Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect o f stationary-phase supernatants from the wild-type strain PAO1 on cell migr ation, actin cytoskeleton distribution, epithelial integrity during and aft er repair of induced wounds, and the balance between matrix metalloproteina ses (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered woun d repair by slowing the migration velocity in association with altered acti n cytoskeleton polymerization in the lamellipodia of migrating airway epith elial cells and delaying or inhibiting the restoration of epithelial integr ity after wound closure. PAO1 virulence factors overactivated two of the ge latinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. Durin g HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 act ivation was associated with decreased rates of its specific inhibitor TIMP- 2, whereas enhanced MMP-9 activation was independent of changes of its spec ific inhibitor TIMP-1. These inhibitory effects were specific to P. aerugin osa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 a nd Escherichia coli strain DH5 alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the pr esence of increasing concentrations of activated MMP-9 or MMP-2. Whereas in creasing concentrations of active MMP-9 accelerated repair, excess activate d MMP-2 generated a lower migration velocity. All these data demonstrate th at P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.