Characterization of Monoamine Oxidase-A in tropical liver fluke, Fasciola gigantica

Abstract

Fasciola gigantica, responsible for the zoonotic disease fasciolosis, pose a great threat to the livestock and human health worldwide. The triclabendazole (TCBZ) has been used for decades as a broad spectrum anthelmintic to control this perilous disease but the emergence of resistance in flukes against TCBZ has prompted researchers across the world to explore for new drugs and antigenic targets. World Health Organization has strongly recommended the utilization of neurobiologically significant biomolecules as new drug/antigenic targets because of their significant role in the physiology of parasites. Monoamine Oxidase (MAO) is an important neurobiological enzyme which catabolizes aminergic neurotransmitters thus preventing prolonged excitation of neurons and in non-neuronal cells it prevents cellular toxicity due to accumulation of toxic monoamines. Owing to the important role of MAO in the survival and perpetuation of parasites, multipronged approaches were undertaken for the characterization of MAO-A in F. gigantica. The activity of MAO was found to be 1.5 times higher in the mitochondrial samples than the whole homogenate samples. The adult worms of the F. gigantica appeared to possess both the isoforms of MAO i.e., MAO-A and MAO-B. The zymographic studies revealed strong enzyme activity in its native state as assessed through prominent dark bands at 250KDa in the zymogram. The enzyme was also found to be highly immunogenic as revealed by high antibody titer at 1:6400 dilution. The immunogenicity of MAO-A enzyme was further established in the Western Blots in which a strong band of 50KDa was distinctly evident. Despite ubiquitous presence of MAO in F. gigantica some regions like tegumental surface and intestinal caecae displayed strong immunofluorescence as compared to other regions. The detection of MAO-A in the F. gigantica samples in Dot-Blot assay indicate a great potential of this molecule for the immunodiagnostics of fasciolosis, particularly in the field conditions. The enzyme activity was sensitive to the specific inhibitor clorgyline in a concentration dependant manner, particularly in the late incubation period. The zymographic results also exhibited similar trend. The strong intensity of spots in Dot-blots indicate high immunogenicity of the MAO protein. The intensity of bands/spots in the samples of worms treated with clorgyline also declined, clearly indicating that the tropical liver fluke possesses prominent MAO-A activity.