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.