Corneal Virulence of Pseudomonas aeruginosa Elastase B and Alkaline Protease Produced by Pseudomonas putida

Purpose: To measure the specific virulence contributions of two Pseudomonas aeruginosa proteases, elastase B and alkaline protease, when expressed separately by Pseudomonas putida in a rabbit model of bacterial keratitis. Methods: P. putida KT2440 was transformed with plasmids that enabled the extracellular production of either elastase or alkaline protease. Protease expression was confirmed by zymography and immunoblotting. P. putida expressing elastase, alkaline protease, or vector alone was injected intrastromally (103 colony forming units [CFU]) into rabbit corneas (n = 6). Infected eyes were graded by slit-lamp examination (SLE) at 20, 24, 28, and 32 hr postinfection (PI). Rabbits were sacrificed at 33 hr PI, and the log CFU (±SEM) per cornea was determined. Results: SLE scores for eyes infected with P. putida producing elastase were significantly higher than those infected with vector alone at all time points (p ≤ 0.008). SLE scores for eyes infected with P. putida producing alkaline protease were not significantly higher than the control (p ≥ 0.1), but small erosions formed in 33% of corneas. At both 24 and 28 hr PI, the SLE scores for corneas infected with P. putida producing elastase were significantly higher than those infected with P. putida producing alkaline protease (p ≤ 0.002). Conclusions: Elastase production by P. putida caused significant increases in SLE scores whereas expression of alkaline protease caused limited corneal erosions. This suggests that the production of elastase during P. aeruginosa keratitis enhances ocular pathology, whereas alkaline protease production contributes to limited corneal erosion.

[1]  M. Marquart,et al.  Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions. , 2005, Investigative ophthalmology & visual science.

[2]  M. Marquart,et al.  Pseudomonas aeruginosa Protease IV: A Corneal Virulence Factor of Low Immunogenicity , 2005, Ocular immunology and inflammation.

[3]  M. Traidej,et al.  Effectiveness of Ciprofloxacin, Levofloxacin, or Moxifloxacin for Treatment of Experimental Staphylococcus aureus Keratitis , 2004, Antimicrobial Agents and Chemotherapy.

[4]  M. Traidej,et al.  Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases. , 2004, Investigative ophthalmology & visual science.

[5]  M. Traidej,et al.  Identification of the Active Site Residues of Pseudomonas aeruginosa Protease IV , 2003, The Journal of Biological Chemistry.

[6]  M. Traidej,et al.  Molecular analysis of Pseudomonas aeruginosa protease IV expressed in Pseudomonas putida. , 2003, Investigative ophthalmology & visual science.

[7]  M. Marquart,et al.  Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor , 2003, Medical Microbiology and Immunology.

[8]  R. O'Callaghan,et al.  Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit. , 2002, Investigative ophthalmology & visual science.

[9]  O. White,et al.  Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. , 2002, Environmental microbiology.

[10]  E. Hume,et al.  Effectiveness of Mupirocin and Polymyxin B in Experimental Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens Keratitis , 2002, Cornea.

[11]  J. A. Hobden.  Pseudomonas aeruginosa proteases and corneal virulence. , 2002, DNA and cell biology.

[12]  M. Marquart,et al.  Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases. , 2001, Analytical biochemistry.

[13]  R. O'Callaghan,et al.  The effectiveness of tobramycin and Ocuflox® in a prophylaxis model of Staphylococcus keratitis , 2001, Current eye research.

[14]  J. Tommassen,et al.  Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex. , 2000, Microbiology.

[15]  S. Lory,et al.  Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen , 2000, Nature.

[16]  W. Pierce,et al.  Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa , 2000, The Journal of Biological Chemistry.

[17]  J. Lyczak,et al.  Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. , 2000, Microbes and infection.

[18]  S. C. Huang,et al.  Microbial keratitis--a late complication of penetrating keratoplasty. , 2000, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[19]  J. Tommassen,et al.  Identification of a Chitin-Binding Protein Secreted by Pseudomonas aeruginosa , 2000, Journal of bacteriology.

[20]  J. A. Hobden,et al.  A Pseudomonas aeruginosa strain isolated from a Contact Lens-Induced Acute Red Eye (CLARE) is protease-deficient , 2000, Current eye research.

[21]  L. Hazlett,et al.  Alkaline protease-deficient mutants of Pseudomonas aeruginosa are virulent in the eye. , 2000, Current eye research.

[22]  A. Karabulut,et al.  Conjunctival impression cytology and tear-film changes in patients with psoriasis. , 1999, Cornea.

[23]  R. O'Callaghan.  Role of exoproteins in bacterial keratitis: the Fourth Annual Thygeson Lecture, presented at the Ocular Microbiology and Immunology Group Meeting, November 7, 1998. , 1999, Cornea.

[24]  James M. Hill,et al.  Protease IV, a Unique Extracellular Protease and Virulence Factor from Pseudomonas aeruginosa * , 1998, The Journal of Biological Chemistry.

[25]  J. Tommassen,et al.  Function of bacterial propeptides. , 1998, Trends in microbiology.

[26]  W. Wee,et al.  Bacterial keratitis after photorefractive keratectomy in a young, healthy man , 1997, Journal of cataract and refractive surgery.

[27]  J. A. Hobden,et al.  Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence. , 1996, Investigative ophthalmology & visual science.

[28]  H. Schweizer,et al.  Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. , 1994, Gene.

[29]  E. Kessler,et al.  The propeptide of Pseudomonas aeruginosa elastase acts an elastase inhibitor. , 1994, The Journal of biological chemistry.

[30]  B. Smith,et al.  SDS Polyacrylamide Gel Electrophoresis of Proteins. , 1984 .

[31]  U. Baumann,et al.  Three‐dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two‐domain protein with a calcium binding parallel beta roll motif. , 1993, The EMBO journal.

[32]  D. Frank,et al.  Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins. , 1993, Investigative ophthalmology & visual science.

[33]  J. A. Hobden,et al.  Pseudomonas aeruginosa keratitis in leukopenic rabbits. , 1993, Current eye research.

[34]  J. A. Hobden,et al.  Ciprofloxacin Ointment Versus Ciprofloxacin Drops for Therapy of Experimental Pseudomonas Keratitis , 1993, Cornea.

[35]  F. Duong,et al.  Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways. , 1992, Gene.

[36]  K. Kenyon,et al.  Proteolytic activation of corneal matrix metalloproteinase by Pseudomonas aeruginosa elastase. , 1992, Current eye research.

[37]  B. Ghebrehiwet,et al.  Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3. , 1992, Clinical immunology and immunopathology.

[38]  J. Reidy,et al.  Ciprofloxacin and prednisolone therapy for experimental Pseudomonas keratitis. , 1992, Current eye research.

[39]  J. Pagés,et al.  Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism , 1991, Journal of bacteriology.

[40]  M. Parmely,et al.  Proteolytic inactivation of cytokines by Pseudomonas aeruginosa , 1990, Infection and immunity.

[41]  D. Galloway,et al.  Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity , 1990, Journal of bacteriology.

[42]  A. Filloux,et al.  Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli , 1990, Journal of bacteriology.

[43]  E. Kessler,et al.  Synthesis, processing, and transport of Pseudomonas aeruginosa elastase , 1988, Journal of bacteriology.

[44]  P. A. Schad,et al.  Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene , 1988, Journal of bacteriology.

[45]  B. Pedersen,et al.  Inhibition of human natural killer cell activity by Pseudomonas aeruginosa alkaline protease and elastase , 1987, Infection and immunity.

[46]  D. Abrahamson,et al.  Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease , 1986, Infection and immunity.

[47]  E. J. Miller,et al.  Specific cleavage of human type III and IV collagens by Pseudomonas aeruginosa elastase , 1986, Infection and immunity.

[48]  B. Iglewski,et al.  The contribution of exoproducts to virulence of Pseudomonas aeruginosa. , 1986, Canadian journal of microbiology.

[49]  B. Iglewski,et al.  Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model , 1984, Infection and immunity.

[50]  K. Botzenhart,et al.  [Extracellular toxins of Pseudomonas aeruginosa. II. Effect of two proteases on human immunoglobulins IgG, IgA and secretory IgA (author's transl)]. , 1981, Zentralblatt fur Bakteriologie. 1. Abt. Originale. A: Medizinische Mikrobiologie, Infektionskrankheiten und Parasitologie.

[51]  T. Liesegang,et al.  Spectrum of microbial keratitis in South Florida. , 1980, American journal of ophthalmology.

[52]  J. Sadoff,et al.  Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization , 1980, Infection and immunity.

[53]  A. Kreger,et al.  Purification of Pseudomonas aeruginosa proteases and microscopic characterization of pseudomonal protease-induced rabbit corneal damage , 1978, Infection and immunity.

[54]  E. Kessler,et al.  The corneal response to Pseudomonas aeruginosa: histopathological and enzymatic characterization. , 1977, Investigative ophthalmology & visual science.

[55]  H. B. Ostler,et al.  The changing pattern of the etiology of central bacterial corneal (hypopyon) ulcer. , 1976, Transactions of the Pacific Coast Oto-Ophthalmological Society annual meeting.

[56]  M. Kawano,et al.  Corneal ulcers caused by protease and elastase from Pseudomonas aeruginosa. , 1974, The Japanese journal of experimental medicine.

[57]  Chunfang ZHANGt.  Pseudomonas aeruginosa. , 1966, Lancet.

[58]  K. Morihara.  PRODUCTION OF ELASTASE AND PROTEINASE BY PSEUDOMONAS AERUGINOSA , 1964, Journal of bacteriology.

[59]  K. Morihara.  Pseudomonas aeruginosa proteinase: I. Purification and general properties , 1963 .