Bacterial strains, plasmids, cell lines, and chicken sera positive for various avian pathogens
MS WVU1853, MG Rlow, and MI 695 were purchased from the China Veterinary Culture Collection Center (CVCC, Beijing, China). MS JS1 and SD1 were isolated from the swollen joints of two diseased chickens from Jiangsu and Shandong provinces in China. MS SH1 and HB1 were isolated from the throat swabs of two diseased chickens from Shanghai and Hubei provinces in China. MG 08, 013, FBH, SGN, and SS were donated by Professor Zhaofeng Sui's research group at Shandong Animal Science and Veterinary College. All strains of MS, MG, and MI were cultured in Mycoplasma Broth Base (Hopebio, China) supplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (Roche, China) and 10% porcine serum (Gibco, USA) at 37 °C in an atmosphere containing 5% CO2. Contiguous cell lines of chicken embryo fibroblasts DF-1 were purchased from Shanghai Institute of Biochemistry and Cell Biology and cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in an atmosphere containing 5% CO2. Chicken sera positive for MS strains (including WVU1853, JS1, HB1, SD1, and SH1), MI 695, and various MG strains (including Rlow, 08, 013, FBH, MG SGN, and SS) were prepared in our laboratory as described previously [55]. Briefly, cultured MS, MG, and MI were collected and inactivated with 0.4% formaldehyde for 24 h, suspended in phosphate buffered saline (PBS), and emulsified with MONTANIDE ISA 71 VG adjuvant (SEPPIC, France) in a ratio of 3:7, then inoculated into 10-day-old SPF chickens [109 color change units (CCUs) per chicken] subcutaneously two times at 2-week intervals. Two weeks after the second immunization, blood samples were collected to separate antisera. The other chicken sera positive for E. coli O1/O2/O78, SPG, PM, STA, NDV, IBDV, and IBV were all obtained from CVCC. The field MS-negative chicken sera (FN-1, FN-2, and FN-3) were collected from a poultry farm in Shanghai, China, and were identified by a MS-antibody ELISA test kit (IDEXX, USA).
Expression and purification of rMSNOX and rMSFBA
Fructose-1,6-bisphosphate aldolase (FBA) of MS was previously identified as a cytoplasmic protein [56] and was used as a cytoplasmic protein control in this research. MSnox and MSfba gene fragments were amplified from MS WVU1853 by overlap PCR with the primers shown in Table S1, which have been described in our previous studies [26, 56]. The MSnox and MSfba fragments were ligated into pET28a ( +) (Novagen, USA), and the recombinant strain E. coli BL21 (pET28a-MSnox) and E. coli BL21 (pET28a-MSfba) were constr0-ucted. Then the His-tagged rMSNOX protein and rMSFBA protein were expressed and purified as described previously [26, 56]. The purified rMSNOX protein and rMSFBA protein were analyzed with 12.5% SDS-PAGE, stained with Coomassie blue-G250 (Solarbio, China), and imaged with an infrared laser scanning imaging system (Ddyssey; LI-COR, USA). The protein concentrations were detected with a BCA protein assay kit (Beyotime, China).
Preparation of rabbit antisera
Two-month-old New Zealand white rabbits were purchased from Songlian Experimental Animal Farm (Shanghai, China), and pre-immune serum was collected as a negative control. To prepare polyclonal antibodies against rMSNOX or rMSFBA, we injected rabbits subcutaneously three times at 2-week intervals with 300 µg of purified rMSNOX or rMSFBA protein mixed with an equal volume of Freund’s adjuvant (Sigma, USA). Complete Freund’s adjuvant was used for the first immunization, and incomplete Freund’s adjuvant was used subsequently. Two weeks after the third immunization, blood samples from immunized rabbits were collected to separate antisera. As described above, rabbit sera against MS or MG were also prepared by immunization of rabbits with 1010 CCU of inactivated MS WVU1853 or MG Rlow whole cells (incubated with 0.4% formaldehyde for 24 h). The antibody titers of the rabbit sera were analyzed with ELISAs using plates coated with purified rMSNOX protein, purified rMSFBA protein, or whole cell proteins of MS WVU1853 or MG Rlow (0.5 µg/well for each protein). Briefly, 96-well ELISA plates (Corning, USA) were coated with 0.5 μg of rMSNOX, rMSFBA, MS or MG whole cell proteins in carbonate coating buffer (2.94 g/L NaHCO3, and 1.6 g/L Na2CO3, pH 9.6) at 37 °C for 2 h. After being washed three times with PBST, the plates were blocked with 5% non-fat milk in PBST and incubated with serially diluted rabbit antiserum (from 1:100 to 1:204,800) at 37 °C for 1.5 h, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (diluted 1:5000; Thermo, USA) at 37 °C for 1 h. In each well,100 μL of soluble TMB substrate solution (Tiangen, China) was added and incubated for 15 min for color reaction, which was then stopped by 50 μL of 2 M H2SO4. Finally, the absorbance values at 450 nm (OD450 nm) were measured with a multi-mode microplatereader (SynergyH1; Biotek, USA). The experiments were performed in triplicate. When the ratio of the OD450 nm value of the antiserum and the pre-immune serum was greater than 2.1, the maximum dilution was the antibody titer of the antiserum.
Immunogenicity, immunoreactivity and specificity analysis of rMSNOX
Purified rMSNOX and rMSFBA (His-tagged) protein (0.5 µg/well) was subjected to 12.5% SDS-PAGE with PageRuler Prestained Protein Ladder (#26,616, Thermo) and transferred to a nitrocellulose filter (NC) membrane (Amersham, USA). The NC membrane was then blocked with 5% skimmed milk at 37 °C for 2 h and incubated with rabbit anti-rMSNOX serum (1:1000) or pre-immune rabbit serum (1:1000) at 37 °C for 1.5 h. After being washed three times with PBST (PBS adding 0.05% Tween-20), the NC membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:5000; Thermo) at 37 °C for 1 h. Then the membranes were visualized with a Basic Luminol-enhanced Chemiluminescence (ECL) kit (Yeasen, China) and imaged with a chemiluminescence imager (Tanon 5200; Tanon, China).
To evaluate the immunoreactivity and specificity of rMSNOX with various chicken sera, we performed western blots and ELISAs. In western blot assays, the rMSNOX proteins (0.5 µg/well) were used to react with sera positive for various MS strains (including MS WVU1853, JS1, HB1, SD1, and SH1; 1:500) for immunoreactivity detection, and chicken sera against other avian pathogens for specificity analysis, including sera positive for various MG strains (Rlow, 08, 013, FBH, SGN, SS), E. coli O1/O2/O78, SPG, PM, STA, NDV, IBDV, and IBV (each diluted 1:500). Three field MS-negative sera (FN-1, FN-2, and FN-3) and SPF chicken serum (CVCC; 1:500) were used as the negative controls. In ELISAs, the procedure was similar to that described above. The 96-well ELISA plates were coated with purified rMSNOX protein (0.5 μg/well), then incubated with chicken sera against various avian pathogens, including all chicken sera used above. After being washed, the plates were incubated with goat anti-chicken IgY-HRP antibody (1:5000; Abbkine, USA), followed by soluble TMB substrate solution (Tiangen); the reaction was stopped with 2 M H2SO4. Finally, the OD450 nm values were measured as described above.
Complement dependent mycoplasmacidal assays
Mycoplasmacidal assays were performed as described previously with some modifications [57]. All rabbit sera were inactivated at 56 °C for 30 min before use. MS WVU1853, MS JS1, MS SD1, and MG Rlow were cultured to mid-logarithmic phase, washed three times with PBS, centrifuged at 5000 g for 15 min at 4 °C, and resuspended with PBS. The reaction mixtures containing 30 µL of MS or MG bacterial suspension (7 × 108 CCU/mL), 10 µL of rabbit anti-rMSNOX, anti-MS/MG serum, or pre-immune rabbit serum, and 10 µL of complement (CVCC) were mixed thoroughly and incubated at 37 °C for 1 h. In addition, the reaction mixtures with 10 µL of PBS instead of complement added were also tested as described above and were considered complement-free controls. Finally, each reaction mixture was diluted by tenfold gradient in mycoplasma broth and spread onto solid medium for colony counting. Rabbit anti-MS/MG serum and pre-immune rabbit serum were considered as positive and negative controls, respectively. Three independent experiments were repeated, and the mycoplasmacidal rates were calculated according to the following formula: [(CFU of pre-immune serum treatment–CFU of antiserum treatment)/(CFU of pre-immune serum treatment)].
Suspension immunofluorescence assays
NOX has been identified to be distributed in both the cytoplasm and cell membrane components of MS in our previous study, according to western blotting assays [26]. To determine the surface localization of NOX on MS, we performed suspension immunofluorescenceassays as previously described [57]. The MS WVU1853 was cultured to mid-logarithmic phase, collected by centrifugation at 5000 g, and washed twice with PBS. The MS cells were then fixed with 4% paraformaldehyde at room temperature for 20 min and washed three times with PBS. The fixed MS cells were re-suspended and incubated with rabbit anti-rMSNOX serum (1:1000 diluted by PBS) at 37 °C for 1 h. The rabbit anti-MS serum (1:1000) was used as a positive control, the rabbit anti-rMSFBA serum and pre-immune rabbit serum (1:1000) were used as negative controls. After being washed three times with PBST, the MS cells were incubated with FITC-conjugate goat anti-rabbit IgG (1:1000, Sigma-Aldrich) at 37 °C for 2 h. After being washed, the MS pellets were spread onto glass slides and observed under a fluorescence microscope (Ni-U; Nikon, Japan).
Immunogold transmission electron microscopy assays
The collected MS cells were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde at room temperature for 2 h, then washed three times with PBS. The fixed MS cells were dehydrated with various concentrations (30, 50, and 70%) of ethanol, and embedded in LR White resin (Sigma, USA). Grids with ultrathin sections were blocked with 5% BSA and then incubated with rabbit anti-MSNOX, anti-rMSFBA serum, or pre-immune rabbit serum (1:1000 diluted by PBST) at 37 °C for 1.5 h. After being washed five times with PBST, the sections were incubated with goat anti-rabbit IgG (whole molecule)-gold antibody (1:100 diluted by PBST; Sigma) at 37 °C for 1 h. After being washed with PBST, the sections were fixed with 2.5% glutaraldehyde for 10 min and stained with uranyl acetate for 5 min and lead citrate for 2 min at room temperature. The dried sections were then visualized with a transmission electron microscope (Tecnai G2 Spirit Biotwin; FEI, USA).
Adherence and inhibition of adherence of rMSNOX to DF-1 cells
To detect the adherence and inhibition of adherence of rMSNOX to DF-1 cells, we performed indirect immunofluorescence assays as described previously, with some modifications [57]. DF-1 cells were propagated on coverslips in 12-well cell culture plates (Corning) for 24 h. After being washed, the DF-1 cells were incubated with 10 µg of freshly purified rMSNOX in 500 µL DMEM for 1 h at 37 °C. The DF-1 cells adhered by His-tagged rMSFBA protein or no protein were used as controls. For assays of inhibition of adherence, 10 µg of freshly prepared rMSNOX was pre-incubated with rabbit anti-rMSNOX serum (1:50), rabbit anti-MG serum (1:50), or pre-immune rabbit serum (1:50) respectively, at 37 °C for 1 h. Then the serum-treated rMSNOX protein was added to the DF-1 cells for incubation as described above. After incubation, cells were washed four times with PBST to remove the unadhered protein, the bound rMSNOX or rMSFBA protein was recognized by rabbit anti-rMSNOX or rabbit anti-rMSFBA serum (1:1000) for 1 h, and then labeled with goat anti-rabbit IgG-FITC (1:1000; Sigma) for 1 h. The cell membranes and nuclei were stained with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanineperchlorate (Dil, Beyotime) and 4’,6-diamidino-2-phenylindole (DAPI, Beyotime), according to methods described previously [57]. Finally, the cellular coverslips were treated with antifade mounting medium (Sangon Biotech, China) and observed under a laser scanning confocal microscope (LSM800; Zeiss, German). The experiments were performed in triplicate. The FI of FITC and DAPI was quantitatively assessed by ImageJ software and the FI ratios of FITC to DAPI were analyzed.
Adherence and inhibition of adherence of MS/MG to DF-1 cells
The inhibition of adherence of MS or MG to DF-1 cells by anti-rMSNOX serum was estimated with indirect immunofluorescence and colony counting assays. DF-1 cells were propagated in DMEM on microscope coverslips in six-well cell culture plates (Corning) for 24 h. MS WVU1853, MS JS1, MS SD1, and MG Rlow were cultured to logarithmic growth phase and collected by centrifugation at 5000 g for 15 min. Then the three MS isolates and MG Rlow (2 × 108 CCU/mL) were incubated with rabbit anti-rMSNOX, anti-MS (for MS isolates only), anti-MG (for MG Rlow only), or pre-immune serum (1:50) at 37 °C for 1 h. The rabbit anti-MS/MG and pre-immune serum were used as positive and negative controls. All rabbit sera were inactivated at 56 °C before use. The DF-1 cells were infected with pre-treated MS or MG cells (6 × 107 CCU/well) at a multiplicity of infection of 200 at 37 °C for 2 h, then were washed thoroughly with PBS to remove the un-adhered mycoplasma. For indirect immunofluorescence assays, adhered mycoplasma cells were recognized by rabbit anti-MS or anti-MG serum (1:1000) for 1 h, and were then labeled with goat anti-rabbit IgG-FITC (1:1000, Sigma) for 1 h. The DF-1 cell membranes and nuclei were stained with Dil (1:100, Beyotime) and DAPI (1:1000, Beyotime) and observed under a fluorescence microscope (Ni-U; Nikon). The FIs of FITC and DAPI from three independent experiments were quantitatively assessed by ImageJ software and the FI ratios of FITC to DAPI were analyzed. For colony counting assays, the DF-1 cells were scraped and then lysed in 1 mL serum-free mycoplasma culture medium for 20 min. The suspension was serially diluted and spread onto mycoplasma agar plates for colony counting. Three independent experiments were performed in triplicate. The inhibition of adherence rate was calculated as [(CFU from pre-immune serum treatment–CFU from antiserum treatment)/CFU from pre-immune serum treatment] × 100%.
Binding activity of rMSNOX to cPlg and hFn
Western blotting and ELISA were used to determine the binding activity of rMSNOX to cPlg and hFn.
For western blot analysis, gradient diluted rMSNOX protein (2, 1, and 0.5 µg) and 2 µg BSA (Sigma) were subjected to 12.5% SDS-PAGE, then transferred to an NC membrane. After blocking with 5% skimmed milk, the NC membrane was incubated with 10 µg/mL of cPlg (Cell Sciences, USA) or hFn (Sigma) for 2 h at 37 °C. After excessive washing with PBST, the membrane was incubated with rabbit anti-cPlg/hFn polyclonal antibody (1:1000; Cell Sciences) for 1 h at 37 °C, then incubated with HRP conjugated goat anti-rabbit IgG antibody (1:5000; Thermo) for 1 h at 37 °C. Membranes were visualized with a Basic Luminol ECL kit (Yeasen).
For ELISA analysis, the 96-well plates (Corning) were coated with 1 µg/well of cPlg, hFn or BSA (negative control). After blocking with 5% skimmed milk, wells were incubated with various amounts of rMSNOX protein (1, 0.5, 0.15, 0.125, 0.0625, and 0.03125 µg/well) at 37 °C for 1.5 h. After being washed, the wells were treated with rabbit anti-rMSNOX serum (1:500) at 37 °C for 1 h, followed by HRP conjugated goat anti-rabbit IgG antibody (1:5000; Thermo) at 37 °C for 1 h. TMB substrate solution and 2 M H2SO4 were added successively and OD450 nm values were measured as described above. The experiments were performed in triplicate.
Statistical analysis
Data are given as the mean with standard deviation for three replicate experiments, and statistical analyses were performed with unpaired T-test and one/two-way ANOVA in GraphPad Prism6 software. Significant differences are denoted ***p < 0.001 or ****p < 0.0001, and ns represents no significance.
