Isolation, Purification And Characterization Of A Novel Exotoxin From Staphylococcus Aureus Isolated From The Eczematous Lesion Of Patient With Atopic Dermatitis

摘要

A novel exotoxin from Staphylococcus aureus isolated from eczematous lesion of patient with atopic dermatitis was isolated, purified, and characterized in this study. These exotoxin has clotting activity (85.5) unit/ml, specific activity (2085.3658) unit/mg and total activity (1282.5) units after (347.56) degree of purification yielded (3.975)% of exotoxin resultant , and the solution of Staphylococcus aureus exotoxin show a very high purified single band protein by using polyacrylamide gel electrophoresis PAGE (7.5%), these band-in comparison with standard protein-has a molecular weight (43.315)kd. Introduction The normal bacterial skin flora in human is composed of three major groups of Gram-positive bacteria, the coryneform bacteria, the micrococci and the staphylococci, with only a minor component of Gram negative bacilli(1). This is chiefly because the skin is a comparatively dry habitat, with available water as the chief factor controlling growth; occlusion of skin is a potent way to increase the number of bacteria on the skin(2). Gram negative bacilli require more available water than Gram-positive bacteria and this probably controls their population density(3). Bacterial counts on unaffected kin are lower than on affected a topic skin(4). The density of Staph. aureus on eczematous lesions has been show to correlate with cutaneous inflammation(5).Chronic skin colonization with Staphylococcus aureus is a characteristic feature of a topic dermatitis (AD)(6), and about 60-90% of Staph. aureus strains isolated from the skin AD patients(7). Up to 65% have been shown to produce exotoxins with superantigenic properties(2). However, the mechanism(s) underlying the effects of this organism in the disease process are unclear.These potent toxins bind directly without antigen-presenting cells (APC) such as macrophages or dendritic cells and to cytokine-induced HLA-DR molecules on non professional APC such as keratinocytes(8). Over half of AD patients have Staph. aureus cultured from their skin that secrete superantigens such as enterotoxins A,B and toxic shock syndrome toxin-1 (TSST-1) and many of unclassified exotoxins(9,10).There are many exotoxins that produce by Staph. aureus isolated from eczematous lesions of AD patients, and we investigate purification and characterization of one of these exotoxin. Materials and Methods Primary screening One isolate of Staph. aureus diagnosed by a routine techniques according to(11) ,were selected from twenty isolates according to highly degrees of chronicity and severity of atopic dermatitis associated with these bacteria.The antibacterial activity against four standard strains of bacteria was studied by culturing Staph. aureus with each of these bacteria(12) .Standard strain are: E coli NCTC 5933, Staph aureus NCTC 6571, Kl. Pneumonia ATCC 10031, and B. subtilis PCI 219. Primary detection of Staph.aureus exotoxin : Two culture media were used to testing ability of Staph. aureus to produce exotoxin (having proteolytic activity): Casein Hydrolysate Agar CHA and Skin Milk Agar (SMA) (Oxoid). A clear zones around the Staph. aureus colonies grew on above media indicate to ability of these bacteria to produce proteolytic enzyme exotoxin(13). Production of exotoxin Estimation of biomass of bacterial growth (gm/100ml) in casein Hydrolysate Broth (CHB) (Oxoid), clotting activity for crude enzyme solution (unite/ml), protein concentration (mg/ml), and exotoxin activity (unit/mg) were carried according to(14). Purification of Staph. aureus exotoxin Three steps of purification were done for Staph. aureus exotoxin Precipitation by Ammonium sulphate salt according to (14).Membranous infiltration (dialysis technique) according to (15) and Gel filtration chromatography by using sephadex G-100 (Pharmacia, Sweden) in column (23 x 2.2 cm) according to (16) evaluate the purity and estimate the molecular weight of Staph. aureus exotoxin were carried by using conventional polyacrylamide gel electrophoresis (LKB