Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase)
Introduction
Pseudomonas aeruginosa is a gram-negative opportunistic bacterial pathogen, which can cause severe infectious disease in compromised hosts (Ryan, 1984). Recently, various antibiotic-resistant and antiseptic-resistant strains were isolated from clinical sources (Nakahara and Kozukue, 1982, Shimizu et al., 1985, Quinn and Dudek, 1986, Watanabe et al., 1991, Poole et al., 1993, Li et al., 1994, Senda et al., 1996), and the mechanisms of resistant gene acquisition have been studied (Kazama et al., 1995, Kazama et al., 1998, Naas et al., 1999). High frequency of detection of these strains in hospital environments increases the cases of opportunistic infection in immunosupressed patients.
P. aeruginosa has various pathogenic factors such as pili, alginate, endotoxin, exotoxin A, exoenzyme S, leukocidine, phospholipase C and proteolytic enzymes: an elastase and an alkaline protease (Ryan, 1984, Iglewski, 1988). Elastase and alkaline protease are active on various substrates involved in host defense mechanisms, and shown to be secreted in vivo over prolonged periods in the airways when P. aeruginosa chronically colonized the airways of patients with cystic fibrosis (Suter, 1994). Elastase has been well characterized by Morihara et al. (1965). This enzyme belongs to the zinc-metalloproteinase family and its primary and three-dimensional structures are homologous to those of thermolysin from Bacillus subtilis (Bever and Iglewski, 1988, Fukushima et al., 1989, Thayer et al., 1991). P. aeruginosa elastase degrades not only elastin and collagen but also several immunologically important proteins such as immunoglobulin G, α1-proteinase inhibitor, γ-interferon, and complement components (Galloway, 1991). This enzyme also possesses damaging effects on respiratory epithelium and may be involved in tissue damage to the airways in cystic fibrosis (Suter, 1994), however, the full in vivo effect of P. aeruginosa elastase has not been clarified sufficiently. In this study, the effect of elastase on muscle tissue is investigated using pathological techniques and the change in creatine phosphokinase activity, which reflects the damage in muscle tissue, is measured. Additionally, the damage to endothelial cells and macrophage cell line is reported.
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Materials
P. aeruginosa strain NPA9809 was isolated from a patient's stool. Dulbecco's modified Eagle medium (DMEM) and RPMI 1640 medium were the products of Nissui Seiyaku, Co. (Tokyo, Japan), and calf serum was from GIBCO BBL (USA). DE52 Cellulose and CM52 Cellulose were purchased from Whatman BioSystems Ltd. (England), and CM-Sephadex and DEAE-Sephacel were from Amersham Pharmacia Biotech. Casein was obtained from MERCK KGaA, azocasein, azoalbumin, hide powder azure and bovine thrombin were from Sigma
Isolation and proteolytic activity
A metalloproteinase was purified from the P. aeruginosa culture supernatant according to the modified method of Elliot and Cohen (1986). Approximately 2.8 mg of sample was obtained from 1 l of culture supernatant, with a yield of 0.13%. The purified sample was identified as an acidic protein with a molecular weight of 34,000. The N-terminal amino acid sequence to residue 25 was determined to be Ala-Glu-Ala-Gly-Gly-Pro-Gly-Gly-Asn-Gln-Lys-Ile-Gly-Lys-Tyr-Thr-Tyr-Gly-Ser-Asp-Tyr-Gly-Pro-Leu-Ile.
Discussion
Elastase is considered to be one of the P. aeruginosa pathogens, and its proteolytic activity on various protein substrates has been well studied in vitro. This B. subtilis thermolysin-like zinc-proteinase possesses wide-range specificity and hydrolyzes collagen (Heck et al., 1986), immunoglobulin G (Fick et al., 1985), α1-proteinase inhibitor (Morihara et al., 1979), γ-interferon (Horvat et al., 1989), and complement components (Galloway, 1991). Blackwood et al. (1983) indicated that elastase
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