烟曲霉中调控胞内铁离子平衡的毒力相关蛋白LeuB和MrsA的功能研究

发布时间：2018-08-01 12:39

【摘要】：近几十年来,随着免疫缺陷人群的不断扩大,侵袭性真菌感染呈逐年上升的趋势。目前,引起广泛关注的致病真菌主要有白念珠菌,隐球菌和烟曲霉,其中烟曲霉是导致侵袭性肺曲霉病的元凶。烟曲霉是一种腐生型的真菌,其分布广,产孢强,在环境中无处不在。由于烟曲霉孢子直径较小,其很容易被人体吸入肺中。对于正常的人体来说,吸入的孢子很容易被杀灭并清除。但是对于免疫缺陷的患者来说,烟曲霉经呼吸道侵染后,体内的免疫细胞若不能及时将其清除,这些逃脱免疫的孢子会在患者体内萌发、繁殖,进而导致侵袭性曲霉病。目前临床上主要的抗真菌药物有伊曲康唑,伏立康唑,两性霉素B以及卡泊芬净等。然而,这些药物都有其局限性,其中两性霉素对宿主的副作用较大;卡泊芬净的抗菌谱窄;而运用最广泛的伊曲康唑和伏立康唑则很容易产生耐药性。因此,开发抗菌活力强,抗菌范围广以及毒副作用小的新型抗真菌药物成为了当务之急,而重中之重是寻找合适的药靶。铁离子作为一种重要的微量元素参与了一系列的生物学过程。由于宿主体内的铁多以血红蛋白以及乳铁蛋白等形式存在,游离的铁浓度很低,病原菌如何成功地从宿主体内获取铁元素是其致病的关键。因此,病原菌中调控铁离子吸收以及平衡的因子经常也是毒力决定子,然而铁离子平衡调控有关的机制在烟曲霉中仍知之甚少。本研究论文以烟曲霉为研究材料,利用基因敲除等手段,对烟曲霉中与铁离子代谢以及毒性都相关的功能基因leuB和mrsA开展了系统的研究,得到了以下结论:1、证实转录因子LeuB不仅调控烟曲霉亮氨酸的合成,同时也调控铁离子吸收。leuB的缺失会显著降低与铁载体合成、转运相关基因(sidG,sit1和mirB)的转录以及低铁载体的产生水平,从而影响烟曲霉在低铁环境下生长。双缺leuB和控制铁离子吸收的转录因子hapX,会加剧ΔleuB对低铁敏感的表型;而双缺leuB和抑制铁离子吸收的转录因子sreA,则会恢复ΔleuB对低铁敏感的表型。这些结果均说明亮氨酸合成调控因子LeuB参与烟曲霉铁离子吸收调控。2、低铁环境下,leuB的缺失会导致细胞铁离子吸收降低,leuA转录下调,进而阻碍亮氨酸的合成。而亮氨酸合成的阻断会使蛋白酶体活力增强从而导致蛋白全局性降解。进一步研究证明LeuB的DNA结合结构域(Zn(Ⅱ)2Cys6)以及其C端对烟曲霉适应低铁环境以及维持蛋白稳定等发面发挥着重要作用。3、证实线粒体铁通道mrsA的缺失会降低烟曲霉适应低铁以及高铁环境的能力。同时,mrsA缺失也会导致sreA转录低调,而受SreA抑制的和铁离子吸收相关的基因如sidA,mirB,ftrA以及fetC则明显高调。这些结果说明,MrsA是烟曲霉调控胞内铁离子平衡必需的。4、进一步研究发现mrsA的缺失提高了烟曲霉对氧化应激以及唑类药物的敏感性,其原因主要是ΔmrsA中ROS积累所致。位点突变实验证实,MrsA中第38,96以及214位保守的组氨酸位点是烟曲霉应对高铁,低铁,氧化应激以及唑类药物等环境所必需的。5、小鼠毒性实验证明烟曲霉中和铁离子代谢相关的功能基因leuB以及mrsA都是维持烟曲霉毒力所必需的。综上所述,本研究阐明了烟曲霉功能基因leuB和mrsA调控铁离子平衡的相关机制;同时也证明了 LeuB和MrsA均和烟曲霉毒力相关,为将来的抗真菌治疗提供了理论支持。
[Abstract]:In recent decades, with the continuous expansion of immune deficiency population, invasive fungal infection has been increasing year by year. At present, the main pathogenic fungi are Candida albicans, Cryptococcus and Aspergillus fumigatus, among which Aspergillus fumigatus is the leading cause of invasive pulmonary aspergillosis. Aspergillus fumigatus is a saprophytic fungus with wide distribution and sporulation. Strong, ubiquitous in the environment. As Aspergillus fumigatus is small in diameter, it is easy to be inhaled in the lungs. For normal human body, the inhaled spores are easily killed and removed. But for the immune deficiency patients, the immune cells in the body can not be removed in time after the infection of the respiratory tract. The degenerated spores will germinate and reproduce in the patients and lead to invasive aspergillosis. The main antifungal drugs are itraconazole, voriconazole, amphotericin B and caspofungin. However, these drugs have their limitations, of which amphotericin has a larger side effect on the host; and the antimicrobial spectrum of caspofungin is narrow; and The use of the most widely used itraconazole and voriconazole is easy to produce resistance. Therefore, the development of a new antifungal agent with strong antibacterial activity, wide antibacterial range and small side effects has become a top priority and the most important is to find the appropriate drug target. Iron is a major trace element involved in a series of biological activities. Because the iron in the host is mostly in the form of hemoglobin and lactoferrin, the concentration of free iron is very low, how the pathogenic bacteria obtain iron elements from the host is the key to its pathogenesis. Therefore, the factors regulating the absorption and balance of iron ions in the pathogenic bacteria are often also the virulence determinants, however, the iron ion balance regulation The related mechanisms are still very little known in Aspergillus fumigatus. This paper studies Aspergillus fumigatus as a material, using gene knockout and other means to study the functional genes related to iron ion metabolism and toxicity in Aspergillus fumigatus, leuB and mrsA. The following conclusions are obtained: 1, the transcriptional factor LeuB not only regulates Aspergillus fumigatus leucine. The synthesis, but also the regulation of the loss of iron ion absorption.LeuB can significantly reduce the iron carrier synthesis, the transcription of transport related genes (sidG, sit1 and mirB) and the production level of the low iron carrier, thus affecting the growth of Aspergillus fumigatus in the low iron environment. Double deficiency leuB and the control of the transcription factor hapX, which control the absorption of iron ions, will aggravate the sensitivity of the delta leuB to the low iron. The phenotypes, while double deficiency of leuB and the transcription factor sreA that inhibit the absorption of iron ions, will restore the delta sensitive phenotype of the low iron. These results suggest that the bright ammonia synthesis regulator LeuB participates in the regulation of.2 of the iron ion absorption of Aspergillus fumigatus, and the absence of leuB in the low iron environment leads to the decrease in the absorption of iron ions in the cells, the downregulation of leuA transcripts and the hindering the brightness of the leuA. The blocking of leucine synthesis can enhance the activity of the proteasome and lead to the global degradation of the protein. Further studies have shown that the DNA binding domain of LeuB (Zn (II) 2Cys6) and its C terminal play an important role in the.3 of the Aspergillus fumigatus to the low iron environment and the maintenance of protein stability, confirming the mrsA of the mitochondrial iron channel. Deletion can reduce the ability of Aspergillus fumigatus to adapt to the low iron and high iron environment. At the same time, the deletion of mrsA can also lead to the low modulation of sreA transcription, while the genes related to SreA inhibition and iron absorption, such as sidA, mirB, ftrA and fetC, are obviously high. These results suggest that MrsA is the necessary.4 for the balance of iron ions in Aspergillus fumigatus. The absence of mrsA increases the sensitivity of Aspergillus fumigatus to oxidative stress and azolic drugs. The reason is mainly due to the accumulation of ROS in the delta mrsA. The site mutation test confirmed that the 38,96 and 214 conservative histidine loci in MrsA are essential for the.5 of Aspergillus fumigatus to respond to the environment of high iron, low iron, oxidative stress and azole, and the toxicity of mice. The experimental results show that the functional genes leuB and mrsA related to the neutralization of Aspergillus fumigatus and iron ions are essential for the maintenance of Aspergillus fumigatus virulence. To sum up, this study illustrates the related mechanisms of the regulation of iron ion balance by Aspergillus fumigatus functional gene leuB and mrsA, and also shows that both LeuB and MrsA are associated with Aspergillus fumigatus virulence, for future antifungal treatment. The treatment provides theoretical support.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R379

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