Figures & data
Table 1. Survival of mycoplasmas preincubated with or without factor H in swine sera.
Table 2. Primers for recombinant expression of ef-tu with a restriction site of BamH I and Xho I based on homologous recombination technology.
Figure 1. The factor H binding ability of M. hyopneumoniae strain 168 and its influence on M. hyopneumoniae survival in swine sera.
(a) The factor H binding ability of M. hyopneumoniae strain 168 incubated with rabbit anti-factor H polyclonal antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibodies. Background values were obtained for the control (no factor H or wells coated with M. hyopneumoniae culture medium). The OD450 was used for the statistical analysis. Each assay was performed in triplicate wells, and repeated independently three times(b) The survival of M. hyopneumoniae cells in the presence of normal swine sera (used as a complement pool) as determined by assaying for CCU (color change unit) after incubating with an equal volume of factor H or PBS. Three samples were assayed in each experiment.Differences were analyzed using the Student’s t- test. A p value ≤ 0.05 was considered highly significant (**). Mhp is short for M. hyopneumoniae.
Table 3. Summary of the analysis performed on the protein spots identified using MALDI-TOF-MS/MALDI-TOF-TOF-MS.
Figure 2. Identification of important factor H binding proteins of M. hyopneumoniae.
(a) Coomassie G-250-stained two-dimensional gel of surface proteins of M. hyopneumoniae strain 168 cultured in KM2 medium (pH 3–10, 13 cm). The numbers on the left indicate the molecular weights of the standards (kDa); the numbers in the profile represent different independent spots.(b) Far-western blot of the corresponding gels of surface proteins from M. hyopneumoniae strain 168 using factor H and anti-factor H antibodies. The numbers on the left indicate the molecular weights of the standards (kDa); the numbers in the profile represent different reactive spots that can bind factor H.(c) Interaction networks of the factor H binding proteins and known virulence factors. Protein-protein interactions derived from String database with a confidence score ≥ 0.4 are displayed. The blue edges show interactions of medium confidence (0.4), gray edges indicate high confidence (0.7), and the confidence (0.9) is displayed as pink edges. Blue nodes represent the known putative virulence factors collected from published papers and pink nodes represent the factor H binding proteins identified in this study. Blue lines represent interactions between two nodes.
Figure 3. The interaction of M. hyopneumoniae elongation factor thermo unstable (EF-Tu) with factor H.
(a) The percentage of adhesive M. hyopneumoniae incubated with factor H was calculated from the ratio of copies recovered from cells incubated with anti-EF-Tu antibodies to those incubated with normal sera, and is illustrated as the fold-change of adhesion along with the standard error of the mean (SEM). Data were analyzed for significance using the one-way analysis of variance (ANOVA) test followed by a Student’s t-test for comparisons between two groups. A p value ≤ 0.05 was considered highly significant (**). Mhp is short for M. hyopneumoniae.(b) Diagram of the three independent domains of EF-Tu.(c) The different recombinant domains purified by nickel affinity chromatography. The first lane (M) comprises protein markers. The second to fifth lanes are the full-length recombinant EF-Tu, the first domain, the second domain, and the third domain of EF-Tu respectively.(d) Binding of different concentrations of factor H to recombinant EF-Tu detected using microplates coated different domains of EF-Tu. The OD450 was obtained using the addition of anti-factor H antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibodies. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicate.(e) Sensorgrams depicting the binding of immobilized factor H to recombinant full-length EF-Tu. Increasing concentrations of EF-Tu (0, 1, 2.5, 5.5, 8.5, and 14 μM) were injected at a flow rate of 30 μL/min for 180 s over immobilized factor H. The arrow indicates the end of the injection period, at which point dissociation of EF-Tu from factor H cold be observed. RU = resonance units.
Figure 4. Detection of C3 deposition on the surface of M. hyopneumoniae using flow cytometry.
(a) The gray line indicates M. hyopneumoniae strain 168 successively treated with preimmune serum, factor H, C3 protein, and fluorescein isothiocyanate (FITC)-conjugated C3 antibody; the blue line indicates M. hyopneumoniae strain 168 treated with preimmune serum, C3 protein, and FITC-conjugated C3 antibody; the red line indicates M. hyopneumoniae strain 168 treated with anti- EF-Tu serum, factor H, C3 protein, and FITC-conjugated C3 antibody; the green line indicates M. hyopneumoniae strain 168 treated with anti-EF-Tu serum, C3 protein, and FITC-conjugated C3 antibody.(b) The mean fluorescence intensity (MFI) of C3 deposition in M. hyopneumoniae incubated without anti-EF-Tu sera or factor H (the blue, gray, and green columns) is expressed as the fold-change of the corresponding strain incubated with anti-EF-Tu serum, factor H, C3 protein, and FITC-conjugated C3 antibody (the red column). The asterisks above the charts indicate statistically significant differences.
Figure 5. M. hyopneumoniae Adhesion influenced by factor H.
Left column: Fold-change of the factor H adhesion rate: The number of bacteria recovered in the cells incubated with factor H/number of bacteria recovered in the group incubated without factor H (middle column) × 100%. Data are expressed as means ± SD of at least three experiments with samples assessed in triplicate.Right column: Fold-change of the adhesion rate: The number of bacteria recovered from the swine tracheal epithelia cells (STECs) incubated with anti-factor H antibodies/number of bacteria recovered from the group incubated without factor H antibodies (middle column)× 100%. Data are expressed as means ± SD of at least three experiments with samples assessed in triplicate. Mhp is short for M. hyopneumoniae; STECs is short for swine tracheal epithelial cells.
Figure 6. Factor H is predominantly present in the M. hyopneumoniae colonization site.
Immunohistochemical staining of porcine bronchiole sections from M. hyopneumoniae-challenged pigs was performed using anti-factor H antibodies (a) or anti-P97 monoclonal antibodies (b). The presence of red staining shows the location of factor H (a) or M. hyopneumoniae strains (b). A widespread distribution of factor H can be observed in the bronchiole sections of all samples, especially in the ciliary borders of the bronchioles that are colocalized by M. hyopneumoniae.
Figure 7. The sequence alignment of EF-Tu proteins from different mycoplasmas.
The amino acid sequence alignment of EF-Tu proteins from different mycoplasmas that showed factor H-mediated complement evasion, including M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. gallisepticum, M. pneumoniae, M. genitalium, M. flocculare, and M. bovis was illustrated using DNAMAN. The color of the frame indicates the identity of the amino acid sequence. The darker the color, the higher the sequence identity.
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