A novel vitronectin-binding protein of Pseudomonas aeruginosa for effective infection of the airways

• Objectives Pseudomonas aeruginosa is a Gram-negative species that causes chronic and acute infections of the lung, skin, urinary tract and eyes. Most P. aeruginosa isolates are highly resistant to antibiotics and difficult to eradicate due to biofilm formation. The bacterium is known to utilize host proteins by diverse strategies in order to enhance its virulence. Vitronectin is a glycoprotein that is abundant in serum and the extracellular matrix, and is involved in cell adhesion, migration, tissue repair and regulation of the complement cascade. The concentration of vitronectin in the lung reflects the level of inflammation in patients with interstitial lung disease. Furthermore, the production is upregulated in patients with cystic fibrosis, which are often chronically colonised with P. aeruginosa. In this study, we analysed the vitronectin-binding capability of clinical strains and identified the P. aeruginosa surface proteins involved in vitronectin binding. Methods P. aeruginosa clinical isolates (n=64) from the airway (n=36), blood (n=15) and urine (n=13), in addition to the reference strain (PAO1) were analysed in a direct binding assay using [125I]-vitronectin. To identify the vitronectin-binding surface proteins of P. aeruginosa, the outer membrane proteins of PAO1 were separated by 2D-SDS-PAGE and western blotting. Vitronectin binding proteins of P. aeruginosa were recombinantly expressed in Escherichia coli and protein-protein interactions were evaluated by ELISA and flow cytometry. P. aeruginosa transposon mutants obtained from the “P. aeruginosa two-allele library” were analysed for vitronectin binding by [125I]-vitronectin or vitronectin coated to a glass surface. Results Our direct binding assay revealed that P. aeruginosa airway isolates bound significantly more vitronectin in comparison to blood (p=0.02) and urine isolates (p=0.04) (Fig. A). Using an approach consisting of 2D-SDS-PAGE and western blotting, we identified two outer membrane proteins that interacted with vitronectin (Fig. B). Expression of one of those (vitronectin binding protein 1; VnBp1) in an E. coli laboratory strain resulted in VnBp1 on the cell surface, and a vitronectin-binding phenotype. In addition, recombinantly expressed and purified VnBP1 showed a dose-dependent interaction with vitronectin in an ELISA (Fig. C). P. aeruginosa with a transposon insert in the vnBp1 gene bound significantly less vitronectin in comparison to the wild type (p=0.0009). Moreover, vnBp1 deficient mutants also showed significant reduced adherence to vitronectin coated glass slide (p≤0.001) in comparison to the wild type (Fig. D). Conclusions P. aeruginosa isolates cultured from the lung bind significantly more vitronectin in comparison to strains cultured from urine or blood. Vitronectin is recruited at the surface via VnBp1. This mechanism is likely to be of great importance for P. aeruginosa adhesion to the airway epithelial and basal lamina of disrupted airway epithelial cell layer and hence for the colonisation of the respiratory tract.

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