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Activation of the epithelial sodium channel (ENaC) by the alkaline protease from Pseudomonas aeruginosa.

Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection. Recent studies suggest that misregulation of protease activity in the cystic fibrosis lung may alter fluid secretion and pathogen clearance by proteolytic activation of the epithelial sodium channel (ENaC). To evaluate the possibility that proteolytic activation of ENaC may contribute to the virulence of Pseudomonas, primary human bronchial epithelial cells were exposed to P. aeruginosa and ENaC function was assessed by short circuit current measurements. Apical treatment with a strain known to express high levels of alkaline protease (AP) resulted in an increase in basal ENaC current and a loss of trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To further characterize this AP-induced ENaC activation, AP was purified, and its folding, activity, and ability to activate ENaC were assessed. AP folding was efficient under pH and calcium conditions thought to exist in the airway surface liquid of normal and cystic fibrosis (CF) lungs. Short circuit measurements of ENaC in polarized monolayers indicated that AP activated ENaC in immortalized cell lines as well as post-transplant, primary human bronchial epithelial cells from both CF and non-CF patients. This activation was mapped to the γ-subunit of ENaC. Based on these data, patho-mechanisms associated with AP in the CF lung are proposed wherein secretion of AP leads to decreased airway surface liquid volume and a corresponding decrease in mucocilliary clearance of pulmonary pathogens.
AuthorsMichael B Butterworth, Liang Zhang, Elisa M Heidrich, Michael M Myerburg, Patrick H Thibodeau
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 287 Issue 39 Pg. 32556-65 (Sep 21 2012) ISSN: 1083-351X [Electronic] United States
PMID22859302 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
Chemical References
  • Bacterial Proteins
  • Epithelial Sodium Channels
  • Endopeptidases
  • alkaline protease
Topics
  • Animals
  • Bacterial Proteins (metabolism)
  • Bronchi (metabolism, microbiology, pathology)
  • Cell Line
  • Cell Polarity
  • Cystic Fibrosis (metabolism, microbiology, pathology)
  • Endopeptidases (metabolism)
  • Epithelial Cells (metabolism, microbiology, pathology)
  • Epithelial Sodium Channels (metabolism)
  • Humans
  • Mice
  • Pseudomonas Infections (metabolism, microbiology, pathology)
  • Pseudomonas aeruginosa (enzymology, pathogenicity)

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