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目的研究COP9信号小体复合体基因缺失对新型隐球酵母菌生长与毒力效应的影响,为研究抗真菌药物作用靶点提供试验数据。方法 2014年4月以临床标准株H99为背景,分别敲除其中一个目前已知的COP9编码基因,构建COP9信号小体复合体基因缺失菌株,通过高温、氧化、NO、高盐、高渗、细胞膜、细胞壁体外应激应答反应;进一步检测生长速度,绘制生长曲线;并以巨噬细胞感染模型、蜡蛾感染模型检测新型隐球酵母菌活体内毒力变化。结果与标准株相比,CSN1201△缺失株对温度、氧化、NO、高盐、高渗、细胞壁及细胞膜应激反应的敏感性显著升高;CSN1201基因缺失对新型隐球酵母菌的生长速率有一定的影响,而且高温条件更加重了其影响;CSN1201△缺失株和CSN7△缺失株的胞内生存率显著下降(PCSN1201△=0.003,PCSN7△<0.001),而其他缺失株则与标准株类似;与标准株H99相比,CSN1201△缺失株和CSN7△缺失株的毒力显著下降,平均生存时间分别为(23.5±0.95)d和(21.2±1.28)d(P<0.05);而CSN4△则表现为毒力显著增强,平均生存时间为(6.4±0.36)d(P<0.05),其余缺失株的毒力与标准株比较,差异无统计学意义。结论 COP9信号小体复合体的基因,尤其是CSN1201对于新型隐球酵母菌的毒力有一定的影响,可能是调控多种应激条件下新型隐球酵母菌毒力的重要基因,有可能成为抗真菌药物靶点。
OBJECTIVE: To study the effect of gene deletion of COP9 signaling complex on the growth and virulence of Cryptococcus neoformans, and provide experimental data for studying the antifungal drug target. Methods In April 2014, one of the currently known COP9-encoding genes was knocked down with the clinical standard strain H99 as a background to construct a gene deletion strain of the COP9 signaling small-body complex, which was characterized by high temperature, oxidation, NO, high salt, hypertonicity, Cell membrane and cell wall in vitro. The growth rate was further detected and the growth curve was drawn. The changes of the virulence of Cryptococcus neoformans in vivo were detected by macrophage infection model and wax moth infection model. Results Compared with the standard strain, the susceptibility of CSN1201 △ deletion strain to temperature, oxidation, NO, high salt, hypertonicity, cell wall and cell membrane stress was significantly increased. The growth of Cryptococcus neoformans (PCSN1201 △ = 0.003, PCSN7 △ <0.001), while the other deletion strains were similar to the standard strains . Compared with the standard strain H99, the virulence of CSN1201 △ deletion strain and CSN7 △ deletion strain decreased significantly (P <0.05), while the average survival time was (23.5 ± 0.95) d and (21.2 ± 1.28) d (6.4 ± 0.36) d (P <0.05). The virulence of the other strains was not significantly different from that of the standard strains. Conclusion The gene of COP9 signaling complex, especially CSN1201, has certain influence on the virulence of Cryptococcus neoformans and may be an important gene regulating virulence of Cryptococcus neoformans under various stress conditions and may become Antifungal drug targets.