Assessment Of The Function Of SUB6 Gene For The Pathogenicity Of Trichophyton Mentagrophytes

Background Trichophyton mentagrophytes is a keratinophilic pathogenic fungus which can infect humans and some animals. T. mentagrophytes secretes a number of proteases to invade the epidermal barrier and obtain nutrients to grow and multiply on the keratin. The secreted proteases induce both cell-mediated and humoral immune reactions of host. A seven-member subtilisin gene (SUB) family, encoding serine proteases, has been identified in T. mentagrophytes.The subtilisin gene (SUB) 6 related Tri r2 derived from Trichophyton exhibits the ability to elicit both immediate and delayed type hypersensitivity. The relationship between SUB6 and its coded Sub6 secretion in vitro and in vivo remains unknown. The effective way to investigate the function of a potential virulence gene depends on reverse genetic approach via disrupting target gene and analysing the mutant strains. At present, Agrobacterium tumefaciens-mediated transformation (ATMT) is a common method for fungal gene transfer. It has been successfully applied to many kinds of filamentous fungi.Objectives To study the function of SUB6 gene, the SUB6 gene of T. mentagrophytes was disrupted and the phenotype, growth rate, proteolytic activity, keratinase gene expression and induced pathological changes in the site-directed mutant strains were explored.Methods A dual-vector containing a hygromycin B resistance gene hph flanked by SUB6 gene homology fragments was constructed. The SUB6 gene was disrupted by the method of Agrobacterium tumefaciens-mediated transformation (ATMT). Polymerase Chain Reaction (PCR) analyses and Southern blot were used to confirm the disruption. The phenotypes, growth rate, proteolytic activity of the mutant strains were observed in vitro. The related keratinase gene expressions were detected by the quantitative real-time PCR analyses. During the infection of guinea pigs, the clinical and histopathological follow-up were performed, and the cytokine levels of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-10, IL-12 in spleen homogenates were tested by Enzyme-Linked Immunosorbnent Assay (ELISA).Results The PCR and Southern blot ensured that the SUB6 gene of T. mentagrophytes was disrupted by homologous recombination and harbored an integrated single copy of T-DNA. The metalloprotease gene (MEP) 4 and SUB3 gene expressions increased significantly, which accounted for the increase of proteolytic activity in △SUB6 strains. The △SUB6 strains infected animals showed lower-degree in clinical symptoms and pathological changes; and the level of immunosuppressive cytokine IL-10 was persistently high, while the increase in delayed-type hypersensitivity (DTH) related cytokines IFN-y, TNF-a and IL-12 were delayed and lower in the △SUB6 strains infected animals when comparing with the wild strains.Conclusions The SUB6 gene of T. mentagrophytes was disrupted successfully by ATMT, and the mutant strains showed weakened pathogenicity in vivo. The genetically-linked regulatory effect may account for the increase in proteolytic activity and the residual pathogenicity of the mutant strain.