Expression, purification andin vitroantifungal activity of acidic mammalian chitinase againstCandida albicans,Aspergillus fumigatusandTrichophyton rubrumstrains

Abstract

Evidence has suggested that acidic mammalian chitinase (AMCase) may be an important candidate in defence against and/or digestion of chitin-containing organisms, especially pathogenic fungi. However, to date, there are no reports on the direct inhibition of hyphal growth activity of AMCase in vitro. Aims. To evaluate the direct inhibition of hyphal growth activity of AMCase in vitro. We cloned and expressed AMCase and extracted natural AMCase protein from the stomach of a BALB/c mouse, using a chitin affinity chromatography column. AMCase activity was determined by the reducing end group N-acetamino-glucose assay. We chose Candida albicans, Aspergillus fumigatus and Trichophyton rubrum strains as experimental targets to evaluate the inhibition of hyphal growth activity of AMCase by the disc diffusion susceptibility test, macrodilution method and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. Natural AMCase from the BALB/c mouse stomach showed higher chitinolytic activity compared with the hydrolytic effects of the recombinant AMCase protein. The growth of all three dermatopathogenic fungi was clearly inhibited by recombinant and natural AMCase, especially the natural AMCase. A good correlation between the disc diffusion susceptibility test and minimum inhibitory concentrations (MICs) was seen. Our results indicate that AMCase plays an important role in both the efficient hydrolysis of chitin and antifungal activity. These findings need to be confirmed in future studies using different clinical pathogenic fungal strains.