Detection of β-Glucuronidase Activity within Actinomadura madurae Grains of Human Actinomycetoma

Alejandro Palma-Ramos, Samantha Reyes-Mayén, Laura Estela Castrillón-Rivera, Silvia Elena Fernández-López, Carmen Padilla-Desgarennes, María Elisa Vega-Memije, Roberto Arenas-Guzmán, María Elisa Drago-Serrano, Teresita Sainz-Espuñes

Department of Biological Systems, Universidad Autónoma Metropolitana-Xochimilco, Mexico City, México.
Dermatology Center “Dr. Ladislao de la Pascua”, Mycology Service, Mexico City Health Secretary, Mexico City, México.
General Hospital “Manuel Gea González”, Dermatology Service, Mexico City Health Secretary, Mexico City, México.
General Hospital “Manuel Gea González”, Mycology Service, Mexico City Health Secretary, Mexico City, México.

DOI: 10.4236/aim.2014.46038                

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.

Keywords

Actinomycetoma, A. madurae, β-Glucuronidase

Conflicts of Interest

The authors declare no conflicts of interest.

References

[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. http://dx.doi.org/10.1016/j.clindermatol.2011.06.027
[3]	Venkatswami, S., Sankarasubramanian, A. and Subramanyam, S. (2012) The Madura Foot: Looking Deep. The International Journal of Lower Extremity Wounds, 11, 31-42. http://dx.doi.org/10.1177/1534734612438549
[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. http://dx.doi.org/10.2217/fmb.12.76
[7]	Petrova, O.E. and Sauer, K. (2012) Sticky Situations: Key Components That Control Bacterial Surface Attachment. Journal of Bacteriology, 194, 2413-2425. http://dx.doi.org/10.1128/JB.00003-12
[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. http://dx.doi.org/10.1021/ac60111a017
[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. http://dx.doi.org/10.1038/ni1275
[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. http://dx.doi.org/10.1083/jcb.201203170
[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. http://dx.doi.org/10.1016/S0001-706X(03)00139-6
[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.