Assessment of the function of SUB6 in the pathogenic dermatophyte Trichophyton mentagrophytes.
Trichophyton mentagrophytes is a keratinophilic pathogenic fungus that infects both humans and animals. Subtilisins are important for T. mentagrophytes virulence, particularly when invading the epidermal barrier of the host. Subtilisin gene SUB6 belongs to a seven-member gene family (SUB1-SUB7) encoding the subtilisin serine proteases. Additionally, the SUB6 gene product Sub6, which is thought to be the major allergen Tri r2 in Trichophyton rubrum, elicits both immediate- and delayed-type hypersensitivity (DTH) reactions in humans. To assess its gene function, SUB6 was disrupted using the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot analyses were used to confirm the disruption. In vitro virulence analyses comparing the mutant with the wild-type strain showed that proteolytic activity was significantly increased in the SUB6 gene disruption strain (SUB6::hph), which corresponded to the significantly increase in MEP4 (metalloprotease gene) and SUB3 expression of SUB6::hph. The SUB6::hph -infected animals showed attenuated clinical symptoms and pathological changes, and because of the persistently high level of immunosuppressive cytokine IL-10, the increase in DTH-related cytokines IFN-γ, TNF-α and IL-12 was delayed and lower than that in animals infected with the wild-type strain. These results suggested that SUB6::hph had attenuated virulence in vivo, and that a genetically-linked regulatory effect may account for the increase in proteolytic activity and the residual pathogenicity of the mutant strain.
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
Yao
Y
Shi
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
Qifang
Q
Niu
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
Xiaoxiao
X
Yu
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
Xiaolin
X
Jia
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
Jing
J
Wang
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China csamalin@sina.com.
Degui
D
Lin
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
Yipeng
Y
Jin
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D004195:Disease Models, Animal; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006168:Guinea Pigs; D020860:Subtilisin / Q000235:genetics / Q000378:metabolism*; D014005:Tinea / Q000382:microbiology / Q000473:pathology; D014170:Transformation, Genetic; D014249:Trichophyton / Q000201:enzymology* / Q000235:genetics; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D037521:Virulence Factors; D020860:Subtilisin
Agrobacterium tumefaciens-mediated transformation; SUB6; Trichophyton mentagrophytes; pathogenicity