Article Figures & Data
- FIG 1
Moraxella catarrhalis adheres to various human collagens. (A) Dose-dependent binding of M. catarrhalis Bc5 to purified collagens (50 nM) as revealed by ELISA. ELISA plates were coated with collagens I, II, III, IV, VI, and VIII, and serial dilutions of M. catarrhalis Bc5 were added. Bound bacteria were detected by anti-Moraxella PAbs. (B) Adherence of M. catarrhalis Bc5 to collagen-coated glass slides. Glass slides were coated with 2 µg collagen or HSA, followed by incubation with 107 CFU/ml bacteria in BHI medium. Bacteria were visualized by Gram staining and light microscopy. (C) Binding of M. catarrhalis BBH18 to purified collagens I, II, III, IV, V, VI, and VIII as analyzed by ELISA. (D) Adherence of M. catarrhalis BBH18 to collagen-coated glass slides. (A and C) Data shown are the means of the results from three independent experiments, and error bars represent standard deviations. (A and B) M. catarrhalis bacteria (3.0 × 105 to 1.0 × 107 CFU added) bound to collagens I, II, III, IV, and VI significantly more than to collagens V and VIII (P value range, 0.05 to 0.001; two-way ANOVA). (E) Moraxella catarrhalis clinical isolates bind to human collagens. The adherence of M. catarrhalis isolates (106 CFU) to various collagens (50 nM) was analyzed by ELISA. The means of the results of three independent experiments performed in triplicates are shown and error bars indicate standard deviations. Multiple comparisons were done with one-way ANOVA. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG 2
UspA2 and UspA2H are collagen-binding proteins of M. catarrhalis. (A) Results from ELISA showing adherence of M. catarrhalis Bc5 and isogenic single and multiple mutants to collagens. (B) Adherence of M. catarrhalis BBH18 and isogenic single and multiple mutants to collagens. The means of the results of three independent experiments are shown here, and error bars represent standard deviations. Multiple comparisons were done by a two-way ANOVA. M. catarrhalis (3.0 × 105 to 1.0 × 107 CFU added) Bc5 ΔuspA2, ΔuspA1 ΔuspA2, and ΔuspA1 ΔuspA2 Δmid mutants had significantly reduced binding to collagens in comparison to the binding of Bc5 WT and the ΔuspA1 and Δmid mutants (P value range, 0.05 to 0.001). In parallel, M. catarrhalis BBH18 ΔuspA2H, ΔuspA1 ΔuspA2H, and ΔuspA1 ΔuspA2H Δmid mutants bound significantly less to collagens than did BBH18 WT and ΔuspA1 and Δmid mutants (P value range, 0.05 to 0.001).
- FIG 3
UspA2 and UspA2H have the same binding sites for fibrillar collagens (I, II, and III) and network-forming collagens (IV and VI). (A) Inhibition of M. catarrhalis Bc5 adhesion to immobilized collagens I, II, IV, and VI by the soluble collagens indicated in the key. (B) Inhibition of M. catarrhalis BBH18 adhesion to immobilized collagens I, II, IV, and VI by various soluble collagens. In A and B, the indicated collagens were coated to microtiter plates followed by ELISA.
- FIG 4
Recombinant M. catarrhalis UspA2 and UspA2H bind to fibrillar collagens I and II. (A and B) Binding of recombinant 125I-labeled UspA1, UspA2, and UspA2H to collagen I (A) or collagen II (B). 125I-labeled UspA2 and UspA2H (28 to 156 kcpm added) showed significantly higher levels of binding to collagens I and II than did UspA1 (P value range, 0.01 to 0.001). Mean values from three independent experiments in triplicates are shown. Error bars indicate standard deviations. Multiple comparisons were performed using two-way ANOVA. (C and D) TEM images showing binding of gold-labeled UspA1, UspA2, and UspA2H to a grid coated with collagen I (C) or collagen II (D). The gold particles, appearing as black dots and indicated by arrows, show UspA2 and UspA2H bound to collagen fibrils.
- FIG 5
Recombinant UspA2 and UspA2H bind to network-forming collagens IV and VI. (A and B) Binding of recombinant 125I-labeled UspA1, UspA2 and UspA2H to collagen IV (A) or collagen VI (B) coated in microtiter plates. The means of the results of three independent experiments in triplicates are shown. The error bars indicate standard deviations. Statistical calculations were performed using two-way ANOVA. The differences between 125I-labeled UspA2 and UspA2H and UspA1 (28 to 156 kcpm added) in binding to collagen IV and VI were statistically significant (P value range, 0.01 to 0.001). (C) TEM images of gold-labeled UspA1, UspA2, and UspA2H binding to a collagen IV-coated grid. The solid arrows indicate gold particles bound to NC domains, and dashed arrows show N7 domains of the collagen IV molecule. (D) Binding of gold-labeled UspA1, UspA2, and UspA2H to a collagen VI-coated grid as revealed by TEM. Gold particles are indicated by arrows.
- FIG 6
Adherence of M. catarrhalis bacteria to primary fibroblasts is dependent on UspA2 and UspA2H, as revealed by confocal microscopy. (A and B) Adherence of M. catarrhalis Bc5 WT (A) and BBH18 WT (B) to fibroblasts. A detailed outline of the experiment, in addition to the results for M. catarrhalis ΔuspA2 and ΔuspA2H mutants, is presented in Fig. S3 in the supplemental material. Fibroblasts were incubated with FM 4-64-labeled bacteria (red). After washing, the ECM, including collagen fibrils, were visualized by FITC-labeled UspA230–539 (green). Overlays resulting in orange color illustrate colocalization of M. catarrhalis and collagens, including other components of the ECM. The size bar represents 10 µm. (C) Adherence of M. catarrhalis bacteria to fibroblasts as determined by CFU. Data shown are the means of the results of three independent experiments done in triplicates, and error bars represent standard deviations. Statistical analyses were performed using one-way ANOVA. ***, P ≤ 0.001.
- FIG 7
M. catarrhalis UspA2- and UspA2H-dependent adhesion to collagens in human lung fibroblasts and trachea. (A) Lung fibroblasts targeted by M. catarrhalis WT and UspA2 and UspA2H mutants were analyzed by SEM. Bacteria are visualized in green pseudocolor. (B) Adherence of M. catarrhalis strains per mm2 of fibroblasts. (C) M. catarrhalis WT and UspA2 and UspA2H mutants adhere to human tracheal tissues. (D) Adherence of M. catarrhalis strains per mm2 of trachea. Error bars indicate standard deviations. Statistical analyses were performed using Student’s t test. ***, P ≤ 0.001.
- FIG 8
Increased adherence of M. catarrhalis to mouse COPD lungs. (A) COPD mice were challenged by intratracheal inoculation of M. catarrhalis Bc5 WT and BBH18 WT and the corresponding UspA mutants. After 30 min, tracheal sections were analyzed by SEM. Bacteria are highlighted in green pseudocolor. (B) Quantitative estimation was performed by counting bacterial cells/mm2 from randomly selected regions comprising epithelial surface, epithelium, and subepithelium. (C) Excised lungs were homogenized, and bacterial counts were determined by serial dilution and plating. Error bars represent standard deviations. Statistical analyses were performed using Student’s t test. ***, P ≤ 0.001.
- FIG 9
Adherence of M. catarrhalis to human tracheal tissue. (A) SEM of section of human trachea incubated with M. catarrhalis Bc5 WT and embedded in paraffin. Magnified view of the epithelial surface shows adherence of bacteria to cilia. (B) SEM showing adherence of M. catarrhalis Bc5 WT to lamina propria. Collagen bundles are targeted by the bacteria. (C) Cartoon depicting a model of how M. catarrhalis adheres and colonizes the human host by using outer membrane proteins for targeting the ECM.
Supplemental Material
Figure S1
Adherence of M. catarrhalis clinical isolates to various collagens. (A and B) Binding of 125I-labeled collagens to M. catarrhalis clinical isolates that express UspA2 (A) or UspA2H (B). The data presented in Fig. 1E are plotted here in order to estimate the binding of individual isolates with different collagens. Download Figure S1, TIF file, 0.3 MB.
Copyright © 2016 Singh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S2
Quantification of gold-labeled UspA1, UspA2, and UspA2H in TEM images shown in Fig. 4C and D and 5C and D. (A) Binding of gold-labeled UspA1, UspA2, and UspA2H to collagens I and II. For quantification, 50 different regions (see Fig. 4C and D) were randomly selected, and gold particles were counted. (B) Quantification of gold particles from TEM images shown in Fig. 5C and D. Error bars indicate standard deviations. Statistical analyses were performed using Student’s t test. ***, P ≤ 0.001. Download Figure S2, TIF file, 0.1 MB.
Copyright © 2016 Singh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S3
Adherence of M. catarrhalis to primary fibroblasts is dependent on UspA2 and UspA2H, as revealed by confocal microscopy. Fibroblasts were grown on coverslips and incubated with FM 4-64-labeled bacteria (red). After incubation, the ECM, including collagen fibrils, were visualized by FITC-labeled UspA230–539 (green). Orange spots in overlays illustrate colocalization of M. catarrhalis and ECM, including collagens. The size bar represents 10 µm. Download Figure S3, TIF file, 1.9 MB.
Copyright © 2016 Singh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Additional Files
Supplementary Data
Supplementary Data
- Figure sf1, TIF - Figure sf1, TIF
- Figure sf2, TIF - Figure sf2, TIF
- Figure sf3, TIF - Figure sf3, TIF
