The role of the elastase of Pseudomonas aeruginosa in experimental infection

Abstract

A characteristic vascular lesion has been described in human systemic infections caused by Pseudomonas aeruginosa (Forkner et al., 1958; Margaretten et al., 1961: Rabin et al., 1961). In an attempt to elucidate its pathogenesis, experimental animals were infected with P. aeruginosa to reproduce this vascular lesion. It was found that destruction of the arterial elastic lamina was frequently associated with the lesion, suggesting a probable role of the elastase of P. aeruginosa in the pathogenesis. Some of our studies on the elastase of P. aeruginosa are reported here. MATERIALS AND METHODS Source of Microorganism. A strain of P. aeruginosa, isolated from an infected burn wound of a patient and previously shown to have high elastolytic activity (Mull and Callahan, 1963)) was employed. Preparation of elastase extract. Elastase was prepared from P. aeruginosa cultured in nutrient broth (37°C for 3 days) by the following fractionation procedure carried out at 4°C. Slime and bacterial cells were removed by filtration and/or centrifugation and ammoni'G sulfate (400 gm/liter) was added to the supernatant. The precipitate that formed overnight was recovered by centrifugation, dissolved in a small volume of distilled water, and dialysed for 48 hours. After inert material was removed by precipitation with streptomycin sulfate (10 gm/liter), protein was reprecipitated from the supernatant by the addition of ammonium sulfate (300 gm/liter) after which the precipitate was redissolved in water, dialysed, and lyophilized. The resulting brown product which was completely water soluble was used in the following assays. Enzyme assay. Twenty mg of elastin-orcein (Worthington) and 3 mg of elastase extract dissolved in 3 ml tris buffer [ tris (hydroxy-methyl) aminomethane] 0.05 ICI pH 7.8 were incubated at 37°C with gentle shaking for 4 hours. Reactions were stopped by the addition of 2 ml Sorensen's phosphate buffer, 0.7 M pH 7.0 (Sacher et al., 1955). Since bacterial pigments present in the enzyme preparation had optical absorption properties similar to that of orcein, the extent of elastolysis was determined indirectly by estimating the remaining undigested elastin-orcein. The undigested

Protease-deficient Mutants of Pseudomonas aeruginosa: Pleiotropic Changes in Activity of Other Extracellular Enzymes

“…One of these enzymes (protease II) possessing elastase activity, is dermonecrotic, corneadamaging, and probably responsible for the destruction of the arterial elastic lamina in patients with Pseudomonas bacteraemia (Kawaharajo et al, 1975;Kreger & Griffin, 1974;Mull & Callahan, 1965).…”

Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity

“…Pseudomonas aeruginosa elastase is recognized as one of several secreted proteins associated with the virulence of this opportunistic pathogen (20). The majority (74 to 99%) of Pseudomonas isolated derived from hospital patients produce the enzyme (10,20,27).…”

Pseudomonas septicaemia: in a case of pancytopenia

“…10 Pseudomonas elastase enzyme destroys the blood vessels, which is responsible for invasive manifestations. 9 In present case the use of filgrastim has improved patient condition drastically. The fundamental principle in treatment of this condition is appropriate use of antibiotics along with management of pancytopenia.…”

“…7 Cause of pancytopenia was initially thought to be low vitamin b12 levels (megaloblastic anaemia), but bone marrow biopsy was suggestive of hypoplastic bone marrow. 9 Inspite of higher antibiotics patient had a poor response to therapy.…”