Isolation, Purification And Characterization Of A Novel Exotoxin From Staphylococcus Aureus Isolated From The Eczematous Lesion Of Patient With Atopic Dermatitis
I Alsaimary, K Mahdi, S Bakr, K Alhamdi
atopic dermatitis, staphylococcus aureus exotoxin
I Alsaimary, K Mahdi, S Bakr, K Alhamdi. Isolation, Purification And Characterization Of A Novel Exotoxin From Staphylococcus Aureus Isolated From The Eczematous Lesion Of Patient With Atopic Dermatitis. The Internet Journal of Dermatology. 2005 Volume 4 Number 2.
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 showed a very high purified single band protein by using polyacrylamide gel electrophoresis PAGE (7.5%), this band has -in comparison with standard protein- a molecular weight of (43.315)kd.
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 mainly 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
Chronic skin colonization with
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
There are many exotoxins that produce by
Materials and Methods
One isolate of
Primary detection of Staph.aureus exotoxin
Two culture media were used to testing ability of
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
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
Trypsin inhibitor, RNA polymerase, Bovine serum albumin, Aldolase, Catalase, Ferritin and Thyroglobulin.
The relative mobility (RM) was calculate by
The molecular weight of
These technique was carried according to (17) .
ANOVA test was carried by using SPSS computer program ver.11.
The growth of
Production assay of
Table (1) illustrates results of purification steps of
The extracted crud exotoxin after precipitation by (NH4)2SO4 gave specific activity (8.1)unit/mg, these activity higher than activity of crud extracted solution (6)unit/mg and (32.2)unit/mg with exotoxin recovery (resultant) (46.50%) after membranous infiltration and 2085.3658 with clotting activity reached (85.5) unit/ml and degrees of purification reached 347.56 with exotoxin recovery (resultant) (3.975%) after gel filtration chromatography.
Table (2) illustrates the relative mobility (Rm) and molecular weight of standard proteins and
The solution of
A technique composed of five steps were used to detection isolation purification, identification, and characterization of
Previous studies agreed and evidenced our finding suggested that a tightly correlation between
Abeck and Mempel, (1998) confirmed our results by findings illustrated that
Other study concluded a relationship between severity of skin lesion and sensitization to staphylococcal exotoxins in adult patients with AD, and showed that 30-60% of