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

Paulsson, Magnus LU orcid ; Ringwood, Tamara ; Su, Shanice Yc LU ; Singh, Birendra LU ; Høiby, Niels and Riesbeck, Kristian LU orcid (2015) 25th European Congress of Clinical Microbiology and Infectious Diseases p.0456-0456
Abstract
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... (More)
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. (Less)
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author
; ; ; ; and
organization
publishing date
type
Contribution to conference
publication status
published
pages
0456 - 0456
conference name
25th European Congress of Clinical Microbiology and Infectious Diseases
conference location
Copenhagen, Denmark
conference dates
2015-04-25 - 2015-04-28
project
The Extracellular Matrix in Patients with CF or COPD as a Basis for Novel Therapeutical Opportunities
language
English
LU publication?
yes
id
6a78ecfb-3fac-456b-88e0-1cf51c06f3da
alternative location
https://www.escmid.org/escmid_publications/escmid_elibrary/material/?mid=22926
date added to LUP
2019-06-20 09:21:00
date last changed
2019-06-25 02:19:30
@misc{6a78ecfb-3fac-456b-88e0-1cf51c06f3da,
  abstract     = {{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.}},
  author       = {{Paulsson, Magnus and Ringwood, Tamara and Su, Shanice Yc and Singh, Birendra and Høiby, Niels and Riesbeck, Kristian}},
  language     = {{eng}},
  month        = {{04}},
  pages        = {{0456--0456}},
  title        = {{A novel vitronectin-binding protein of Pseudomonas aeruginosa for effective infection of the airways}},
  url          = {{https://www.escmid.org/escmid_publications/escmid_elibrary/material/?mid=22926}},
  year         = {{2015}},
}