Bacterial strains, plasmids, growth conditions, and chemicals
All of the bacterial strains and plasmids used in this study are shown in Table 3. E. faecalis ATCC 47077 (OG1RF; GenBank accession number CP002621.1) and ATCC 29212 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). E. faecalis strains were cultured in tryptic soya broth (TSB; Oxoid, Basingstoke, UK) at 37℃ with shaking at 220 rpm. Glucose was added to the TSB medium at a concentration of 0.25% for biofilm formation detection. Electroporation was used for plasmid transformation, and B2 medium (1% casein hydrolysate, 2.5% yeast extract, 0.5% glucose, 2.5% NaCl, 0.1% K2HPO4, pH 7.5) was used for bacteria recovery. The antibiotics used in this study were
purchased from Sigma Chemical Co. (St Louis, MO, USA) and used at concentrations of 20 mg/L for chloramphenicol and 750 or 25 mg/L for erythromycin.
Construction of △clpP mutants and complemented strains
The clpP deletion mutant of the OG1RF strain was constructed by in-frame deletion using the temperature-sensitive plasmid pJRS233 as previously described [48]. Briefly, the upstream and downstream fragments of OG1RF_10505 (gene: clpP; product: ATP-dependent Clp protease proteolytic subunit), which is highly homologous (86.8%) to SA0723 (product as the ClpP protease) of S. aureus N315 strain [23], were amplified from OG1RF by PCR and separately cloned into the pJRS233 vector to generate pJRS233-ΔclpP. The recombinant plasmid pJRS233-ΔclpP was successively transferred into E. coli DH5α and OG1RF by electroporation, and then the transformants selected at 30°C on Erm. Chromosomal integrants were selected by growth at 42°C in the presence of Erm. Selection for excision of the integrated plasmid by homologous recombination was accomplished by growing the bacteria at 30°C in the absence of Erm. The complemented ΔclpP mutant strain was constructed using the E. coli -Streptococcus shuttle vector pIB166. The clpP gene was amplified by PCR and cloned into the pIB166 vector to produce pIB166:: clpP. The recombinant plasmid pIB166:: clpP was transformed by electroporation into the ΔclpP mutant strain, forming the complemented ΔclpP/pIB166:: clpP strain. The ΔclpP strain containing the empty vector pIB166 was designated the ΔclpP/pIB166 mutant. The ΔclpP mutant and complemented ΔclpP mutant strain were identified by PCR, RT-qPCR, and direct sequencing. The primers used in this assay are listed in Table 4.
Growth analysis of the △clpP mutant strain
The OG1RF, ΔclpP, ΔclpP/pIB166:: clpP, and ΔclpP/pIB166 strains were cultured in TSB at 37°C with shaking for 12 h and diluted in the same medium to an OD600 value of 1.5, then 50 μL aliquot of the diluted suspension was inoculated into 10 mL fresh TSB and incubated at either 37°C, 45°C or 20°C with circular agitation (220 rpm). The diluted suspension was also inoculated into fresh TSB with 5% NaCl pH 5.5 or 2 mM H2O2 and incubated at 37°C with circular agitation (220 rpm). OD600 values for the cultures were determined using an Eppendorf Biospectrometer (Eppendorf, Hamburg, Germany) at 1-h intervals. Three independent experiments were performed.
The sensitivity of the △clpP mutant strain to SDS
Overnight cultures of E. faecalis strains were diluted 1:200 in fresh TSB medium and incubated at 37°C for 4 h until an OD600 of 1.0 was reached. After 10-fold serial dilution, 5 μL of the aliquot was spotted onto a TSB agar plate containing 0.008% SDS and incubated at 37°C for 24 h. Bacterial colonies on the plates were photographed and counted [28]. Three independent experiments were performed, and representative results are shown.
Microtiter plate assay of biofilm formation
The biofilm-forming ability of E. faecalis isolates was detected as previously described with modifications [49]. Overnight cultures were diluted 1:200 in 200 μL of TSBG (TSB with 0.25% glucose) and inoculated into 96-well polystyrene microtiter plates. After 12, 24, or 48 h of static incubation at 37°C, the supernatant was discarded, and plates were washed thrice with deionized water to remove unattached cells, stained with 1% CV for 20 min at room temperature, and rinsed with distilled water. Finally, the CV was solubilized in ethanol-acetone (80:20, vol/vol), and absorbance at OD570 was determined. Three independent experiments were performed.
Quantification of eDNA
eDNA was quantified as described previously [50]. Overnight cultures of E. faecalis strains were diluted to OD600 = 0.001 in AB medium supplemented with 0.5% glucose, 0.05 mM propidium iodide (PI) and 10% TSB. The diluted cultures were transferred to polystyrene microtiter plates (200 μL/well) and incubated for 24 h at 37°C. The cell density was measured at OD600 using a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA, USA). The fluorescence of PI-bound eDNA was measured by a VarioskanTM LUX multimode microplate reader (Thermo Fisher, Waltham, MA, USA) with the excitation/emission wavelength at 535/610 nm. Relative amounts of eDNA per OD600 unit were determined. Three independent experiments were performed.
Determination of MIC and antimicrobial tolerance of strains
The MICs of the antimicrobials against E. faecalis isolates were determined by the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guideline CLSI-M100-S26 with CLSI-recommended MIC breakpoints. E. faecalis ATCC29212 served as the quality control standard strain. The antimicrobial-tolerance of strains was detected as described previously with modifications [28]. Antimicrobials (at 50× MIC) were added to the stationary-phase cultures (16 h) of the E. faecalis strains, then the cultures were incubated at 37°C for 120 h without shaking. Every 24 h, 1-mL aliquots were sampled and washed twice with ice-cold saline. Ten-fold dilutions were then plated on Muller-Hinton agar, and the numbers of CFUs were determined. Three independent experiments were performed.
Virulence of E. faecalis in G. mellonella
Infection of G. mellonella larvae with E. faecalis strains was performed as described previously for other pathogens [51]. G. mellonella larvae in groups of 40 were infected in the left posterior proleg with 20 μL inocula of E. faecalis strains containing 5 × 106 CFU/mL. Survival of G. mellonella larvae was recorded at 12 h intervals for 72 h p.i. Every trial included a group of 20 G. mellonella larvae injected with saline as a control. Experiments were performed in at least three independent tests, and representative results are shown.
Protein extraction and detection by a mass spectrometer with TMT labeling
E. faecalis strain OG1RF and the ΔclpP mutant were inoculated into TSB and cultured at 37°C for 4 h to logarithmic phase or for 12 h to stationary phase. The cells were harvested at 4°C centrifugation, minced individually with liquid nitrogen, lysed in lysis buffer, and ultrasonicated for 5 min on ice. Protein concentration was determined again with Bradford protein assays. The supernatant from each sample, containing precisely 0.1 mg of protein, was digested with Trypsin Gold (Promega, Madison, WI, USA) at 1:50 enzyme-to-substrate ratio. After 16 h of digestion at 37°C, peptides were desalted with a C18 cartridge to remove urea, and desalted peptides were dried by vacuum centrifugation. Desalted peptides were labeled with TMT6/10-plex reagents (TMT6/10plex™ Isobaric Label Reagent Set, Thermo Fisher) following the manufacturer’s instructions. For 0.1 mg of the peptide, 1 unit of labeling reagent was used. Peptides were dissolved in 100 μL of 0.1 M tetraethylammonium bromide, and the labeling reagent was dissolved in 41 μL of acetonitrile. After incubation for 1 h, the reaction was stopped with ammonium hydroxide. Differently labeled peptides were mixed equally and then desalted in peptide desalting spin columns (Thermo Fisher, 89852). TMT-labeled peptide mix was fractionated using a C18 column (Waters BEH C18 4.6 × 250 mm, 5 μm; Waters Corporation, Milford, MA, USA) on a Rigol L3000 high-performance liquid chromatographer operating at 1 mL/min, and the column oven was set at 50°C. Shotgun proteomics analyses were performed using an EASY-nLCTM 1200 ultra high-performance liquid chromatography system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operated in the data-dependent acquisition mode. The Q Exactive HF-X mass spectrometer was operated in positive polarity mode with a spray voltage of 2.3 kV and capillary temperature of 320°C. Two independent experiments were performed.
Global protein abundance analysis
The resulting spectra from each fraction were searched separately against the NCBI E. faecalis strains OG1RF (CP002621.1) database (https://www.ncbi.nlm.nih.gov/nuccore/CP002621.1) using the search engine Proteome Discoverer 2.2 (PD 2.2, Thermo). The searched parameters were as follows: mass tolerance of 10 ppm for precursor ion scans and mass tolerance of 0.02 Da for production scans. Carbamidomethyl was specified in PD 2.2 as a fixed modification. Oxidation of methionine, acetylation of the N-terminus, and TMT of lysine were specified in PD 2.2 as variable modifications. A maximum of 2 miscleavage sites was allowed. For protein identification, a protein with at least one unique peptide was identified at a false discovery rate FDR <1.0% on peptide and protein levels. Proteins containing similar peptides that could not be distinguished based on MS/MS analysis were grouped as separate protein groups. The protein quantitation results were statistically analyzed by Mann-Whitney tests, and the significance ratios defined as P<0.05 and ratio >1.2 or <0.83 (FC) were used to screen DAPs. GO and InterPro (IPR) analyses were conducted using the interproscan-5 program against the non-redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSiteProfiles, and PANTHER). The databases of COG (Clusters of Orthologous Groups) and KEGG were used to analyze protein families and pathways. The enrichment pipeline was used to perform the enrichment analyses of GO, IPR, and KEGG.
RNA isolation and RT-qPCR
RNA isolation of E. faecalis strains was performed as described previously with some modifications [28]. The E. faecalis strain OG1RF and the ΔclpP mutant were inoculated into TSB and cultured at 37°C for 4 h to logarithmic phase or for 12 h to stationary phase, and the following operations were performed at 4°C for centrifugation or on ice. Bacterial cultures were centrifuged at 12,000 rpm for 5 min, and then the pellets were washed twice with 0.9% saline; the culture was homogenized 5 times using 0.1-mm zirconia-silica beads in a mini-BeadBeater (Biospec, Bartlesville, OK, USA) at 5,000 rpm for 60 s at 1-min intervals; the samples were centrifuged at 15,000 rpm, and the bacterial RNA in the supernatant was purified using an RNeasy minikit (Qiagen, Hilden, Germany) and quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA samples that had a 260/280 ratio between 2.0 and 2.2 were used for RT-qPCR.
Total RNA extracted from strains OG1RF and the ΔclpP mutant were reverse transcribed with the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Dalian, China), and RT-qPCR was performed with the SYBR Premix Ex Taq II Kit (TaKaRa Biotechnology) on the Mastercycler ep realplex system (Eppendorf), with an initial incubation at 95°C for 2 min, followed by 40 cycles of 15 s at 95 °C, and 60 s at 60°C. Each sample was analyzed in triplicate. For all samples, the internal control gene recA was used to normalize the abundance of E. faecalis strains OG1RF genes [52]. The threshold cycle (Ct) numbers were confirmed by the detection system software, and the data were analyzed based on the 2−△△Ct method. The RT-qPCR primers are listed in Table S2.
Statistical analysis
Experimental data were analyzed with SPSS software (version 16.0; SPSS, Chicago, IL, USA) and compared using Student’s t tests, one-way analysis of variance, Mann-Whitney tests, or the log-rank tests. Differences with a P value <0.05 were considered statistically significant.