Bacterial strains, plasmid, and grow conditions.
The bacterial strains and plasmids used in this study were listed in Table 1. Staphylococcus aureus strain SA75 was isolated from a patient with a purulent skin infection at the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). Identification of the isolates was carried out using a VITEK-2 microbiology analyzer according to the manufacturer’s instructions (bioMérieux, Marcy l’Etoile, France). S. aureus and its derivative strains were grown in tryptic soy broth (TSB, BD) medium with 10 mg/l chloramphenicol at 37℃ with shaking at 220 rpm. Escherichia coli was grown in Luria broth (LB, Oxoid) medium with appropriate antibiotics (ampicillin at 100 mg/l and anhydrotetracycline at 50 ng/ml).
Construction of S. aureus sarX Mutant (SA75ΔsarX) and Complemented strain (SA75ΔsarX-C)
The sarX deletion mutant of the SA75 strain was constructed by allelic replacement using the temperature-sensitive plasmid pKOR1. The upstream and downstream fragments of sarX were amplified from SA75 genomic DNA using the sarX-UP-F/sarX-UP-R and sarX-DOWN-F/sarX-DOWN-R sets of primers (Table 2). The amplified products were digested with KpnⅠ and then ligated with T4 DNA ligase to yield the homologous arm fragment with the deletion of sarX gene and then cloned into pKOR1. The recombinant plasmid pKOR1-ΔsarX was successively transferred into E. coli DH5α and DC10B, ultimately electroporated into the S. aureus SA75 competent cells. The allelic replacement mutants were selected using a previously described method and were further confirmed by PCR and sequencing .
To construct the sarX chromosomal complementation strain, the fragments covering the truncated region in the mutant strains were amplified from S. aureus strain SA75 genomic DNA using the sarX-F/sarX-R sets of primers (Table 2). The fragments of each gene were digested with restriction enzymes and then cloned into PRB473. The resulting plasmids, PRB473-sarX were electroporated into the mutant strain. The allelic replacement complementation strains were selected using the same method described above and were further confirmed by PCR and sequencing.
RNA isolation and quantitative real-time RT-PCR analysis
RNA isolation was performed as previously described. Overnight cultures were inoculated to an optical density of 0.01 into fresh TSB medium. Total RNA was isolated and purified using a PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, United States); then was reverse transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan), according to the manufacturer’s protocol using the oligonucleotides shown in Table 2. The real-time PCR was carried out using a SsoFas EvaGreen Supermix kit (Bio-Rad, United States) with the Bio-Rad CFX96 Manager software. SA75 wild type strain was used as a control (relative expression = 1), and gyrB was used as a reference gene to investigate genes of interest. RNA transcript levels were calculated by the method of delta delta Ct (ΔΔCt). Data analysis was carried out using Bio-Rad CFX software. Each reaction was performed in triplicate.
Biofilm formation and analysis
Biofilm formation was determined by the microtiter plate assay based on a previously described method. The overnight cultures were diluted 1:200 in fresh TSB. Two hundred microliter of the diluted cultures of different bacteria were pipetted into sterile 96-well polystyrene plates (BD Biosciences) with three attached wells of each bacteria, then incubated overnight at 37℃ without shaking. Then, the wells were washed gently three times with phosphate-buffered saline (PBS) (to remove nonadherent cells). Methanol (99.5%) was used to stabilized biofilms. The wells were stained with 200 μl of a 1% (w/v) crystal violet (Sigma) for 10 min, and then again washed three times with water. After drying, 30% glacial acetic acid was used to release the biofilm into the solution. The optical density at 600 (OD600) was recorded.
Scanning Electron Microscopy
Biofilm formed on glass coverslips (10 mm in diameter) was observed by scanning electron microscopy (SEM) as previously described. Briefly, overnight cultures were diluted 1:200 with TSB. A glass disk was put in advance into the bottom of the sterile flat bottomed polystyrene plates (Costar 3524; Corning, NY, United States) and 1 ml of the cell suspensions was added into the wells, with static culture at 37℃ for 24 h. The bacteria were cultured under the same conditions for colony counting to ensure that the amount of bacteria was consistent and the next experiment was carried out. Each well was rinsed three times with sterile PBS, each time for 10 min with slight shaking. Biofilms formed on the glasses were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) by incubation for 2 h at room temperature. Next, the coverslips were washed three times with sterile PBS for 15 min. Thereafter, 1% osmium tetroxide was used for a post-fixation step for 2 h at 4℃, followed by three times washed with distilled water for 10 min each, and then dehydrated in a series of ascending ethanol baths (25, 50, 75, 95 and 100%) for 10 min each. The coverslips were placed in a freeze-dried apparatus for 5 h, and the gold spray was taken out for 3 min. The observations were usually performed with a scanning electron microscope (FEI Quanta 200; United States). The experiment was repeated three times.
Triton X-100 Induced Autolysis
Autolysis assays were performed as described by Brunskill and Bayles. The overnight cultures were cultured in 50 ml TSB containing 1 M NaCl until they reached the early exponential phase. Following centrifugation (4000g, 20min), the cells were washed twice with 50 ml of ice-cold water and resuspended into 50 ml of Tris-HCl (pH 7.2) containing 0.05% (vol/vol) TritonX-100. The OD600 value was adjusted to 1.0, followed by shaking of the culture at 37 ℃ for 3 h and was measured at 30 min intervals. The experiment was repeated three times.
Western Blot analysis
The protein expression level of Staphylococcus protein A (SPA) was determined by western blot analysis as previously described. Culture supernatant was collected and washed twice with sterile PBS. Then, supernatant was suspended in 100 μl of PBS incubation at 37℃ for 2 h with 3 μl of lysostaphin (1 mg/ml) and centrifuged (8000 g, 30 min, 4 ℃). The protein concentration of each sample was determined by Bradford dye binding method. The samples were separated by 12% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat milk in PBST buffer at room temperature for 1 h, the membrane was incubated with Anti-Protein A (HRP) antibody at a 1/5000 dilution. The image was visualized using the ECL Western blotting assay kit (GE Healthcare) and the Chemi doc TM XRS system (Bio rad, USA).
Polysaccharide intercellular adhesion (PIA) extracted from S. aureus was detected by a dot blot assay with a germ agglutinin horseradish peroxidase (WGA-HRP) conjugate according to a previously described protocol. Briefly, overnight cultures were diluted to 107 colony-forming units (CFU) and added to 2 ml TSB at a ratio of 1:100 in the six-well polystyrene plates. An equal number of cells from each culture was resuspended in 0.5 M EDTA (pH 8.0) and incubated for 5 min at 100°C and centrifuged, and 40µl of the supernatant was incubated with 10µl of proteinase K (20 mg/ml) (Sangon Biotech) at 37°C for 2 h to minimize nonspecific background. Pipette 10 μl of the extracted PIA sample onto the PVDF membrane after formaldehyde treatment, keep the PVDF membrane moist during the spotting process, and dry the membrane at room temperature after spotting. The membrane was blocked with 3.5% bovine serum albumin (BSA) in PBS with 0.1% Tween 20. The membrane was placed in a dish containing wheat germ agglutinin conjugated to WGA-HRP, and incubated at 37 °C for 1 h. Then color developed with Pierce enhanced chemiluminescence (ECL) Western blotting substrate (Thermo Scientific, Rockford, IL).
All experiments were performed in biological triplicate. Statistical analysis was performed using SPSS statistical software (version23; IBM SPSS Statistic) and GraphPad Prism 8 (version 8.00, La Jolla, CA, United States). For the comparison of two groups, unpaired t test was used; for three or more groups, 1-way or 2-way analysis of variance was used, as appropriate. A value of P < 0.05 was considered statistically significant.