A respiratory streptococcus strain inhibits Acinetobacter baumannii from causing inflammatory damage through ferroptosis

A respiratory streptococcus strain inhibits Acinetobacter baumannii from causing inflammatory damage through ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A respiratory streptococcus strain inhibits Acinetobacter baumannii from causing inflammatory damage through ferroptosis Ye Sun, Shuyin Li, Yuchen Che, Hao Liang, Yi Guo, Chunling Xiao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4692224/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2024 Read the published version in BMC Microbiology → Version 1 posted 18 You are reading this latest preprint version Abstract Background Microecological equilibrium is essential for human health. Previous research has demonstrated that Streptococcus strain A, the main bacterial group in the respiratory tract, can suppress harmful microbes and protect the body. In this study, Streptococcus strain D19 T was isolated from the oral and pharyngeal cavities of healthy children. Its antibacterial mechanism against Acinetobacter baumannii was examined, as well as its potential to prevent inflammatory damage to cells. We evaluated the effect of the fermentation conditions of D19 T on inhibition of Acinetobacter baumannii growth; Isolation and purification of antibacterial active components of strain D19 T and molecular mechanism of inhibition of Acinetobacter baumannii; Molecular mechanism of D19 T bacteriostatic protein reversing cellular inflammatory injury induced by Acinetobacter baumannii. Results The supernatant of fermentation broth of Streptococcus D19T was the active component against Acinetobacter baumannii, but the bacteria had no antibacterial activity. The supernatant of D19 T fermentation broth was precipitated by (NH 4 ) 2 SO 4 solution, and the protein was the active antibacterial component. After gel filtration chromatography and anion gel filtration chromatography, the molecular weight of antibacterial protein was 53kD. D19 T antibacterial protein can improve cell membrane permeability, limit extracellular soluble protein release, inhibit Acinetobacter baumannii biofilm formation, and prevent Acinetobacter baumannii adhesion. Acinetobacter baumannii induces inflammatory damage to respiratory cells via ferroptosis, and the D19 T antibacterial protein can counteract this damage, protecting the respiratory tract. Conclusion Streptococcus strain D19 T , as a potential probiotic, inhibits the growth of Acinetobacter baumannii and the inflammatory damage of respiratory cells, playing a protective role in human respiratory health. Streptococcus strain Acinetobacter baumannii antibacterial ferroptosis inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Human body contains billions of microbes that serve critical roles in digestion and absorption, vitamin production, immunity, and metabolism [ 1 , 2 ] . The microbial community has created a symbiotic relationship with the human body, and microecology must be stable in order to preserve physical health. However, several adverse variables, such as environmental pollution, hospital infections, antibiotic usage, excessive cleaning, and improper food habits, disrupt the microbial community, resulting in a variety of health issues [ 3 – 5 ] . Multidrug-resistant Acinetobacter baumannii infections have emerged as a major hospital infection pathogen as a result of antibiotic overuse, placing a significant strain on international healthcare systems [ 6 ] . The two primary ways that Acinetobacter baumannii spreads are through hospital infections and community transmission [ 7 ] . Acinetobacter baumannii infections in hospitals have the ability to adhere to the surfaces of medical personnel, equipment, and ward items, so spreading the illness to more patients [ 8 – 10 ] . Acinetobacter baumannii mostly affects the elderly and physically frail populations, increasing the death rate of patients through a variety of routes that induce respiratory inflammation and damage. The term "dominant microbial communities" describes the microbial populations that are predominant in a particular host or environment and are crucial to preserving the microecological equilibrium. In the respiratory system, for instance, type A hemolytic Streptococcus strain can fend off the adhesion and invasion of pathogens like Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, helping to keep the microbiota in the body in balance [ 11 – 13 ] . There is currently no information available regarding the connection between Acinetobacter baumannii-induced inflammation and type A hemolytic Streptococcus strain. The purpose of this study is to investigate Streptococcus strain D19 T as a dominant microbial community in the respiratory tract, analyze its anti-inflammatory effects on pathogenic bacteria, and lay the foundation for the clinical development of probiotics. Results Streptococcus strain D19 T inhibited the growth of Acinetobacter baumannii As shown in Fig. 1 -A, both Streptococcus strain D19 T fermentation broth and supernatant could inhibit the growth of Acinetobacter baumannii, but neither live or inactivated bacteria of D19 T could inhibit the growth of Acinetobacter baumannii. Therefore, we speculated that the supernatant of fermentation broth of strain D19 T was the active antibacterial component of Acinetobacter baumannii. In addition, we also optimized the fermentation conditions of strain D19 T . When the pH value of the culture medium was 7.0, the fermentation temperature was 37℃, and the fermentation time was 24h, the supernatant of the fermentation liquid of strain D19 T obtained the greatest antibacterial effect, and the diameter of its inhibitory Acinetobacter baumannii was 22.09 ± 21mm (Fig. 1 -B,C,D). D19 T antibacterial components Identification and purification D19 T fermentation broth was centrifuged to remove bacteria to obtain the supernatant, adding (NH 4 ) 2 SO 4 solution of different saturation to obtain the supernatant and precipitation. As shown in Fig. 2 -A, the supernatant after the salting out of (NH 4 ) 2 SO 4 solution of different saturation has no antibacterial activity, while the precipitation after the salting out of (NH 4 ) 2 SO 4 solution of 30%~70% saturation has antibacterial activity. (NH 4 ) 2 SO 4 solution saturation of 50% had the strongest antibacterial activity. The fermentation supernatant was precipitated with 50% saturated (NH 4 ) 2 SO 4 solution and redissolved in buffer. After dialysis, the protein samples were loaded on Sephadex G-15 chromatography resin. As shown in Fig. 2 -B and C, a total of three protein detection peaks appeared in the elution curve. Each protein peak was tested for activity against Acinetobacter baumannii, and it can be seen that protein peak a has antibacterial activity. Peak a was dialyzed and purified by anion-exchange chromatography. Three peaks were collected again after chromatography. Peak b showed antibacterial activity and a single protein band with a molecular weight of 53 KD was determined by SDS-PAGE electrophoresis (Fig. 2 -D,E,F). D19 T antibacterial protein's inhibition mechanism against Acinetobacter baumannii The minimum inhibitory concentration (MIC) of D19 T was determined to be 15 mg/mL. Then, Acinetobacter baumannii extravasated DNA and RNA levels increased significantly at D19 T antibacterial protein concentrations ≥ 1/2MIC, as measured by the protein gel imaging system( p < 0.05) (Fig. 3 -A). In addition, we also found that the soluble protein expression of Acinetobacter baumannii was significantly reduced at the concentration of D19 T antibacterial protein ≥ 1/2MIC( p < 0.05) (Fig. 3 -B). Finally, we found that Acinetobacter baumannii's biofilm formation and adhesion were significantly reduced at the concentration of D19 T antibacterial protein ≥ 1/2MIC ( p < 0.05) (Fig. 3 -C and D). Acinetobacter baumannii induces ferroptosis in respiratory epithelial cells Human bronchial epithelial cells BEAS-2B and 16HBE were infected with Acinetobacter baumannii. The ferroptosis of BEAS-2B and 16HBE cells was detected. As shown in Fig. 4 -A, BEAS-2B and 16HBE cells infected with Acinetobacter baumannii showed significant decreases in the expression of ferroptosis-related regulatory proteins GPX4, SLC7A11, and SLC3A2 ( p < 0.05). In addition, Fluorescence microscopy revealed that Acinetobacter baumannii infected BEAS-2B and 16HBE cells have increased ferroptosis fluorescence signals (Fig. 4 -B). Acinetobacter baumannii causes inflammatory damage to respiratory epithelial cells Previous studies have confirmed that ferroptosis is closely related to inflammation, so we further studied the inflammatory damage of cells after Acinetobacter baumannii infection. As shown in Fig. 5 -A to C, expressions of inflammatory factors TNF-α, IL-6 and IL-8 were significantly increased in BEAS-2B and 16HBE cells infected with Acinetobacter baumannii. ( p < 0.05). In addition, we also examined the expression of intracellular regulatory proteins of inflammatory factors. As shown in Fig. 5 -D, the expression levels of NF-κB, ASCL4, COX2 and LOX proteins were significantly increased in BEAS-2B and 16HBE cells infected with Acinetobacter baumannii ( p < 0.05). D19 T antibacterial protein reverses the ferroptosis of respiratory epithelial cells induced by Acinetobacter baumannii The effect of D19 T antibacterial protein on the ferroptosis of BEAS-2B and 16HBE cells infected with Acinetobacter baumannii was analyzed in vitro. As shown in Fig. 6-A, D19 T antibacterial protein could inhibit the ferroptosis of BEAS-2B and 16HBE cells caused by Acinetobacter baumannii in a concentration-dependent manner. When the concentration of antibacterial protein was ≥ 1/2MIC, the expression levels of ferroptosis-related regulatory proteins GPX4, SLC7A11 and SLC3A2 were significantly increased ( p < 0.05). In addition, when the concentration of antibacterial protein was ≥ 1/2MIC, the intensity of intracellular ferroptosis fluorescence signals were significantly decreased in Acinetobacter baumannii infected BEAS-2B and 16HBE cells ( p < 0.05)(Fig. 6 -B). By electron microscopy, the antibacterial protein could restore the damage caused by Acinetobacter baumannii, such as the decrease of mitochondria, the increase of membrane density and the decrease of mitochondrial ridge(Fig. 6 -C). D19 T antibacterial protein reverses the inflammatory injury of respiratory epithelial cells induced by Acinetobacter baumannii Similarly, we also analyzed the effect of D19 T antibacterial protein on inflammatory factors in BEAS-2B and 16HBE cells infected with Acinetobacter baumannii. As shown in Fig. 7 -A to C, D19 T antibacterial protein could inhibit the inflammatory damage of BEAS-2B and 16HBE cells caused by Acinetobacter baumannii in a concentration-dependent manner. When the concentration of D19 T antibacterial protein was ≥ 1/2MIC, the expression levels of TNF-α, IL-6 and IL-8 were significantly inhibited ( p < 0.05). In addition, the expression levels of NF-κB, ASCL4, COX2 and LOX proteins in Acinetobacter baumannii infected BEAS-2B and 16HBE cells were significantly decreased when the concentration of D19 T antibacterial protein was ≥ 1/2MIC ( p < 0.05)(Fig. 7 -D). Discussion Acinetobacter baumannii is an opportunistic pathogen with strong adhesion, which is a common pathogen of nosocomial infection. According to statistics, infections caused by Acinetobacter baumannii account for about 2% of all health care-associated infections in the United States and Europe, while the rate is significantly higher in Asia [ 14 ] . Acinetobacter baumannii is mainly distributed in ICU and respiratory departments of hospitals, and the objects of infection are mainly immunocompromised people, critically ill patients caused by invasive procedures, and patients treated with broad-spectrum antibiotics [ 15 ] . Acinetobacter baumannii mainly causes respiratory tract infection, and complications include bacteremia, urinary tract infection, meningitis, and ventilator-associated pneumonia [ 16 ] . Biofilm refers to the bacteria secretes aggregated membrane-like substances such as polysaccharide matrix, fibrin, and lipid proteins to enclose the whole bacterial community [ 17 ] . Biofilms can resist the antibacterial effects of antibiotics, immune-clearing cells, and immune effector substances. Biofilm is the main pathogenic factor and an important antibacterial index of Acinetobacter baumannii. In this study, we first confirmed that the proteins in the supernatant of D19 T fermentation broth were the antibacterial components by antibacterial experiments. We also found that D19 T protein increased the extravasation of macromolecules such as DNA and RNA in Acinetobacter baumannii broth. This suggests that the D19 T antibacterial protein affects the stability of Acinetobacter baumannii cell membrane and increases its permeability. In addition, we found that the D19 T antibacterial protein was able to inhibit the secretion of soluble proteins and reduce the efflux of toxic substances from Acinetobacter baumannii. Finally, our results showed that D19 T antibacterial protein significantly inhibited the biofilm formation and adhesion ability of Acinetobacter baumannii. Ferroptosis is an iron-dependent, non-apoptotic form of cell death accompanied by increased glutathione peroxidase activity and lipid peroxidation [ 18 ] . It has been confirmed that ferroptosis is closely related to the occurrence and development of many diseases, such as tumor, neurodegenerative diseases, ischemia-reperfusion injury, and so on [ 19 ] . Recent studies have found that the process of ferroptosis is often accompanied by inflammatory manifestations.When cells undergo ferroptosis, inflammation-related molecules are produced to stimulate the innate immune system, and immune cells trigger inflammatory responses by producing cytokines [ 20 ] . Lipoxygenase and epoxidation products play an important role in inflammatory response and may be closely related to ferroptosis. Our results show that Acinetobacter baumannii can cause inflammatory injury in human respiratory epithelial cells through ferroptosis pathway. D19 T antibacterial protein inhibits Acinetobacter baumannii induced ferroptosis in respiratory epithelial cells BEAS-2B and 16HBE by up-regulating GPX4, SLC7A11 and SLC3A2 protein expression. We also found that D19 T antibacterial protein inhibited the expression of TNF-α, IL-6 and IL-8 in Acinetobacter baumannii infected BEAS-2B and 16HBE cells. These results suggest that D19 T antibacterial protein can reverse the inflammatory injury of respiratory epithelial cells induced by Acinetobacter baumannii. Streptococcus strain D19 T is the dominant bacteria in human respiratory tract colonization and has the ability to resist pathogenic microorganisms. D19 T and its metabolites can be used as potential microecological agents to inhibit the growth of Acinetobacter baumannii and inflammatory damage of respiratory cells, and play a protective role in human respiratory health. Conclusions Acinetobacter baumannii is an important pathogen of nosocomial infection. It causes inflammatory damage to the respiratory tract through ferroptosis. Streptococcus strain D19 T is the dominant bacteria in human respiratory tract colonization, which can inhibit the growth of Acinetobacter baumannii and reverse inflammatory damage, and play a protective role in human respiratory system health. Materials and methods Antibodies The primary antibodies include mouse monoclonal anti-GPX4 (1:3000, Cat No. 67763-1-Ig), rabbit polyclonal anti-SLC7A11 (1:1000, Cat No. 26864-1-AP), rabbit polyclonal anti-SLC3A2 (1:20000, Cat No. 15193-1-AP), mouse monoclonal anti-NF-κB p65 (1:1000, Cat No. 66535-1-Ig), rabbit polyclonal anti-ASCL4 (1:6000, Cat No. 22401-1-AP), rabbit polyclonal anti-COX2 (1:2000, Cat No. 12375-1-AP), rabbit polyclonal anti-LOX (1:600, Cat No. 17958-1-AP)(Proteintech Group Inc., Rosemont, IL, USA), goat anti-rabbit IgG (1:50000, H + L, Cat No. RGAR001), and goat anti-mouse IgG (1:20000, H + L, Cat No. RGAM001) were purchased from Proteintech Group, Inc (Rosemont, IL, USA), Strains and cells and their culture Streptococcus strain D19 T was collected from the oropharynx of healthy children and cultured in bacto brain heart infusion medium(Solarbio, Beijing, China) at 37℃, 180r/min, for 18hours. Acinetobacter baumannii S2009-4 originates from the Laboratory of Shenyang Medical University Affiliated Central Hospital and is cultured in broth medium(Solarbio, Beijing, China) under conditions at 37 ℃, 180 r/min, for 18 hours. Human bronchial epithelium BEAS-2B cells and 16HBE cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., LTD. BEAS-2B and 16HBE cells were cultured in DMEM(Hyclone, Logan, UT, USA) medium containing 10% fetal bovine serum(Hyclone, Logan, UT, USA), 100 units/mL penicillin(Genview, Australia), and 100 units/mL streptomycin solution(Genview, Australia) at 37℃ and 5% CO 2 . Antibacterial assay The concentration of Acinetobacter baumannii bacterial solution in the logarithmic phase was adjusted to 1×10 6 CFU/mL, and 100µL was removed and evenly spread on nutrient AGAR plates. Using the disk diffusion method, 200µL of the sample solution to be tested was added to the disk and placed in a bacterial incubator for 18 h at 37°C. The diameter of inhibition was recorded with a vernier caliper. Fermentation broth conditions determination D19 T single colonies were inoculated into brain heart infusion medium and incubated at 37°C, 180 r/min, for 18h. The inhibitory diameter was measured under different fermentation conditions with different pH (3–11), fermentation temperature (28–43℃) and fermentation time (12-60h). Antibacterial components determination Antibacterial component determination: D19 T fermentation broth was centrifuged at 4 ℃ and 12000 r/min for 10 minutes to remove bacterial cells and obtain the supernatant of the fermentation broth. Add (NH 4 ) 2 SO 4 solution to the supernatant to a saturation of 30%, and precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12000 r/min for 10 minutes. The protein precipitate was dissolved in 50 mmol/L phosphate buffer and desalinated using a dialysis bag(Yeasen Biotechnology, Shanghai, China). Measure the antibacterial effects of the precipitated protein and supernatant separately. The determination of the optimal concentration of (NH 4 ) 2 SO 4 : The method for obtaining the supernatant of D19 T fermentation broth is consistent with the method mentioned earlier. Add (NH 4 ) 2 SO 4 solution to the supernatant, and the saturation of (NH 4 ) 2 SO 4 in the final supernatant ranges from 20–80%. Precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12000 r/min for 10 minutes. The protein precipitate was dissolved in 50 mmol/L phosphate buffer and desalinated using a dialysis bag. Measure the antibacterial effect of proteins precipitated with different saturation levels (NH 4 ) 2 SO 4 separately. Antibacterial proteins Isolation and purification The protein solution was loaded on a Sephadex G-15(Sigma, Louis, MO, USA) column and eluted with PBS solution at a flow rate of 1.5 mL/min. The elution peaks of each protein were detected and collected under ultraviolet light at 280nm to determine the inhibitory diameter. The bacteriostatic active fractions were collected and concentrated and then processed by cellulose DE-52 chromatography(Biosharp, Anhui, China). The flow rate was 1.5 mL/min and equilibrated in PBS solution until the baseline was stable. Linear gradient elution was carried out with 0 ~ 1.0 mol/L NaCL in PBS buffer, and the flow rate was controlled at 0.5 mL/min. The elution peaks of each protein were collected to determine the inhibitory diameter. Minimum inhibitory concentration (MIC) determination The antibacterial protein was diluted to 2, 4, 8, 16, 32 and 64 times with bacto brain heart infusion medium. 100µL protein solution was added to 96-well plate, and 10µL pathogen solution (1×10 6 CFU/mL) was added to each well. After mixing, the 96-well plate was cultured in a bacterial incubator at 37℃ for 24 hours. The minimum drug concentration without bacterial growth was read by the plate viable bacteria count method, which was the minimum inhibitory concentration (MIC) of the bacteria to the drug. DNA and RNA exosmosis levels determination DNA and RNA exosmosis levels of Acinetobacter baumannii were measured using Thremo Nanodrop 2000 detector(Thermo Fisher Scientific, Waltham, MA, USA). The operation procedure is summarized as follows: Run the Nanodrop when the sample measuring arm is off, add 2µL of distilled water to the optical fiber surface, lower the measuring arm, and set to zero. Add 1.0µL of the sample to be measured successively, and repeat the measurement for each sample to be measured 3 times. Soluble protein levels determination The level of soluble protein in Acinetobacter baumannii was determined by SDS-PAGE. The operation steps are summarized as follows: the suspension of Acinetobacter baumannii after treatment in the control group and the experimental group was centrifuge and the supernatant was removed to collect the bacteria. Acinetobacter baumannii in each group of samples were washed with pre-cooled PBS solution twice, the supernatant was removed by centrifuge and the bacteria were collected, then re-suspended in PBS solution and adjusted to the same bacterial density. The bacteria were treated in a metal bath at 100℃ for 10min, mixed upside down once every 2min, and the lysed cells released soluble proteins. Protein samples of 20µL were added for SDS-PAGE electrophoresis, dyed with Coomassie bright blue (Beyotime Biotechnology, Shanghai, China) for 40min, decolorized with distilled water, and photographed for analysis of protein expression. Biofilm formation determination 100 µL Acinetobacter baumannii solution at a concentration of 1×10 6 CFU/mL was added to 96-well plate, and then antibacterial protein solution was added to the final concentration of 0, 1/2MIC and MIC, respectively, and cultured at 37℃ for 24 hours. The culture medium and non-adherent bacteria were washed with PBS buffer, methanol was fixed for 15min, the biofilm was stained with 2% crystal violet solution for 15min, and the cells were decolorized with 33% glacial acetic acid. The absorbance was determined at 630 nm. The broth medium without bacteria was used as a negative control. Adhesion ability determination 500 µL of BEAS-2B and 16-HBE cells at a concentration of 5×10 4 cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37℃ and 5%CO 2 overnight. The cells were first cultured in serum-containing DMEM for 3 days, and then starved in serum-free DMEM culture for 12h. After washing with PBS, 1mL of Acinetobacter Baumannii suspensions with a concentration of 1×10 8 CFU/mL were added to each well, and 1mL of DMEM culture solution was added, mixed evenly, and incubated together for 2h. The cells were washed with PBS and fixed with 4% paraformaldehyde for 30min. The number of Acinetobacter baumannii adherens on the cell surface was observed and counted under the microscope. Non-adhesive bacteria (≤ 40 bacteria), adhesive bacteria (41–100 bacteria), strongly adhesive bacteria (> 100 bacteria). Cell iron death determination 500 µL of BEAS-2B and 16-HBE cells at a concentration of 5×10 4 cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37℃ and 5%CO 2 overnight. Add 500µL DMEM containing ammonium ferric sulfate (II) into each well (the final concentration of ammonium ferric sulfate (II) is 100 mol/L), and incubate in the incubator for 30 minutes. After washing the cells with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then treated with 1% Triton X-100 for 20 minutes. The cells were washed with PBS and incubated in an incubator with 500µL FerroGreen solution for 30 minutes. The intensity of each fluorescence signal was detected by GFP filter and BF filter in fluorescence microscope. Inflammatory cytokines detection The supernatant was obtained by centrifugation at 1000g for 20 minutes. Standard and sample holes were set up according to TNF-α, IL-6 and IL-8 instructions(Mibio, Shanghai, China). Add 50µL of standard product with different concentration to the standard product hole, and add 50µL of sample to the sample hole. Horseradish peroxidase (HRP) labeled detection antibody 100µL was added to each well and incubated in an incubator at 37℃ for 60min. Add 300µL washing solution to each well and repeat washing for 5 times. Add 50µL of substrate A and substrate B to each well and incubate at 37℃ for 15min without light. The absorbance of each hole was measured at 450nm wavelength by adding 50µL terminating solution to each hole. Western Blot Wash the cells once with a pre-cooled PBS solution to remove as much excess fluid as possible. 500µL RIPA lysate(Beyotime Biotechnology, Shanghai, China) containing 1mM PMSF(Beyotime Biotechnology, Shanghai, China) was added to the cells, and the entire lysate process was performed on ice. The protein samples were separated by SDS-PAGE gel electrophoresis at 80V, 20 min, 120V, 50 min. The protein on the gel was transferred to the PVDF membrane(Millipore, Boston, MA, USA) by wet transfer method at 300mA for 2 hours. The transferred PVDF film was washed with TBST and sealed overnight with 5% skim milk(Sigma, Louis, MO, USA) powder solution at 4℃. On the second day, they were incubated at room temperature for 1 hour with first antibody diluent and second antibody diluent, respectively. The protein was developed with enhanced ECL chemiluminescence reagent (Vazyme, Shanghai, China), and the gray values of the protein bands were calculated by Image J software. Ferroptosis was detected by electron microscopy Cells were collected and added to the fixative precooled at 4°C and placed at 4°C overnight. The fixative was decanted, rinsed with phosphate buffer, and samples were fixed with 1% osmic acid solution for 2h. "The samples were dehydrated with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) and finally treated with acetone. The samples were embedded in the embedding agent and cut by a microtome. The sections were stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 10min, respectively, and observed under a transmission electron microscope. Statistical analysis Since each experiment was performed three times, the data is given as mean ± SD. A two-tailed Student 'st test was used to assess the differences between the two groups. Analysis of Variance (ANOVA) is used to assess differences between multiple sets of data. P value lower than 0.05 was considered significant. SPSS 19.0 was used to analyze the data. Abbreviations potential of hydrogen (pH), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), minimum inhibitory concentration (MIC), tumor necrosis factor-α (TNF-α), Interleukin (IL), nuclear factor kappa-B (NF-κB), achaete-scute family bHLH transcription factor 4 (ASCL4), cyclooxygenase-2 (COX2), lectin-type oxidized LDL receptor 1(LOX), glutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11), solute carrier family 3, member 3 (SLC3A2), Intensive Care Unit (ICU), phosphate-buffered saline (PBS). Declarations Consent for publication Not applicable. Availability of data and materials All data included in this study are available on request from the corresponding author. Competing interests The authors declare that they have no competing interests. Funding This work was financially supported by Scientific Research Issues and Medical Technical Problems of China Medical Education Association (Grant No.2022KTM026) and Ministry of Education Higher Education Scientific Research Project (Grant No.ZJXF2022012). Authors' contributions CX: Experimental design (lead); funding support (lead). YS: Experimental operation (lead); data analysis (lead); article writing (lead). SL: Experimental design. YC: Experimental operation. HL: Experimental design. YG: Data analysis. D ata availablity All data included in this study are available on request from the corresponding author Acknowledgements The author thanks the China Medical Education Association for funding this study. Authors' information Teacher of Shenyang Medical College Compliance with ethical standards Ethics approval —The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Shenyang Medical College. Informed consent —Written informed consent was obtained from individual or guardian participants. References Guo XY, Liu XJ, Hao JY. Gut microbiota in ulcerative colitis: insights on pathogenesis and treatment. J Dig Dis. 2020 Mar;21(3):147-159. Fu Q, Song T, Ma X, et al. Research progress on the relationship between intestinal microecology and intestinal bowel disease. Animal Model Exp Med. 2022 Dec;5(4):297-310. Wang J, Liang J, He M, et al. Chinese expert consensus on intestinal microecology and management of digestive tract complications related to tumor treatment (version 2022). J Cancer Res Ther. 2022 Dec;18(7):1835-1844. Isolauri E. Microbiota and Obesity. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4692224","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":339586035,"identity":"44e3957b-c7c9-490b-9393-1e1e580de09c","order_by":0,"name":"Ye Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYNCCCijNQ7yWMyRrYWwjRYv8jNxjEh/n1SWubT/A+OBtG4O8OSEtBjfy0iRnbjucuO1MArPh3DYGw50NhLRI5JhJ8247kLjtBgObNG8bQ4LBAYIOA2r5O6cOpIX9N1FaGG4AtTA2MINtYSZKi8GZN8aWPccOG287k9gsOeechOEGgg5rzzG88aOmTnbb8cMHP7wps5En7DAGBhYJIOHYwMDYAKQlCKsHAuYPQMKeKKWjYBSMglEwMgEA52pAhTH27lwAAAAASUVORK5CYII=","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":true,"prefix":"","firstName":"Ye","middleName":"","lastName":"Sun","suffix":""},{"id":339586036,"identity":"e4c915fd-c34b-45ec-a492-baa458cfa555","order_by":1,"name":"Shuyin Li","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shuyin","middleName":"","lastName":"Li","suffix":""},{"id":339586037,"identity":"57374aed-c92c-497d-9560-7d72334dd62f","order_by":2,"name":"Yuchen Che","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Che","suffix":""},{"id":339586038,"identity":"4a867d03-6be7-44c0-a714-c4c03cb1f79d","order_by":3,"name":"Hao Liang","email":"","orcid":"","institution":"Shenyang Vocational and Technical College","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Liang","suffix":""},{"id":339586041,"identity":"8998fba5-1b24-4c70-876e-b6edb52a364d","order_by":4,"name":"Yi Guo","email":"","orcid":"","institution":"Shenyang Vocational and Technical College","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Guo","suffix":""},{"id":339586043,"identity":"c7f87a41-9ed4-487e-9d00-efa34535140b","order_by":5,"name":"Chunling Xiao","email":"","orcid":"","institution":"Shenyang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Chunling","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-07-05 12:22:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4692224/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4692224/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-024-03589-7","type":"published","date":"2024-10-28T16:20:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62733096,"identity":"4f2b1788-86ea-4a4f-b5d3-12358b09f492","added_by":"auto","created_at":"2024-08-18 23:52:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":277581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD19\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e fermentation broth antibacterial assay and optimization of fermentation conditions.\u003c/strong\u003e (A) Detection of antibacterial components in D19\u003csup\u003eT\u003c/sup\u003e fermentation broth. (B) Effect of fermentation broth pH on the bacteriostasis of D19\u003csup\u003eT\u003c/sup\u003e. (C) Effect of fermentation temperature on the bacteriostasis of D19\u003csup\u003eT\u003c/sup\u003e. (D) Effect of fermentation time on the bacteriostasis of D19\u003csup\u003eT\u003c/sup\u003e. (a) Fermentation broth. (b) Fermentation broth supernatant. (c) Live bacterial body. (d) Inactivated bacterial body.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/cbca28b6e4a1c8df469b6629.jpg"},{"id":62733790,"identity":"fe25ec17-42e5-421e-a2b3-f6fb2d8b9c35","added_by":"auto","created_at":"2024-08-19 00:00:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and purification of antibacterial active components of D19\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Antibacterial test of fermentation broth supernatant precipitated by (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at different saturation. (B) Sephadex G-15 molecular sieve was used to isolate and purify the protein and to detect its antibacterial activity. (C) Antibacterial activity was detected by disk dispersion method. (D) Isolation and purification of protein by cellulose DE-52 chromatography and bacteriostatic detection. (E) Molecular weight determination of bacteriostatic protein. (F) Antibacterial activity was detected by disk dispersion method. pa：peak a, pb：peak b, pc：peak c.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/5221e6882b36cb8267dcc26f.jpg"},{"id":62733093,"identity":"94b3eaba-b53e-4315-a3ce-16649bfdc177","added_by":"auto","created_at":"2024-08-18 23:52:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":216429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism of D19\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e antibacterial protein inhibiting the growth of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcinetobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e baumannii.\u003c/strong\u003e (A) Effect of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein on \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii DNA and RNA extravasation. (B) Effect of D19\u003csup\u003eT\u003c/sup\u003e bacteriostatic protein on soluble proteins of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. (C) Effect of D19\u003csup\u003eT\u003c/sup\u003e bacteriostatic protein on adhesion of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. (D) Effect of D19\u003csup\u003eT\u003c/sup\u003e bacteriostatic protein on \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii biofilm.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/c061c49f8c28cabd20e9139b.jpg"},{"id":62733789,"identity":"f555a62b-5f5b-4c3e-a359-ceab80780f63","added_by":"auto","created_at":"2024-08-19 00:00:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":365790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAcinetobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e baumannii induces ferroptosis in respiratory epithelial cells. \u003c/strong\u003e(A) Western blot detection of iron death related regulatory proteins. (B)Fluorescence labeling method to detect iron cell death.Full-length gels are presented in Supplementary Figure S1 and Figure S2.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/701332cf13e69cdc67437946.jpg"},{"id":62733097,"identity":"3ea2c4c0-0d90-4836-adfb-2fd8afb124ed","added_by":"auto","created_at":"2024-08-18 23:52:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":465269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAcinetobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e baumannii induces inflammatory injury of respiratory epithelial cells.\u003c/strong\u003e ELISAmethod to detect the (A) TNF-α, (B) IL-6, (C) IL-8 levels. (D) Western blot detection of inflammatory injuryrelated regulatory proteins. Full-length gels are presented in Supplementary Figure S3 and Figure S4.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/09c3a3d9e6f8454c496434d5.jpg"},{"id":62733098,"identity":"42bc888b-2102-40d6-8b8e-d54e3a0a8bea","added_by":"auto","created_at":"2024-08-18 23:52:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1452528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of D19\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e antibacterial protein on ferroptosis of respiratory epithelial cells induced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcinetobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e baumannii.\u003c/strong\u003e (A) Western blot detection of iron death related regulatory proteins. (B) Fluorescence labeling method to detect iron cell death. (C) Transmission electron microscopy method to detect iron cell death. Full-length gels are presented in Supplementary Figure S5 and Figure S6.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/9c5fca58aba12d5a90b28af9.png"},{"id":62733100,"identity":"f9219cdc-97b0-4d48-9e90-a414686859a5","added_by":"auto","created_at":"2024-08-18 23:52:53","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":571914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of D19\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e antibacterial protein on inflammatory injury of respiratory epithelial cells induced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcinetobacter\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e baumannii.\u003c/strong\u003e ELISA method to detect the (A) TNF-α, (B) IL-6, (C) IL-8 levels. (D) Western blot detection of inflammatory injury related regulatory proteins. Full-length gels are presented in Supplementary Figure S7 and Figure S8.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/cf2b6ee0afbba14b772ea9e7.jpg"},{"id":68207137,"identity":"657ce5b1-fda9-42f9-85c8-d66f90cba077","added_by":"auto","created_at":"2024-11-04 16:35:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4613650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/728acf74-b759-4eca-b164-39da572c0ba5.pdf"},{"id":62733101,"identity":"87c4c328-702a-4fe5-ae1e-92a24af01be1","added_by":"auto","created_at":"2024-08-18 23:52:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9671309,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4692224/v1/7c660777628ac0246600fc71.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A respiratory streptococcus strain inhibits Acinetobacter baumannii from causing inflammatory damage through ferroptosis","fulltext":[{"header":"Background","content":"\u003cp\u003eHuman body contains billions of microbes that serve critical roles in digestion and absorption, vitamin production, immunity, and metabolism\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The microbial community has created a symbiotic relationship with the human body, and microecology must be stable in order to preserve physical health. However, several adverse variables, such as environmental pollution, hospital infections, antibiotic usage, excessive cleaning, and improper food habits, disrupt the microbial community, resulting in a variety of health issues\u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMultidrug-resistant \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infections have emerged as a major hospital infection pathogen as a result of antibiotic overuse, placing a significant strain on international healthcare systems\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The two primary ways that \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii spreads are through hospital infections and community transmission\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infections in hospitals have the ability to adhere to the surfaces of medical personnel, equipment, and ward items, so spreading the illness to more patients\u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii mostly affects the elderly and physically frail populations, increasing the death rate of patients through a variety of routes that induce respiratory inflammation and damage. The term \"dominant microbial communities\" describes the microbial populations that are predominant in a particular host or environment and are crucial to preserving the microecological equilibrium. In the respiratory system, for instance, type A hemolytic \u003cem\u003eStreptococcus\u003c/em\u003e strain can fend off the adhesion and invasion of pathogens like Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, helping to keep the microbiota in the body in balance\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. There is currently no information available regarding the connection between \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii-induced inflammation and type A hemolytic \u003cem\u003eStreptococcus\u003c/em\u003e strain. The purpose of this study is to investigate \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e as a dominant microbial community in the respiratory tract, analyze its anti-inflammatory effects on pathogenic bacteria, and lay the foundation for the clinical development of probiotics.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eStreptococcus\u003c/b\u003e \u003cb\u003estrain D19\u003c/b\u003e\u003csup\u003e\u003cb\u003eT\u003c/b\u003e\u003c/sup\u003e \u003cb\u003einhibited the growth of\u003c/b\u003e \u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003ebaumannii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e-A, both \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e fermentation broth and supernatant could inhibit the growth of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii, but neither live or inactivated bacteria of D19\u003csup\u003eT\u003c/sup\u003e could inhibit the growth of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. Therefore, we speculated that the supernatant of fermentation broth of strain D19\u003csup\u003eT\u003c/sup\u003e was the active antibacterial component of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. In addition, we also optimized the fermentation conditions of strain D19\u003csup\u003eT\u003c/sup\u003e. When the pH value of the culture medium was 7.0, the fermentation temperature was 37℃, and the fermentation time was 24h, the supernatant of the fermentation liquid of strain D19\u003csup\u003eT\u003c/sup\u003e obtained the greatest antibacterial effect, and the diameter of its inhibitory \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii was 22.09\u0026thinsp;\u0026plusmn;\u0026thinsp;21mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e-B,C,D).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eD19\u003csup\u003eT\u003c/sup\u003e antibacterial components Identification and purification\u003c/h2\u003e \u003cp\u003eD19\u003csup\u003eT\u003c/sup\u003e fermentation broth was centrifuged to remove bacteria to obtain the supernatant, adding (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution of different saturation to obtain the supernatant and precipitation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e-A, the supernatant after the salting out of (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution of different saturation has no antibacterial activity, while the precipitation after the salting out of (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution of 30%~70% saturation has antibacterial activity. (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution saturation of 50% had the strongest antibacterial activity. The fermentation supernatant was precipitated with 50% saturated (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution and redissolved in buffer. After dialysis, the protein samples were loaded on Sephadex G-15 chromatography resin. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e-B and C, a total of three protein detection peaks appeared in the elution curve. Each protein peak was tested for activity against \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii, and it can be seen that protein peak a has antibacterial activity. Peak a was dialyzed and purified by anion-exchange chromatography. Three peaks were collected again after chromatography. Peak b showed antibacterial activity and a single protein band with a molecular weight of 53 KD was determined by SDS-PAGE electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e-D,E,F).\u003c/p\u003e \u003cp\u003e \u003cb\u003eD19\u003c/b\u003e \u003csup\u003e \u003cb\u003eT\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eantibacterial protein's inhibition mechanism against\u003c/b\u003e \u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003ebaumannii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) of D19\u003csup\u003eT\u003c/sup\u003e was determined to be 15 mg/mL. Then, \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii extravasated DNA and RNA levels increased significantly at D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein concentrations\u0026thinsp;\u0026ge;\u0026thinsp;1/2MIC, as measured by the protein gel imaging system(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A). In addition, we also found that the soluble protein expression of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii was significantly reduced at the concentration of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein\u0026thinsp;\u0026ge;\u0026thinsp;1/2MIC(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e-B). Finally, we found that \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii's biofilm formation and adhesion were significantly reduced at the concentration of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein\u0026thinsp;\u0026ge;\u0026thinsp;1/2MIC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e-C and D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003ebaumannii induces ferroptosis in respiratory epithelial cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHuman bronchial epithelial cells BEAS-2B and 16HBE were infected with \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. The ferroptosis of BEAS-2B and 16HBE cells was detected. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A, BEAS-2B and 16HBE cells infected with \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii showed significant decreases in the expression of ferroptosis-related regulatory proteins GPX4, SLC7A11, and SLC3A2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, Fluorescence microscopy revealed that \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infected BEAS-2B and 16HBE cells have increased ferroptosis fluorescence signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e-B).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003ebaumannii causes inflammatory damage to respiratory epithelial cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies have confirmed that ferroptosis is closely related to inflammation, so we further studied the inflammatory damage of cells after \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A to C, expressions of inflammatory factors TNF-α, IL-6 and IL-8 were significantly increased in BEAS-2B and 16HBE cells infected with \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, we also examined the expression of intracellular regulatory proteins of inflammatory factors. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e-D, the expression levels of NF-κB, ASCL4, COX2 and LOX proteins were significantly increased in BEAS-2B and 16HBE cells infected with \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eD19\u003c/b\u003e \u003csup\u003e \u003cb\u003eT\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eantibacterial protein reverses the ferroptosis of respiratory epithelial cells induced by\u003c/b\u003e \u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003ebaumannii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein on the ferroptosis of BEAS-2B and 16HBE cells infected with \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii was analyzed in vitro. As shown in Fig.\u0026nbsp;6-A, D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein could inhibit the ferroptosis of BEAS-2B and 16HBE cells caused by \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii in a concentration-dependent manner. When the concentration of antibacterial protein was \u0026ge;\u0026thinsp;1/2MIC, the expression levels of ferroptosis-related regulatory proteins GPX4, SLC7A11 and SLC3A2 were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, when the concentration of antibacterial protein was \u0026ge;\u0026thinsp;1/2MIC, the intensity of intracellular ferroptosis fluorescence signals were significantly decreased in \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infected BEAS-2B and 16HBE cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e-B). By electron microscopy, the antibacterial protein could restore the damage caused by Acinetobacter baumannii, such as the decrease of mitochondria, the increase of membrane density and the decrease of mitochondrial ridge(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e-C).\u003c/p\u003e \u003cp\u003e \u003cb\u003eD19\u003c/b\u003e \u003csup\u003e \u003cb\u003eT\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eantibacterial protein reverses the inflammatory injury of respiratory epithelial cells induced by\u003c/b\u003e \u003cb\u003eAcinetobacter\u003c/b\u003e \u003cb\u003ebaumannii\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSimilarly, we also analyzed the effect of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein on inflammatory factors in BEAS-2B and 16HBE cells infected with \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e-A to C, D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein could inhibit the inflammatory damage of BEAS-2B and 16HBE cells caused by \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii in a concentration-dependent manner. When the concentration of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein was \u0026ge;\u0026thinsp;1/2MIC, the expression levels of TNF-α, IL-6 and IL-8 were significantly inhibited (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the expression levels of NF-κB, ASCL4, COX2 and LOX proteins in \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infected BEAS-2B and 16HBE cells were significantly decreased when the concentration of D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein was \u0026ge;\u0026thinsp;1/2MIC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e-D).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii is an opportunistic pathogen with strong adhesion, which is a common pathogen of nosocomial infection. According to statistics, infections caused by \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii account for about 2% of all health care-associated infections in the United States and Europe, while the rate is significantly higher in Asia\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii is mainly distributed in ICU and respiratory departments of hospitals, and the objects of infection are mainly immunocompromised people, critically ill patients caused by invasive procedures, and patients treated with broad-spectrum antibiotics\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii mainly causes respiratory tract infection, and complications include bacteremia, urinary tract infection, meningitis, and ventilator-associated pneumonia\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBiofilm refers to the bacteria secretes aggregated membrane-like substances such as polysaccharide matrix, fibrin, and lipid proteins to enclose the whole bacterial community\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Biofilms can resist the antibacterial effects of antibiotics, immune-clearing cells, and immune effector substances. Biofilm is the main pathogenic factor and an important antibacterial index of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. In this study, we first confirmed that the proteins in the supernatant of D19\u003csup\u003eT\u003c/sup\u003e fermentation broth were the antibacterial components by antibacterial experiments. We also found that D19\u003csup\u003eT\u003c/sup\u003e protein increased the extravasation of macromolecules such as DNA and RNA in \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii broth. This suggests that the D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein affects the stability of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii cell membrane and increases its permeability. In addition, we found that the D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein was able to inhibit the secretion of soluble proteins and reduce the efflux of toxic substances from \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. Finally, our results showed that D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein significantly inhibited the biofilm formation and adhesion ability of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii.\u003c/p\u003e \u003cp\u003eFerroptosis is an iron-dependent, non-apoptotic form of cell death accompanied by increased glutathione peroxidase activity and lipid peroxidation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. It has been confirmed that ferroptosis is closely related to the occurrence and development of many diseases, such as tumor, neurodegenerative diseases, ischemia-reperfusion injury, and so on\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Recent studies have found that the process of ferroptosis is often accompanied by inflammatory manifestations.When cells undergo ferroptosis, inflammation-related molecules are produced to stimulate the innate immune system, and immune cells trigger inflammatory responses by producing cytokines\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Lipoxygenase and epoxidation products play an important role in inflammatory response and may be closely related to ferroptosis. Our results show that \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii can cause inflammatory injury in human respiratory epithelial cells through ferroptosis pathway. D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein inhibits \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii induced ferroptosis in respiratory epithelial cells BEAS-2B and 16HBE by up-regulating GPX4, SLC7A11 and SLC3A2 protein expression. We also found that D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein inhibited the expression of TNF-α, IL-6 and IL-8 in \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii infected BEAS-2B and 16HBE cells. These results suggest that D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein can reverse the inflammatory injury of respiratory epithelial cells induced by \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e is the dominant bacteria in human respiratory tract colonization and has the ability to resist pathogenic microorganisms. D19\u003csup\u003eT\u003c/sup\u003e and its metabolites can be used as potential microecological agents to inhibit the growth of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii and inflammatory damage of respiratory cells, and play a protective role in human respiratory health.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii is an important pathogen of nosocomial infection. It causes inflammatory damage to the respiratory tract through ferroptosis. \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e is the dominant bacteria in human respiratory tract colonization, which can inhibit the growth of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii and reverse inflammatory damage, and play a protective role in human respiratory system health.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe primary antibodies include mouse monoclonal anti-GPX4 (1:3000, Cat No. 67763-1-Ig), rabbit polyclonal anti-SLC7A11 (1:1000, Cat No. 26864-1-AP), rabbit polyclonal anti-SLC3A2 (1:20000, Cat No. 15193-1-AP), mouse monoclonal anti-NF-κB p65 (1:1000, Cat No. 66535-1-Ig), rabbit polyclonal anti-ASCL4 (1:6000, Cat No. 22401-1-AP), rabbit polyclonal anti-COX2 (1:2000, Cat No. 12375-1-AP), rabbit polyclonal anti-LOX (1:600, Cat No. 17958-1-AP)(Proteintech Group Inc., Rosemont, IL, USA), goat anti-rabbit IgG (1:50000, H\u0026thinsp;+\u0026thinsp;L, Cat No. RGAR001), and goat anti-mouse IgG (1:20000, H\u0026thinsp;+\u0026thinsp;L, Cat No. RGAM001) were purchased from Proteintech Group, Inc (Rosemont, IL, USA),\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStrains and cells and their culture\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e was collected from the oropharynx of healthy children and cultured in bacto brain heart infusion medium(Solarbio, Beijing, China) at 37℃, 180r/min, for 18hours. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii S2009-4 originates from the Laboratory of Shenyang Medical University Affiliated Central Hospital and is cultured in broth medium(Solarbio, Beijing, China) under conditions at 37 ℃, 180 r/min, for 18 hours.\u003c/p\u003e \u003cp\u003eHuman bronchial epithelium BEAS-2B cells and 16HBE cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., LTD. BEAS-2B and 16HBE cells were cultured in DMEM(Hyclone, Logan, UT, USA) medium containing 10% fetal bovine serum(Hyclone, Logan, UT, USA), 100 units/mL penicillin(Genview, Australia), and 100 units/mL streptomycin solution(Genview, Australia) at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eAntibacterial assay\u003c/h3\u003e\n\u003cp\u003eThe concentration of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii bacterial solution in the logarithmic phase was adjusted to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL, and 100\u0026micro;L was removed and evenly spread on nutrient AGAR plates. Using the disk diffusion method, 200\u0026micro;L of the sample solution to be tested was added to the disk and placed in a bacterial incubator for 18 h at 37\u0026deg;C. The diameter of inhibition was recorded with a vernier caliper.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFermentation broth conditions determination\u003c/h2\u003e \u003cp\u003eD19\u003csup\u003eT\u003c/sup\u003e single colonies were inoculated into brain heart infusion medium and incubated at 37\u0026deg;C, 180 r/min, for 18h. The inhibitory diameter was measured under different fermentation conditions with different pH (3\u0026ndash;11), fermentation temperature (28\u0026ndash;43℃) and fermentation time (12-60h).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial components determination\u003c/h2\u003e \u003cp\u003eAntibacterial component determination: D19\u003csup\u003eT\u003c/sup\u003e fermentation broth was centrifuged at 4 ℃ and 12000 r/min for 10 minutes to remove bacterial cells and obtain the supernatant of the fermentation broth. Add (NH\u003csub\u003e4\u003c/sub\u003e) \u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution to the supernatant to a saturation of 30%, and precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12000 r/min for 10 minutes. The protein precipitate was dissolved in 50 mmol/L phosphate buffer and desalinated using a dialysis bag(Yeasen Biotechnology, Shanghai, China). Measure the antibacterial effects of the precipitated protein and supernatant separately.\u003c/p\u003e \u003cp\u003eThe determination of the optimal concentration of (NH\u003csub\u003e4\u003c/sub\u003e) \u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e: The method for obtaining the supernatant of D19\u003csup\u003eT\u003c/sup\u003e fermentation broth is consistent with the method mentioned earlier. Add (NH\u003csub\u003e4\u003c/sub\u003e) \u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution to the supernatant, and the saturation of (NH\u003csub\u003e4\u003c/sub\u003e) \u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in the final supernatant ranges from 20\u0026ndash;80%. Precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12000 r/min for 10 minutes. The protein precipitate was dissolved in 50 mmol/L phosphate buffer and desalinated using a dialysis bag. Measure the antibacterial effect of proteins precipitated with different saturation levels (NH\u003csub\u003e4\u003c/sub\u003e) \u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e separately.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial proteins Isolation and purification\u003c/h2\u003e \u003cp\u003eThe protein solution was loaded on a Sephadex G-15(Sigma, Louis, MO, USA) column and eluted with PBS solution at a flow rate of 1.5 mL/min. The elution peaks of each protein were detected and collected under ultraviolet light at 280nm to determine the inhibitory diameter. The bacteriostatic active fractions were collected and concentrated and then processed by cellulose DE-52 chromatography(Biosharp, Anhui, China). The flow rate was 1.5 mL/min and equilibrated in PBS solution until the baseline was stable. Linear gradient elution was carried out with 0\u0026thinsp;~\u0026thinsp;1.0 mol/L NaCL in PBS buffer, and the flow rate was controlled at 0.5 mL/min. The elution peaks of each protein were collected to determine the inhibitory diameter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration (MIC) determination\u003c/h2\u003e \u003cp\u003eThe antibacterial protein was diluted to 2, 4, 8, 16, 32 and 64 times with bacto brain heart infusion medium. 100\u0026micro;L protein solution was added to 96-well plate, and 10\u0026micro;L pathogen solution (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) was added to each well. After mixing, the 96-well plate was cultured in a bacterial incubator at 37℃ for 24 hours. The minimum drug concentration without bacterial growth was read by the plate viable bacteria count method, which was the minimum inhibitory concentration (MIC) of the bacteria to the drug.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDNA and RNA exosmosis levels determination\u003c/h2\u003e \u003cp\u003eDNA and RNA exosmosis levels of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii were measured using Thremo Nanodrop 2000 detector(Thermo Fisher Scientific, Waltham, MA, USA). The operation procedure is summarized as follows: Run the Nanodrop when the sample measuring arm is off, add 2\u0026micro;L of distilled water to the optical fiber surface, lower the measuring arm, and set to zero. Add 1.0\u0026micro;L of the sample to be measured successively, and repeat the measurement for each sample to be measured 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSoluble protein levels determination\u003c/h2\u003e \u003cp\u003eThe level of soluble protein in \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii was determined by SDS-PAGE. The operation steps are summarized as follows: the suspension of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii after treatment in the control group and the experimental group was centrifuge and the supernatant was removed to collect the bacteria. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii in each group of samples were washed with pre-cooled PBS solution twice, the supernatant was removed by centrifuge and the bacteria were collected, then re-suspended in PBS solution and adjusted to the same bacterial density. The bacteria were treated in a metal bath at 100℃ for 10min, mixed upside down once every 2min, and the lysed cells released soluble proteins. Protein samples of 20\u0026micro;L were added for SDS-PAGE electrophoresis, dyed with Coomassie bright blue (Beyotime Biotechnology, Shanghai, China) for 40min, decolorized with distilled water, and photographed for analysis of protein expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation determination\u003c/h2\u003e \u003cp\u003e100 \u0026micro;L \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii solution at a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL was added to 96-well plate, and then antibacterial protein solution was added to the final concentration of 0, 1/2MIC and MIC, respectively, and cultured at 37℃ for 24 hours. The culture medium and non-adherent bacteria were washed with PBS buffer, methanol was fixed for 15min, the biofilm was stained with 2% crystal violet solution for 15min, and the cells were decolorized with 33% glacial acetic acid. The absorbance was determined at 630 nm. The broth medium without bacteria was used as a negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion ability determination\u003c/h2\u003e \u003cp\u003e500 \u0026micro;L of BEAS-2B and 16-HBE cells at a concentration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37℃ and 5%CO\u003csub\u003e2\u003c/sub\u003e overnight. The cells were first cultured in serum-containing DMEM for 3 days, and then starved in serum-free DMEM culture for 12h. After washing with PBS, 1mL of \u003cem\u003eAcinetobacter\u003c/em\u003e Baumannii suspensions with a concentration of 1\u0026times;10\u003csup\u003e8\u003c/sup\u003eCFU/mL were added to each well, and 1mL of DMEM culture solution was added, mixed evenly, and incubated together for 2h. The cells were washed with PBS and fixed with 4% paraformaldehyde for 30min. The number of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii adherens on the cell surface was observed and counted under the microscope. Non-adhesive bacteria (\u0026le;\u0026thinsp;40 bacteria), adhesive bacteria (41\u0026ndash;100 bacteria), strongly adhesive bacteria (\u0026gt;\u0026thinsp;100 bacteria).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell iron death determination\u003c/h2\u003e \u003cp\u003e500 \u0026micro;L of BEAS-2B and 16-HBE cells at a concentration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37℃ and 5%CO\u003csub\u003e2\u003c/sub\u003e overnight. Add 500\u0026micro;L DMEM containing ammonium ferric sulfate (II) into each well (the final concentration of ammonium ferric sulfate (II) is 100 mol/L), and incubate in the incubator for 30 minutes. After washing the cells with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then treated with 1% Triton X-100 for 20 minutes. The cells were washed with PBS and incubated in an incubator with 500\u0026micro;L FerroGreen solution for 30 minutes. The intensity of each fluorescence signal was detected by GFP filter and BF filter in fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory cytokines detection\u003c/h2\u003e \u003cp\u003eThe supernatant was obtained by centrifugation at 1000g for 20 minutes. Standard and sample holes were set up according to TNF-α, IL-6 and IL-8 instructions(Mibio, Shanghai, China). Add 50\u0026micro;L of standard product with different concentration to the standard product hole, and add 50\u0026micro;L of sample to the sample hole. Horseradish peroxidase (HRP) labeled detection antibody 100\u0026micro;L was added to each well and incubated in an incubator at 37℃ for 60min. Add 300\u0026micro;L washing solution to each well and repeat washing for 5 times. Add 50\u0026micro;L of substrate A and substrate B to each well and incubate at 37℃ for 15min without light. The absorbance of each hole was measured at 450nm wavelength by adding 50\u0026micro;L terminating solution to each hole.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eWash the cells once with a pre-cooled PBS solution to remove as much excess fluid as possible. 500\u0026micro;L RIPA lysate(Beyotime Biotechnology, Shanghai, China) containing 1mM PMSF(Beyotime Biotechnology, Shanghai, China) was added to the cells, and the entire lysate process was performed on ice. The protein samples were separated by SDS-PAGE gel electrophoresis at 80V, 20 min, 120V, 50 min. The protein on the gel was transferred to the PVDF membrane(Millipore, Boston, MA, USA) by wet transfer method at 300mA for 2 hours. The transferred PVDF film was washed with TBST and sealed overnight with 5% skim milk(Sigma, Louis, MO, USA) powder solution at 4℃. On the second day, they were incubated at room temperature for 1 hour with first antibody diluent and second antibody diluent, respectively. The protein was developed with enhanced ECL chemiluminescence reagent (Vazyme, Shanghai, China), and the gray values of the protein bands were calculated by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFerroptosis was detected by electron microscopy\u003c/h2\u003e \u003cp\u003eCells were collected and added to the fixative precooled at 4\u0026deg;C and placed at 4\u0026deg;C overnight. The fixative was decanted, rinsed with phosphate buffer, and samples were fixed with 1% osmic acid solution for 2h. \"The samples were dehydrated with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) and finally treated with acetone. The samples were embedded in the embedding agent and cut by a microtome. The sections were stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 10min, respectively, and observed under a transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSince each experiment was performed three times, the data is given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. A two-tailed Student 'st test was used to assess the differences between the two groups. Analysis of Variance (ANOVA) is used to assess differences between multiple sets of data. P value lower than 0.05 was considered significant. SPSS 19.0 was used to analyze the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003epotential of hydrogen (pH), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), minimum inhibitory concentration (MIC), tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), Interleukin (IL), nuclear factor kappa-B (NF-\u0026kappa;B), achaete-scute family bHLH transcription factor 4 (ASCL4), cyclooxygenase-2 (COX2), lectin-type oxidized LDL receptor 1(LOX), glutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11), solute carrier family 3, member 3 (SLC3A2), Intensive Care Unit (ICU), phosphate-buffered saline (PBS).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Scientific Research Issues and Medical Technical Problems of China Medical Education Association (Grant No.2022KTM026) and Ministry of Education Higher Education Scientific Research Project (Grant No.ZJXF2022012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCX:\u0026nbsp;Experimental design\u0026nbsp;(lead); funding support\u0026nbsp;(lead).\u003c/p\u003e\n\u003cp\u003eYS:\u0026nbsp;Experimental operation\u0026nbsp;(lead); data analysis\u0026nbsp;(lead); article writing\u0026nbsp;(lead).\u003c/p\u003e\n\u003cp\u003eSL: Experimental design.\u003c/p\u003e\n\u003cp\u003eYC: Experimental operation.\u003c/p\u003e\n\u003cp\u003eHL: Experimental design.\u003c/p\u003e\n\u003cp\u003eYG: Data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eata availablity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available on request from the corresponding author\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks the China Medical Education Association for funding this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTeacher\u0026nbsp;of\u0026nbsp;Shenyang Medical College\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u0026mdash;The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Shenyang Medical College.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u0026mdash;Written informed consent was obtained from individual or guardian participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGuo XY, Liu XJ, Hao JY. Gut microbiota in ulcerative colitis: insights on pathogenesis and treatment. J Dig Dis. 2020 Mar;21(3):147-159.\u003c/li\u003e\n \u003cli\u003eFu Q, Song T, Ma X, et al. Research progress on the relationship between intestinal microecology and intestinal bowel disease. Animal Model Exp Med. 2022 Dec;5(4):297-310.\u003c/li\u003e\n \u003cli\u003eWang J, Liang J, He M, et al. Chinese expert consensus on intestinal microecology and management of digestive tract complications related to tumor treatment (version 2022). J Cancer Res Ther. 2022 Dec;18(7):1835-1844.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIsolauri E. Microbiota and Obesity. Nestle Nutr Inst Workshop Ser. 2017;88:95-105.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTennyson CA, Friedman G. Microecology, obesity, and probiotics. Curr Opin Endocrinol Diabetes Obes. 2008 Oct;15(5):422-7.\u003c/li\u003e\n \u003cli\u003eLee CR, Lee JH, Park M, et al. Biology of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options. Front Cell Infect Microbiol. 2017 Mar 13;7:55.\u003c/li\u003e\n \u003cli\u003eGiamarellou H, Antoniadou A, Kanellakopoulou K. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii: a universal threat to public health? Int J Antimicrob Agents. 2008 Aug;32(2):106-19.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePalethorpe S, Farrow JM, Wells G, et al. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System. J Bacteriol. 2022 Feb 15;204(2):e0049421.\u003c/li\u003e\n \u003cli\u003eRamirez MS, Bonomo RA, Tolmasky ME. Carbapenemases: Transforming \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii into a Yet More Dangerous Menace. Biomolecules. 2020 May 6;10(5):720-750.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYang CH, Su PW, Moi SH, et al. Biofilm Formation in \u003cem\u003eAcinetobacter\u003c/em\u003e Baumannii: Genotype-Phenotype Correlation. Molecules. 2019 May 14;24(10):1849.\u003c/li\u003e\n \u003cli\u003eZhang WX, Xiao CL. \u003cem\u003eStreptococcus\u003c/em\u003e strain D19T as a probiotic candidate to modulate oral health. BMC Microbiol. 2023 Nov 16;23(1):339-346.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLiu D, Xiao C, Li X, et al. \u003cem\u003eStreptococcus\u003c/em\u003e shenyangsis sp. nov., a New Species Isolated from the Oropharynx of a Healthy Child from Shenyang China. Curr Microbiol. 2021 Jul;78(7):2821-2827.\u003c/li\u003e\n \u003cli\u003eQi H, Liu D, Zou Y, et al. Description and genomic characterization of \u003cem\u003eStreptococcus\u003c/em\u003e symci sp. nov., isolated from a child\u0026apos;s oropharynx. Antonie Van Leeuwenhoek. 2021 Feb;114(2):113-127.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAlAmri AM, AlQurayan AM, Sebastian T, et al. Molecular Surveillance of Multidrug-Resistant \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii. Curr Microbiol. 2020 Mar;77(3):335-342.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTuan Anh N, Nga TVT, Tuan HM, et al. Molecular epidemiology and antimicrobial resistance phenotypes of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii isolated from patients in three hospitals in southern Vietnam. J Med Microbiol. 2017 Jan;66(1):46-53.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStrateva T, Sirakov I, Stoeva T, et al. Carbapenem-resistant \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii: Current status of the problem in four Bulgarian university hospitals (2014-2016). J Glob Antimicrob Resist. 2019 Mar;16:266-273.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRabin N, Zheng Y, Opoku-Temeng C, et al. Biofilm formation mechanisms and targets for developing antibiofilm agents. Future Med Chem. 2015;7(4):493-512.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021 Apr;22(4):266-282.\u003c/li\u003e\n \u003cli\u003eTang D, Chen X, Kang R, et al. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021 Feb;31(2):107-125.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDeng L, He S, Guo N, et al. Molecular mechanisms of ferroptosis and relevance to inflammation. Inflamm Res. 2023 Feb;72(2):281-299.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Streptococcus strain, Acinetobacter baumannii, antibacterial, ferroptosis, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-4692224/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4692224/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMicroecological equilibrium is essential for human health. Previous research has demonstrated that \u003cem\u003eStreptococcus\u003c/em\u003e strain A, the main bacterial group in the respiratory tract, can suppress harmful microbes and protect the body. In this study, \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e was isolated from the oral and pharyngeal cavities of healthy children. Its antibacterial mechanism against \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii was examined, as well as its potential to prevent inflammatory damage to cells. We evaluated the effect of the fermentation conditions of D19\u003csup\u003eT\u003c/sup\u003e on inhibition of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii growth; Isolation and purification of antibacterial active components of strain D19\u003csup\u003eT\u003c/sup\u003e and molecular mechanism of inhibition of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii; Molecular mechanism of D19\u003csup\u003eT\u003c/sup\u003e bacteriostatic protein reversing cellular inflammatory injury induced by \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe supernatant of fermentation broth of \u003cem\u003eStreptococcus\u003c/em\u003e D19T was the active component against \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii, but the bacteria had no antibacterial activity. The supernatant of D19\u003csup\u003eT\u003c/sup\u003e fermentation broth was precipitated by (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e solution, and the protein was the active antibacterial component. After gel filtration chromatography and anion gel filtration chromatography, the molecular weight of antibacterial protein was 53kD. D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein can improve cell membrane permeability, limit extracellular soluble protein release, inhibit \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii biofilm formation, and prevent \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii adhesion. \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii induces inflammatory damage to respiratory cells via ferroptosis, and the D19\u003csup\u003eT\u003c/sup\u003e antibacterial protein can counteract this damage, protecting the respiratory tract.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStreptococcus\u003c/em\u003e strain D19\u003csup\u003eT\u003c/sup\u003e, as a potential probiotic, inhibits the growth of \u003cem\u003eAcinetobacter\u003c/em\u003e baumannii and the inflammatory damage of respiratory cells, playing a protective role in human respiratory health.\u003c/p\u003e","manuscriptTitle":"A respiratory streptococcus strain inhibits Acinetobacter baumannii from causing inflammatory damage through ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-18 23:52:48","doi":"10.21203/rs.3.rs-4692224/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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