Shi Yao, Niu Qifang, Yu Xiaoxiao, Jia Xiaolin, Wang Jing, Lin Degui, Jin Yipeng
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China.
The Clinical Department, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China
Med Mycol. 2016 Jan;54(1):59-71. doi: 10.1093/mmy/myv071. Epub 2015 Sep 1.
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.
须癣毛癣菌是一种嗜角蛋白的致病性真菌，可感染人类和动物。枯草杆菌蛋白酶对须癣毛癣菌的毒力很重要，尤其是在侵入宿主表皮屏障时。枯草杆菌蛋白酶基因SUB6属于一个由七个成员组成的基因家族（SUB1 - SUB7），该家族编码枯草杆菌蛋白酶丝氨酸蛋白酶。此外，SUB6基因产物Sub6被认为是红色毛癣菌中的主要变应原Tri r2，可在人类中引发速发型和迟发型超敏反应（DTH）。为了评估其基因功能，使用根癌农杆菌介导的转化方法破坏了SUB6。采用聚合酶链反应和Southern印迹分析来确认破坏情况。将突变体与野生型菌株进行体外毒力分析，结果显示SUB6基因破坏菌株（SUB6::hph）的蛋白水解活性显著增加，这与SUB6::hph中MEP4（金属蛋白酶基因）和SUB3表达的显著增加相对应。感染SUB6::hph的动物表现出减轻的临床症状和病理变化，并且由于免疫抑制细胞因子IL - 10持续高水平，与DTH相关的细胞因子IFN - γ、TNF - α和IL - 12的增加延迟且低于感染野生型菌株的动物。这些结果表明SUB6::hph在体内毒力减弱，并且一种基因连锁的调节作用可能解释了蛋白水解活性的增加和突变菌株的残余致病性。
