Combination of Antibiotic, Efflux Pump Inhibitor and Autolysis Inhibitor Protects Mice from S. aureus Peritonitis by Inhibiting α-hemolysin-induced MAPKs/NF-κB/NLRP3 Activation CURRENT STATUS: POSTED

Background: The infection of staphylococcus aureus (S. aureus) is difficult to treat, our aim was to investigate the antibacterial abilities of a combination of antibiotic cloxacillin, efflux pump inhibitor thioridazine and autolysis inhibitor tetracycline and its anti-inflammation properties through inhibiting α-hemolysin induced MAPKs/NF-κB/NLRP3 activation in vitro and in vivo. Methods: The antibacterial susceptibility test and three-dimensional checkerboard method were utilized to investigate the synergistic antibacterial activity of the three-drug combination cloxacillin/thioridazine/tetracycline, α-hemolysin test and scanning electron microscope were used to assay the inhibition effects of the combination on the secretion of α-hemolysin and membrane-derived vesicles production from S. aureus, Western blot and pharmacological inhibition assays were performed to test α-hemolysin -induced MAPKs/NF-κB/NLRP3 activation in macrophage in vitro. Results: In vitro, the three-drug combination showed synergistic antistaphylococcal activity; the subinhibitory concentration of the combination significantly inhibited the secretion of α-hemolysin related to the number of vesicles produced by S. aureus and significantly inhibited the expression of MAPKs/NF-κB/NLRP3 proteins in the macrophages induced by S. aureus α-hemolysin. In vivo, the drug combination significantly reduced bacterial colony-forming unit counts of the viscera in mouse peritonitis model of S. aureus infection, the combination therapy reduced the primary inflammatory pathology and the release of bacterial-stimulated cytokines such as IL-1β and TNF-α, and inhibited the expression of MAPKs/NF-κB/NLRP3 proteins in peritoneal macrophage. Conclusions: Combination of antibiotic, efflux pump inhibitor and autolysis inhibitor owns good antistaphylococcal activity and significantly inhibit staphylococcal α-hemolysin and its inflammation, thus, the combination is a novel strategy of antibacterial and anti-inflammation. The results of this study show that the three-drug combination can effectively inhibit the expression of Hlα at a lower concentration than the single drug, indicating that the three-drug combination can effectively reduce the risk of diseases caused by S. aureus Hlα. The drug combination inhibits the hemolytic activity bacterial protein expression levels of Hlα at concentrations below the MIC all these results indicate that the three-drug combination used here inhibits the bacteria and simultaneously inhibits the expression of the bacterial virulence protein Hlα.

Veterinary Diseases, College of Veterinary Medicine, Jilin University. Macrophage RAW264.7, purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). CXN, TZ and TC were purchased from Sigma-Aldrich and dissolved in sterile water at a concentration of 40,960 μg/ml under sterile conditions and stored at -20°C until use.

Antibacterial susceptibility test
To determine the minimum inhibitory concentration (MIC) of 8325-4, DU1090 versus CXN, TZ and TC, a micro-broth dilution method was used according to the CLSI (formerly known as NCCLS) guidelines.
Synergistic assays were performed in 96-well microtiter plates containing two antibacterial agents distributed along the transverse and longitudinal wells in a checkerboard pattern on a dioptric dilution basis. Each well contained 0.1 mL of a separate antimicrobial composition or broth control. Other conditions are the same as the MIC measurement conditions to determine the combined drug results.

Hemolysis test
S. aureus 8325-4/DU1090 was incubated in tryptic soy broth (TSB) at 37°C to the exponential growth phase (OD 600 nm = 2.5) with or without drug or drug combination. The culture was centrifuged and the supernatant was collected. 100 μl containing different concentrations of supernatant were preincubated in Eppendorf tubes with defibrin rabbit red blood cells (25 μL) and 875 μl hemolysis buffer (20 mM CaCl 2 , 0.125 M NaCl) for 30 min at 37°C. Hemolysis buffer was used as a negative control.
After centrifugation, the supernatant was removed and the absorbance was measured at 450 nm.
S. aureus strain 8325-4/DU1090 was incubated in tryptic soy broth (TSB) at 37°C to the exponential growth phase (OD 600 nm = 2.5) with or without drug combination. The culture was centrifuged at 8,500 g for 30 min at 4°C, and the culture supernatant (900 μL) was incubated with 100 μL of LTCA for 12 h at 4°C. The supernatant protein was collected and centrifuged at 8,500 g for 70 min at 4°C.
The extracted SMVs is then repeatedly frozen and thawed until broken. Protein Hlα was tested by Western blot analysis. The primary antibody recognizing Hlα was purchased from Sigma-Aldrich.

Scanning electron microscope.
Scanning electron microscopy (SEM) was used to analyze the number of spherical vesicles associated with the surface of S. aureus. Fixation of the cells should be fixed with 2.5% glutaraldehyde for 30 minutes at -4°C. Then, fixed with 1% osmium tetroxide at -4°C for 30 min, dehydrated with a graded ethanol series, dried at a critical point, and covered with a gold film by sputtering. Floor. The sample was then analyzed by a scanning electron microscope.

Vesicle extraction separation
S. aureus 8325-4/DU1090 was incubated with drug-pretreated S. aureus 8325-4/DU1090. It was overnight at 37°C at 200 rpm in an orbital shaker. The S. aureus strain was grown in 200 ml of LB medium and grown to an optical density (OD 600 ) of 2.0. Then, after a series of gradient centrifugation and filtration, the remaining supernatant was passed through a Beckman cryogenic ultracentrifuge (Beckman, USA) at 100,000 g for ultra-centrifugation at 4°C for 1 h to obtain membrane vesicles.

Western blot analysis
Bubble-treated RAW264.7 cells (1 × 10 6 ) obtained from S. aureus 8325-4/DU1090 (E:T ratio, 5:1) or pure Hlα (100 μg/ml, Sigma Aldrich) or ultracentrifuge). The drug combination (CXN: 0.015625 μg/ml, TZ: 0.25 μg/ml, TC: 0.03125 μg/ml) was present or not at 37°C for 6 h. Total protein was collected and the total protein concentration was determined by the BCA method. The same amount of protein was subjected to SDS-PAGE gel electrophoresis, and an ECL imaging system was used for image acquisition.

Mouse S. aureus peritonitis model
We used the previously described and widely used mouse peritonitis model to measure in vivo antibacterial effects (14,15). In short, 6-week-old BALB/c female mice were inoculated with resuspended S. aureus (per 50 μL 2 × 10 8 colony-forming unit (CFU). Each experimental group contained 30 mice. To further investigate the therapeutic effect of the drug combination in vivo, 100 μL of drug or PBS was administered subcutaneously at 2 hours after infection with S. aureus 8325-4 / DU1090, and then administered at 12 hour intervals. After 72 h, the infected mice were euthanized. In vivo, the concentration of the drug combination treatment group was: CXN (1.6125 mg/kg/d), TC (3.125 mg/kg/d), TZ (25 mg/kg/d). Peritoneal macrophages were extracted and counted. Ascites colony counts of each group were performed. For histopathological analysis, formalin-fixed liver and spleen lung tissues were stained with hematoxylin and eosin and visualized using an Olympus BX53 fluorescence microscope with a 20 × objective. Western blot analysis of protein extracted from peritoneal macrophages.

Statistical analysis
Comparisons of mean values from three experiments were statistically evaluated by analysis of variance, followed by One-Way ANOVA analysis. All statistical analyses were performed using SPSS software (version 11.5; SPSS).

Drug MIC and FICI measurement results
To test the respective MICs of drug CXN, TZ and TC against S. aureus strains in vitro, we conducted a drug sensitivity test. The results showed that the MICs of CXN against S. aureus strain 8325-4 and DU1090 (an Hlα-deleted strain) were 0.125 μg/mL and 0.125 μg/mL respectively; the MICs of TZ against strain 8325-4 and DU1090 were 16 μg/mL and 32 μg/mL, respectively; the MICs of TC against strain 8325-4 and DU1090 were 0.125 μg/mL and 0.125 μg/mL, respectively (Table 1). To test the synergistic antibacterial activity of combination CXN, TZ and TC in vitro, we conducted a threedimensional checkerboard method. The FICI results of the drug combination showed the two-drug combination of a certain concentration of CXN and TC, CXN and TZ, TZ and TC showed an additive effect on strain 8325-4, DU1090 (Table 2). Whereas the three-drug combination of a certain concentration of CXN, TZ and TC showed synergistic effect on strain 8325-4, and DU1090 (Table 2), for their sum FICI is less than 1.0 [19,20] , among them, we chose the synergistic three-drug combination of MIC CXN : MIC TZ : MIC TC = 0.03125 μg/mL: 0.0625 μg/mL: 0.5 μg/mL as a candidate for further screen of three-drug prescription having simultaneous antibacterial and hemolysis-inhibiting activities.

Subinhibitory concentration of three-drug combination inhibits hemolytic activity of Hlα in vitro
To test the effect of drug combination on S. aureus Hlα production, but not inhibiting the bacterial growth, we selected a subinhibitory concentration of three-drug combination (1/2 MIC CXN : 1/2 MIC TZ : 1/2 MIC TC = 0.015625 μg/mL: 0.03125 μg/mL: 0.25 μg/mL) for further experiments including Western blotting, hemolytic activity, and so on. Western blot analysis showed that CXN, TZ or TC alone at the concentration of subinhibitory three-drug combination inhibited the expression of Hlα of S. aureus strain 8325-4 strain to varying degrees, the subinhibitory concentration of three-drug combination of CXN, TZ and TC significantly inhibited the expression of Hlα (Fig. 1A -B). Further blood plate experiment also verified that subinhibitory concentration of three-drug combination that of CXN, TZ and TC had best hemolysis-inhibiting activities (Fig. 1E). Moreover, By measuring the absorbance of the supernatant at 450 nm with a spectrophotometer (Fig. 1C -D), we found that CXN, TZ or TC alone had a certain of hemolysis-inhibiting activities, we also found subinhibitory concentration of threedrug combination had best hemolysis-inhibiting effect.

Subinhibitory concentration of three-drug combination inhibits the production of SMVs
Recent research evidence indicates that S. aureus secretes Hlα-containing SMVs to the outside of the cell, and Hlα is closely related to SMVs. In our study, we observed S. aureus strain 8325-4 produced much more SMVs on the surface than that of strain DU1090, wheras the drug CXN, TZ or TC alone inhibited the production of SMVs to varying degrees, especially, the subinhibitory concentration of three-drug combination treatment significantly inhibited the production of vesicles in strain 8325-4 than that of drugs alone treatment ( Fig. 2A, 2D). In addition, we compared the expression of Hlα in the SMVs extracted from strain 8325-4 treated by different drug treatments, the results showed that Hlα was expressed in the SMVs isolated from strain 8325-4, and the subinhibitory concentration of three-drug combination significantly inhibited the expression of Hlα, however, the expression of Hlα in SMVs was not found in strain DU1090 (Fig. 2B -C). These results suggest that Hlα is associated with SMVs, and that the subinhibitory concentration of three-drug combination suppresses the production of SMVs and the expression of Hlα mediated by SMVs.

Subinhibitory concentration of three-drug combination inhibits MAPKs, NF-κB and NLRP3 pathways in macrophage induced by S. aureus, SMVs or Hlα
In order to detect the effects of the three-drug combinations on the inflammatory pathway in S. aureus, Hlα or vesicle-induced RAW264.7 cells, we performed Western blot analysis on the expression of MAPKs (Fig. 3A), NF-κB (Fig. 3B) and NLRP3 (Fig. 3C)

MAPKs (P38 and ERK1/2) mediates Hlα-stimulated activation of NF-κB and NLRP3 inflammasome in macrophage
Further experiments were performed to examine the upstream and downstream relationship between the MAPKs, NF-кB and NLRP3 in the Hlα-activated inflammation pathways, we tested the expression of each pathway proteins in macrophages treated with Hlα under pretreatment using the P38 inhibitor SB 203580, the ERK1/2 inhibitor PD 98059 and the JNKs inhibitor SP 600125, the NF-кB inhibitor BAY increase was significantly decreased after three-drug combination treatment (Fig. 5C).
Next, we performed Western blot analysis on the extracted mouse peritoneal macrophages (Fig. 6A).
The results showed that the three-drug combination significantly reduced the levels of p-JNK, p-ERK1/2, p-P38, NF-κB, IKK, ASC, IL-18, IL-1β, NLRP3 and caspase-1 (Fig. 6B). Serum ELISA analysis showed that the expression of IL-1β and the expression of TNF-α in the serum of the 8325-4-infected group were higher than those in strain DU1090-infected group and the blank group at 72 h and at 144 h, respectively, and the values of strain DU1090-infected group were higher than those in the blank group, however, the counterpart of the three-drug combination treatment were significantly decreased in strain 8325-4-infected group and in strain DU1090-infected group (Fig. 6C -F

Discussion
A number of studies have shown that combined treatment can not only improve the antibacterial effect, but also reduce the toxicity caused by excessive concentration of a single drug (16). In this study, a three-drug combination consist of CXN, TZ and TC, was used to develop an efficient antistaphylococcal prescription. The results of this study indicate that the combination of CXN, TZ and TC has a synergistic effect against S. aureus (Table 1,  S. aureus produces potent hemolysins (17) and leukotoxins. S. aureus secretes membrane-derived vesicles (SMVs) that deliver virulence factors to host cells and induce cytopathology (18). Thus, the screen of anti-Hlα drugs is of great importance for the elimination of bacterial toxicity. The results of this study show that the three-drug combination can effectively inhibit the expression of Hlα at a lower concentration than the single drug, indicating that the three-drug combination can effectively reduce the risk of diseases caused by S. aureus Hlα. The drug combination inhibits the hemolytic activity and bacterial protein expression levels of Hlα at concentrations below the MIC (Fig. 1  MAPKs mediates activation of NF-κB and NLRP3 inflammasome induced by Hlα in MФ.
Upstream and downstream relationship between MAPKs, NF-кB and NLRP3 pathways.

Supplementary Files
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