Mhp strains and growth conditions
All five Mhp strains were thawed from frozen Mhp bacterial stocks and subcultured for three generations before use for subsequent analysis. The strains were cultured in modified Friis medium, designated KM2 cell-free medium, which contained 20% (v/v) pig serum (sterilized by irradiation from a clean snatch-farrowed porcine colostrum-deprived piglet and produced in our lab) cultivated in a humidified incubator at 37°C [17]. Mhp strain 168 was isolated and cultured from a pig exhibiting representative characteristics of mycoplasma pneumonia of swine (MPS) in China[18]. This field strain was gradually attenuated through continuous passage to the 380th generation, resulting in the low-virulence strain 168L [19]; the Mhp 168L strain used in this study was passage 353. Strain JS is a virulent strain that can induce typical characteristics of MPS with a lung lesion score of approximately 15, as mentioned previously [17]. Strain LH is a virulent clinical strain that was isolated in our lab. Strain J (ATCC 25934) was passaged once from the ATCC stock to yield frozen stocks. The titers of Mhp strains were quantified using the 50% color change unit (CCU50) assay [20], which was modified from the CCU assay [21] and tested by quantitative PCR.
RNA transcriptional analysis
The five Mhp strains were cultured in the abovementioned culture conditions at 37°C for 48 h. Then, total RNA was extracted using a Total RNA Extraction Kit (Cat No. R6834, Omega Biotek, Guangzhou, China). HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (Cat No. R223-01, Vazyme, Nanjing, China) was used to reverse transcribe at least 1 µg of total RNA in a 10-µL reaction volume before performing qRT-PCR on an ABI 7500 Real Time PCR System with the help of a HiScript® II One Step qRT-PCR SYBR® Green Kit (Cat No. Q221-01, Vazyme, Nanjing, China). qRT-PCR was run using cDNA for the NFOR gene under specific conditions. The P46 gene of Mhp was selected as the internal control. The PCR primers used in the quantitative assays are listed in Table 1. The fold changes in mRNA expression of the NFOR genes of various Mhp strains differing in virulence were determined using the 2-ΔΔCT method as described previously [22].
Multiple sequence alignment
Seventeen amino acid sequences of the NFOR protein from Mhp were retrieved from the National Center for Biotechnology Information and UniProt protein databases, and their homologies were analyzed. All sequences were aligned with the CLUSTAL W program. Phylogenetic inference according to the neighbor-joining criterion was performed through Molecular Evolutionary Genetics Analysis version 10 (MEGA 10). A total of 2000 nonparametric bootstrap analyses were used to test the robustness of the hypothesis.
Protein expression and preparation of polyclonal antibody
The Mhp NFOR gene (MHP168_RS01740) was synthesized by GenScript Biotech Corp. (Nanjing, China) and was expressed in the BL21(DE3) E. coli strain through the pET32a vector, purified using High Affinity Ni-Charged Resin FF affinity columns (Cat No. L00666, GenScript, Nanjing, China), and identified by Western blot analysis. Purified proteins were determined with the BCA Protein Assay Kit (Cat No. P0012S, Beyotime, Shanghai, China), and the protein concentration was calculated before storage at -70°C. Polyclonal antibodies against Mhp NFOR were obtained by subcutaneously immunizing 1-month-old New Zealand white rabbits with 1.2 mg of recombinant protein (rNFOR) emulsified in Freund's complete adjuvant for the first immunization (Cat No. F5881, Sigma-Aldrich, St Louis, MO, USA). Each rabbit was then immunized with 1.2 mg of rNFOR emulsified in Freund's incomplete adjuvant (Cat No. F5506, Sigma-Aldrich, St Louis, MO, USA) twice at 2-week intervals. Booster immunization was performed once a week after three immunizations before sera were collected.
Enzymatic activity assays
The enzymatic activity of purified rNFOR was determined by calculating the oxidation of NADH to NAD+ at 25°C. In brief, 5 μg/mL rNFOR, 0.1 M potassium phosphate buffer (pH 7.5, containing 1 mM dithiothreitol), 10 μM flavin mononucleotide (FMN) (Cat No. F107158, Aladdin, Shanghai, China) and 0.5 mM NADH (Cat No. N106933, Aladdin, Shanghai, China) were used in this study. The reaction system was 2 mL, and the purified rNFOR was preincubated with FMN for 5 min before NADH was incubated. The optical density (OD) was measured at 340 nm (OD340). The specific activity was calculated by the following equation:
Surface-exposed NFOR detection by flow cytometry
Flow cytometry analysis was used to test whether NFOR is located on the surface of Mhp strains and to probe NFOR surface distributions between the high-virulence strain 168 and the low-virulence strain 168L. Mhp strains 168 and 168L (titers of which were 1 × 108 CCU/mL) were incubated with anti-rNFOR serum or with preimmune serum (negative control) at a 1:100 dilution, as reported previously [23, 24]. Fluorescein isothiocyanate (FITC)-conjugated anti-IgG (Cat No. BA1105, Boster, Wuhan, China) was then used to stain the above Mhp strains, and a BD Accuri C6 flow cytometer was used to measure the fluorescence intensity.
Surface-exposed NFOR detection by immunoelectron microscopy
Mhp strains were cultured and grown to mid-log phase at 10 000 × g by centrifugation at 10°C for 20 min before each Mhp bacterium was harvested. A total of 1×108 CCU of bacterial suspension was washed three times and finally resuspended in a volume of 50 µL of 0.1 M phosphate-buffered saline (PBS, pH 7.4). Immunoelectron microscopy was performed according to previous studies [25, 26] with some modifications. Briefly, 5 µL of the sample was added to a 400 mesh formvar-coated nickel grid and allowed to stand for 5 min. Then, the grid was fixed with 2% paraformaldehyde in PBS for 5 min at room temperature (RT) followed by blocking with 1% negative rabbit serum and blocking buffer (1% (w/v) BSA in PBS) for 1 h. The samples were then incubated with anti-rNFOR antibody or preimmune serum (negative control) at a 1:10 dilution in blocking buffer for another 1 h (PBS as a blank control). After washing five times in blocking buffer, the samples were incubated with a secondary gold-conjugated antibody (goat anti-rabbit IgG, 10 nm-gold particles, Cat No. GA1014, Boster, Wuhan, China) at a dilution of 1:20 for 1 more hour. The samples were washed 5 times with PBS for 5 min each time before being fixed in 2% paraformaldehyde in PBS for 5 min. Then, the grids were washed eight times in distilled water and stained with 1% phosphotungstic acid (pH 6.5) for 15 s. After the samples were dried by an infrared lamp, they were observed under a Tecnai high-field transmission electron microscope.
Indirect immunofluorescence assay (IFA)
Immortalized porcine bronchial epithelial cells (hTERT-PBECs) were established and cultured to a density of 80% in 24-well cell plates with Dulbecco's modified Eagle’s medium:nutrient mixture F-12 (DMEM/F12) medium plus 2% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) supplemented with growth factors (Cat No. CC-4175, Lonza, Basel, Switzerland), as we previously made and reported [22]. Cells were washed three times with cold PBS before being fixed with 4% paraformaldehyde for 10 min at RT. Subsequently, 0.2% Triton X-100 was used at RT for 3 min, followed by blocking for 2 h using 3% (w/v) BSA in PBS. Cells were incubated with 100 μg of purified rNFOR for 1 h at 37°C in a cell incubator before they were washed three times with PBS and incubated with anti-rNFOR antibody at a 1:250 dilution for another 2 h at 37°C. After three washes with PBS, the cells were then incubated with a 1:100 dilution of tetraethyl rhodamine isothiocyanate (TRITC)-conjugated anti-IgG (Cat No. SA00007-2, Proteintech, Rosemont, IL, USA) for 1 h in a 37°C incubator. 6-Diamidino-2-phenylindole (DAPI, Cat No. D8417, Sigma-Aldrich, St Louis, MO, USA) was used for nuclear staining before the cells were observed using a fluorescence microscope (Zeiss, Tokyo, Japan). Instead of rNFOR, BSA was selected as a negative control.
Antibody-mediated adhesion inhibition
Mhp strains (high-virulence 168 and low-virulence 168L, the titers of which were 1 × 107 CCU/mL) were collected by centrifugation at 10 000 × g for 20 min at 10°C and resuspended in 500 µL of PBS after washing three times with PBS. The samples were preincubated with polyclonal antibody against rNFOR or preimmune serum at a 1:20 dilution for 30 min in a 37°C incubator. Mhp bacteria were suspended in DMEM/F12 before being added to confluent hTERT-PBECs seeded in 24-well cell plates. Plates were then centrifuged at 1 000 × g for 10 min before being placed at 4°C for 2 h. After washing three times with PBS, hTERT-PBECs were collected after digestion with 0.125% trypsin (twice diluted with Hanks medium with 0.25% trypsin, Cat No. 25200072, Gibco, Grand Island, NY, USA), and the cells were centrifuged at 1 300 rpm for 10 min after adding DMEM/F12 containing 10% FBS to stop cell digestion. Following Mhp bacterial genome extraction, quantitative real-time PCR was then performed as previously reported [27]; the real-time PCR primers are shown in Table 1. Experiments were performed in triplicate, and data were analyzed using SPSS 20.0. Mhp titers were quantified using the CCU50 assay mentioned above.
Surface plasmon resonance (SPR) analysis
SPR analysis was performed according to our previous study [26] using a Biacore X100 Plus instrument (GE Healthcare, Boston, MA, USA). Fibronectin and plasminogen were diluted to 50 µg/mL before they were linked covalently to the CM5 sensor chip as a ligand using an amine coupling kit (Biacore AB, Cytiva, Guangzhou, China). The immobilization of soluble fibronectin and plasminogen produced approximately 2000 resonance units (RUs). The binding kinetics were measured by increasing the concentration (0-4000 nmol/L) of the analyte (Mhp NFOR) in running buffer (HBS-EP), which consisted of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% (v/v) surfactant P20 (Biacore AB, Cytiva, Guangzhou, China), with a flow rate of 30 μL/min, passing through immobilized Mhp NFOR at 20°C for 3 min. The dissociation phase was monitored for 1000 seconds by allowing buffer to flow through the chip. Biacore X100 control software was used to manually analyze the binding kinetics.
Far-Western blot (Far-WB) analysis
Fifteen micrograms of rNFOR was resolved by 10% SDS-PAGE and then transferred to a PVDF membrane (Cat No. IPFL00010, Millipore, Darmstadt, Germany). After three washes with PBS, the membrane was blocked with 5% skimmed milk in TBST (TBS containing 0.5% Tween 20), which served as the blocking buffer, before being placed in a 37°C incubator for 2 h with gentle shaking. Then, the membrane was incubated with 15 μg/mL fibronectin (Cat No. F1056, Sigma-Aldrich, Darmstadt, Germany) or plasminogen (Cat No. SRP6518, Sigma-Aldrich, Darmstadt, Germany) at 37°C for another 2 h. After another three washes with TBST, membranes were subsequently incubated with anti-fibronectin antibody (Cat No. ab299, Abcam, Cambridge, UK) at a 1:1000 dilution or anti-plasminogen antibody (Cat No. ABP55618, Annkine, Wuhan, China) at a 1:400 dilution in blocking buffer for 2 h in a 37°C incubator. The membranes were then incubated with the secondary antibody (HRP-conjugated goat anti-rabbit IgG) (Cat No. BA1055, Boster, Wuhan, China) at a 1:2000 dilution for 2 h in a 37°C incubator after three washes with TBST. Finally, the membranes were developed with Electro-Chemi-Luminescence (ECL) substrate using a ChemiDoc XRS+ system (Bio-Rad). Instead of rNFOR, BSA was used as a negative control.
Quantification of lactate dehydrogenase (LDH) release
hTERT-PBECs were seeded in 24-well cell plates one night before cell growth reached 80% confluence. First, the cells were incubated with purified rNFOR protein at different concentrations (5 μg, 10 μg, 15 μg, 20 μg). Six hours later, culture supernatants were collected, and LDH activity was measured with a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Cat No. G1780, Promega, Madison, WI, USA) following the manufacturer’s instructions. The corrected values in the formula below were used to calculate the percentage of cytotoxicity: percent cytotoxicity = 100 × experimental LDH release (OD490)/maximum LDH release (OD490). PBS was used instead of rNFOR as a negative control, and three Mhp strains (strains JS and J and 168L, 1 × 108 CCU/mL) that differed in virulence were used as positive controls.
Reactive oxygen species (ROS) detection
hTERT-PBECs were seeded in 24-well plates one day before cell growth reached 80% confluence. Cells were incubated with 20 μg of purified rNFOR in a 37°C incubator for 6 h. ROS detection was measured using a ROS-Glo™ H2O2 Assay (Cat No. G8820, Promega, Madison, WI, USA), and relative luminescence units (RLU) were recorded using a plate reader. PBS was used as a negative control, and positive controls were the RLUs from three Mhp strains (strain JS, J and 168 L, 1 × 108 CCU/mL).
Apoptosis assay
hTERT-PBECs were seeded one night before at 2×105 cells/well in culture medium of a total volume of 500 µL in 24-well cell plates. hTERT-PBECs were then incubated with 20 μg of purified rNFOR at 37°C for 12 h, with cells grown from culture medium DMEM/F12 plus 2% FBS and epithelial growth factors to maintain the DMEM/F12 without adding the above reagents as a negative control, and Mhp strains that differed in virulence (high-virulence strain JS and low-virulence strain 168L, 1 × 108 CCU/mL) were used as positive controls. To explore whether antiserum to rNFOR could block and reduce the apoptosis induced by Mhp in hTERT-PBECs, we preincubated Mhp strains (JS and 168L, 1 × 108 CCU/mL) with rabbit polyclonal antibody raised against rNFOR at a 1:20 dilution at 37°C for half an hour before they were applied to hTERT-PBECs seeded in 24-well plates in a 37°C incubator for 12 h. The apoptosis rate was detected and calculated using a dual apoptosis detection kit (Cat No. A211, Vazyme, Nanjing, China) with Annexin V-FITC/PI.
Statistical analysis
Data for the adhesion rate from quantitative real-time PCR analysis and the apoptosis rate were analyzed by GraphPad Prism 6 software and FlowJo software v7.6. Relative NFOR mRNA expression levels between the moderate-virulence strain J or the low-virulence strain 168L and the high-virulence Mhp strains 168, JS, and LH were assessed via multiple comparisons of analysis of variance (ANOVA). The adhesion rates between anti-NFOR serum or negative serum and groups of the high-virulence strain 168 or between low-virulence strain 168L and anti-NFOR serum or negative serum were compared by the multiple t test. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.