title: In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection.
abstract: OBJECTIVE
  To establish if active pseudomonal proteases are present in vivo during corneal infection with Pseudomonas aeruginosa and to determine if the mouse strains used in these and previous studies have the ability to mount a nonocular antibody response to the purified proteases because antibodies to the bacterial proteases were not detected previously during in vivo ocular infection.
  
  
  METHODS
  At certain times after corneal infection with P. aeruginosa, corneas were harvested and supernatants from the corneal homogenates were analyzed for proteolytic activity by zymography and immunoreactivity by immunoblotting. The efficiency of the extraction procedures used in these studies was determined by incubating uninfected corneal homogenates with the purified proteases. The resultant supernatants were analyzed for alkaline protease and elastase activity. Additionally, mice were immunized intraperitoneally with the purified proteases with and without adjuvant to determine if the animals could mount a nonocular antibody response.
  
  
  RESULTS
  Corneas infected with P. aeruginosa demonstrated the presence of alkaline protease, but not elastase, by the two methods examined. The kinetics of the in vivo alkaline protease response closely parallels previously reported bacterial clearance studies in that peak alkaline protease activity was detected in corneal tissue when peak bacterial numbers also were observed in the eye, and it was absent when the eyes were sterile or nearly sterile. In addition, C57BL/6J mice were capable of mounting a nonocular antibody response to microgram quantities of both proteases only in the presence of adjuvant.
  
  
  CONCLUSIONS
  In the model described, enzymatically active alkaline protease, but not elastase, was demonstrated in corneal tissues during in vivo infection. Concentrations of these proteases were much lower than those required to stimulate an antibody response.
authors.affiliation: Department of Immunology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Kernacki
authors.forename: J A
authors.initials: JA
authors.lastname: Hobden
authors.forename: L D
authors.initials: LD
authors.lastname: Hazlett
authors.forename: R
authors.initials: R
authors.lastname: Fridman
authors.forename: R S
authors.initials: RS
authors.lastname: Berk
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D003315:Cornea / Q000201:enzymology; D015818:Eye Infections, Bacterial / Q000201:enzymology* / Q000276:immunology; D007114:Immunization; D015151:Immunoblotting; D007634:Keratitis / Q000201:enzymology* / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D010196:Pancreatic Elastase / Q000096:biosynthesis / Q000276:immunology; D011552:Pseudomonas Infections / Q000201:enzymology* / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000276:immunology; D011817:Rabbits; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; C032923:microbial serine proteinases