Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase)

Abstract

Pseudomonas aeruginosa elastase, a Bacillus subtilis thermolysin-like zinc-proteinase was examined for hemorrhagic activity and its effect on muscle and endothelial cells. Subcutaneous and intramuscular injections of elastase into mice caused severe hemorrhage with an acute increase of creatine phosphokinase activity in serum. The elastase also possessed fibrinogenolytic and fibrinolytic activities. The Aα and Bβ chains of fibrinogen were completely hydrolyzed as demonstrated by their electrophoretic disappearance on SDS polyacrylamide gels. The pathological study indicates that elastase induces changes in the structure of the vascular wall and causes leakage of the plasma component and red and white blood cells into the extravascular tissue. This is further supported by results showing injury to cultured endothelial cells and macrophages. These data indicate that P. aeruginosa elastase directly affects endothelial cells and destroys the basement membrane of blood vessels to cause hemorrhage. Since fibrinogenolytic activity is an additional component of this elastase and this activity induces the hemorrhagic tendency, the damage in tissues could become increasingly severe.

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.