated with 0.1% DMSO serving as a control. Results are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from triplicate measurements.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Detection of Anti-biofilm Activity of the LLY-507\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Microtiter plate assays of biofilm formation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA detailed protocol for the inhibition of biofilm formation assay has been previously reported. Isolates of Staphylococcus aureus were cultured overnight in Tryptic Soy Broth (TSB) at 37\u0026deg;C with shaking at 220 rpm. The overnight cultures were then diluted 1:200 and inoculated into a polystyrene 96-well plate containing 200 \u0026micro;L of TSBG (TSB supplemented with 2% glucose) at concentrations of 1/8 \u0026times;, 1/4 \u0026times;, or 1/2 \u0026times; MIC of LLY-507. For the biofilm eradication assay, plates were incubated at 37\u0026deg;C for 24 hours to allow the formation of mature biofilms. Following incubation, the medium was carefully removed, and the wells were washed gently three times with phosphate-buffered saline (PBS). Fresh medium containing various concentrations of LLY-507 was then added to each well and incubated for an additional 24 hours. Biofilms were stained with crystal violet and assessed by measuring the optical density at 570 nm (OD\u003csub\u003e570\u003c/sub\u003e). Data were obtained from at least two independent experiments, yielding consistent results.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Adhesion cell counts in bacterial biofilm\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBiofilm inhibitory effects and mature biofilm removal effects were determined. Briefly, 1 mL of TSB medium containing 2% glucose with a dilution of 500 times was added to each well of a 24-well plate. For biofilm inhibitory effect assay, LLY-507 at different concentrations (1 \u0026times; MIC, 1/2 \u0026times; MIC, 1/4 \u0026times; MIC, and 1/8 \u0026times; MIC) were cultured overnight and added to the bacterial solution and incubated at 37℃ for 24 hours. After removing the culture solution carefully, wells were washed twice with sterile saline before being suspended, and then incubated in TSB plate and cells were counted. The 0.1% DMSO was treated as the negative control group.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Determination of cell viability in mature biofilms by CLSM\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe effect of LLY-507 on cell viability in mature biofilms (24 hours) was assessed using the Live/Dead Bacterial Viability method (Live/Dead BacLight, Molecular Probes, USA), employing SYTO 9 and propidium iodide (PI) dyes to stain live and dead cells within the biofilms. Staphylococcus aureus YUSA145 was cultured in cell-culture dishes (Fluorodish, FD35-100) using TSBG medium and incubated at 37\u0026deg;C for 24 hours. After incubation, planktonic bacteria were removed and discarded, and fresh TSBG containing LLY-507 (at 1/2 \u0026times; MIC concentrations) was added. The cultures were then incubated at 37\u0026deg;C for an additional 24 hours. Following staining, the mature biofilms were visualized using a confocal laser scanning microscope (CLSM, Leica) with an oil-immersion objective. Image editing and analysis were performed using IMARIS 7.0.0 software (Bitplane). Green fluorescence indicated viable cells, while red fluorescence represented dead cells. The proportion of dead cells within the mature biofilm was quantified as the PI/total percentage using ImageJ software (Rawak Software Inc., Stuttgart, Germany). This assay was conducted in triplicate, yielding consistent results.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Proteomics analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProteomics analysis was performed following a previously reported method with modifications. Staphylococcus aureus YUSA145 in the exponential growth phase was treated with 1/2 \u0026times; MIC of LLY-507 or DMSO at 37\u0026deg;C for 2 hours on a shaker set to 200 rpm, with each treatment group consisting of three biological replicates. Bacteria were harvested by centrifugation at 5,000 g for 10 minutes at 4\u0026deg;C and washed three times with 1 \u0026times; PBS. The cell pellets were suspended in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime) supplemented with a protease inhibitor cocktail (P1065, Beyotime). Zirconia/Silica beads (0.1 mm diameter, catalog number: 11079101z) were added, and the samples were homogenized at 4\u0026deg;C using a Cell Disruption Device (JXFSTPRP-24L, Shanghai Jingxin Industrial Development Co., Ltd.) with a lysis protocol of 70 Hz for 1 minute, followed by 10-second pauses over four cycles.\u003c/p\u003e \u003cp\u003eThe samples were centrifuged at 4\u0026deg;C and 12,000 rpm for 10 minutes, and the supernatant was collected. Protein concentrations were determined using the BCA assay and SDS-PAGE. For protein reduction, 50 mg of total protein was transferred into 10 KD ultrafilter tubes and treated with 200 \u0026micro;L of 8 M urea solution containing 10 mM DTT for 2 hours at 37\u0026deg;C. The urea solution was removed by centrifugation (12,000 rpm at 20\u0026deg;C for 10 minutes), and then 200 \u0026micro;L of 8 M urea solution with 50 mM iodoacetamide (IAA) was added to the pellet and incubated in the dark for 15 minutes at room temperature.\u003c/p\u003e \u003cp\u003eSamples were desalted using Amicon ultracentrifugal filters (10 KD, Millipore, Billerica, MA, USA), centrifuged at 12,000 g for 10 minutes at room temperature, and washed three times with 200 \u0026micro;L of 8 M urea solution containing 25 mM ammonium bicarbonate. Subsequently, 100 \u0026micro;L of 25 mM ammonium bicarbonate with 1 \u0026micro;g of trypsin (protein ratio of 50:1) was added to each filter tube and incubated at 37\u0026deg;C for 12 hours. The filter tubes were washed twice with 100 \u0026micro;L of 25 mM ammonium bicarbonate, and the resulting flow-through peptide fractions were collected and lyophilized.\u003c/p\u003e \u003cp\u003eThe lyophilized peptide fractions were re-suspended in 40 \u0026micro;L of ddH2O containing 0.1% formic acid, with 30 \u0026micro;L placed in the inner cannula of the autosampler vial and a 4 \u0026micro;L aliquot loaded into a C18 trap column (PepMap Neo Trap Cartridge, 164946, Thermo Scientific). Online chromatographic separation was performed on an Ultimate 3000 RSLCnano (Thermo Scientific) using 20 \u0026micro;L of 100% solvent A (0.1% formic acid) for trapping and desalting. An elution gradient of 5\u0026ndash;38% solvent B (80% acetonitrile, 0.1% formic acid) over 60 minutes was applied to an analytical column (Acclaim PepMap RSLC, 75 mm \u0026times; 25 cm C18-2 \u0026micro;m, 164941, Thermo Scientific). Data-dependent acquisition (DDA) mass spectrometry techniques were utilized to obtain tandem mass spectrometry (MS) data on a Thermo Fisher Q Exactive Plus mass spectrometer equipped with a Nano Flex ion source. Data were collected using an ion spray voltage of 1.9 kV and an interface heater temperature of 320\u0026deg;C.\u003c/p\u003e \u003cp\u003eProtein identification and quantitative analysis of MS/MS data were conducted using Thermo Proteome Discoverer. After searching the target database, the local false discovery rate for peptides was set at 1.0%, allowing a maximum of two missed cleavages. In addition to a p-value threshold of \u0026lt;\u0026thinsp;0.05, a two-fold cutoff value was employed to identify upregulated and downregulated proteins. Differentially expressed proteins were uploaded to the OMICSBEAN database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omicsbean.com\u003c/span\u003e\u003cspan address=\"http://www.omicsbean.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for volcano plot generation, heatmap visualization, gene ontology (GO) annotation (encompassing biological processes, cellular components, and molecular functions), KEGG pathway analysis, and protein-protein interaction (PPI) network analysis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Membrane permeability and depolarization assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the membrane permeability assay, mid-log phase cells of Staphylococcus aureus YUSA145 were washed three times with sterile PBS and adjusted to a turbidity of 0.5 McFarland. The cell suspension was then diluted 1:10. Following this, the cells were incubated with 2 \u0026micro;M propidium iodide (PI) at 37\u0026deg;C for 10 minutes. Subsequently, the cell suspension was treated with varying concentrations of candesartan cilexetil, 0.1% Triton X-100, or 0.1% DMSO. The treated cell suspensions were placed in a 96-well plate with a black border and transparent bottom. Fluorescence intensity was dynamically monitored at an excitation wavelength of 493 nm and an emission wavelength of 534 nm. This assay allowed for the assessment of alterations in cell membrane integrity based on the fluorescence intensity of PI-stained cells in response to different treatments.\u003c/p\u003e \u003cp\u003eFor the determination of membrane potential, mid-log phase cells of S. \u003cem\u003eaureus\u003c/em\u003e YUSA145 were similarly washed three times with PBS, adjusted to a 0.5 McFarland turbidity, and further diluted 1:10. The cells were then incubated with 1 \u0026micro;M DiBaC4(3) at 37\u0026deg;C for 10 minutes. Following incubation, the cell suspension was treated with different concentrations of candesartan cilexetil (12.5\u0026ndash;100 \u0026micro;M) or 0.1% DMSO as a control. The treated cell suspensions were again placed in a 96-well plate with a black border and transparent bottom. Fluorescence intensity was dynamically monitored at an excitation wavelength of 492 nm and an emission wavelength of 515 nm. This assay facilitated the evaluation of changes in cell membrane potential based on the fluorescence intensity of DiBaC4(3)-stained cells in response to various treatments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Antibacterial activity of LLY-507 with phospholipids and fatty acids\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eVarious phospholipids and fatty acids, including phosphatidylglycerol (PG, Aladdin, China), phosphatidylethanolamine (Y0001953, Sigma-Aldrich, USA), cardiolipin (CL, Sigma-Aldrich, C0563, USA), and arachidonic acid (A5837, Sigma-Aldrich, USA), were dissolved in methanol. The effects of these phospholipids (concentrations ranging from 8 to 128 \u0026micro;g/mL) and fatty acids (concentrations ranging from 7.8 to 500 \u0026micro;M) on the minimum inhibitory concentration (MIC) values of LLY-507 in TSB medium were evaluated using the checkerboard method, as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7. In vitro induction of LLY-507-tolerant isolates of S. aureus\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStaphylococcus aureus YUSA145 was serially subcultured in TSB containing LLY-507, with initial inducing concentrations ranging from 6.25 \u0026micro;M to 200 \u0026micro;M. Each strain was cultured for 10 days at each concentration before being exposed to the subsequent concentration. On day 60, three separate isolated clones were selected for culture on TSB agar plates, and the biofilm formation of these isolated clones was assessed. Wild-type (WT) strains of S. aureus YUSA145 were utilized as controls. The isolated clones were preserved at -80\u0026deg;C in glycerol containing 50% TSB for future studies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Whole-genome sequencing detection of mutations in LLY-507-tolerant clones\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eChromosomal DNA extracted from LLY-507-induced resistant strains of Staphylococcus aureus YUSA145 was prepared for whole-genome sequencing. Nextera libraries were constructed, and sequencing was performed on the Illumina HiSeq platform by Novogene Co. Ltd. (Beijing, China). The resulting sequences were aligned to the reference genome of S. \u003cem\u003eaureus\u003c/em\u003e YUSA145 using bwa-mem software (version 0.7.5a) with standard parameters. Single nucleotide polymorphisms and indels in the resistant strains YUSA145 were identified using MUMmer (version 3.23). The data have been deposited in the National Center for Biotechnology Information (NCBI) under accession number PRJNA755754 (NCBI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/bioproject/1166673\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/bioproject/1166673\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Biolayer interferometry assay (BLI)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBiotinylated cardiolipin (L-C16B, Echelon Biosciences, USA) was utilized to assess the binding affinities between LLY-507 and these lipids using a biolayer interferometry (BLI) assay employing Gatorprime instrumentation (Gator Bio, San Francisco, USA). Streptavidin biosensor tips were first conditioned with kinetic buffer (PBS, 0.05% bovine serum albumin, 0.01% Tween 20) and then immobilized with biotin-labeled cardiolipin. Various concentrations of LLY-507 were subsequently applied to the streptavidin biosensors. Duplicate sets of biosensors were employed as background controls, incubated in buffer without proteins to account for nonspecific binding and signal variability. The assays were conducted in 96-well black plates at 30\u0026deg;C with a total volume of 300 \u0026micro;L per well, following standardized protocols. Data analysis utilized Gatorprime software, employing a double reference subtraction method for accurate determination of binding kinetics.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.10. In vivo efficacy in Galleria mellonella model\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate the potential toxic effects of LLY-507, G. \u003cem\u003emellonella\u003c/em\u003e larvae were injected with LLY-507 at concentrations of 25, 50, 100 and 200 \u0026micro;M. Post-injection, the larvae were maintained at 37\u0026deg;C in the dark, and their phenotypic changes, including melanization and mobility, were observed every 24 hours for a total of 96 hours. Control larvae were injected with 1\u0026times;PBS. To assess the in vivo efficacy of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e LLY-507 infection, groups of G. \u003cem\u003emellonella\u003c/em\u003e larvae were infected with a predetermined lethal dose of \u003cem\u003eS. aureus\u003c/em\u003e SA113. Thirty minutes post-infection, LLY-507 was administered at a concentration of 50 and 100 \u0026micro;M and 12 \u0026micro;M vancomycin. PBS-injected larvae served as negative controls. After injection, all larvae were placed in clean petri dishes and incubated at 37\u0026deg;C for 3 days. Larvae viability was monitored and recorded at 6 to 12-hour intervals.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Quantification and statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism software (version 8.0), employing analysis of variance (ANOVA) or Tukey's test for multiple comparisons. The Log-rank (Mantel-Cox) test was utilized to assess survival rates. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, with a p-value of \u0026lt;\u0026thinsp;0.05 deemed statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Most experiments were conducted at least twice, yielding similar results.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Antimicrobial activity of LLY-507 in vitro\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur data indicated that the MIC50/MIC90 values of LLY-507 against MRSA and MSSA strains were 25/50 \u0026micro;M and 25/25 \u0026micro;M (14.37 \u0026micro;g/mL), respectively (Table\u0026nbsp;1), Whereas no antibacterial activities of this agent towards gram-negative bacteria, including \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eK. peneumoniae\u003c/em\u003e and \u003cem\u003eA. baumanii\u003c/em\u003e were found in this study. In addition, the planktonic growth curve analysis using an automatic bacterial growth analyzer (Bioscreen C) showed that LLY-507 could significantly inhibit the growth of \u003cem\u003eS. aureus\u003c/em\u003e planktonic cells at 1/2 \u0026times; MIC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the bactericidal effect of LLY-507 on \u003cem\u003eS. aureus\u003c/em\u003e, time-killing curve studies were carried out. As shown in the logarithmic growth phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), LLY-507 could completely kill the \u003cem\u003eS. aureus\u003c/em\u003e cells at 2 \u0026times; MIC value and the CFUs were rapidly decreased to be undetectable within 2 h, suggesting its bactericidal activity was significantly stronger than vancomycin and linezolid at 2 \u0026times; MIC. Moreover, LLY-507 also displayed a strong bactericidal effect on \u003cem\u003eS. aureus\u003c/em\u003e isolates at 2 \u0026times; MIC value in the stationary phase, further demonstrating the potent bactericidal activity of LLY-507 might significantly surpass that of clinically traditional antibiotics such as linezolid and vancomycin. The specific MIC values for various bacteria could be found in the supplementary materials (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Antibiofilm activity of LLY-507\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLLY-507 with 1/4 \u0026times; MIC can cause a significant decrease in the biofilm formation of tested \u003cem\u003eS. aureus\u003c/em\u003e isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, LLY-507 decreased at least 80% biofilm formation of all MSSA isolates tested at the concentration of 1/2 \u0026times; MIC, which indicated that LLY-507 significantly inhibited the biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e. Moreover, adhesion viable cell counts can be significantly decreased by sub-MIC concentration of LLY-507 through confocal laser scanning microscopy (CLSM) using live/dead staining, further suggesting a noticeable \u003cem\u003eS. aureus\u003c/em\u003e biofilm reduction by LLY-507 with the concentration of 1/2 \u0026times; MIC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1.\u003c/b\u003e MICs values of LLY-507 against various bacterial species\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Determination of genetic mutation in LLY-507-induced S. aureus by whole-genome sequencing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eYUSA145 was induced with the continuous passage under LLY-507 pressure for 60 generations and then, the LLY-507-induced \u003cem\u003eS. aureus\u003c/em\u003e, which was renamed with YUSA145N60, and its MIC was elevated to 200 \u0026micro;M increased from YUSA145 with MIC of 25 \u0026micro;M, were selected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Whole genome sequencing of YuSA145N60 was performed and the whole genome comparison indicated 6 SNPs, including 5 non-synonymous mutations and 1 synonymous mutation, were found (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of particular note is the non-synonymous mutation in the fatty acid kinase binding subunit FakB1. Fatty acid kinase generates acyl-phosphate, which is utilized in synthesizing membrane phospholipids in Gram-positive bacterial pathogens, suggesting the possible interaction between LLY-507 and the cell membrane and phospholipids of \u003cem\u003eS. aureus\u003c/em\u003e. Phosphatidate cytidylyltransferase catalyzes the synthesis of the phospholipid intermediate cytidine diphosphate (CDP)-diacylglycerol, which serves as a precursor for the production of phosphatidylglycerol (PG) and cardiolipin (CL). This further suggests that the resistance gene mutations induced by long-term exposure to LLY-507 may be associated with alterations in the cell membrane.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Proteomic response of S. aureus to LLY-507\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eQuantitative label-free proteomic analysis was performed to understand the impact of LLY-507 on the proteomic level of the \u003cem\u003eS. aureus\u003c/em\u003e YUSA145 strain. The proteomic response of \u003cem\u003eS. aureus\u003c/em\u003e treated with 1/2 \u0026times; MIC LLY-507 or DMSO alone was analyzed by mass spectrometry. Overall, 1382 proteins were confidently identified (matched peptides\u0026thinsp;\u0026ge;\u0026thinsp;1, and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Among the 1382 proteins quantified, 51 proteins showed significantly different expression levels (\u0026ge;|1.5|-fold change, P\u0026thinsp;\u0026le;\u0026thinsp;0.05) compared with that of the control, containing 22 upregulated and 29 downregulated proteins in the LLY-507 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The proteins that are significantly upregulated or downregulated are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Gene ontology (GO) annotations of the differential proteins according to biological processes, molecular functions, and cellular components were done using the Omicsbean online database (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. EGG analysis revealed that the differential proteins were mainly enriched in the degradation of xylene, epithelial cell invasion, ribosomal component proteins, biosynthesis of peptidoglycan, sulfur metabolism, degradation of chloroalkanes, ascorbate and aldarate metabolism, β-ketone metabolism, selenium compound metabolism, and interconversion between pentose and glucuronate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of genetic mutations inYUSA145N60 by whole-genome sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRef_gene_ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMutation Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC mutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAA mutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSubject Description\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYUSA145_GM000364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enonsense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT439A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK147X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYkyA family protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYUSA145_GM000551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enonsyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC765A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD255E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATPase/histidine kinase/DNA gyrase B/HSP90 domain protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYUSA145_GM001001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enonsyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG686A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS229L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ephosphatidate cytidylyltransferase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYUSA145_GM001209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enonsyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT285A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS95R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003efatty acid kinase binding subunit FakB1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYUSA145_GM001478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enonsyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC944T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG315D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNa(+)/H(+) antiporter subunit A1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYUSA145_GM001937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enonsyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA669T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE223D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epur operon repressor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUsing the KEGG database, a protein-protein interaction network of differentially expressed proteins was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The results indicated that interactions among downregulated proteins primarily centred around SAOUHSC_00139, SAOUHSC_02654, FemA protein, FemX protein, and iron-sulfur lipoyl synthase LipM. These proteins are mainly involved in the metabolic pathways of xylene degradation, sulfur metabolism, biosynthesis of peptidoglycan, and selenium compound metabolism. Upregulated proteins were enriched in ribosomal component proteins such as rpsE, rpsG, rpsQ, and rpsK, fibronectin-binding protein FnbA, and aldehyde dehydrogenase (SAOUHSC_02363). These proteins primarily participate in metabolic pathways associated with protein biosynthesis, epithelial cell invasion, and interconversion between pentose and glucuronate.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Antibacterial mechanism of LLY-507 against S. aureus\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThrough proteomic analysis, we observed downregulation of femA expression after treatment with LLY-507. Previous studies have linked femA to cell wall or membrane biosynthesis. Additionally, whole-genome sequencing revealed mutations in genes associated with membrane phospholipid synthesis in MRSA strains, suggesting that LLY-507 may exert its antibacterial activity by disrupting cell surface integrity. Propidium iodide (PI) fluorescent dye is used to detect membrane permeability. When it enters bacteria and binds to nucleic acids through damaged cell membranes, the fluorescence intensity increases. We found that treatment of \u003cem\u003eS. aureus\u003c/em\u003e with LLY-507 resulted in a significant increase in PI fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After stabilization of LLY-507 treatment (56 min later), the membrane permeability of the 2\u0026times;MIC and 1\u0026times;MIC groups attained 70% and 60% of the positive control group, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). DiBaC4(3) fluorescent dye reflects changes in membrane potential. A significant increase in DiBaC4(3) fluorescence intensity was observed in \u003cem\u003eS. aureus\u003c/em\u003e treated with LLY-507, indicating membrane depolarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results suggest that LLY-507 induces membrane damage in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe further validated the impact of LLY-507 on the \u003cem\u003eS. aureus\u003c/em\u003e cell membrane. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, when different concentrations of various types of membrane phospholipids, especially cardiolipin, were added, the MIC values of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e YUSA145 increased multiplicatively, up to 16 \u0026times; MIC. The result of BLI kinetic analysis between LLY-507 and cardiolipin further confirmed our speculation that there exists binding affinity between LLY-507 and CL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Phospholipids are major constituents of cell membranes, and their addition neutralized the antibacterial activity of LLY-507. This further suggests that cardiolipin in the cell membrane may be the primary target of LLY-507's antibacterial activity.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effects of exogenous fatty acids on the antibacterial activity of LLY-507\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePrevious results indicate that LLY-507 may exert its antibacterial impacts by disrupting the cell membrane permeability of \u003cem\u003eS. aureus\u003c/em\u003e. To further explore the potential antibacterial mechanism of LLY-507, we investigated the effect of the additional mixture of exogenous fatty acid components with various concentrations in the bacterial culture medium on the antibacterial activity of LLY-507. Fatty acids are crucial components in phospholipid synthesis. By observing the impact of fatty acids on the antibacterial activity of LLY-507, we aim to further elucidate LLY's antibacterial mechanism. Checkerboard assay results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) showed that the unsaturated long-chain fatty acids palmitoleic acid, oleic acid, linoleic acid, α-linoleic acid, and arachidonic acid could decrease the antibacterial activity of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e, with a significant rise in MIC values with the concentration-dependent manner. Therefore, we propose that LLY-507 might exert its antibacterial activity by affecting the fatty acid-related pathways in \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. In vivo efficacy studies of LLY-507 in G. mellonella infection model\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAntimicrobial medication toxicity has been extensively tested in vivo using Galleria mellonella as a reliable model. We studied the toxicity of LLY-507 against larvae of G. \u003cem\u003emellonella\u003c/em\u003e and their capacity to shield the larvae from a fatal dosage of \u003cem\u003eS. aureus\u003c/em\u003e SA113 infection. A pre-experiment was first conducted to determine that LLY-507 showed no toxic effect in G. \u003cem\u003emellonella\u003c/em\u003e at either 2 \u0026times; MIC or 4 \u0026times; MIC concentrations. The SA113 infective dose was determined as being 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/larvae. In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, larvae infected with SA113 and no treatment (PBS as negative control) rapidly died in 24h and the death rate reached 100% at 32h. However, infected larvae treated with 4 \u0026times; MIC LLY-507 (100 \u0026micro;M) or 4 \u0026times; MIC vancomycin (12 \u0026micro;M) showed 50% and 20% survival after 32 h. According to these findings, LLY-507 demonstrated an anti-infection protective action against this clinically significant drug-resistant pathogen in the in vivo model and markedly increased the survival of G. \u003cem\u003emellonella\u003c/em\u003e larvae infected with a lethal dosage of \u003cem\u003eS. aureus\u003c/em\u003e SA113 bacteria.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLLY-507, a cell-active and selective inhibitor of SMYD2, serves as a valuable chemical probe for elucidating the role of SMYD2 in cancer and other biological processes. Our results demonstrate that LLY-507 exhibits significant antimicrobial activity against clinically isolated strains of \u003cem\u003eS. aureus\u003c/em\u003e and E.faecalis, with MIC50/MIC90 values of 25 \u0026micro;M. The bactericidal activity of antimicrobial agents is a crucial determinant of their clinical efficacy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, LLY-507 exhibits more pronounced bactericidal activity than vancomycin and linezolid, particularly against MRSA. Furthermore, the formation of biofilms by \u003cem\u003eS. aureus\u003c/em\u003e is a critical virulence factor contributing to chronic infections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Currently, clinically available antibiotics have limited efficacy against \u003cem\u003eS. aureus\u003c/em\u003e biofilms. LLY-507 effectively inhibits the formation of \u003cem\u003eS. aureus\u003c/em\u003e biofilms and can penetrate mature biofilms, killing \u003cem\u003eS. aureus\u003c/em\u003e persisters within them. These findings suggest the potential clinical application of LLY-507 in the treatment of biofilm-associated Gram-positive bacterial infections.\u003c/p\u003e \u003cp\u003eElucidating the mechanism of action of antimicrobial compounds is beneficial for the modification of promising derivatives for improved efficacy and provides new targets for the development of novel antimicrobial agents. To clarify the bactericidal mechanism of LLY-507 against planktonic \u003cem\u003eS. aureus\u003c/em\u003e, we employed proteomics analysis to identify antimicrobial targets. Proteomics analysis has emerged as a commonly used research method in recent years and has shown promising prospects in elucidating the mechanism of action of novel antimicrobial drugs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, we utilized proteomics technology to preliminarily find that LLY-507 affects multiple metabolic pathways in \u003cem\u003eS. aureus\u003c/em\u003e, as evidenced by the inhibition of the expression of various oxidative stress-related proteins, including proteins involved in vanillin degradation (SAOUHSC_00139), iron-sulfur sulfur transferase LipM, and selenium compound metabolism-related protein SAOUHSC_02654. Vanillin is a natural substance known for its anti-inflammatory, antioxidant, and antimicrobial properties [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Iron-sulfur sulfur transferase LipM is an essential cofactor for mitochondrial oxidative metabolism, involved in the catalysis and regulation of various enzyme complexes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Inorganic selenium is passively absorbed in metabolism and primarily participates in redox processes. The inhibition of the expression of these proteins suggests that LLY-507-induced stress leads to a decrease in bacterial reducing power and an imbalance in redox processes. Furthermore, the upregulation of the pathway involved in the conversion between pentoses and glucuronic acid indicates that LLY-507-induced stress promotes normal carbohydrate and energy metabolism in \u003cem\u003eS. aureus\u003c/em\u003e, thereby reducing damage caused by stress [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, proteins related to protein synthesis and sorting systems are upregulated by LLY-507 activation, possibly due to the inhibition of bacterial primary metabolism. In response to the inhibited protein synthesis, bacteria accelerate the renewal and turnover of ribosomes to ensure the synthesis of essential proteins for maintaining basic life activities.\u003c/p\u003e \u003cp\u003eThe protein product of the FemA gene is a 48kDa protein closely associated with methicillin resistance in \u003cem\u003eS. aureus\u003c/em\u003e and is intimately involved in the biosynthesis of the cell wall or membrane of \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. FemX, a member of the FemA family, is an essential component of the cell wall synthesized only in a subset of Gram-positive bacteria and serves as a target for pathogen-specific antibiotics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Proteomic analysis results showed a decrease in the expression of FemA and FemX, indicating that the antimicrobial activity of LLY-507 may be related to the disruption of cell surface integrity. Using fluorescent dyes, we observed that LLY-507 could depolarize the cell membrane, increasing its permeability, and demonstrating its ability to damage the cell membrane of \u003cem\u003eS. aureus\u003c/em\u003e. The cell membrane of \u003cem\u003eS. aureus\u003c/em\u003e consists of a phospholipid bilayer, which can be divided into phosphatidylglycerol and sphingomyelin according to the glycerol backbone. In this experiment, mutations were observed in the FakB1 gene associated with membrane phospholipid synthesis in the methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e strain YuSA145N60, which may be a target for the antimicrobial activity of LLY-507. Our results showed that the addition of cardiolipin increased the MIC value of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e, a phenomenon commonly found in drugs that need to penetrate the bacterial cell membrane to exert their effects. Phospholipids in the cell membrane of \u003cem\u003eS. aureus\u003c/em\u003e might, at least partly, neutralize the antibacterial activity of LLY-507 by blocking its penetration from entering the \u003cem\u003eS. aureus\u003c/em\u003e cell.\u003c/p\u003e \u003cp\u003eMoreover, phospholipids are also important components of the bacteria membrane, and studies have shown that some antimicrobial peptides can penetrate the membrane, affect the activity of enzymes inside the bacteria, disrupt substance metabolism, and ultimately lead to bacterial death [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, LLY-507 may disrupt the membrane of bacteria, affecting the activity of various enzymes, and thereby exerting antimicrobial activity, but further experimental evidence is still needed. It is worth noting that our results showed a gene mutation in the DNA gyrase B subunit in the MRSA strain YuSA145N60. DNA gyrase is a member of bacterial type IIA topoisomerases [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which controls the topological structure of DNA by introducing transient breaks in both DNA strands during transcription, replication, and recombination processes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The DNA gyrase B subunit in bacteria is a promising target for the discovery and development of novel antibiotics [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. According to our research results, another potential target for the antimicrobial activity of LLY-507 is DNA gyrase B, but further experimental validation is still needed.\u003c/p\u003e \u003cp\u003eWe evaluated the in vivo efficacy of LLY-507 and its interaction with exogenous fatty acids to investigate its possible antibacterial mechanism. The production of phospholipids, which are essential to bacterial cell membranes, depends on fatty acids. The MIC values of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e were dramatically enhanced in a concentration-dependent manner upon the addition of unsaturated long-chain fatty acids, including arachidonic acid, oleic acid, palmitoleic acid, linoleic acid, and α-linolenic acid. According to this, LLY-507 may interact with pathways linked to fatty acids, possibly causing disruptions in the synthesis of membrane phospholipids and jeopardizing the integrity of bacterial cell membranes. Fatty acids play a well-documented function in the formation of membrane phospholipids, and numerous antibacterial substances work to suppress bacterial growth by targeting fatty acid synthesis.\u003c/p\u003e \u003cp\u003eIn the in vivo efficacy studies using the G. \u003cem\u003emellonella\u003c/em\u003e infection model, LLY-507 exhibited a significant protective effect against a lethal dosage of \u003cem\u003eS. aureus\u003c/em\u003e SA113. Larvae treated with 4 \u0026times; MIC of LLY-507 demonstrated a 50% survival rate after 32 hours, compared to 20% for those treated with vancomycin, highlighting LLY-507's potential as an effective antimicrobial agent. The G. \u003cem\u003emellonella\u003c/em\u003e model is a reliable and widely-used in vivo system for assessing antimicrobial toxicity and efficacy, further validating our findings. However, the survival rate of the larvae indicates that while LLY-507 is effective, there is still a need for improvement. Future studies should focus on optimizing the dosing regimen and exploring the pharmacokinetics and pharmacodynamics of LLY-507 in more complex mammalian models.\u003c/p\u003e \u003cp\u003eDespite LLY-507's promising activity, the observed MIC values remain relatively high, indicating that there is room for optimization. Future research should focus on modifying the structure of LLY-507 to improve its potency and reduce the effective concentration required to inhibit bacterial growth. Exploring combination treatments with other antimicrobial agents that target different pathways may also enhance the overall antibacterial efficacy of LLY-507.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe present study demonstrates that LLY-507 exhibits antimicrobial activity against Gram-positive bacteria, significantly inhibiting biofilm formation and effectively killing bacteria within mature biofilms. Furthermore, quantitative proteomic analysis identified differentially expressed proteins and metabolic pathways closely associated with the inhibition of \u003cem\u003eS. aureus\u003c/em\u003e growth by LLY-507. Fluorescent staining confirmed LLY-507's ability to disrupt bacterial cell membranes. These results suggest the potential of LLY-507 for treating the infectious diseases caused by gram-positive bacteria. However, the relatively high MIC of LLY-507 compared to clinically used antibiotics may limit its clinical application. Further exploration to identify the key pharmacophores of LLY-507, based on its molecular structure, is necessary to obtain derivatives with improved antimicrobial activity. And in vivo tests utilizing the G. \u003cem\u003emellonella\u003c/em\u003e infection model demonstrated the efficacy of LLY-507. Therefore, further elucidating the direct targets of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e is crucial for advancing the development of antimicrobial drugs based on LLY-507.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Conceptualization, T.H., P.L., Y.T., S.H., and Z.Y.; methodology, Y.T., P.L., Z.X., and Y.L.; validation, Y.T., T.H., P.L.; formal analysis, Y.T., P.L., Z.X., Y.L. and J.Z.; investigation, Y.T., Z.X., Y.L., J.Z., Z.W., and Z.C.; resources, Y.T., Z.X., and Y.L.; data curation, Y.T., P.L.; writing\u0026mdash;original draft preparation, Y.T.; writing\u0026mdash;review and editing, T.H, P.L, Y.T., and S.H.; visualization, Y.T., P.L; supervision, T.H., P.L., and S.H.; project administration, T.H., P.L., Y.T., and S.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the National Natural Science Foundation of China (82172283); Guangdong Basic and Applied Basic Research Foundation (2023A1515220082, 2022A1515110096, ); Provincial medical funds of Guangdong (A2023468, A2022497); Shenzhen Key Medical Discipline Construction Fund (SZXK06162); Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20230807115814030, JCYJ20220530142006015) and the Shenzhen Nanshan District Scientific Research Program of the People\u0026apos;s Republic of China (NS2024007, NSZD2024023, NSZD2023035, NS2022046, NSZD2023019, NS2023049, NS2023027, NS2023097; The Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTD 2219).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The whole-genome sequencing data were deposited in the National Center for Biotechnology Information (NCBI) with accession number PRJNA1166673 (NCBI: http://www.ncbi.nlm.nih.gov/bioproject/1166673).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were carried out in accordance with relevant guidelines and regulations and were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital. Bacterial strains were collected as part of the routine clinical management of patients, according to the national guidelines in China (Clinical trial number: not applicable). Therefore, informed consent was not sought, and informed consent waiver was approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Weiguang Pan (Department of Laboratory Medicine, Shenzhen Nanshan People\u0026rsquo;s Hospital and the 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen 518052, China) for helping identify and preserve the bacterial strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDisclaimer/Publisher\u0026rsquo;s Note:\u003c/strong\u003e All methods were carried out in accordance with relevant guidelines and regulations and were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital. Bacterial strains were collected as part of the routine clinical management of patients, according to the national guidelines\u0026nbsp;in China. Therefore, informed consent was not sought, and informed consent\u0026nbsp;waiver was approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi, G.; Walker, M. J.; De Oliveira, D. M. Vancomycin resistance in Enterococcus and\u003cem\u003e Staphylococcus aureus\u003c/em\u003e. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e11\u003c/em\u003e, 24.\u003c/li\u003e\n\u003cli\u003eSubramaniam, G.; Girish, M. 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Chem\u003c/em\u003e. \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e35\u003c/em\u003e, 1781-1799.\u003c/li\u003e\n\u003cli\u003eCollin, F.; Karkare, S.; Maxwell, A. Exploiting bacterial DNA gyrase as a drug target: current state and perspectives. \u003cem\u003eAppl. Microbiol. Biotechnol\u003c/em\u003e. \u003cstrong\u003e2011\u003c/strong\u003e, \u003cem\u003e92\u003c/em\u003e, 479-497.\u003c/li\u003e\n\u003cli\u003eBarbee, L. A.; Soge, O. O.; Holmes, K. K.; Golden, M. R. In vitro synergy testing of novel antimicrobial combination therapies against Neisseria gonorrhoeae. \u003cem\u003eJ. Antimicrob. Chemother\u003c/em\u003e. \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e69\u003c/em\u003e, 1572-1578.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"LLY-507, Staphylococcus aureus, Antimicrobial activity, Biofilm","lastPublishedDoi":"10.21203/rs.3.rs-5353061/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5353061/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe emergence of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) as a prevalent antibiotic-resistant pathogen underscores the urgent need for novel antibacterial agents. This study investigates the potential of LLY-507 against gram-positive bacteria, particularly \u003cem\u003eS. aureus\u003c/em\u003e, focusing on its antibacterial and anti-biofilm properties. Here, our data exhibited favorable antibacterial activity of LLY-507 with MIC50 and MIC90 values of 25 \u0026micro;M against \u003cem\u003eS. aureus\u003c/em\u003e. Additionally, LLY-507 at sub-MIC concentrations effectively reduced the planktonic growth and biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e. Proteomic analysis of \u003cem\u003eS. aureus\u003c/em\u003e treated with LLY-507 revealed the classification of the functional proteins with significant expression level alterations in bacterial metabolism, particularly amino acid biosynthesis. Furthermore, we demonstrated the disruption of \u003cem\u003eS. aureus\u003c/em\u003e cell integrity by LLY-507 through membrane permeability assays and direct binding experiments between LLY-507 and cardiolipin. Lastly, the effectiveness of LLY-507 was proven in vivo using the \u003cem\u003eG. mellonella\u003c/em\u003e infection model. Overall, these findings highlight the promising antibacterial and anti-biofilm activities of LLY-507 against \u003cem\u003eS. aureus\u003c/em\u003e and provide insights into its mechanism of action, implicating its potential as a lead compound for developing novel antibacterial agents targeting Gram-positive bacteria.\u003c/p\u003e","manuscriptTitle":"Inhibition of Growth and Biofilm Formation in Staphylococcus aureus by LLY-507","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 13:54:15","doi":"10.21203/rs.3.rs-5353061/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9aabceb5-cc88-4e0b-acc9-1fe695e2aaf0","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-15T08:24:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-13 13:54:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5353061","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5353061","identity":"rs-5353061","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}