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

BMC Microbiology volume 24, Article number: 437 (2024) Cite this article

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 D19T 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 D19T on inhibition of Acinetobacter baumannii growth; Isolation and purification of antibacterial active components of strain D19T and molecular mechanism of inhibition of Acinetobacter baumannii; Molecular mechanism of D19T antibacterial 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 D19T fermentation broth was precipitated by (NH4)2SO4 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. D19T 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 D19T antibacterial protein can counteract this damage, protecting the respiratory tract.

Conclusion

Streptococcus strain D19T, 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.

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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 [3,4,5].

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 [8,9,10]. 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 [11,12,13]. 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 D19T 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 D19T inhibited the growth of Acinetobacter baumannii

As shown in Fig. 1-A, both Streptococcus strain D19T fermentation broth and supernatant could inhibit the growth of Acinetobacter baumannii, but neither live or inactivated bacteria of D19T could inhibit the growth of Acinetobacter baumannii. Therefore, we speculated that the supernatant of fermentation broth of strain D19T was the active antibacterial component of Acinetobacter baumannii. In addition, we also optimized the fermentation conditions of strain D19T. When the pH value of the culture medium was 7.0, the fermentation temperature was 37℃, and the fermentation time was 24 h, the supernatant of the fermentation liquid of strain D19T obtained the greatest antibacterial effect, and the diameter of its inhibitory Acinetobacter baumannii was 22.09 ± 21 mm (Fig. 1-B, C, D).

Fig. 1
figure 1

D19T fermentation broth antibacterial assay and optimization of fermentation conditions. (A) Detection of antibacterial components in D19T fermentation broth. (B) Effect of fermentation broth pH on the bacteriostasis of D19T. (C) Effect of fermentation temperature on the bacteriostasis of D19T. (D) Effect of fermentation time on the bacteriostasis of D19T. (a) Fermentation broth. (b) Fermentation broth supernatant. (c) Live bacterial body. (d) Inactivated bacterial body

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D19T antibacterial components identification and purification

D19T fermentation broth was centrifuged to remove bacteria to obtain the supernatant, adding (NH4)2SO4 solution of different saturation to obtain the supernatant and precipitation. As shown in Fig. 2-A, the supernatant after the salting out of (NH4)2SO4 solution of different saturation has no antibacterial activity, while the precipitation after the salting out of (NH4)2SO4 solution of 30%~70% saturation has antibacterial activity. (NH4)2SO4 solution saturation of 50% had the strongest antibacterial activity. The fermentation supernatant was precipitated with 50% saturated (NH4)2SO4 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).

Fig. 2
figure 2

Isolation and purification of antibacterial active components of D19T. (A) Antibacterial test of fermentation broth supernatant precipitated by (NH4)2SO4 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 antibacterial detection. (E) Molecular weight determination of antibacterial protein. (F) Antibacterial activity was detected by disk dispersion method. (G) D19T growth and production of antibacterial protein activity curve. (H) Culture medium supernatant and antibacterial protein activity detection at 37 °C. (I) Salt concentration against antibacterial protein activity detection. p.a.: peak a, pb peak b, pc peak c

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D19T entered a stable growth phase at 18 h and commenced the production of antibacterial proteins. By 24 h, the levels of the antibacterial protein peaked (Fig. 2-G). The antibacterial activity exhibited by the antibacterial protein was sustained for five days at 37 °C, with no statistically significant difference observed between the antibacterial protein and the culture supernatant (Fig. 2-H). Low concentrations of salt did not affect antibacterial properties of the antibacterial protein; however, a significant reduction in their efficacy was noted at salt concentrations exceeding 0.2 mol/L (Fig. 2-I).

D19T antibacterial protein’s inhibition mechanism against Acinetobacter baumannii

The minimum inhibitory concentration (MIC) of D19T was determined to be 15 mg/mL. Then, Acinetobacter baumannii extravasated DNA and RNA levels increased significantly at D19T 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 D19T 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 D19T antibacterial protein ≥ 1/2MIC (p < 0.05) (Fig. 3-C and D).

Fig. 3
figure 3

Mechanism of D19T antibacterial protein inhibiting the growth of Acinetobacter baumannii. (A) Effect of D19T antibacterial protein on Acinetobacter baumannii DNA and RNA extravasation. (B) Effect of D19T antibacterial protein on soluble proteins of Acinetobacter baumannii. (C) Effect of D19T antibacterial protein on adhesion of Acinetobacter baumannii. (D) Effect of D19T antibacterial protein on Acinetobacter baumannii biofilm

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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).

Fig. 4
figure 4

Acinetobacter baumannii induces ferroptosis in respiratory epithelial cells. (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 Figures S1 and S2

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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).

Fig. 5
figure 5

Acinetobacter baumannii induces inflammatory injury of respiratory epithelial cells. 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 Figures S3 and S4

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D19T antibacterial protein reverses the ferroptosis of respiratory epithelial cells induced by Acinetobacter baumannii

The effect of D19T 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, D19T 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).

Fig. 6
figure 6

Effect of D19T antibacterial protein on ferroptosis of respiratory epithelial cells induced by Acinetobacter baumannii. (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 Figures S5 and S6

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D19T antibacterial protein reverses the inflammatory injury of respiratory epithelial cells induced by Acinetobacter baumannii

Similarly, we also analyzed the effect of D19T antibacterial protein on inflammatory factors in BEAS-2B and 16HBE cells infected with Acinetobacter baumannii. As shown in Fig. 7-A to C, D19T 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 D19T 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 D19T antibacterial protein was ≥ 1/2MIC (p < 0.05)(Fig. 7-D).

Fig. 7
figure 7

Effect of D19T antibacterial protein on inflammatory injury of respiratory epithelial cells induced by Acinetobacter baumannii. 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 Figures S7 and S8

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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 initially confirmed the inhibitory effect of the D19T mixture on Acinetobacter baumannii through antibacterial experiments. Subsequently, we isolated the fermented bacterial broth and assessed the antibacterial activity of each fraction. The results indicated that neither live nor inactivated D19T cells exhibited any inhibitory effect on Acinetobacter baumannii; however, the supernatant obtained after cell removal demonstrated significant inhibitory activity against this pathogen. We then proceeded to separate and purify both protein components and other chemical constituents using ammonium sulfate precipitation followed by gel chromatography. Notably, a specific protein within the D19T fermentation broth was identified as being active against Acinetobacter baumannii. We investigated the biological characteristics of this antibacterial protein and discovered that it was produced during the stable growth phase of D19T, with optimal inhibitory effects observed at 24 h post-cultivation. Furthermore, we found that the antibacterial activity of this protein could be sustained for up to five days at 37 °C. While variations in salt concentration within the antibacterial protein solution did not enhance its antimicrobial efficacy, an increase in salt concentration adversely affected its activity. Consequently, purified D19T protein underwent dialysis to eliminate excess salt prior to further analysis. We also found that D19T antibacterial protein increased the extravasation of macromolecules such as DNA and RNA in Acinetobacter baumannii broth. This suggests that the D19T antibacterial protein affects the stability of Acinetobacter baumannii cell membrane and increases its permeability. In addition, we found that the D19T 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 D19T 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. D19T 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 D19T 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 D19T antibacterial protein can reverse the inflammatory injury of respiratory epithelial cells induced by Acinetobacter baumannii.

In this study, we confirmed that D19T, as the dominant bacteria in the normal human respiratory tract, could be used as a microecological agent for clinical treatment and prevention of Acinetobacter baumannii infection in the future. Although we have successfully isolated and purified the bacteriostatic protein from D19T fermentation broth, the biological characteristics of the protein, such as amino acid composition, spatial conformation and active site, need to be further explored.

Conclusions

Acinetobacter baumannii is an important pathogen of nosocomial infection. It causes inflammatory damage to the respiratory tract through ferroptosis. Streptococcus strain D19T 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 D19T was collected from the oropharynx of healthy children and cultured in bacto brain heart infusion medium(Solarbio, Beijing, China) at 37 °C, 180 r/min, for 18 h. 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 °C, 180 r/min, for 18 h.

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 °C and 5% CO2.

Antibacterial assay

The concentration of Acinetobacter baumannii bacterial solution in the logarithmic phase was adjusted to 1 × 106 CFU/mL, and 100 µL was removed and evenly spread on nutrient AGAR plates. Using the disk diffusion method, 200 µL 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

D19T single colonies were inoculated into brain heart infusion medium and incubated at 37 °C, 180 r/min, for 18 h. The inhibitory diameter was measured under different fermentation conditions with different pH (3–11), fermentation temperature (28–43 °C) and fermentation time (12–60 h).

Antibacterial components determination

Antibacterial component determination: D19T fermentation broth was centrifuged at 4 °C and 12,000 r/min for 10 min to remove bacterial cells and obtain the supernatant of the fermentation broth. Add (NH4) 2SO4 solution to the supernatant to a saturation of 30%, and precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12,000 r/min for 10 min. 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 (NH4) 2SO4: The method for obtaining the supernatant of D19T fermentation broth is consistent with the method mentioned earlier. Add (NH4) 2SO4 solution to the supernatant, and the saturation of (NH4) 2SO4 in the final supernatant ranges from 20 to 80%. Precipitate overnight at 4 °C. The next day, the fermentation broth was centrifuged at 12,000 r/min for 10 min. 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 (NH4) 2SO4 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 280 nm to determine the inhibitory diameter. The antibacterial 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. 100 µL protein solution was added to 96-well plate, and 10 µL pathogen solution (1 × 106 CFU/mL) was added to each well. After mixing, the 96-well plate was cultured in a bacterial incubator at 37 °C for 24 h. 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 2 µL of distilled water to the optical fiber surface, lower the measuring arm, and set to zero. Add 1.0 µL 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 10 min, mixed upside down once every 2 min, and the lysed cells released soluble proteins. Protein samples of 20 µL were added for SDS-PAGE electrophoresis, dyed with Coomassie bright blue (Beyotime Biotechnology, Shanghai, China) for 40 min, decolorized with distilled water, and photographed for analysis of protein expression.

Biofilm formation determination

100 µL Acinetobacter baumannii solution at a concentration of 1 × 106 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 °C for 24 h. The culture medium and non-adherent bacteria were washed with PBS buffer, methanol was fixed for 15 min, the biofilm was stained with 2% crystal violet solution for 15 min, 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 × 104 cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37 °C and 5% CO2 overnight. The cells were first cultured in serum-containing DMEM for 3 days, and then starved in serum-free DMEM culture for 12 h. After washing with PBS, 1 mL of Acinetobacter Baumannii suspensions with a concentration of 1 × 108 CFU/mL were added to each well, and 1mL of DMEM culture solution was added, mixed evenly, and incubated together for 2 h. The cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. 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 × 104 cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37 °C and 5% CO2 overnight. Add 500 µL 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 min. After washing the cells with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and then treated with 1% Triton X-100 for 20 min. The cells were washed with PBS and incubated in an incubator with 500 µL FerroGreen solution for 30 min. 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 1000 g for 20 min. Standard and sample holes were set up according to TNF-α, IL-6 and IL-8 instructions(Mibio, Shanghai, China). Add 50 µL of standard product with different concentration to the standard product hole, and add 50 µL of sample to the sample hole. Horseradish peroxidase (HRP) labeled detection antibody 100 µL was added to each well and incubated in an incubator at 37 °C for 60 min. Add 300 µL washing solution to each well and repeat washing for 5 times. Add 50 µL of substrate A and substrate B to each well and incubate at 37 °C for 15 min without light. The absorbance of each hole was measured at 450 nm wavelength by adding 50 µL 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. 500 µL 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 80 V, 20 min, 120 V, 50 min. The protein on the gel was transferred to the PVDF membrane(Millipore, Boston, MA, USA) by wet transfer method at 300 mA for 2 h. 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 h 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 2 h. 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 10 min, 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’s t-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.

Data availability

All data included in this study are available on request from the corresponding author.

Abbreviations

pH:

Potential of hydrogen

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

MIC:

Minimum inhibitory concentration

TNF-α:

Tumor necrosis factor-α

IL:

Interleukin

NF-κB:

Nuclear factor kappa-B

ASCL4:

Achaete-scute family bHLH transcription factor 4

COX2:

Cyclooxygenase-2

LOX:

Lectin-type oxidized LDL receptor 1

GPX4:

Glutathione peroxidase 4

SLC7A11:

Solute carrier family 7, member 11

SLC3A2:

Solute carrier family 3, member 3

ICU:

Intensive Care Unit

PBS:

Phosphate-buffered saline

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Acknowledgements

The author thanks the China Medical Education Association for funding this study.

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).

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Authors and Affiliations

  1. Key Lab of Environmental Pollution and Microecology of Liaoning Province, Shenyang Medical College, 146 Huanghe North Street, Yuhong District, Shenyang, 110034, China

    Ye Sun, Shuyin Li, Yuchen Che, Hao Liang & Chunling Xiao

  2. School of Health Management, Shenyang Vocational and Technical College, 32 Laodong Road, Dadong District, Shenyang, 110045, China

    Yi Guo

Authors
  1. Ye Sun
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  2. Shuyin Li
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  3. Yuchen Che
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  4. Hao Liang
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  5. Yi Guo
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  6. Chunling Xiao
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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.

Corresponding author

Correspondence to Chunling Xiao.

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Ethical 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.

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Written informed consent was obtained from individual or guardian participants.

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The authors declare no competing interests.

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Sun, Y., Li, S., Che, Y. et al. A respiratory Streptococcus strain inhibits Acinetobacter baumannii from causing inflammatory damage through ferroptosis. BMC Microbiol 24, 437 (2024). https://doi.org/10.1186/s12866-024-03589-7

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Keywords

BMC Microbiology

ISSN: 1471-2180

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