Detection of <i>β</i>-Glucuronidase Activity within <i>Actinomadura madurae</i> Grains of Human Actinomycetoma

Abstract

Actinomycetoma syndrome by Actinomadura (A.) madurae is characterized by a subcutaneous chronic lesion that affects fascia, muscle and bone. A. madurae produces colonies that form grains of less than 1 mm in diameter. Grains are surrounded and infiltrated by neutrophils involved in the grain disruption by enzymes like β-glucuronidase released after the neutrophil degranulation. The aim of this work was to evaluate the polysaccharide degradation of grains treated with β-glucuronidase and to detect the presence and activity of β-glucuronidase within the A. madurae grains. Actinomadura madura grains from patients infected were processed to quantify the total content of polysaccharide with the phenol-sulfuric acid reaction. Grains were treated with β-glucuronidase at different conditions to evaluate the optimal polysaccharide degradation. Grains were analyzed to detect the enzyme by using anti-human β-glucuronidase antibody while enzymatic activity was assessed by evaluating the release of reduced sugars and by in situ enzymatic activity. Optimal degradation of polysaccharide in the grains treated with β-glucuronidase was found with 300 units/ml of enzyme and 24 hr of incubation at 37˚C. Presence and activity of β-glucuronidase enzyme within the grains were detected. Results suggested that β-glucuronidase present within A. madurae grain resulted from degranulated neutrophils surrounding and/or infiltrated within the grain.