Targeting Phospholipids: Fingolimod's Antibacterial Mechanism Against Staphylococcus aureus

Targeting Phospholipids: Fingolimod's Antibacterial Mechanism Against Staphylococcus aureus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting Phospholipids: Fingolimod's Antibacterial Mechanism Against Staphylococcus aureus Yongpeng Shang, Yu Huang, Qingyin Meng, Zhijian Yu, Zewen Wen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5223352/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 It’s urgently needed to find new repurposed antibacterial drugs as the desired novel choices to counter biofilms and persister of gram-positive bacteria. Several reports have supported that Fingolimod, which was approved by FDA as a novel drug for the treatment of relapsing multiple sclerosis, can kill the bacteria by selectively disrupting the cell membrane of bacteria. However, the action mode and mechanism of Fingolimod against gram-positive bacteria remains elusive. Our data indicated that Fingolimod exerted the bactericidal activity against a wide spectrum of gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae et al . Moreover, Fingolimod could significantly eliminate the persister , inhibit biofilm formation, eradicate mature biofilm in vitro against S. aureus . Fingolimod rapidly eradicated S. aureus by pH-dependent disruption of the bacterial cell membrane's permeability and integrity, with its minimum inhibitory concentration (MIC) increasing up to 16-fold in response to elevated concentrations of phospholipids CL, PG, and PE. After four months of Fingolimod exposure, the MIC values of S. aureus showed a slight increase, and three genetic mutations related to phospholipid metabolism—PhoP, AcpP, and PhoU2—were identified in Fingolimod-induced clones, suggesting that Fingolimod may disrupt the cell membrane by targeting phospholipids. Overall, Fingolimod kills S. aureus by disrupting the bacteria membrane and targeting the phospholipids within the cell membrane. This study first reveals that Fingolimod kills S. aureus by targeting cell membrane phospholipids, a mechanism similar to cationic bactericides. Fingolimod Staphylococcus aureus Phospholipids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Staphylococcus aureus has been widely regarded as a common cause of hospital and community-acquired infection. S. aureus can cause serious invasive and life-threatening infections, such as endocarditis, osteomyelitis, necrotic pneumonia, sepsis, septic arthritis et al [ 1 , 2 ]. With the rapid spread of methicillin-resistant S. aureus (MRSA) and the widespread use of last-line agents such as vancomycin, linezolid, and daptomycin, the emergence of multi-drug resistant S. aureus has been increasingly reported and gradually posed a threat for the clinical outcome of S. aureus infection[ 3 , 4 ]. Moreover, antibiotic resistance of S. aureus against several new anti-MRSA drugs, such as dalbavancin, telavancin, oritavancin, and tigecycline, also emerged shortly after their introduction[ 5 ]. Bacteria biofilm is a complex structure of extracellular polymeric and can protect the cells against antibiotics and hostile conditions, and moreover, the sub-population of the biofilm-embedded bacteria exists in the form of dormant cells with metabolically inactive to antibiotic-tolerant persisters phenotype. A high frequency of clinical S. aureus isolates can form the biofilm. Biofilm formation and persisters pose tremendous challenges for clinicians in treating S. aureus infections[ 6 – 9 ]. Therefore, it’s urgent to discover the novel anti- S. aureus agents which can overcome drug resistance, kill the persisters and eradicate the biofilm. A multitude of bacterial vital biochemical processes, such as selective permeability, nutrients transportation and aerobic respiration, takes place on the cell membrane. The bacterial membrane plays an essential role in the survival and growth, and can be regarded as an ideal biological target to discover novel anti-microbial agents[ 10 ]. Several commonly used antibiotics, such as daptomycin, polymyxin, and bedaquiline, play their bactericidal activity by targeting bacterial membranes[ 11 – 13 ]. The membrane-targeting antibiotics against bacteria might perturb the mammalian membrane. Several reports have reported the antibacterial activity of Fingolimod (FTY720) against a wide range of bacteria, including Staphylococcus aureus , Staphylococcus epidermidis , Acinetobacter baumannii , Escherichia coli , and Pseudomonas aeruginosa. Moreover, the inhibition of biofilm formation of S. aureus by Fingolimod was demonstrated[ 14 ]. In addition, Fingolimod could inhibit quorum sensing of Chromobacterium violaceum and it is postulated the bactericidal activity against some Gram-negative strains by targeting the QS activity[ 15 ]. Therefore, the action mode and bactericidal characteristics of Fingolimod (FTY720) against bacterial growth and biofilm formation need to be further studied. Here, the bactericidal activity of Fingolimod (FTY720) against a wide range of gram-positive bacteria was evaluated. the inhibition and eradication of biofilm formation by Fingolimod (FTY720) were demonstrated. Fingolimod rapidly killed the bacteria by disrupting the permeability and integrity of the bacterial cell membrane of S. aureus with PH dependence. Fingolimod kills S. aureus by disrupting the bacteria membrane and interfering with phospholipids. This study offers a new mechanism of action for Fingolimod in the treatment of bacterial infections. Materials and methods Bacterial strains, antibiotics, and chemicals The strains used in this study were listed in Supplementary Table 1. S. aureus SA113 and NCTC 8325 strains were originally from American Type Culture Collection (ATCC). The clinical isolates of S. aureus, E. faecalis and S. agalactiae were collected from Shenzhen Nanshan People's Hospital and used in this study and grown in Tryptic Soya Broth (TSB) or Mueller–Hinton broth (MHB) (OXOID, Basingstoke, UK) at 37℃. The Oxacillin (MB5519-1, Meilunbio, Dalian, China), Vancomycin (MB1260-1, Meilunbio, Dalian, China), Linezolid (MB1469, Meilunbio, Dalian, China) and Fingolimod (CAS No. 162359-56-0, MCE, Shanghai, China) were purchased. The dimethylsulfoxide (DMSO) was used to dissolve Fingolimod and the highest DMSO concentration in the incubation medium was 0.5%, which was not affecting the bacterial growth and biofilm formation [ 16 ]. MIC, growth curve, and time-kill curve of bacteria MIC was determined by broth microdilution according to CLSI guidelines using the MHB. Bacteria were grown overnight and adjusted to 5.0 × 10 5 c.f.u./ mL mixed with varying concentrations of test antibiotics in 96-well microtiter plates and incubated at 37°C for 20–24 h. The bacteria strains were diluted 1:200 in TSB with or without drugs and grown for 24 h shaking at 37°C with 220 rpm, the OD 600 was detected at 1 h intervals by Bioscreen C (Turku, Finland). Bacterial growth curves in TSB without Fingolimod were used as an untreated control. A time-kill curve study was carried out to measure the tolerance of S. aureus to varying antibiotics. Overnight MSSA SA113 and MRSA CHS350 were diluted (10 8 CFU/ml) and incubated with 4×MIC drugs including Fingolimod (12.5 µg/mL), vancomycin (2 µg/mL), cefazolin (only used on MSSA SA113) (2 µg/mL), and linezolid (2 µg/mL) Samples were diluted in saline, spread on TSB agar plates and the CFU was counted at indicated time points for planktonic cells. Multiple comparisons among varying antibiotics group mean differences were checked using Dunnett’s test. All of the experiments were repeated in triplicate at least three times. The biofilm assays The inhibition and clearance of biofilm by Fingolimod was performed by crystal violet assay and CLSM according to previously reported[ 17 ]. Simply say, the bacteria isolates were incubated in TSBG (TSB with 0.5% glucose) individually into 96 polystyrene microtiter plates or a cell culture dish inlaying a glass coverslip (World Precision Instruments, USA) containing various concentrations of Fingolimod for indicated times. Then the biofilm was washed three times with saline and determined using crystal violet staining measured at 570 nm or stained with LIVE/DEAD reagents (1 µM SYTO9 and 1 µM propidium iodide [PI]; Thermo Fisher Scientific, Houston, TX) and acquired images using a Confocal Laser Scanning Microscope (FV3000, OLYMPUS, Japan) with 60× oil immersion objective. All of the experiments were repeated in triplicate at least three times. Hemolytic activity assay The hemolytic activity of S. aureus impacted by Fingolimod was performed as previously described[ 18 ]. S. aureus strains were inoculated with diverse concentrations of Fingolimod at 37°C for 24 h. The supernatant was harvested by centrifugation, filter sterilized through a 0.22µm filter (Millipore), and mixed with 1% rabbit erythrocytes (SBJ-RBC-RAB003, Sbjbio, China) at a volume ratio of 1:1. Then, the mixture was incubated at 37°C for 30 min. The supernatant was harvested by centrifugation and measured the OD 550 . The 0.1% Triton X-100 and saline were served as the positive control of 100% and the negative control of 0% hemolysis. All of the experiments were repeated in triplicate. Phospholipids impair the antibacterial activity of Figolimod Fingolimod's antibacterial efficacy was investigated using a modified version of previously published methods[ 19 ]. Sigma-Aldrich provided cardiolipin (CL), phosphocholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). Using a checkerboard assay, we investigated the impact of phospholipid concentration (0 to 128 g/mL) on Fingolimod's antibacterial activity against MSSA SA113 and MRSA CHS350. Permeability and integrity of cell membrane The cell membrane permeability of bacteria and mammalian cell were determined according to the previous report [ 20 ]. The SYTOX green penetrates into the cell and binds to intracellular DNA when the membrane is damaged. The exponentially growing S. aureus SA113, USA300 E. coli ATCC 25922, and human monocyte cell line THP-1 were centrifugated and resuspended in 0.9% NaCl, stained for 30 mins by SYTOX green (1 µM), then treated with 0.9% NaCl (negative control), 1% Triton X-100 (positive control), 2×MIC and 4×MIC of Fingolimod, daptomycin and linezolid only for S. aureus , polymixin and levofloxacin only for E. coli for 30 mins. The fluorescence was monitored for 20 minutes at 37°C using a BioTek multifunctional microplate reader under the conditions of excitation wavelength of 490 nm and emission wavelength of 520 nm, respectively. All of the experiments were repeated in triplicate. The cell membrane integrity of S. aureus was observed by transmission electron microscopy. The exponentially growing S. aureus SA113 cells were treated with 2×MIC Fingolimod for 30 mins. Cells were collected, washed three times with PBS (PH = 7.4), and fixed with 2.5% paraformaldehyde/PBS solution. The cells were washed with 0.1M phosphoric, fixed with acid 1% osmic acid for 2 h at 4 ℃, dehydrated, and embedded in paraffin. Then the samples were sliced into 70 nm sections, stained with 3% uranium acetate-citric acid, and imaged by a transmission electron microscope (HT7800, HITACHI, Japan). In vitro induction and the whole-genome sequencing of Fingolimod resistance S. aureus isolates S. aureus isolates (CH101 and SA113) were re-inoculated with 0.5x, 1x, 2x, and 4× of Fingolimod MIC at 1:100 every day until MIC changed (40 and 44 consecutive days). The individual derivative clone was picked and isolated in TSB plates. The chromosomal DNA of wild and resistance to Fingolimod of CH101 and SA113 were extracted using the MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (Takara Biotechnology, Dalian, China) for whole-genome sequencing (Novogene, Beijing, China). The genome of S. aureus NCTC 8325 (NCBI Reference Sequence: NC_007795.1) as standard parameters was used to map the sequencing files. Using the MUMmer comparison software, the sequence of resistance strains was serially compared with the wild strain and screened for SNPs (Single Nucleotide Polymorphisms) according to our previous reports [ 21 ]. Proteomic Screening of Fingolimod’s Bacterial Targets Exponentially growing S. aureus cells were adjusted to OD 600 ≈ 0.5, washed three times with PBS, and centrifuged at 6000 g. Bacterial cells were then incubated with Fingolimod at 4× MIC for 60 minutes at 37°C. The cells were disrupted with 0.1 mm glass beads through high-speed vortexing. The lysate was treated with RNase (0.5 mg/mL) and DNase (0.75 mg/mL) on ice for 1 hour, followed by the addition of 5 mM TBP and alkylation with 15 mM iodoacetamide. Total protein concentration was measured, and 100 µg of total protein was digested with trypsin and labeled with TMT 10-plex reagents. Proteomic analysis was conducted using a Q Exactive Plus mass spectrometer (Thermo Scientific), followed by data analysis. Statistical analysis The data were analyzed GraphPad Prism 9 and P values < 0.05 were considered statistically significant. Data availability The whole-genome sequencing files of Fingolimod resistance CH101 and SA113 clone were deposited in the NCBI database with the biosample accession SAMN18385243/18385244 and SAMN18385245/183852436 and the reference sequence the parenteral isolate CH101 and SA113 with the biosample accession SAMN15745752/15745753 and SAMN15745758/15745759. Data will be made available on request. Results Bactericidal activity of Fingolimod against the common pathogen. The MICs distribution of Fingolimod against the common pathogen clinical isolates from China was determined by broth microdilution. The range of MIC of Fingolimod against S. aureus , E. faecalis , S. agalactiae , E. coli , K. pneumoniae , and A. baumannii was 6.25 µg/mL, 12.5 µg/mL, 3.125 µg/ml, 3.25 ~ > 200 µg/mL, 6.52 ~ > 200 µg/mL and 50 ~ > 200 µg/mL (Table 1 and Fig. 1S )), suggesting that robust bactericidal activity to Gram-positive bacteria and powerless bactericidal activity to Gram-negative bacteria was different. Subsequently, the time-kill assay of Fingolimod with or without vancomycin, cefazolin, and linezolid was performed with two S. aureus , including SA113 (MSSA) and CHS350 (MRSA), proving its bactericidal activity against the S. aureus persisters with its monotherapy superior to vancomycin, cefazolin (only used on MSSA SA113), and linezolid ( Fig. 1 ) and suggesting a synergistic bactericidal effect to MRSA CHS350 exerted combination of Fingolimod and vancomycin or linezolid. The inhibition and eradication of S. aureus biofilm formation by Fingolimod. The inhibition of Fingolimod with subinhibitory concentrations of MIC on the biofilm formation of S. aureus was investigated in six biofilm-positive S. aureus clinical isolates that were previously reported [ 22 ], including MSSA strains of SE13、SE16、SA113、YUSA10 and MRSA of CHS350、CHS655. The biofilm formation of six S. aureus strains was significantly inhibited by 1/2×MIC Fingolimod (1.56 µg/mL) ( Fig. 2A ). This finding was further observed and confirmed by laser scanning confocal microscopy in S. aureus SA113 ( Fig. 2B ). Furthermore, the eradication activity of Fingolimod against S. aureus mature biofilm was evaluated by crystal violet staining and laser scanning confocal microscopy, indicating the monotherapy of Fingolimod with ≥ 4×MIC could more strongly eradicate the mature biofilm Fingolimod when compared with vancomycin, linezolid, and daptomycin which were first-line drug for clinical Staphylococcus aureus therapy ( Fig. 2C and D ). The selective disruption of Fingolimod against the S. aureus cell membrane The cell membrane permeability of two S. aureus strains, one E. coli strain, and the human monocyte cell line THP-1staining with SYTOX green were assessed with Fingolimod, daptomycin, polymyxin, linezolid, and levofloxacin. The 1% TritonX-100 was used as a positive control for the increased membrane permeability of both bacteria and the THP-1 cell line. Our data indicated Fingolimod effectively damaged the bacterial membrane and enhanced its permeability of S. aureus and E. coli but not the THP-1 cell line ( Fig. 3 ). Moreover, the significantly increased membrane permeability of S. aureus and E. coli was found in daptomycin and polymyxin which are targeted to the cell membrane, but no change in linezolid and levofloxacin which aren’t targeted to the cell membrane, indicating that Fingolimod might selectivity target bacterial cell membrane and rapidly induce the increased permeability. The cell membrane damage and disruption were observed in S. aureus SA113 which was grown planktonically to the mid-log phase and treated with Fingolimod for 30 mins by electron microscopy ( Fig. 4 ), indicating that bactericidal activity of Fingolimod might be due to disruption of the bacterial cell membrane integrity. A previous study proved that the chemical structure of Fingolimod contained a side-chain -NH 2 , which could be protonated to present in the form of –NH 3 + under the slightly acidic condition, as found in the normal airway surface liquid[ 23 ]. Therefore, whether the protonation of NH 2 was required for the bactericidal effects of Fingolimod was validated by assaying the growth curve of bacteria under various PH conditions. Our data showed the complete suppression of bacteria planktonic growth by Fingolimod at the concentration of 6.25 µg/ml under pH 6 not PH 8, suggesting the protonation of the NH 2 might impact the antibacterial effect of Fingolimod ( Fig. 5 ). Phospholipids as a Target for Fingolimod Fingolimod can kill the bacteria by selectively disrupting the cell membrane with the PH-dependent protonation, therefore, we hypothesized that Fingolimod with positively charged in the slightly acidic solution could bind to negatively charged membrane components of bacteria during the protonation process. Cardiolipin, which is absent in the membrane structure of mammalian cells, plays a key physiological role in the cell membrane of bacteria. A previous study showed that the bactericidal mechanism of sphingosine, which is an analogue of Fingolimod, required binding to cardiolipin in plasma membranes of E. coli or P. aeruginosa . In this study, the impact of cardiolipin synthase ( cls ) on Fingolimod susceptibility against bacteria was investigated by constructing the cls gene knockout strain in E. coli ATCC 25922 (Δ cls ). The planktonic growth of wild-type or Δ cls strains under exposure to Fingolimod, polymyxin, and levofloxacin were detected and the result showed that Fingolimod and polymyxin, which destroyed the cell membrane targeting to the lipid A, inhibited the growth of Δ cls strain but not wild-type strain, while the growth between wild-type and Δ cls strains showed no significant difference with levofloxacin, which inhibited DNA synthesis and have no impact on bacteria membrane ( Fig. 6 ), demonstrating knockout of the cls gene could enhance the sensitivity to Fingolimod and polymyxin[ 24 , 25 ]. The Δ cls strain increased sensitivity to Fingolimod as same as polymyxin, not levofloxacin, suggesting Fingolimod possibly has a similar role to polymyxin ( Fig. 6 ). We evaluated the antibacterial activity of Fingolimod by using checkerboard assays on its target phospholipids. This assay employed three common bacterial membrane phospholipids, namely phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). After 20 hours, the MICs of Fingolimod were measured against clinically isolated strains of SA113 and CH350. The MIC of Fingolimod increased with phospholipid concentration, reaching a 16-fold increase with CL, PG and PE for both isolates ( Fig. 7 ). Among these phospholipids, CL has a particularly strong influence on increasing the MIC compared to the other two. In order to identify the potential target site of Fingolimod, the Fingolimod-induced resistant S. aureus clones were selected by in vitro serial passaging under the pressure of Fingolimod. The MIC value of SA113 and CHS101 were elevated from 3.125 to 6.25 µg/mL after 40 or 45 passages ( Fig. 8 ). A comparison of whole-genome sequencing between the Fingolimod parental isolates and Fingolimod-induced S. aureus clones showed the four coding genetic mutations in the acyl carrier protein synthase (AcpS), inorganic phosphate transport regulatory protein (PhoP and PhoU2) respectively. The function of AcpS, PhoP, and PhoU2 were closely correlated with the phospholipid metabolism, and moreover, AcpS participated in bacteria autolysin (Table 2 ). To explore Fingolimod's potential targets in S. aureus , this study compared the proteomes of Fingolimod-treated and DMSO-treated bacteria. The results identified 147 differentially expressed proteins with over two-fold changes, 76 upregulated and 71 downregulated ( Fig. 9A ). GO analysis showed these proteins were mainly linked to redox functions and cell membrane composition and integrity ( Fig. 9B ), supporting the idea that Fingolimod disrupts the cell membrane. ClueGO analysis found 42 proteins involved in membrane composition ( Fig. 9C ), and protein localization within S. aureus was mapped ( Fig. 9D ), with enriched cell component proteins visualized (Fig. 9E). Table 2 Genetic mutations in the resistant strains of SA113 and CHS101 detecting by the whole-genome sequencing. Ref_gene_ID NA mutations AA mutations Subject description CHS101_GM000826 A269C V90G response regulator transcription factor,PhoP CHS101_GM000905 T347C D116G autolysin SA113_GM001858 G94T Q32K Acyl carrier protein, AcpP SA113_GM000155 G506A R169K distantly related to PhoU, PhoU2 Discussion The presence of persisters and biofilm formation is closely linked to the recalcitrance of chronic infections and often results in a bad clinical outcome that is associated with significant mortality[ 26 – 29 ]. The conventional antibiotic treatment is frequently difficult to eradicate chronic infections caused by S. aureus , in particular MRSA[ 30 ]. Previous studies have demonstrated the various physiological uses of survival bacteria cells require the participation of intact membranes. Several reports supported the bacteria membrane is a promising potential target to counter the persisters and biofilm for the development of new drugs. To shorten research time and costs, the drug repurposing targeting bacteria membrane by screening libraries of FDA-approved drugs could streamline the need of pharmacokinetic and toxicity[ 31 , 32 ]. In the present study, our data indicated that Fingolimod can efficiently inhibit the planktonic growth of S. aureus with the MIC range from 1.56µg/mL to 6.25 µg/mL. Moreover, Fingolimod can eradicate the persisters and mature biofilms. Time-killing assay demonstrated the stronger bactericidal activity of Fingolimod when compared with vancomycin or linezolid. However, the influence of Fingolimod on the hemolytic activity of S. aureus was also analyzed in 5 clinical S. aureus strains using the rabbit erythrocytes, indicating no significant impact on the hemolytic activity by Fingolimod exposure. This effectiveness provides clues that Fingolimod had the potential to provide a novel choice for the antimicrobial treatment of chronic infections of S. aureus . Fingolimod has been approved as the first-line drug for the treatment of the relapsing forms of multiple sclerosis. The clinical application practices have demonstrated Fingolimod is well-tolerated with a favorable safety profile. In eukaryotic, the main mechanism of Fingolimod is involved in the inhibition of S1P signaling and selectively retained lymphocytes in the lymphoid organs by targeting sphingosine 1-phosphate (S1P) receptors[ 33 , 34 ]. Additionally, several recent studies have also shown the antifungal, antiviral, and antibacterial effects of Fingolimod and its chemical homologues. Lu-Qi Wei et al. reported that Fingolimod exhibited a synergistic effect with Amphotericin B against diverse fungal pathogens due to the excessive accumulation of reactive oxygen species[ 35 ]. ZengZi Zhou et al. showed that Fingolimod inhibited Chlamydia dissemination from the upper genital tract to the gastrointestinal tract[ 36 ]. Recent two studies have demonstrated the robust inhibitory activity of Fingolimod and its homologues derivatives against a wide range of gram-positive bacteria and several gram-negative bacteria species. Moreover, the impact of these drugs on both planktonic growth and biofilm formation of S. aureus has been found. The chemical structure of Fingolimod was similar with to LuxR family quorum sensing (QS) and the mechanism of Fingolimod against Gram-negative bacteria has been speculated by inhibiting the QS [ 14 , 15 ]. No alterations of MIC of Fingolimod in the wild type and luxS deficiency of Staphylococcus epidermidis were examined, suggesting the impact of Fingolimod on S. aureus can’t be explained by the hypothesis as different QS systems between Gram-positive and Gram-negative bacteria[ 37 ]. Sphingosine, which is a structural analog of Fingolimod, exists in human upper airway epithelial cells and displays antibacterial efficacy against pathogens including P. aeruginosa , S. aureus , and E. coli . Recent reports demonstrated the inhibition of bacteria growth of sphingosine by binding to the cardiolipin and destroying the cell membranes. Moreover, the E. coli or P. aeruginosa strains with the knockout of cardiolipin synthase are resistant to sphingosine[ 38 ]. In this study, our data indicated that similar to sphingosine, Fingolimod disrupted the integrity and permeability of S. aureus cell membranes with cation-dependence. However, the cardiolipin knockout strain of E. coli is showed more sensitive to Fingolimod, suggesting the bactericidal mechanism of Fingolimod is different from sphingosine. To elucidate the action mode of Fingolimod against S. aureus , cell membrane disruption of S. aureus was further validated by electron microscopy during Fingolimod exposure. The membrane disruption of S. aureus by Fingolimod is PH-dependent, suggesting the cationic is important for its antibacterial effect of Fingolimod. Moreover, Fingolimod can results in the enhanced membrane permeability in S. aureus SA113 and E. coli 25922, hypothesizing its bactericidal mechanism is similar to daptomycin and polymyxin that targets the lipid in cell membranes [ 39 ]. Furthermore, in E. coli strain, the knockout of cardiolipin synthesis could enhance the sensitivity to Fingolimod as same as polymyxin, suggesting the action mode of Fingolimod might target on lipid similar to polymyxin. Comparison of whole-genome sequencing showed that three genes, including AcpS, PhoP and PhoU2, were shown with genetic mutations in Fingolimod-induced resistant S. aureus clones when compared with the parental isolates. The function of AcpS, PhoP and PhoU2 closely participated in the phospholipids, supporting the impact of Fingolimod on the lipid of cell membrane[ 40 ]. In summary, our data further supported the excellent antibacterial against planktonic cells and persisters in clinical strains of S. aureus, E. faecalis , and S. agalactiae with very low MIC values of 1.56–6.25 µg/mL. Fingolimod could significantly eliminate the persisters , inhibit biofilm formation and eradicate mature biofilm. In addition, disruption of the bacterial cell membrane permeability and integrity were demonstrated by Fingolimod. In the presence of phospholipids, particularly cardiolipin, the antibacterial efficacy of the drug is significantly reduced. Fingolimod exposure can results in the genetic mutations in the critical genes (AcpS, PhoP and PhoU2) correlated with phospholipid metabolism. therefore, phospholipids might be an important target of Fingolimod against bacteria growth. These data firstly demonstrate the mechanism of Fingolimod during killing S. aureus by targeting the lipid of cell membrane, which is similar to cationic bactericides. Declarations Author information Authors and Affiliations Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China Yongpeng Shang, Yu Huang, Fangyou Yu Department of Infectious Diseases and the Key Lab of Endogenous Infection, Shenzhen Nanshan People's Hospital, the 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen 518052, China. Qingyin Meng, Zhijian Yu, Zewen Wen Contributions Yongpeng Shang: conceptualization (lead); formal analysis (equal); supervision (lead); writing original draft (equal). Yu Huang : formal analysis (equal) supervision (equal); writing— review and editing (equal). Fangyou Yu: Conceptualization (equal); formal analysis (equal) supervision (equal); writing— review and editing (equal). Qingyin Meng: methodology (equal). Zhijian Yu: practical methodology (equal); writing— review and editing (equal). Zewen Wen: methodology (equal); writing— review and editing (equal). Corresponding author Correspondence to Fangyou Yu. Acknowledgements Special thanks to Zhanwen Wang, Junwen Chen, Chengchun Chen and Jingxin Zheng for their assistance with the experiments. Funding This work was supported by a grant from the National Natural Science Foundation of China (82302592), and the Guangdong Basic and Applied Basic Research Foundation (2021Al515110114), and the Shenzhen Nanshan District Scientific Research Program (NSZD2024002，NS2024009). Ethics approval and consent to participate Ethical review and approval were performed by the Medical Ethics Committee of Shanghai Pulmonary Hospital, School of Medicine (Ethical no. K23-088Y). References David, M.Z. and R.S. Daum, Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev, 2010. 23(3): p. 616-87. Gupta, A.K., D.C. Lyons and T. 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Zheng, J.X., et al., In vitro activities of telithromycin against Staphylococcus aureus biofilms compared with azithromycin, clindamycin, vancomycin and daptomycin. J Med Microbiol, 2020. 69(1): p. 120-131. Zajac, M., et al., Airway Surface Liquid pH Regulation in Airway Epithelium Current Understandings and Gaps in Knowledge. Int J Mol Sci, 2021. 22(7). Andrade, F.F., et al., Colistin Update on Its Mechanism of Action and Resistance, Present and Future Challenges. Microorganisms, 2020. 8(11). Bardet, L. and J.M. Rolain, Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains. Can J Infect Dis Med Microbiol, 2018. 2018: p. 3095249. Gagliotti, C., et al., Staphylococcus aureus bloodstream infections: diverging trends of meticillin-resistant and meticillin-susceptible isolates, EU/EEA, 2005 to 2018. Euro Surveill, 2021. 26(46). Mairi, A., A. Touati and J.P. Lavigne, Methicillin-Resistant Staphylococcus aureus ST80 Clone: A Systematic Review. Toxins (Basel), 2020. 12(2). McConville, T.H., et al., In-Vitro Cytotoxicity and Clinical Correlates of MRSA Bacteremia. Antimicrob Agents Chemother, 2021: p. AAC0155921. Vazquez-Rosas, G.J., et al., Molecular Characterization of Staphylococcus aureus Obtained from Blood Cultures of Paediatric Patients Treated in a Tertiary Care Hospital in Mexico. Infect Drug Resist, 2021. 14: p. 1545-1556. Maisuria, V.B., et al., Proanthocyanidin Interferes with Intrinsic Antibiotic Resistance Mechanisms of Gram-Negative Bacteria. Adv Sci (Weinh), 2019. 6(15): p. 1802333. Fernandes, P. and E. Martens, Antibiotics in late clinical development. Biochem Pharmacol, 2017. 133: p. 152-163. Savoia, D., New Antimicrobial Approaches: Reuse of Old Drugs. Curr Drug Targets, 2016. 17(6): p. 731-8. Chun, J. and H.P. Hartung, Mechanism of action of oral fingolimod (FTY720) in multiple sclerosis. Clin Neuropharmacol, 2010. 33(2): p. 91-101. Kappos, L., et al., A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med, 2010. 362(5): p. 387-401. Wei, L.Q., et al., Fingolimod Potentiates the Antifungal Activity of Amphotericin B. Front Cell Infect Microbiol, 2021. 11: p. 627917. Zhou, Z., et al., Effects of Immunomodulatory Drug Fingolimod (FTY720) on Chlamydia Dissemination and Pathogenesis. Infection and Immunity, 2020. 88(11): p. e00281-20. Mukherjee, S. and B.L. Bassler, Bacterial quorum sensing in complex and dynamically changing environments. Nat Rev Microbiol, 2019. 17(6): p. 371-382. Verhaegh, R., et al., Sphingosine kills bacteria by binding to cardiolipin. J Biol Chem, 2020. 295(22): p. 7686-7696. Andrade, F.F., et al., Colistin Update on Its Mechanism of Action and Resistance, Present and Future Challenges. Microorganisms, 2020. 8(11). Bechinger, B. and S.U. Gorr, Antimicrobial Peptides: Mechanisms of Action and Resistance. J Dent Res, 2017. 96(3): p. 254-260. Supplementary Table 1 Supplemental Table 1 is not included with this version Additional Declarations No competing interests reported. Supplementary Files figureS1.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5223352","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":366873256,"identity":"2e56c8ff-1286-40f9-ab74-78d9fd3796df","order_by":0,"name":"Yongpeng Shang","email":"","orcid":"","institution":"Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"Yongpeng","middleName":"","lastName":"Shang","suffix":""},{"id":366873257,"identity":"33447d9b-7cac-4883-a060-59896cefdc9c","order_by":1,"name":"Yu Huang","email":"","orcid":"","institution":"Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Huang","suffix":""},{"id":366873258,"identity":"18f5ab2e-cc94-4866-948b-991185e9fcc2","order_by":2,"name":"Qingyin Meng","email":"","orcid":"","institution":"Department of Infectious Diseases and the Key Lab of Endogenous Infection, Shenzhen Nanshan People's Hospital, the 6th Affiliated Hospital of Shenzhen University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Qingyin","middleName":"","lastName":"Meng","suffix":""},{"id":366873259,"identity":"3172893b-314d-4e57-b449-478a41d53197","order_by":3,"name":"Zhijian Yu","email":"","orcid":"","institution":"Department of Infectious Diseases and the Key Lab of Endogenous Infection, Shenzhen Nanshan People's Hospital, the 6th Affiliated Hospital of Shenzhen University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Zhijian","middleName":"","lastName":"Yu","suffix":""},{"id":366873260,"identity":"99e965d6-265f-4ae3-a3b8-1cfc1a11690c","order_by":4,"name":"Zewen Wen","email":"","orcid":"","institution":"Department of Infectious Diseases and the Key Lab of Endogenous Infection, Shenzhen Nanshan People's Hospital, the 6th Affiliated Hospital of Shenzhen University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Zewen","middleName":"","lastName":"Wen","suffix":""},{"id":366873261,"identity":"a582db1f-46e4-4f67-b881-6501f4f62e24","order_by":5,"name":"Fangyou Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYHCCBAYGGwY5Nvb2A6RoSWMw5uM5k0CKRWkMifMkHAyIU2xw/sAzaZ6Ew+ltEkDrflRsI0LLgQPJxkAtuW3SjQcYe87cJqzF7GBD4mPeH0AtMgcSmBnbiNFymCHhMMhhbBIJBkRqOcaQ+BioJYF4LfZnGJIN5ySkG7YBA/kgUX6R7D+TJvEmwVpevr394IMfFURoYWDgSWDiYWgGMw8Qox4I2A8w/mCoI1LxKBgFo2AUjEgAAMNiPfzKvyrbAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China","correspondingAuthor":true,"prefix":"","firstName":"Fangyou","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-10-08 08:23:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5223352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5223352/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66982750,"identity":"10137e1d-a577-4acd-823e-ddd7f005895d","added_by":"auto","created_at":"2024-10-18 18:14:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":814889,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5223352/v1/842a7f26d734681eb43e9efa.png"},{"id":66983351,"identity":"b37cda22-6044-4d8c-aa4c-ee1e95904738","added_by":"auto","created_at":"2024-10-18 18:30:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2116057,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included 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version\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5223352/v1/bc46030ddbda8a9c7458562b.png"},{"id":66982758,"identity":"57ad31a0-f9b1-442e-9b15-8d5be757137b","added_by":"auto","created_at":"2024-10-18 18:14:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":529322,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5223352/v1/88b0e2533cdd59d573200569.png"},{"id":67398753,"identity":"e87bf3ec-e7c3-4c61-a99b-9b4351b2d6ae","added_by":"auto","created_at":"2024-10-24 12:46:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8529924,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5223352/v1/bc9e0f8e-5a45-4895-80c2-c14f6b205966.pdf"},{"id":66982898,"identity":"7ecdb9e3-edef-462f-b2e0-3f9ff9376afb","added_by":"auto","created_at":"2024-10-18 18:22:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":63521,"visible":true,"origin":"","legend":"","description":"","filename":"figureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5223352/v1/b9d2c7b397307723b99b9168.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting Phospholipids: Fingolimod's Antibacterial Mechanism Against Staphylococcus aureus","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e has been widely regarded as a common cause of hospital and community-acquired infection. \u003cem\u003eS. aureus\u003c/em\u003e can cause serious invasive and life-threatening infections, such as endocarditis, osteomyelitis, necrotic pneumonia, sepsis, septic arthritis \u003cem\u003eet al\u003c/em\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With the rapid spread of methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) and the widespread use of last-line agents such as vancomycin, linezolid, and daptomycin, the emergence of multi-drug resistant \u003cem\u003eS. aureus\u003c/em\u003e has been increasingly reported and gradually posed a threat for the clinical outcome of \u003cem\u003eS. aureus\u003c/em\u003e infection[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, antibiotic resistance of \u003cem\u003eS. aureus\u003c/em\u003e against several new anti-MRSA drugs, such as dalbavancin, telavancin, oritavancin, and tigecycline, also emerged shortly after their introduction[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bacteria biofilm is a complex structure of extracellular polymeric and can protect the cells against antibiotics and hostile conditions, and moreover, the sub-population of the biofilm-embedded bacteria exists in the form of dormant cells with metabolically inactive to antibiotic-tolerant \u003cem\u003epersisters\u003c/em\u003e phenotype. A high frequency of clinical \u003cem\u003eS. aureus\u003c/em\u003e isolates can form the biofilm. Biofilm formation and \u003cem\u003epersisters\u003c/em\u003e pose tremendous challenges for clinicians in treating \u003cem\u003eS. aureus\u003c/em\u003e infections[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it\u0026rsquo;s urgent to discover the novel anti-\u003cem\u003eS. aureus\u003c/em\u003e agents which can overcome drug resistance, kill the \u003cem\u003epersisters\u003c/em\u003e and eradicate the biofilm.\u003c/p\u003e \u003cp\u003eA multitude of bacterial vital biochemical processes, such as selective permeability, nutrients transportation and aerobic respiration, takes place on the cell membrane. The bacterial membrane plays an essential role in the survival and growth, and can be regarded as an ideal biological target to discover novel anti-microbial agents[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Several commonly used antibiotics, such as daptomycin, polymyxin, and bedaquiline, play their bactericidal activity by targeting bacterial membranes[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The membrane-targeting antibiotics against bacteria might perturb the mammalian membrane. Several reports have reported the antibacterial activity of Fingolimod (FTY720) against a wide range of bacteria, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and Pseudomonas \u003cem\u003eaeruginosa.\u003c/em\u003e Moreover, the inhibition of biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e by Fingolimod was demonstrated[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, Fingolimod could inhibit quorum sensing of \u003cem\u003eChromobacterium violaceum\u003c/em\u003e and it is postulated the bactericidal activity against some Gram-negative strains by targeting the QS activity[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, the action mode and bactericidal characteristics of Fingolimod (FTY720) against bacterial growth and biofilm formation need to be further studied.\u003c/p\u003e \u003cp\u003eHere, the bactericidal activity of Fingolimod (FTY720) against a wide range of gram-positive bacteria was evaluated. the inhibition and eradication of biofilm formation by Fingolimod (FTY720) were demonstrated. Fingolimod rapidly killed the bacteria by disrupting the permeability and integrity of the bacterial cell membrane of \u003cem\u003eS. aureus\u003c/em\u003e with PH dependence. Fingolimod kills \u003cem\u003eS. aureus\u003c/em\u003e by disrupting the bacteria membrane and interfering with phospholipids. This study offers a new mechanism of action for Fingolimod in the treatment of bacterial infections.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eBacterial strains, antibiotics, and chemicals\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe strains used in this study were listed in Supplementary Table\u0026nbsp;1. \u003cem\u003eS. aureus\u003c/em\u003e SA113 and NCTC 8325 strains were originally from American Type Culture Collection (ATCC). The clinical isolates of \u003cem\u003eS. aureus, E. faecalis\u003c/em\u003e and \u003cem\u003eS. agalactiae\u003c/em\u003e were collected from Shenzhen Nanshan People's Hospital and used in this study and grown in Tryptic Soya Broth (TSB) or Mueller\u0026ndash;Hinton broth (MHB) (OXOID, Basingstoke, UK) at 37℃. The Oxacillin (MB5519-1, Meilunbio, Dalian, China), Vancomycin (MB1260-1, Meilunbio, Dalian, China), Linezolid (MB1469, Meilunbio, Dalian, China) and Fingolimod (CAS No. 162359-56-0, MCE, Shanghai, China) were purchased. The dimethylsulfoxide (DMSO) was used to dissolve Fingolimod and the highest DMSO concentration in the incubation medium was 0.5%, which was not affecting the bacterial growth and biofilm formation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMIC, growth curve, and time-kill curve of bacteria\u003c/h3\u003e\n\u003cp\u003eMIC was determined by broth microdilution according to CLSI guidelines using the MHB. Bacteria were grown overnight and adjusted to 5.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e c.f.u./ mL mixed with varying concentrations of test antibiotics in 96-well microtiter plates and incubated at 37\u0026deg;C for 20\u0026ndash;24 h. The bacteria strains were diluted 1:200 in TSB with or without drugs and grown for 24 h shaking at 37\u0026deg;C with 220 rpm, the OD\u003csub\u003e600\u003c/sub\u003e was detected at 1 h intervals by Bioscreen C (Turku, Finland). Bacterial growth curves in TSB without Fingolimod were used as an untreated control. A time-kill curve study was carried out to measure the tolerance of \u003cem\u003eS. aureus\u003c/em\u003e to varying antibiotics. Overnight MSSA SA113 and MRSA CHS350 were diluted (10\u003csup\u003e8\u003c/sup\u003e CFU/ml) and incubated with 4\u0026times;MIC drugs including Fingolimod (12.5 \u0026micro;g/mL), vancomycin (2 \u0026micro;g/mL), cefazolin (only used on MSSA SA113) (2 \u0026micro;g/mL), and linezolid (2 \u0026micro;g/mL) Samples were diluted in saline, spread on TSB agar plates and the CFU was counted at indicated time points for planktonic cells. Multiple comparisons among varying antibiotics group mean differences were checked using Dunnett\u0026rsquo;s test. All of the experiments were repeated in triplicate at least three times.\u003c/p\u003e\n\u003ch3\u003eThe biofilm assays\u003c/h3\u003e\n\u003cp\u003eThe inhibition and clearance of biofilm by Fingolimod was performed by crystal violet assay and CLSM according to previously reported[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Simply say, the bacteria isolates were incubated in TSBG (TSB with 0.5% glucose) individually into 96 polystyrene microtiter plates or a cell culture dish inlaying a glass coverslip (World Precision Instruments, USA) containing various concentrations of Fingolimod for indicated times. Then the biofilm was washed three times with saline and determined using crystal violet staining measured at 570 nm or stained with LIVE/DEAD reagents (1 \u0026micro;M SYTO9 and 1 \u0026micro;M propidium iodide [PI]; Thermo Fisher Scientific, Houston, TX) and acquired images using a Confocal Laser Scanning Microscope (FV3000, OLYMPUS, Japan) with 60\u0026times; oil immersion objective. All of the experiments were repeated in triplicate at least three times.\u003c/p\u003e\n\u003ch3\u003eHemolytic activity assay\u003c/h3\u003e\n\u003cp\u003eThe hemolytic activity of \u003cem\u003eS. aureus\u003c/em\u003e impacted by Fingolimod was performed as previously described[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e strains were inoculated with diverse concentrations of Fingolimod at 37\u0026deg;C for 24 h. The supernatant was harvested by centrifugation, filter sterilized through a 0.22\u0026micro;m filter (Millipore), and mixed with 1% rabbit erythrocytes (SBJ-RBC-RAB003, Sbjbio, China) at a volume ratio of 1:1. Then, the mixture was incubated at 37\u0026deg;C for 30 min. The supernatant was harvested by centrifugation and measured the OD\u003csub\u003e550\u003c/sub\u003e. The 0.1% Triton X-100 and saline were served as the positive control of 100% and the negative control of 0% hemolysis. All of the experiments were repeated in triplicate.\u003c/p\u003e\n\u003ch3\u003ePhospholipids impair the antibacterial activity of Figolimod\u003c/h3\u003e\n\u003cp\u003eFingolimod's antibacterial efficacy was investigated using a modified version of previously published methods[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Sigma-Aldrich provided cardiolipin (CL), phosphocholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). Using a checkerboard assay, we investigated the impact of phospholipid concentration (0 to 128 g/mL) on Fingolimod's antibacterial activity against MSSA SA113 and MRSA CHS350.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePermeability and integrity of cell membrane\u003c/h2\u003e \u003cp\u003eThe cell membrane permeability of bacteria and mammalian cell were determined according to the previous report [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The SYTOX green penetrates into the cell and binds to intracellular DNA when the membrane is damaged. The exponentially growing \u003cem\u003eS. aureus\u003c/em\u003e SA113, USA300 \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, and human monocyte cell line THP-1 were centrifugated and resuspended in 0.9% NaCl, stained for 30 mins by SYTOX green (1 \u0026micro;M), then treated with 0.9% NaCl (negative control), 1% Triton X-100 (positive control), 2\u0026times;MIC and 4\u0026times;MIC of Fingolimod, daptomycin and linezolid only for \u003cem\u003eS. aureus\u003c/em\u003e, polymixin and levofloxacin only for \u003cem\u003eE. coli\u003c/em\u003e for 30 mins. The fluorescence was monitored for 20 minutes at 37\u0026deg;C using a BioTek multifunctional microplate reader under the conditions of excitation wavelength of 490 nm and emission wavelength of 520 nm, respectively. All of the experiments were repeated in triplicate.\u003c/p\u003e \u003cp\u003eThe cell membrane integrity of \u003cem\u003eS. aureus\u003c/em\u003e was observed by transmission electron microscopy. The exponentially growing \u003cem\u003eS. aureus\u003c/em\u003e SA113 cells were treated with 2\u0026times;MIC Fingolimod for 30 mins. Cells were collected, washed three times with PBS (PH\u0026thinsp;=\u0026thinsp;7.4), and fixed with 2.5% paraformaldehyde/PBS solution. The cells were washed with 0.1M phosphoric, fixed with acid 1% osmic acid for 2 h at 4 ℃, dehydrated, and embedded in paraffin. Then the samples were sliced into 70 nm sections, stained with 3% uranium acetate-citric acid, and imaged by a transmission electron microscope (HT7800, HITACHI, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro induction and the whole-genome sequencing of Fingolimod resistance\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e isolates (CH101 and SA113) were re-inoculated with 0.5x, 1x, 2x, and 4\u0026times; of Fingolimod MIC at 1:100 every day until MIC changed (40 and 44 consecutive days). The individual derivative clone was picked and isolated in TSB plates. The chromosomal DNA of wild and resistance to Fingolimod of CH101 and SA113 were extracted using the MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (Takara Biotechnology, Dalian, China) for whole-genome sequencing (Novogene, Beijing, China). The genome of \u003cem\u003eS. aureus\u003c/em\u003e NCTC 8325 (NCBI Reference Sequence: NC_007795.1) as standard parameters was used to map the sequencing files. Using the MUMmer comparison software, the sequence of resistance strains was serially compared with the wild strain and screened for SNPs (Single Nucleotide Polymorphisms) according to our previous reports [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProteomic Screening of Fingolimod’s Bacterial Targets\u003c/h3\u003e\n\u003cp\u003eExponentially growing \u003cem\u003eS. aureus\u003c/em\u003e cells were adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;0.5, washed three times with PBS, and centrifuged at 6000 g. Bacterial cells were then incubated with Fingolimod at 4\u0026times; MIC for 60 minutes at 37\u0026deg;C. The cells were disrupted with 0.1 mm glass beads through high-speed vortexing. The lysate was treated with RNase (0.5 mg/mL) and DNase (0.75 mg/mL) on ice for 1 hour, followed by the addition of 5 mM TBP and alkylation with 15 mM iodoacetamide. Total protein concentration was measured, and 100 \u0026micro;g of total protein was digested with trypsin and labeled with TMT 10-plex reagents. Proteomic analysis was conducted using a Q Exactive Plus mass spectrometer (Thermo Scientific), followed by data analysis.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed GraphPad Prism 9 and P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe whole-genome sequencing files of Fingolimod resistance CH101 and SA113 clone were deposited in the NCBI database with the biosample accession SAMN18385243/18385244 and SAMN18385245/183852436 and the reference sequence the parenteral isolate CH101 and SA113 with the biosample accession SAMN15745752/15745753 and SAMN15745758/15745759. Data will be made available on request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBactericidal activity of Fingolimod against the common pathogen.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MICs distribution of Fingolimod against the common pathogen clinical isolates from China was determined by broth microdilution. The range of MIC of Fingolimod against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003eA. baumannii\u003c/em\u003e was 6.25 \u0026micro;g/mL, 12.5 \u0026micro;g/mL, 3.125 \u0026micro;g/ml, 3.25\u0026thinsp;~\u0026thinsp;\u0026gt;\u0026thinsp;200 \u0026micro;g/mL, 6.52\u0026thinsp;~\u0026thinsp;\u0026gt;\u0026thinsp;200 \u0026micro;g/mL and 50\u0026thinsp;~\u0026thinsp;\u0026gt;\u0026thinsp;200 \u0026micro;g/mL (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cstrong\u003eFig.\u0026nbsp;1S\u003c/strong\u003e)), suggesting that robust bactericidal activity to Gram-positive bacteria and powerless bactericidal activity to Gram-negative bacteria was different.\u003c/p\u003e\n\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1729254744.png\"\u003e\u003c/div\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, the time-kill assay of Fingolimod with or without vancomycin, cefazolin, and linezolid was performed with two \u003cem\u003eS. aureus\u003c/em\u003e, including SA113 (MSSA) and CHS350 (MRSA), proving its bactericidal activity against the \u003cem\u003eS. aureus persisters\u003c/em\u003e with its monotherapy superior to vancomycin, cefazolin (only used on MSSA SA113), and linezolid (\u003cstrong\u003eFig.\u0026nbsp;1\u003c/strong\u003e) and suggesting a synergistic bactericidal effect to MRSA CHS350 exerted combination of Fingolimod and vancomycin or linezolid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe inhibition and eradication of\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003ebiofilm formation by Fingolimod.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibition of Fingolimod with subinhibitory concentrations of MIC on the biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e was investigated in six biofilm-positive \u003cem\u003eS. aureus\u003c/em\u003e clinical isolates that were previously reported [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e], including MSSA strains of SE13、SE16、SA113、YUSA10 and MRSA of CHS350、CHS655. The biofilm formation of six \u003cem\u003eS. aureus\u003c/em\u003e strains was significantly inhibited by 1/2\u0026times;MIC Fingolimod (1.56 \u0026micro;g/mL) (\u003cstrong\u003eFig.\u0026nbsp;2A\u003c/strong\u003e). This finding was further observed and confirmed by laser scanning confocal microscopy in \u003cem\u003eS. aureus\u003c/em\u003e SA113 (\u003cstrong\u003eFig.\u0026nbsp;2B\u003c/strong\u003e). Furthermore, the eradication activity of Fingolimod against \u003cem\u003eS. aureus\u003c/em\u003e mature biofilm was evaluated by crystal violet staining and laser scanning confocal microscopy, indicating the monotherapy of Fingolimod with \u0026ge;\u0026thinsp;4\u0026times;MIC could more strongly eradicate the mature biofilm Fingolimod when compared with vancomycin, linezolid, and daptomycin which were first-line drug for clinical \u003cem\u003eStaphylococcus aureus\u003c/em\u003e therapy (\u003cstrong\u003eFig.\u0026nbsp;2C and D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe selective disruption of Fingolimod against the\u003c/strong\u003e \u003cstrong\u003eS. aureus\u003c/strong\u003e \u003cstrong\u003ecell membrane\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell membrane permeability of two \u003cem\u003eS. aureus\u003c/em\u003e strains, one \u003cem\u003eE. coli\u003c/em\u003e strain, and the human monocyte cell line THP-1staining with SYTOX green were assessed with Fingolimod, daptomycin, polymyxin, linezolid, and levofloxacin. The 1% TritonX-100 was used as a positive control for the increased membrane permeability of both bacteria and the THP-1 cell line. Our data indicated Fingolimod effectively damaged the bacterial membrane and enhanced its permeability of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e but not the THP-1 cell line (\u003cstrong\u003eFig.\u0026nbsp;3\u003c/strong\u003e). Moreover, the significantly increased membrane permeability of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e was found in daptomycin and polymyxin which are targeted to the cell membrane, but no change in linezolid and levofloxacin which aren\u0026rsquo;t targeted to the cell membrane, indicating that Fingolimod might selectivity target bacterial cell membrane and rapidly induce the increased permeability. The cell membrane damage and disruption were observed in \u003cem\u003eS. aureus\u003c/em\u003e SA113 which was grown planktonically to the mid-log phase and treated with Fingolimod for 30 mins by electron microscopy (\u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e), indicating that bactericidal activity of Fingolimod might be due to disruption of the bacterial cell membrane integrity.\u003c/p\u003e\n\u003cp\u003eA previous study proved that the chemical structure of Fingolimod contained a side-chain -NH\u003csub\u003e2\u003c/sub\u003e, which could be protonated to present in the form of \u0026ndash;NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e under the slightly acidic condition, as found in the normal airway surface liquid[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, whether the protonation of NH\u003csub\u003e2\u003c/sub\u003e was required for the bactericidal effects of Fingolimod was validated by assaying the growth curve of bacteria under various PH conditions. Our data showed the complete suppression of bacteria planktonic growth by Fingolimod at the concentration of 6.25 \u0026micro;g/ml under pH 6 not PH 8, suggesting the protonation of the NH\u003csub\u003e2\u003c/sub\u003e might impact the antibacterial effect of Fingolimod (\u003cstrong\u003eFig.\u0026nbsp;5\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003ePhospholipids as a Target for Fingolimod\u003c/h2\u003e\n \u003cp\u003eFingolimod can kill the bacteria by selectively disrupting the cell membrane with the PH-dependent protonation, therefore, we hypothesized that Fingolimod with positively charged in the slightly acidic solution could bind to negatively charged membrane components of bacteria during the protonation process. Cardiolipin, which is absent in the membrane structure of mammalian cells, plays a key physiological role in the cell membrane of bacteria. A previous study showed that the bactericidal mechanism of sphingosine, which is an analogue of Fingolimod, required binding to cardiolipin in plasma membranes of \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e. In this study, the impact of cardiolipin synthase (\u003cem\u003ecls\u003c/em\u003e) on Fingolimod susceptibility against bacteria was investigated by constructing the \u003cem\u003ecls\u003c/em\u003e gene knockout strain in \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 (\u0026Delta;\u003cem\u003ecls\u003c/em\u003e). The planktonic growth of wild-type or \u0026Delta;\u003cem\u003ecls\u003c/em\u003e strains under exposure to Fingolimod, polymyxin, and levofloxacin were detected and the result showed that Fingolimod and polymyxin, which destroyed the cell membrane targeting to the lipid A, inhibited the growth of \u0026Delta;\u003cem\u003ecls\u003c/em\u003e strain but not wild-type strain, while the growth between wild-type and \u0026Delta;\u003cem\u003ecls\u003c/em\u003e strains showed no significant difference with levofloxacin, which inhibited DNA synthesis and have no impact on bacteria membrane (\u003cstrong\u003eFig.\u0026nbsp;6\u003c/strong\u003e), demonstrating knockout of the \u003cem\u003ecls\u003c/em\u003e gene could enhance the sensitivity to Fingolimod and polymyxin[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. The \u0026Delta;\u003cem\u003ecls\u003c/em\u003e strain increased sensitivity to Fingolimod as same as polymyxin, not levofloxacin, suggesting Fingolimod possibly has a similar role to polymyxin (\u003cstrong\u003eFig.\u0026nbsp;6\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eWe evaluated the antibacterial activity of Fingolimod by using checkerboard assays on its target phospholipids. This assay employed three common bacterial membrane phospholipids, namely phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). After 20 hours, the MICs of Fingolimod were measured against clinically isolated strains of SA113 and CH350. The MIC of Fingolimod increased with phospholipid concentration, reaching a 16-fold increase with CL, PG and PE for both isolates (\u003cstrong\u003eFig.\u0026nbsp;7\u003c/strong\u003e). Among these phospholipids, CL has a particularly strong influence on increasing the MIC compared to the other two.\u003c/p\u003e\n \u003cp\u003eIn order to identify the potential target site of Fingolimod, the Fingolimod-induced resistant \u003cem\u003eS. aureus\u003c/em\u003e clones were selected by \u003cem\u003ein vitro\u003c/em\u003e serial passaging under the pressure of Fingolimod. The MIC value of SA113 and CHS101 were elevated from 3.125 to 6.25 \u0026micro;g/mL after 40 or 45 passages (\u003cstrong\u003eFig.\u0026nbsp;8\u003c/strong\u003e). A comparison of whole-genome sequencing between the Fingolimod parental isolates and Fingolimod-induced \u003cem\u003eS. aureus\u003c/em\u003e clones showed the four coding genetic mutations in the acyl carrier protein synthase (AcpS), inorganic phosphate transport regulatory protein (PhoP and PhoU2) respectively. The function of AcpS, PhoP, and PhoU2 were closely correlated with the phospholipid metabolism, and moreover, AcpS participated in bacteria autolysin (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). To explore Fingolimod\u0026apos;s potential targets in \u003cem\u003eS. aureus\u003c/em\u003e, this study compared the proteomes of Fingolimod-treated and DMSO-treated bacteria. The results identified 147 differentially expressed proteins with over two-fold changes, 76 upregulated and 71 downregulated (\u003cstrong\u003eFig.\u0026nbsp;9A\u003c/strong\u003e). GO analysis showed these proteins were mainly linked to redox functions and cell membrane composition and integrity (\u003cstrong\u003eFig.\u0026nbsp;9B\u003c/strong\u003e), supporting the idea that Fingolimod disrupts the cell membrane. ClueGO analysis found 42 proteins involved in membrane composition (\u003cstrong\u003eFig.\u0026nbsp;9C\u003c/strong\u003e), and protein localization within \u003cem\u003eS. aureus\u003c/em\u003e was mapped (\u003cstrong\u003eFig.\u0026nbsp;9D\u003c/strong\u003e), with enriched cell component proteins visualized (Fig. 9E).\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eGenetic mutations in the resistant strains of SA113 and CHS101 detecting by the whole-genome sequencing.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRef_gene_ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNA mutations\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAA mutations\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSubject description\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHS101_GM000826\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA269C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eV90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eresponse regulator transcription factor,PhoP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHS101_GM000905\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT347C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD116G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eautolysin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSA113_GM001858\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG94T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQ32K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcyl carrier protein, AcpP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSA113_GM000155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG506A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR169K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edistantly related to PhoU, PhoU2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe presence of \u003cem\u003epersisters\u003c/em\u003e and biofilm formation is closely linked to the recalcitrance of chronic infections and often results in a bad clinical outcome that is associated with significant mortality[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The conventional antibiotic treatment is frequently difficult to eradicate chronic infections caused by \u003cem\u003eS. aureus\u003c/em\u003e, in particular MRSA[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previous studies have demonstrated the various physiological uses of survival bacteria cells require the participation of intact membranes. Several reports supported the bacteria membrane is a promising potential target to counter the \u003cem\u003epersisters\u003c/em\u003e and biofilm for the development of new drugs. To shorten research time and costs, the drug repurposing targeting bacteria membrane by screening libraries of FDA-approved drugs could streamline the need of pharmacokinetic and toxicity[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the present study, our data indicated that Fingolimod can efficiently inhibit the planktonic growth of \u003cem\u003eS. aureus\u003c/em\u003e with the MIC range from 1.56\u0026micro;g/mL to 6.25 \u0026micro;g/mL. Moreover, Fingolimod can eradicate the \u003cem\u003epersisters\u003c/em\u003e and mature biofilms. Time-killing assay demonstrated the stronger bactericidal activity of Fingolimod when compared with vancomycin or linezolid. However, the influence of Fingolimod on the hemolytic activity of \u003cem\u003eS. aureus\u003c/em\u003e was also analyzed in 5 clinical \u003cem\u003eS. aureus\u003c/em\u003e strains using the rabbit erythrocytes, indicating no significant impact on the hemolytic activity by Fingolimod exposure. This effectiveness provides clues that Fingolimod had the potential to provide a novel choice for the antimicrobial treatment of chronic infections of \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFingolimod has been approved as the first-line drug for the treatment of the relapsing forms of multiple sclerosis. The clinical application practices have demonstrated Fingolimod is well-tolerated with a favorable safety profile. In eukaryotic, the main mechanism of Fingolimod is involved in the inhibition of S1P signaling and selectively retained lymphocytes in the lymphoid organs by targeting sphingosine 1-phosphate (S1P) receptors[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, several recent studies have also shown the antifungal, antiviral, and antibacterial effects of Fingolimod and its chemical homologues. Lu-Qi Wei \u003cem\u003eet al.\u003c/em\u003e reported that Fingolimod exhibited a synergistic effect with Amphotericin B against diverse fungal pathogens due to the excessive accumulation of reactive oxygen species[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. ZengZi Zhou \u003cem\u003eet al.\u003c/em\u003e showed that Fingolimod inhibited Chlamydia dissemination from the upper genital tract to the gastrointestinal tract[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Recent two studies have demonstrated the robust inhibitory activity of Fingolimod and its homologues derivatives against a wide range of gram-positive bacteria and several gram-negative bacteria species. Moreover, the impact of these drugs on both planktonic growth and biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e has been found. The chemical structure of Fingolimod was similar with to LuxR family quorum sensing (QS) and the mechanism of Fingolimod against Gram-negative bacteria has been speculated by inhibiting the QS [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. No alterations of MIC of Fingolimod in the wild type and \u003cem\u003eluxS\u003c/em\u003e deficiency of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e were examined, suggesting the impact of Fingolimod on \u003cem\u003eS. aureus\u003c/em\u003e can\u0026rsquo;t be explained by the hypothesis as different QS systems between Gram-positive and Gram-negative bacteria[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Sphingosine, which is a structural analog of Fingolimod, exists in human upper airway epithelial cells and displays antibacterial efficacy against pathogens including \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e. Recent reports demonstrated the inhibition of bacteria growth of sphingosine by binding to the cardiolipin and destroying the cell membranes. Moreover, the \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e strains with the knockout of cardiolipin synthase are resistant to sphingosine[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, our data indicated that similar to sphingosine, Fingolimod disrupted the integrity and permeability of \u003cem\u003eS. aureus\u003c/em\u003e cell membranes with cation-dependence. However, the cardiolipin knockout strain of \u003cem\u003eE. coli\u003c/em\u003e is showed more sensitive to Fingolimod, suggesting the bactericidal mechanism of Fingolimod is different from sphingosine.\u003c/p\u003e \u003cp\u003eTo elucidate the action mode of Fingolimod against \u003cem\u003eS. aureus\u003c/em\u003e, cell membrane disruption of \u003cem\u003eS. aureus\u003c/em\u003e was further validated by electron microscopy during Fingolimod exposure. The membrane disruption of \u003cem\u003eS. aureus\u003c/em\u003e by Fingolimod is PH-dependent, suggesting the cationic is important for its antibacterial effect of Fingolimod. Moreover, Fingolimod can results in the enhanced membrane permeability in \u003cem\u003eS. aureus\u003c/em\u003e SA113 and \u003cem\u003eE. coli\u003c/em\u003e 25922, hypothesizing its bactericidal mechanism is similar to daptomycin and polymyxin that targets the lipid in cell membranes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Furthermore, in \u003cem\u003eE. coli\u003c/em\u003e strain, the knockout of cardiolipin synthesis could enhance the sensitivity to Fingolimod as same as polymyxin, suggesting the action mode of Fingolimod might target on lipid similar to polymyxin. Comparison of whole-genome sequencing showed that three genes, including AcpS, PhoP and PhoU2, were shown with genetic mutations in Fingolimod-induced resistant \u003cem\u003eS. aureus\u003c/em\u003e clones when compared with the parental isolates. The function of AcpS, PhoP and PhoU2 closely participated in the phospholipids, supporting the impact of Fingolimod on the lipid of cell membrane[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, our data further supported the excellent antibacterial against planktonic cells and \u003cem\u003epersisters\u003c/em\u003e in clinical strains of \u003cem\u003eS. aureus, E. faecalis\u003c/em\u003e, and \u003cem\u003eS. agalactiae\u003c/em\u003e with very low MIC values of 1.56\u0026ndash;6.25 \u0026micro;g/mL. Fingolimod could significantly eliminate the \u003cem\u003epersisters\u003c/em\u003e, inhibit biofilm formation and eradicate mature biofilm. In addition, disruption of the bacterial cell membrane permeability and integrity were demonstrated by Fingolimod. In the presence of phospholipids, particularly cardiolipin, the antibacterial efficacy of the drug is significantly reduced. Fingolimod exposure can results in the genetic mutations in the critical genes (AcpS, PhoP and PhoU2) correlated with phospholipid metabolism. therefore, phospholipids might be an important target of Fingolimod against bacteria growth. These data firstly demonstrate the mechanism of Fingolimod during killing \u003cem\u003eS. aureus\u003c/em\u003e by targeting the lipid of cell membrane, which is similar to cationic bactericides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Author information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China\u003c/p\u003e\n\u003cp\u003eYongpeng Shang, Yu Huang, Fangyou Yu\u003c/p\u003e\n\u003cp\u003eDepartment of Infectious Diseases and the Key Lab of Endogenous Infection, Shenzhen Nanshan People\u0026apos;s Hospital, the 6th Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen 518052, China.\u003c/p\u003e\n\u003cp\u003eQingyin Meng, Zhijian Yu, Zewen Wen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYongpeng Shang: conceptualization (lead); formal analysis (equal); supervision (lead); writing original draft (equal).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eYu Huang\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eformal analysis (equal) supervision (equal); writing\u0026mdash; review and editing (equal).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFangyou Yu: Conceptualization (equal); formal analysis (equal) supervision (equal); writing\u0026mdash; review and editing (equal).\u0026nbsp;Qingyin Meng: methodology (equal).\u0026nbsp;Zhijian Yu: practical methodology (equal); writing\u0026mdash; review and editing (equal).\u0026nbsp;Zewen Wen: methodology (equal); writing\u0026mdash; review and editing (equal).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence to Fangyou Yu.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecial thanks to Zhanwen Wang, Junwen Chen, Chengchun Chen and Jingxin Zheng for their assistance with the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Natural Science Foundation of China (82302592), and the Guangdong Basic and Applied Basic Research Foundation (2021Al515110114), and the Shenzhen Nanshan District Scientific Research Program (NSZD2024002，NS2024009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical review and approval were performed by the Medical Ethics Committee of\u0026nbsp;Shanghai Pulmonary Hospital, School of Medicine\u0026nbsp;(Ethical no. K23-088Y).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDavid, M.Z. and R.S. Daum, Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev, 2010. 23(3): p. 616-87.\u003c/li\u003e\n\u003cli\u003eGupta, A.K., D.C. Lyons and T. Rosen, New and emerging concepts in managing and preventing community-associated methicillin-resistant Staphylococcus aureus infections. Int J Dermatol, 2015. 54(11): p. 1226-32.\u003c/li\u003e\n\u003cli\u003eLiu, W.T., et al., Emerging resistance mechanisms for 4 types of common anti-MRSA antibiotics in Staphylococcus aureus: A comprehensive review. 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Horswill, Staphylococcal Biofilm Development: Structure, Regulation, and Treatment Strategies. Microbiol Mol Biol Rev, 2020. 84(3).\u003c/li\u003e\n\u003cli\u003eYan, J. and B.L. Bassler, Surviving as a Community: Antibiotic Tolerance and Persistence in Bacterial Biofilms. Cell Host Microbe, 2019. 26(1): p. 15-21.\u003c/li\u003e\n\u003cli\u003eHurdle, J.G., et al., Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Microbiol, 2011. 9(1): p. 62-75.\u003c/li\u003e\n\u003cli\u003eChen, H., et al., The Mycobacterial Membrane: A Novel Target Space for Anti-tubercular Drugs. Front Microbiol, 2018. 9: p. 1627.\u003c/li\u003e\n\u003cli\u003ePujol, M., et al., Daptomycin Plus Fosfomycin Versus Daptomycin Alone for Methicillin-resistant Staphylococcus aureus Bacteremia and Endocarditis: A Randomized Clinical Trial. Clin Infect Dis, 2021. 72(9): p. 1517-1525.\u003c/li\u003e\n\u003cli\u003eTrimble, M.J., et al., Polymyxin: Alternative Mechanisms of Action and Resistance. Cold Spring Harb Perspect Med, 2016. 6(10).\u003c/li\u003e\n\u003cli\u003eGilbert-Girard, S., et al., Screening of FDA-Approved Drugs Using a 384-Well Plate-Based Biofilm Platform: The Case of Fingolimod. Microorganisms, 2020. 8(11).\u003c/li\u003e\n\u003cli\u003eZore, M., et al., Synthesis and Biological Evaluation of Fingolimod Derivatives as Antibacterial Agents. ACS Omega, 2021. 6(28): p. 18465-18486.\u003c/li\u003e\n\u003cli\u003eZheng, J., et al., Diclazuril Inhibits Biofilm Formation and Hemolysis of Staphylococcus aureus. ACS Infect Dis, 2021. 7(6): p. 1690-1701.\u003c/li\u003e\n\u003cli\u003eShang, Y., et al., Clemastine Inhibits the Biofilm and Hemolytic of Staphylococcus aureus through the GdpP Protein. Microbiol Spectr, 2022: p. e0054121.\u003c/li\u003e\n\u003cli\u003eBae, I.G., et al., Presence of genes encoding the panton-valentine leukocidin exotoxin is not the primary determinant of outcome in patients with complicated skin and skin structure infections due to methicillin-resistant Staphylococcus aureus: results of a multinational trial. J Clin Microbiol, 2009. 47(12): p. 3952-7.\u003c/li\u003e\n\u003cli\u003eWeng, Z.; et al., Antimicrobial activities of lavandulylated flavonoids in Sophora flavences against methicillin-resistant Staphylococcus aureus via membrane disruption. J.Adv. Res. 2023, No. 18033\u003c/li\u003e\n\u003cli\u003ePorto, W.F., et al., In silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design. Nat Commun, 2018. 9(1): p. 1490.\u003c/li\u003e\n\u003cli\u003eLin, Z., et al., Omadacycline Efficacy against Enterococcus faecalis Isolated in China: In Vitro Activity, Heteroresistance, and Resistance Mechanisms. Antimicrob Agents Chemother, 2020. 64(3).\u003c/li\u003e\n\u003cli\u003eZheng, J.X., et al., In vitro activities of telithromycin against Staphylococcus aureus biofilms compared with azithromycin, clindamycin, vancomycin and daptomycin. J Med Microbiol, 2020. 69(1): p. 120-131.\u003c/li\u003e\n\u003cli\u003eZajac, M., et al., Airway Surface Liquid pH Regulation in Airway Epithelium Current Understandings and Gaps in Knowledge. Int J Mol Sci, 2021. 22(7).\u003c/li\u003e\n\u003cli\u003eAndrade, F.F., et al., Colistin Update on Its Mechanism of Action and Resistance, Present and Future Challenges. Microorganisms, 2020. 8(11).\u003c/li\u003e\n\u003cli\u003eBardet, L. and J.M. Rolain, Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains. Can J Infect Dis Med Microbiol, 2018. 2018: p. 3095249.\u003c/li\u003e\n\u003cli\u003eGagliotti, C., et al., Staphylococcus aureus bloodstream infections: diverging trends of meticillin-resistant and meticillin-susceptible isolates, EU/EEA, 2005 to 2018. Euro Surveill, 2021. 26(46).\u003c/li\u003e\n\u003cli\u003eMairi, A., A. Touati and J.P. Lavigne, Methicillin-Resistant Staphylococcus aureus ST80 Clone: A Systematic Review. Toxins (Basel), 2020. 12(2).\u003c/li\u003e\n\u003cli\u003eMcConville, T.H., et al., In-Vitro Cytotoxicity and Clinical Correlates of MRSA Bacteremia. Antimicrob Agents Chemother, 2021: p. AAC0155921.\u003c/li\u003e\n\u003cli\u003eVazquez-Rosas, G.J., et al., Molecular Characterization of Staphylococcus aureus Obtained from Blood Cultures of Paediatric Patients Treated in a Tertiary Care Hospital in Mexico. Infect Drug Resist, 2021. 14: p. 1545-1556.\u003c/li\u003e\n\u003cli\u003eMaisuria, V.B., et al., Proanthocyanidin Interferes with Intrinsic Antibiotic Resistance Mechanisms of Gram-Negative Bacteria. Adv Sci (Weinh), 2019. 6(15): p. 1802333.\u003c/li\u003e\n\u003cli\u003eFernandes, P. and E. Martens, Antibiotics in late clinical development. Biochem Pharmacol, 2017. 133: p. 152-163.\u003c/li\u003e\n\u003cli\u003eSavoia, D., New Antimicrobial Approaches: Reuse of Old Drugs. Curr Drug Targets, 2016. 17(6): p. 731-8.\u003c/li\u003e\n\u003cli\u003eChun, J. and H.P. Hartung, Mechanism of action of oral fingolimod (FTY720) in multiple sclerosis. Clin Neuropharmacol, 2010. 33(2): p. 91-101.\u003c/li\u003e\n\u003cli\u003eKappos, L., et al., A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med, 2010. 362(5): p. 387-401.\u003c/li\u003e\n\u003cli\u003eWei, L.Q., et al., Fingolimod Potentiates the Antifungal Activity of Amphotericin B. Front Cell Infect Microbiol, 2021. 11: p. 627917.\u003c/li\u003e\n\u003cli\u003eZhou, Z., et al., Effects of Immunomodulatory Drug Fingolimod (FTY720) on Chlamydia Dissemination and Pathogenesis. Infection and Immunity, 2020. 88(11): p. e00281-20.\u003c/li\u003e\n\u003cli\u003eMukherjee, S. and B.L. Bassler, Bacterial quorum sensing in complex and dynamically changing environments. Nat Rev Microbiol, 2019. 17(6): p. 371-382.\u003c/li\u003e\n\u003cli\u003eVerhaegh, R., et al., Sphingosine kills bacteria by binding to cardiolipin. J Biol Chem, 2020. 295(22): p. 7686-7696.\u003c/li\u003e\n\u003cli\u003eAndrade, F.F., et al., Colistin Update on Its Mechanism of Action and Resistance, Present and Future Challenges. Microorganisms, 2020. 8(11).\u003c/li\u003e\n\u003cli\u003eBechinger, B. and S.U. Gorr, Antimicrobial Peptides: Mechanisms of Action and Resistance. J Dent Res, 2017. 96(3): p. 254-260.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Table 1","content":"\u003cp\u003eSupplemental Table 1 is not included with this version\u003c/p\u003e\n"}],"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":"Fingolimod, Staphylococcus aureus, Phospholipids","lastPublishedDoi":"10.21203/rs.3.rs-5223352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5223352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIt\u0026rsquo;s urgently needed to find new repurposed antibacterial drugs as the desired novel choices to counter biofilms and \u003cem\u003epersister\u003c/em\u003e of gram-positive bacteria. Several reports have supported that Fingolimod, which was approved by FDA as a novel drug for the treatment of relapsing multiple sclerosis, can kill the bacteria by selectively disrupting the cell membrane of bacteria. However, the action mode and mechanism of Fingolimod against gram-positive bacteria remains elusive. Our data indicated that Fingolimod exerted the bactericidal activity against a wide spectrum of gram-positive bacteria, including \u003cem\u003eStaphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae et al\u003c/em\u003e. Moreover, Fingolimod could significantly eliminate the \u003cem\u003epersister\u003c/em\u003e, inhibit biofilm formation, eradicate mature biofilm \u003cem\u003ein vitro against S. aureus\u003c/em\u003e. Fingolimod rapidly eradicated \u003cem\u003eS. aureus\u003c/em\u003e by pH-dependent disruption of the bacterial cell membrane's permeability and integrity, with its minimum inhibitory concentration (MIC) increasing up to 16-fold in response to elevated concentrations of phospholipids CL, PG, and PE. After four months of Fingolimod exposure, the MIC values of \u003cem\u003eS. aureus\u003c/em\u003e showed a slight increase, and three genetic mutations related to phospholipid metabolism\u0026mdash;PhoP, AcpP, and PhoU2\u0026mdash;were identified in Fingolimod-induced clones, suggesting that Fingolimod may disrupt the cell membrane by targeting phospholipids. Overall, Fingolimod kills \u003cem\u003eS. aureus\u003c/em\u003e by disrupting the bacteria membrane and targeting the phospholipids within the cell membrane. This study first reveals that Fingolimod kills \u003cem\u003eS. aureus\u003c/em\u003e by targeting cell membrane phospholipids, a mechanism similar to cationic bactericides.\u003c/p\u003e","manuscriptTitle":"Targeting Phospholipids: Fingolimod's Antibacterial Mechanism Against Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 18:13:59","doi":"10.21203/rs.3.rs-5223352/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":"793d56d8-f7c9-4f6c-8393-7cec3ce9f8ee","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-24T12:38:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-18 18:13:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5223352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5223352","identity":"rs-5223352","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}