产酶溶杆菌C3菌株多环四胺酸大环内酰胺类化合物的分离与抗真菌作用机制研究

发布时间：2018-06-21 15:22

本文选题：产酶溶杆菌C3 + HSAF　；参考：《山东大学》2016年博士论文

【摘要】：随着放射疗法、化学疗法、免疫抑制剂、抗生素和内置医疗器械的广泛应用,导致了发生严重的感染,尤其是真菌感染。其中,白色念珠菌在免疫低下的病人体内成为最易感染的真菌,也是许多重大疾病导致死亡的直接原因。治疗白色念珠菌感染需要长期大量地使用药物,导致耐药菌不断出现,加剧了临床治疗的难度。发现新结构和新作用机制的抗真菌药物,是控制白色念珠菌感染的重要途径。具有活性的化合物大部分来源于微生物。长期以来人们主要关注革兰氏阳性菌和真菌来源的活性天然产物,而革兰氏阴性细菌蕴藏的丰富天然产物资源没有得到应有的关注。近些年人们对溶杆菌的开发,得到了系列结构新颖的活性化合物,溶杆菌正成为活性天然产物的新资源。本学位论文对产酶溶杆菌C3菌株的发酵条件进行筛选,在其次级代谢产物中分离得到了4个多环四胺酸大环内酰胺(PTM)类化合物；开展了2个PTM类化合物的体内外抗真菌活性评价,探讨了其抗真菌的作用机制。本论文第一章概述了当前真菌感染的严重性和临床抗真菌药物品种的匮乏。实际上,目前临床抗真菌药物的作用靶点主要集中在细胞壁和细胞膜,并且导致大量耐药菌株的出现。从新资源溶杆菌中寻找新颖的抗真菌化合物,是一条行之有效的途径。本章综述了溶杆菌活性天然产物及其生物活性和生物合成的研究现状。本论文第二章是产酶溶杆菌C3菌株发酵培养条件的优化,对该菌株中得到的代谢产物进行了结构鉴定。产酶溶杆菌C3菌株的平板发酵培养基筛选结果表明,C3菌株在TSB培养基中次级代谢产物的产量最高。同时我们对TSB的用量进行考察,发现随着TSB用量的减少,在C3菌株中得到的产物的量也相应降低。最终我们选用1/10 TSB培养基进行大规模发酵,得到4个PTM类化合物。其中,3个已知化合物(HSAF、3-deOH-HSAF和3-deOH-alteramide B)和1个新化合物alteramide B(ATB)。本论文第三章是HSAF抑制丝状真菌和白色念珠菌的活性研究。首先以水稻稻瘟病菌为研究模型,进行转录组差异分析,发现HSAF对细胞的凋亡途径有重要影响。由于细胞壁具有成分比较复杂的外层和结构比较致密的内层结构,能够影响小分子物质转运到细胞内。我们研究在给药过程中小分子药物对于细胞各组分起到的作用及其作用机制,其中首要的是获得原生质体,进而以白色念珠菌为模型,在原生质体水平,考察了HSAF作用方式。利用DCFH-DA荧光染色,检测HSAF作用后细胞内的活性氧(reactive oxygen species,ROS)的水平。加入四种活性氧清除剂：抗坏血酸(AA)、硫脲(TU)、乙酰半胱氨酸(NAC)和谷胱甘肽(GSH)后,HSAF的抗真菌活性明显降低,表明HSAF通过诱导细胞内ROS上调抑制真菌生长。通过荧光倒置显微镜、流式细胞分析仪和western blotting等检测,发现HSAF引起线粒体膜发生变化,其电位呈现出下降的趋势,引起DNA损伤和细胞周期分布异常,停留在G2/M期,最终导致细胞发生早期和晚期凋亡。本论文第四章是新化合物ATB抗白色念珠菌的作用机制研究。ATB对白念珠菌的体外抑制较好,不仅能抑制白念珠菌酵母态的生长,也能抑制其菌丝体的生长。ATB能够诱导白色念珠菌细胞内ROS水平的上升,引起细胞内线粒体膜电位的下降。进一步研究发现,ATB能够引起白色念珠菌细胞G2/M期停滞,导致细胞发生早期和晚期凋亡。AA可以抑制ATB对白色念珠菌细胞的生长周期阻滞和凋亡诱导作用,因此,ATB通过诱导白色念珠菌细胞内ROS的产生和积累而发挥其抗真菌活性。作用靶点研究结果表明,在HeLa细胞中,ATB能够与微管蛋白结合；在体外,ATB能够抑制微管蛋白的聚集；分子模拟表明,ATB能与白色念珠菌β-tubulin的12个关键氨基酸相互作用；点突变实验结果证明,ATB与β-tubulin的可能结合位点分别为L215、L217、L273、T274和R282,其中R282是关键作用位点。小鼠体内实验结果表明,ATB发挥了良好的体内抑制白色念珠菌的作用。本论文研究发现了新化合物ATB,探讨了其抗真菌作用靶点和作用机制,首次发现微管蛋白有望成为抗真菌药物的新靶点,为临床治疗白色念珠菌感染提供了新思路,对开发新型抗真菌药物有重要参考意义。
[Abstract]:With the extensive use of radiation therapy, chemotherapy, immunosuppressants, antibiotics, and built-in medical instruments, serious infections, especially fungal infections, are caused by Candida albicans, the most susceptible fungi in immunocompromised patients, and a direct cause of death in many major diseases. Treatment of white beads Bacteria infection requires long and large use of drugs, resulting in the continuous emergence of drug-resistant bacteria, which exacerbates the difficulty of clinical treatment. Discovering new structures and antifungal agents for new mechanisms of action is an important way to control Candida albicans infection. Most of the active compounds are derived from microorganism. The natural products of the bacteria and fungi are natural products, and the rich natural products of the Gram-negative bacteria have not been paid attention to. In recent years, a series of novel active compounds have been obtained for the development of Bacillus lysin. The bacilli have become a new resource of the active natural products. The fermentation conditions were screened and 4 polycyclic four amino acid macrocyclic amides (PTM) were isolated from its secondary metabolites, and the antifungal activity of 2 PTM compounds was evaluated in vivo and in vitro. The first chapter of this paper summarized the severity of fungal infection and the clinical antifungal infection. In fact, the targets of clinical antifungal agents are mainly concentrated in the cell walls and cell membranes and lead to the emergence of a large number of resistant strains. It is an effective way to find novel antifungal compounds from the new resources of bacilli. In this chapter, the natural products and biological activities of the bacilli are described in this chapter. The second chapter of this paper is the optimization of fermentation conditions for the strain of C3 producing Bacillus lysin. The structure of the metabolites obtained in this strain was identified. The screening results of the plate fermentation medium of C3 producing strain of Bacillus lysin showed that the secondary metabolites of the C3 strain were the highest in the TSB medium. We investigated the amount of TSB and found that as the amount of TSB decreased, the amount of products obtained in the C3 strain also decreased accordingly. Finally, we selected the 1/10 TSB medium for large-scale fermentation and obtained 4 PTM compounds. Among them, 3 known compounds (HSAF, 3-deOH-HSAF and 3-deOH-alteramide B) and 1 new compounds alteramide B. The third chapter of this thesis is the study of HSAF inhibition of filamentous fungi and Candida albicans. First of all, rice blast fungus was used as a study model to analyze the difference of transcriptional groups. It was found that HSAF had an important influence on the apoptosis pathway of cells. Molecular substances are transported to the cells. We study the role and mechanism of small molecular drugs in the process of drug delivery. The first is to obtain protoplasts, and then with Candida albicans as a model, the HSAF action formula is examined at the level of protoplast. DCFH-DA fluorescence staining is used to detect the effect of HSAF. The level of intracellular active oxygen (reactive oxygen species, ROS). After adding four kinds of active oxygen scavengers: ascorbic acid (AA), thiourea (TU), acetylcysteine (NAC) and glutathione (GSH), the antifungal activity of HSAF decreased obviously, indicating that HSAF is up to inhibit the growth of fungi by inducing the ROS up cell in the cell. By fluorescence inverted microscope, flow cells The analysis of the analyzer and Western blotting, found that HSAF caused the mitochondrial membrane changes, its potential showed a downward trend, causing DNA damage and abnormal cell cycle distribution, staying in the G2/M stage, and eventually leading to the early and late apoptosis of the cells. The fourth chapter of this paper is the study of the mechanism of the action mechanism of the new compound ATB against Candida albicans,.ATB In vitro inhibition of Candida albicans can not only inhibit the growth of Candida albicans, but also inhibit the growth of the mycelial growth of Candida albicans, which can induce the increase of ROS level in the cells of Candida albicans, causing the decrease of the mitochondrial membrane potential in the cells. Further studies have found that ATB can cause the stagnation of the G2/M phase of Candida albicans cells, leading to the refinement of the cells of Candida albicans. Early and late apoptotic.AA can inhibit the growth cycle arrest and apoptosis inducing effect of ATB on Candida albicans cells. Therefore, ATB plays its antifungal activity by inducing the production and accumulation of ROS in Candida albicans. The target point study shows that in HeLa cells, ATB can be combined with microtubule protein; In addition, ATB can inhibit the aggregation of microtubule protein; molecular simulation shows that ATB can interact with 12 key amino acids of Candida albicans beta -tubulin, and point mutation experimental results show that the possible binding sites of ATB and beta -tubulin are L215, L217, L273, T274 and R282 respectively, and R282 is the key site. TB has played a good role in inhibiting Candida albicans in vivo. In this paper, a new compound, ATB, was found to explore the target and mechanism of its antifungal action. It was found that microtubulin could be the new target of antifungal drugs for the first time. It provides a new idea for the clinical treatment of Candida albicans infection and the development of new antifungal drugs. Important reference significance.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R915

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