Figure 1.
Interaction of A. baumannii with immobilized fibronectin.
(A) A. baumannii binding to fibronectin. ATCC 19606, 77 or 113-16 strains were incubated in BSA or in fibronectin-coated wells for 3 h at room temperature. Adherent bacteria were quantified by serial dilutions as described in materials and methods. (B) Inhibition of A. baumannii adherence to immobilized fibronectin by free fibronectin. ATCC 19606, 77 or 113-16 strain were incubated in fibronectin-coated wells containing increasing concentrations of free fibronectin (0, 10, 100 and 1,000 µg/mL) or BSA (1,000 µg/mL). Adherent bacteria were quantified by serial dilutions as described in materials and methods. Results were expressed as the percentage of total untreated A. baumannii adhered to immobilized fibronectin. Representative results of three independent experiments are shown and data are the means ± SEM. P<0.05: * between untreated and treated groups. Fn: fibronectin and BSA: bovine serum albumin.
Figure 2.
Immunodetection of binding of fibronectin to outer membrane proteins of A. baumannii and rOMPA.
Immunodetection of FBPs outer membrane of A.baumannii ATCC 19606, 77 or 113-16 strain (A) and FBP rOMPA (B). OMPs were extracted from outer membrane and rOMPA was produced in E. coli as described in materials and methods and stained with SimplyBlue™ SafeStain (SDS-PAGE) or electrotransferred onto nitrocellulose membrane, and incubated with Fn. FBPs were probed with rabbit anti- human fibronectin and HRP-conjugated goat anti-rabbit IgG (WB). Molecular mass standards (kDa) are shown on the right. FBPs: fibronectin binding proteins, Fn: fibronectin, MW: molecular weight, WB: western blot.
Table 1.
Identification of FBPs from A. baumannii ATCC 19606 by MALDI-TOF-TOF (MS-MS/MS) of selected bands.
Figure 3.
Involvement of fibronectin in the adherence of A. baumannii to human lung epithelial cells.
A549 cells were pretreated with RGD (0.5 and 5 mg/mL) (A), rabbit anti-human fibronectin (1∶25), rat anti-human E-cadherin (1∶25) or control mouse IgG (1∶25) (B) and infected with 108 cfu/mL of A. baumannii ATCC 19606, 77 or 113-16 strain for 2 h. Adherence assay was performed as described in materials and methods. The effect of treatment on A. baumannii adherence to A549 cells is expressed as the percentage of total untreated A. baumannii adhered to A549 cells. Immunostaining for A549 cells fibronectin and A. baumannii OMPs in infected A549 cells with 108 cfu/mL A. baumannii ATCC 19606, 77 or 113 strains for 2 h (C) and 24 h (D) were performed and imaged by immunofluorescence microscopy. Fibronectin of A549 cells and OMPs of A. baumannii strains were detected by rabbit anti-human fibronectin and mouse anti-A. baumannii OMPs antibodies and labeled with Alexa594 and Alexa488-tagged the secondary antibodies that appeared red and green, respectively. Blue staining shows the location of A549 cells nucleus. Representative results of three independent experiments are shown and data are the means ± SEM. P<0.05: * between untreated and treated groups. Fn: fibronectin and Ab: antibody.
Figure 4.
Involvement of A. baumannii OMPA in the adherence of A. baumannii to human lung epithelial cells.
A549 cells were infected for 2 h with 108 cfu/mL of A. baumannii ATCC 19606, 77 or 113-16 strain previously incubated for 1 h with mouse anti-OMPA of A. baumannii (1∶1,000 and 1∶250), rabbit anti-CarO of A. baumannii (1∶25) and control mouse IgG (1∶25). Adherence assay was performed as described in materials and methods. The effect of treatment on A. baumannii adherence to A549 cells is expressed as the percentage of total untreated A. baumannii adhered to A549 cells. Representative results of three independent experiments are shown and data are the means ± SEM. P<0.05: * between untreated and treated groups. OMPA: outer membrane protein A and Ab: antibody.