AiM  Vol.4 No.6 , May 2014
Detection of β-Glucuronidase Activity within Actinomadura madurae 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.
Cite this paper: Palma-Ramos, A. , Reyes-Mayén, S. , Castrillón-Rivera, L. , Fernández-López, S. , Padilla-Desgarennes, C. , Vega-Memije, M. , Arenas-Guzmán, R. , Drago-Serrano, M. and Sainz-Espuñes, T. (2014) Detection of β-Glucuronidase Activity within Actinomadura madurae Grains of Human Actinomycetoma. Advances in Microbiology, 4, 317-323. doi: 10.4236/aim.2014.46038.

[1]   Lavalle, A.P., Padilla, D.M., Perez, G.J., Rivera, I. and Reynoso, R.S. (2000) Micetomas por Actinomadura madurae en Mexico. Revista del Centro Dermatologico Pascua, 9, 19-24.

[2]   Welsh, O., Vera-Cabrera, L., Welsh, E. and Salinas, M.C. (2012) Actinomycetoma and Advances in Its Treatment. Clinics in Dermatology, 30, 372-381.

[3]   Venkatswami, S., Sankarasubramanian, A. and Subramanyam, S. (2012) The Madura Foot: Looking Deep. The International Journal of Lower Extremity Wounds, 11, 31-42.

[4]   Palma, R.A., Castrillon, L.R., Encinas, M.G., Padilla, C.D. and Guzman, R.A. (2009) Participacion De Los Queratinocitos En La Respuesta Inmunitaria Contra El Actinomicetoma. Dermatologia Revista Mexicana, 53, 225-233.

[5]   Mollinedo, F. (2003) Human Neutrophil Granules and Exocytosis Molecular Control. Immunologia, 22, 340-358.

[6]   Mah, T.F. (2012) Biofilm-Specific Antibiotic Resistance. Future Microbiology, 7, 1061-1072.

[7]   Petrova, O.E. and Sauer, K. (2012) Sticky Situations: Key Components That Control Bacterial Surface Attachment. Journal of Bacteriology, 194, 2413-2425.

[8]   Dubois, M., Gilles, K., Hamilton, J., Rebers, P. and Smith, F. (1956) Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry, 28, 350-356.

[9]   Goldstein, G. (1961) Serum Β-Glucuronidase Assay by the Phenolphthalein Mono-Β-Glucuronide Method. Clinical Chemistry, 7, 136-142.

[10]   Fishman, W.H., Springer, B. and Brunettei, R. (1948) Application of an Improved Glucuronidase Assay Method to the Study of Human Blood Beta-Glucuronidase. Journal of Biological Chemistry, 173, 449-456.

[11]   Talalay, P., Fishman, W.H. and Huggins, C. (1946) Chromogenic Substrates; Phenolphthalein Glucuronic Acid as Substrate for the Assay of Glucuronidase Activity. Journal of Biological Chemistry, 166, 757-772.

[12]   Lorbacher, P., Yam, L. and Mitus, W. (1967) Cytochemical Demonstration of Β-Glucuronidase Activity in Blood and Bone Marrow Cells. Journal of Histochemistry & Cytochemistry, 15, 680-687.

[13]   Palma, R.A., Castrillon, L.R., Padilla, D.M. and Reyes, F.F. (2005) Caracterizacion Histoquimica De Micetomas Por Actinomadura Madurae, Nocardia Brasiliensis Y Madurella Mycetomatis. Dermatologia Revista Mexicana, 49, 51-58.

[14]   Palma, R.A., Castrillon, R.L., Padilla, D.M., Rosas, H.L. and Marquez, C. (2006) Purificacion Y Determinacion De La Estructura De Los Polisacaridos Que Forman El Cemento De Union En Granos De Actinomicetomas Ocasionados Por Actinomadura Madurae Y Nocardia Brasiliensis. Dermatologia Revista Mexicana, 50, 165-173.

[15]   Luster, A.D., Alon, R. and Von Andrian, U.H. (2005) Immune Cell Migration in Inflammation: Present and Future Therapeutic Targets. Nature Immunology, 6, 1182-1190.

[16]   Brinkmann, V. and Zychlinsky, A. (2012) Neutrophil Extracellular Traps: Is Immunity the Second Function of Chromatin? The Journal of Cell Biology, 198, 773-783.

[17]   Guimaraes, C.C., Castro, L.G. and Sotto, M.N. (2003) Lymphocyte Subsets, Macrophages and Langerhans Cells in Actinomycetoma and Eumycetoma Tissue Reaction. Acta Tropica, 87, 377-384.

[18]   Castrillon, R.L., Palma, R.A. and Padilla, D.M. (2010) Importancia De Las Bio-Peliculas En La Practica Medica. Dermatologia Revista Mexicana, 54, 14-24.