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. 1984 Mar;43(3):1058–1063. doi: 10.1128/iai.43.3.1058-1063.1984

Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model.

T R Howe, B H Iglewski
PMCID: PMC264293  PMID: 6421735

Abstract

Mutants of Pseudomonas aeruginosa are described which are markedly deficient in alkaline protease production. Characterization of these mutants in vitro suggests that the mutations in two of these strains are specific for alkaline protease production. Examination of these mutants in a mouse eye model demonstrates that alkaline protease is required for the establishment of corneal infections with P. aeruginosa PA103. Mutants deficient in alkaline protease production could not colonize traumatized cornea and did not produce the corneal damage characteristic of infection by the parental strain. Addition of subdamaging amounts of alkaline protease to eyes infected with the protease-deficient mutants resulted in infections which were indistinguishable from infections caused by the parental strain.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bohigian G. M., Escapini H. Corneal ulcer due to Pseudomonas aeruginosa. A comparison of the disease in California and El Salvador. Arch Ophthalmol. 1971 Apr;85(4):405–409. doi: 10.1001/archopht.1971.00990050407002. [DOI] [PubMed] [Google Scholar]
  2. Cicmanec J. F., Holder I. A. Growth of Pseudomonas aeruginosa in normal and burned skin extract: role of extracellular proteases. Infect Immun. 1979 Aug;25(2):477–483. doi: 10.1128/iai.25.2.477-483.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cryz S. J., Iglewski B. H. Production of alkaline protease by Pseudomonas aeruginosa. J Clin Microbiol. 1980 Jul;12(1):131–133. doi: 10.1128/jcm.12.1.131-133.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. FISHER E., Jr, ALLEN J. H. Corneal ulcers produced by cell-free extracts of Pseudomonas aeruginosa. Am J Ophthalmol. 1958 Jul;46(1 Pt 2):21–27. doi: 10.1016/0002-9394(58)90030-8. [DOI] [PubMed] [Google Scholar]
  6. Farmer J. J., 3rd, Herman L. G. Epidemiological fingerprinting of Pseudomonas aeruginosa by the production of and sensitivity of pyocin and bacteriophage. Appl Microbiol. 1969 Nov;18(5):760–765. doi: 10.1128/am.18.5.760-765.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gerke J. R., Magliocco M. V. Experimental Pseudomonas aeruginosa Infection of the Mouse Cornea. Infect Immun. 1971 Feb;3(2):209–216. doi: 10.1128/iai.3.2.209-216.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanne L. F., Howe T. R., Iglewski B. H. Locus of the Pseudomonas aeruginosa toxin A gene. J Bacteriol. 1983 Apr;154(1):383–386. doi: 10.1128/jb.154.1.383-386.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirao Y., Homma J. Y. Therapeutic effect of immunization with OEP, protease toxoid and elastase toxoid on corneal ulcers in mice due to Pseudomonas aeruginosa infection. Jpn J Exp Med. 1978 Feb;48(1):41–51. [PubMed] [Google Scholar]
  10. Holt R. J. The detection of bacterial esterases and lipases. Med Lab Technol. 1971 Apr;28(2):208–210. [PubMed] [Google Scholar]
  11. Homma J. Y., Abe C., Tanamoto K., Hirao Y., Morihara K., Tsuzuki H., Yanagawa R., Honda E., Aoi Y., Fujimoto Y. Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), protease and elastase toxoids of Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink due to P. aeruginosa. Jpn J Exp Med. 1978 Apr;48(2):111–133. [PubMed] [Google Scholar]
  12. KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
  13. Kawaharajo K., Abe C., Homma J. Y., Kawano M., Goto E. Corneal ulcers caused by protease and elastase from Pseudomonas aeruginosa. Jpn J Exp Med. 1974 Oct;44(5):435–442. [PubMed] [Google Scholar]
  14. Kawaharajo K., Homma J. Y. Pathogenesis of the mouse keratitis produced with Pseudomonas aeruginosa. Jpn J Exp Med. 1975 Dec;45(6):515–524. [PubMed] [Google Scholar]
  15. Kearney J. F., Radbruch A., Liesegang B., Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol. 1979 Oct;123(4):1548–1550. [PubMed] [Google Scholar]
  16. Kreger A. S., Gray L. D. Purification of Pseudomonas aeruginosa proteases and microscopic characterization of pseudomonal protease-induced rabbit corneal damage. Infect Immun. 1978 Feb;19(2):630–648. doi: 10.1128/iai.19.2.630-648.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kreger A. S., Griffin O. K. Physicochemical fractionation of extracellular cornea-damaging proteases of Pseudomonas aeruginosa. Infect Immun. 1974 May;9(5):828–834. doi: 10.1128/iai.9.5.828-834.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laibson P. R. Cornea and sclera. Arch Ophthalmol. 1972 Nov;88(5):553–574. doi: 10.1001/archopht.1972.01000030555018. [DOI] [PubMed] [Google Scholar]
  19. Liu P. V. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. 3. Identity of the lethal toxins produced in vitro and in vivo. J Infect Dis. 1966 Oct;116(4):481–489. doi: 10.1093/infdis/116.4.481. [DOI] [PubMed] [Google Scholar]
  20. MORIHARA K. PRODUCTION OF ELASTASE AND PROTEINASE BY PSEUDOMONAS AERUGINOSA. J Bacteriol. 1964 Sep;88:745–757. doi: 10.1128/jb.88.3.745-757.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ohman D. E., Burns R. P., Iglewski B. H. Corneal infections in mice with toxin A and elastase mutants of Pseudomonas aeruginosa. J Infect Dis. 1980 Oct;142(4):547–555. doi: 10.1093/infdis/142.4.547. [DOI] [PubMed] [Google Scholar]
  22. Ohman D. E., Sadoff J. C., Iglewski B. H. Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun. 1980 Jun;28(3):899–908. doi: 10.1128/iai.28.3.899-908.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rinderknecht H., Geokas M. C., Silverman P., Haverback B. J. A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Acta. 1968 Aug;21(2):197–203. doi: 10.1016/0009-8981(68)90127-7. [DOI] [PubMed] [Google Scholar]
  24. Snell K., Holder I. A., Leppla S. A., Saelinger C. B. Role of exotoxin and protease as possible virulence factors in experimental infections with Pseudomonas aeruginosa. Infect Immun. 1978 Mar;19(3):839–845. doi: 10.1128/iai.19.3.839-845.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sokol P. A., Ohman D. E., Iglewski B. H. A more sensitive plate assay for detection of protease production by Pseudomanas aeruginosa. J Clin Microbiol. 1979 Apr;9(4):538–540. doi: 10.1128/jcm.9.4.538-540.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stern G. A., Weitzenkorn D., Valenti J. Adherence of Pseudomonas aeruginosa to the mouse cornea. Epithelial v stromal adherence. Arch Ophthalmol. 1982 Dec;100(12):1956–1958. doi: 10.1001/archopht.1982.01030040936014. [DOI] [PubMed] [Google Scholar]
  27. Thompson M. R., Miller R. D., Iglewski B. H. In vitro production of an extracellular protease by Legionella pneumophila. Infect Immun. 1981 Oct;34(1):299–302. doi: 10.1128/iai.34.1.299-302.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  29. Woods D. E., Sokol P. A., Iglewski B. H. Modulatory effect of iron on the pathogenesis of Pseudomonas aeruginosa mouse corneal infections. Infect Immun. 1982 Feb;35(2):461–464. doi: 10.1128/iai.35.2.461-464.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Woods D. E., Straus D. C., Johanson W. G., Jr, Bass J. A. Role of fibronectin in the prevention of adherence of Pseudomonas aeruginosa to buccal cells. J Infect Dis. 1981 Jun;143(6):784–790. doi: 10.1093/infdis/143.6.784. [DOI] [PubMed] [Google Scholar]
  31. Wretlind B., Sjöberg L., Wadström T. Protease-deficient mutants of Pseudomonas aeruginosa: pleiotropic changes in activity of other extracellular enzymes. J Gen Microbiol. 1977 Dec;103(2):329–336. doi: 10.1099/00221287-103-2-329. [DOI] [PubMed] [Google Scholar]
  32. Young L. S. The role of exotoxins in the pathogenesis of Pseudomonas aeruginosa infections. J Infect Dis. 1980 Oct;142(4):626–630. doi: 10.1093/infdis/142.4.626. [DOI] [PubMed] [Google Scholar]

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