转录因子UME6调控氧化应激相关基因SOD2表达介导热带假丝酵母菌毒力增强的机制研究

Candida tropicalis has been one of the most important pathogens in invasive fungal infection, and those with strong virulence will cause serious system infection. A close relationship between overexpression of oxidative stress-related gene SOD2 and increased virulence was observed in our previous study, but transcription regulator of SOD2 and its role in the enhancement of virulence were still not clear. Morphological changes were observed in C. tropicalis isolates with strong virulence, and in our previous experiment, overexpression of morphological transcription factor UME6 was detected. The expression level of SOD2 decreased correspondingly in ume6Δ/ume6Δ C. tropicalis. In this project, effects of SOD2 and UME6 genes on virulence of C. tropicalis will be investigated by constructing the gene knock-out, reversed and overexpressed isolates, and analyzing their levels of ROS and SOD expression. Transcriptional regulation of UME6 on SOD2 gene will be detected by pGL3-basic luciferase reporter vector. Finally, the role of the transcriptional regulation in counteracting ROS and enhancing virulence of C. tropicalis will be explored. This project will provide new aspect of investigation on the mechanism of enhanced virulence of C. tropicalis.

热带假丝酵母菌已成为侵袭性真菌感染的重要病原体，强毒力株更易引起免疫缺陷患者严重的全身感染。本课题组前期研究发现，氧化应激相关基因SOD2的表达上调与热带假丝酵母菌的毒力增强密切相关，但调控SOD2表达的转录因子及其增强菌株毒力的作用机制尚未明了。毒力增强的假丝酵母菌常伴有细胞形态的转变，我们前期发现与假丝酵母菌形态改变相关的调控基因UME6在临床分离的毒力强株中表达升高，而ume6Δ/ume6Δ菌株中SOD2基因的表达相应降低。本课题将构建SOD2和UME6基因缺失株、回复株和过表达株，观察菌株的活性氧、SOD水平及毒力强弱间的关系，明确UME6和SOD2表达对菌株毒力的影响；通过荧光素酶等实验验证UME6对SOD2的调控作用；并进一步观察UME6对SOD2基因表达调控与热带假丝酵母菌抵抗内源性ROS、增强其毒力间的关联性，最终解析热带假丝酵母菌毒力增强的可能机制。

热带念珠菌已成为侵袭性真菌感染的重要病原体，强毒力株更易引起免疫缺陷患者严重的全身感染。热带念珠菌存在三种细胞形态，分别为酵母样、假菌丝样和菌丝样，在培养过程中我们发现毒力强的热带念珠菌往往较毒力弱的菌株更易形成菌丝。我们通过构建了转录因子UME6的敲除及过表达菌株，明确了其在热带念珠菌菌丝生成及毒力当中发挥着重要作用。同时，通过分析毒力强和弱的两组菌株的转录组，我们发现氧化应激相关基因SOD2的表达上调与热带念珠菌的毒力增强密切相关。通过构建SOD2敲除菌株，我们发现了SOD2基因缺失可大大减轻小鼠感染程度，从而证明了其在毒力当中具有重要作用。然而，转录因子UME6敲除及过表达菌株中的SOD2表达水平并无显著变化，提示转录因子UME6对SOD2无直接调控作用。为了进一步了解热带念珠菌的毒力因子，我们还探究了低浓度氟康唑对于热带念珠菌重要致病因子之一分泌型天冬氨酸蛋白酶（Sap）表达的影响。通过检测不同浓度氟康唑刺激下菌株Sap的活性及SAP基因的表达情况，我们发现了低浓度氟康唑刺激可提高Sap的分泌水平，然而这一现象具有菌株特异性，其中耐药性较强的菌株更容易出现此类现象。以上研究将有利于我们进一步了解热带念珠菌毒力及致病机制，为日后临床预防感染、指导治疗及研发治疗药物有重要意义。