title: Alkaline protease-deficient mutants of Pseudomonas aeruginosa are virulent in the eye.
abstract: OBJECTIVE
  Alkaline protease has been associated with virulence in Pseudomonas aeruginosa corneal infections. To define the role of this enzyme in such infections, isogenic mutants of P. aeruginosa deficient in alkaline protease production were constructed. This study examines the ability of these mutants to adhere to scarified corneal tissue in vitro and to establish corneal infections in vivo.
  
  
  METHODS
  Mutants were constructed by allelic exchange in two phenotypically different wild type strains, PAO1 (invasive) and ATCC 19660 (cytotoxic). Alkaline protease-deficient mutants were characterized by zymography and western blot analysis of bacterial culture supernatants. Allelic exchange was confirmed by PCR analysis of the disrupted aprA gene of the mutants. Adherence of wild type and mutant strains to scarified corneal epithelium was assessed by an in vitro organ culture assay, while ocular virulence of the strains was determined in vivo using a mouse scarification model of bacterial keratitis.
  
  
  RESULTS
  Being isogenic, phenotypes of mutants were identical to their respective parents with the exception of the loss of alkaline protease production. The absence of alkaline protease did not alter corneal adherence or ocular virulence of the organisms when compared to similar wild type strains.
  
  
  CONCLUSIONS
  These data provide evidence that alkaline protease produced by P. aeruginosa is not essential in the pathogenesis of P. aeruginosa keratitis.
authors: Department of Immunology and Microbiology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
authors: C M
authors: CM
authors: Pillar
authors: L D
authors: LD
authors: Hazlett
authors: J A
authors: JA
authors: Hobden
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion; D001426:Bacterial Proteins; D015153:Blotting, Western; D003315:Cornea / Q000382:microbiology*; D003320:Corneal Ulcer / Q000382:microbiology* / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D010450:Endopeptidases / Q000235:genetics; D015818:Eye Infections, Bacterial / Q000382:microbiology* / Q000473:pathology; D005260:Female; D005822:Genetic Vectors; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000382:microbiology* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000472:pathogenicity*; D012697:Serine Endopeptidases / Q000172:deficiency / Q000502:physiology*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D010450:Endopeptidases; D012697:Serine Endopeptidases; C032923:microbial serine proteinases; C087822:alkaline protease