多聚泛素UBI4低表达对新生隐球菌生长与毒力的功能影响

中国真菌学杂志　 2015, Vol. 10 　Issue (1): 16-21.

多聚泛素UBI4低表达对新生隐球菌生长与毒力的功能影响

孟云芳¹, 周兆婧¹, 杨雅骊¹, 赵静宇^1,2, 张超¹, 法振宗¹, 桑军军¹, 方伟¹, 廖万清¹

1. 上海长征医院皮肤病与真菌病研究所全军真菌病重点实验室第二军医大学附属长征医院皮肤科, 上海 200003;
2. 上海市皮肤病医院, 上海 200050

收稿日期:2015-01-04 出版日期:2015-02-28 发布日期:2015-02-28
通讯作者: 廖万清,E-mail:Liaowanqing@sohu.com;方伟,E-mail:weifang081782@163.com E-mail:Liaowanqing@sohu.com;weifang081782@163.com
作者简介:孟云芳,女(汉族),博士研究生在读.E-mail:mengyunfang6.12@163.com;周兆婧,女(汉族),硕士研究生在读.E-mail:zhouzhaojing320@163.com
基金资助:
国家973项目(2013CB531601);上海市自然科学基金(12ZR1454400,12JC1411000);上海市医学真菌分子生物学实验室基金(14DZ2272900)

Effects of polyubiquitin UBI4 downregulation on growth and pathogenesis of Cryptococcus neoformans

MENG Yun-fang¹, ZHOU Zhao-jing¹, YANG Ya-li¹, ZHAO Jing-yu^1,2, ZHANG Chao¹, FA Zhen-zong¹, SANG Jun-jun¹, FANG Wei¹, LIAO Wan-qing¹

1. Shanghai Key Laboratory of Molecular Medical Mycology and PLA Key Laboratory of Fungal Diseases, Department of Dermatology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China;
2. Shanghai Dermatology Hospital, Shanghai 200003, China

Received:2015-01-04 Online:2015-02-28 Published:2015-02-28

摘要/Abstract

摘要： 目的构建新生隐球菌的多聚泛素UBI4低表达菌株,检测UBI4低表达对新生隐球菌生长和毒力的功能影响.方法采用融合PCR方法,扩增含有UBI4基因的片段,将其与含有组蛋白启动子的质粒融合.通过基因枪将重组质粒片段转化入新生隐球菌,应用PCR筛选、DNA序列测序以及实时定量PCR方法对基因重建株进行鉴定与验证.检测多聚泛素UBI4低表达菌株对多种应激耐受力、生长曲线以及对蜡蛾感染的毒力影响.结果成功构建了新生隐球菌多聚泛素UBI4低表达.表型检测结果提示:较之标准株,UBI4低表达株生长速率下降,对高温、H₂O₂和NO应激以及多种抗真菌药物敏感性显著增强;同时,UBI4下调表达后隐球菌对蜡蛾的毒力显著下调.结论我们的研究初步证实多聚泛素蛋白UBI4对新生隐球菌的生长增殖、应激应答以及感染能力具有重要的调控作用,为后续深入探讨新生隐球菌泛素系统的功能机制奠定基础.

关键词: 新生隐球菌, 多聚泛素, 致病机制, 应激应答

Abstract: Objective To construct Cryptococcus neoformans strain with downregulated UBI4 expression and examine the effects of UBI4 downregulation on growth and pathogenesis of C.neoformans.Methods UBI4 DNA fragment was amplified and infused into linear plasmid pCH20 containing histone promoter.The fused plasmid was then transformed into C.neoformans through gene gun.The positive transformants were screened by diagnostic PCR and DNA sequencing.Various phenotype of C.neoformans reconstituted strain H99^His-UBI4 were examined.Results Cryptococcus neoformans strain with low expression of UBI4 were constructed and phenotypic assays demonstrated that down-regulated UBI4 lead to slower growth rate and enhanced susceptibility to various stresses such as H₂O₂,NO and antifungal agents in C.neoformans.Moreover,C.neoformans with down-regulated UBI4 was also attenuated dramatically in wax moth infection model.Conclusion Our study suggested that polyubiquitin UBI4 played a critical role in cell proliferation,stress response and pathogenesis in C.neoformans.Our work laid a foundation for following studies about functions and mechanisms of ubiquitin system.

Key words: Cryptococcus neoformans, polyubiquitin, pathogenesis, stress response

中图分类号:

R379.5

孟云芳, 周兆婧, 杨雅骊, 赵静宇, 张超, 法振宗, 桑军军, 方伟, 廖万清. 多聚泛素UBI4低表达对新生隐球菌生长与毒力的功能影响[J]. 中国真菌学杂志, 2015, 10(1): 16-21.

MENG Yun-fang, ZHOU Zhao-jing, YANG Ya-li, ZHAO Jing-yu, ZHANG Chao, FA Zhen-zong, SANG Jun-jun, FANG Wei, LIAO Wan-qing. Effects of polyubiquitin UBI4 downregulation on growth and pathogenesis of Cryptococcus neoformans[J]. Chinese Journal of Mycology, 2015, 10(1): 16-21.

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