幽门螺杆菌在NO压力下的巯基亚硝基化修饰及蛋白质组反应特征

发布时间：2018-08-17 14:36

【摘要】： 幽门螺杆菌(Helicobacter pylori,H.pylori)是一种革兰氏阴性、螺杆状的微需氧菌,定植于人胃粘膜上皮细胞,可导致慢性胃炎、消化性溃疡,胃粘膜相关淋巴组织(MALT)淋巴瘤及胃癌的发生,1994年世界卫生组织国际癌症研究机构(IARC)已将其列为Ⅰ类致癌因子。幽门螺杆菌在定植胃粘膜后可以激活人体免疫系统,包括局部性和全身性抗体反应、细胞介导的免疫反应等。临床资料及动物实验表明这些先天性和继发性免疫反应对幽门螺杆菌具有一定的清除能力,如果能够充分地理解它们对幽门螺杆菌的免疫杀伤作用,就可以为新型抗幽门螺杆菌制剂的开发提供重要线索。一氧化氮(nitric oxide,NO)分子是具有抗菌作用和免疫调节活性的重要的效应分子,它的L-精氨酸合成途径有赖于一氧化氮合酶(NOS)的催化作用,机体内的NOS有结构型一氧化氮合酶(cNOS)和诱导型一氧化氮合酶(iNOS)两类。其中,在感染、炎症以及一些生理状态下,iNOS可表达于多种细胞。当iNOS表达后,能持续生成大量的NO,在机体的免疫过程中,尤其是抗感染免疫中发挥重要的作用。因此,NO是当今生物学和医学领域中比较热门的研究课题之一,在微生物学领域,NO的研究也十分活跃。众所周知,半胱氨酸(含巯基,结构式为HS-)在多种蛋白功能中发挥重要作用。NO与巯基之间形成共价键的过程被称为巯基亚硝基化修饰。这种修饰与多种生理和病理过程密切相关,例如平滑肌舒张、神经递质传递和免疫防御等。目前比较常用的分析巯基亚硝基化蛋白质特征的方法是生物素转换法,并借助抗体法或质谱技术来进一步研究发生巯基亚硝基化的蛋白质。将活性巯基进行亚硝基化修饰是NO损伤病原体的重要机制,因此,针对巯基亚硝基化的分析方法非常适合研究免疫分子NO对微生物的作用靶点。基因组学和蛋白质组学技术都是整体的高通量的生物技术体系,它们的兴起推动了生命科学各个分支突飞猛进的发展。基因是遗传信息的携带者,蛋白质才是全部生物功能的执行者,由于转录后调控和翻译的影响,mRNA水平的变化不能完全反映蛋白质丰度的变化。而蛋白质组学是在整体水平上高通量地对不同时间和空间发挥功能的特定蛋白质群体进行研究的技术体系,已被证实是研究细菌应对各种环境压力的适应反应的有力工具。幽门螺杆菌的两个菌株(H.pylori26695和H.pyloriJ99)的全基因组序列已经测序完成,为了充分利用这些生物信息学资源,本研究应用生物素转换法和高通量的比较蛋白质组学方法分析了NO对幽门螺杆菌蛋白质的巯基亚硝基化修饰和幽门螺杆菌在NO压力下的蛋白质表达的变化情况,旨在发现幽门螺杆菌抗逆生存的新机制。该课题主要研究内容及实验结果如下: 一、一氧化氮对幽门螺杆菌菌体蛋白巯基亚硝基化修饰及其抑菌作用当幽门螺杆菌感染人体后,它可以激活宿主的先天性和继发性免疫反应,其中,NO是一种重要的非特异性免疫效应分子,目前尚无研究分析NO等活性氮类物质(RNS)对幽门螺杆菌的抑菌活性,而RNS对抗病原微生物的一个主要的作用机制是将重要生命蛋白的活性半胱氨酸氧化和亚硝基化,使菌体蛋白功能弱化或丢失。然而,RNS对幽门螺杆菌的靶蛋白尚不清楚。本实验首次在体外实验检测了NO对幽门螺杆菌的较强的抑菌和杀菌活性,分析了幽门螺杆菌在应用NO供体GSNO处理后蛋白质发生巯基亚硝基化的情况。即将幽门螺杆菌的菌体蛋白质与GSNO孵育后,用生物素转换法(BSM)标记发生巯基亚硝基化的蛋白,然后再进行纯化、凝胶电泳分离,最后对巯基亚硝基化蛋白进行MALDI-TOF-TOF质谱鉴定。明确地鉴定了五种发生巯基亚硝基化的蛋白质,分别为:伴侣分子及热休克蛋白(GroEL)、尿素酶α亚基(UreA)、烷基氢过氧化物还原酶(TsaA)、HP0721和HP0129,这初步揭示了RNS对幽门螺杆菌的可能的抑菌机制:通过对蛋白质的巯基亚硝基化修饰靶定、抑制细菌的抗氧化防御功能以及粘附、定植能力等。二、幽门螺杆菌在一氧化氮压力下的蛋白质组反应特征幽门螺杆菌具有多种策略逃逸先天性免疫反应,经由NOS途径合成的NO等活性氮类物质是先天性免疫反应中的重要效应分子。然而,幽门螺杆菌如何在NO压力下存活的机制至今尚未完全阐明。本实验应用蛋白质组学的方法分析了幽门螺杆菌在亚硝基化压力下的蛋白质反应特征。利用比较蛋白质组学的方法筛选到38个受NO供体(硝普钠)调节的蛋白质。这些蛋白质分别参与蛋白质处理过程、抗氧化、一般性压力反应、毒力因子以及一些未知功能等。值得注意的是在我们所筛选到的蛋白质中,一些蛋白质参与铁代谢,综合相关的文献报道的结果,推测它们很可能处在高铁携带调节元(Fur)的控制下。虽然可能由于受蛋白质低丰度的限制,在比较蛋白质组学中没有捕获到Fur蛋白,但实时定量PCR的结果表明亚硝基化压力可以诱导Fur在转录水平的上调。此外,在38种压力蛋白中,着重对硫氧还蛋白还原酶(TrxR)进行了分析。为了证实Fur或TrxR在幽门螺杆菌耐受NO压力中的作用,我们构建了Fur和TrxR的基因缺失突变株,经检测fur或trxR基因缺失突变后幽门螺杆菌对亚硝基化压力更加敏感,这表明Fur调节元和TrxR在NO压力调节中发挥重要的作用。
[Abstract]:Helicobacter pylori (H. pylori) is a gram-negative, screw-like microaerobic bacterium that colonizes human gastric epithelial cells and can cause chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. It was listed by the International Agency for Research on Cancer (IARC) of the World Health Organization in 1994. Class I carcinogenic factors. Helicobacter pylori can activate the human immune system after colonization of gastric mucosa, including local and systemic antibody reactions, cell-mediated immune responses, etc. Clinical data and animal experiments show that these congenital and secondary immune reactions have certain clearance of Helicobacter pylori, if fully geographically Understanding their immuno-killing effect on Helicobacter pylori may provide important clues for the development of new anti-Helicobacter pylori agents.
Nitric oxide (NO) molecule is an important effector molecule with antimicrobial and immunoregulatory activities. Its L-arginine synthesis pathway depends on the catalysis of nitric oxide synthase (NOS). There are two types of NOS in the body: structural nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS). INOS can be expressed in many kinds of cells under some physiological conditions. When iNOS is expressed, it can continuously produce a large number of NO, which plays an important role in the immune process of the body, especially in anti-infective immunity. Therefore, NO is one of the hottest research topics in the field of biology and medicine. In the field of microbiology, the study of NO is also very active. Jump.
It is well known that cysteine (HS-) plays an important role in many protein functions. The formation of covalent bonds between NO and thiols is called mercaptonitrosation modification. This modification is closely related to a variety of physiological and pathological processes, such as smooth muscle relaxation, neurotransmitter delivery and immune defense. Biotin conversion method is used to analyze the characteristics of mercapto-nitroso proteins, and the mercapto-nitroso proteins are further studied by antibody or mass spectrometry. Nitroso modification of active mercapto is an important mechanism of NO damage to pathogens. Therefore, the analysis method for mercapto-nitroso is very suitable. Objective to study the target of immune molecule NO on microorganisms.
Genomics and proteomics are high-throughput biotechnology systems as a whole, and their rise has promoted the rapid development of various branches of life sciences. Genes are carriers of genetic information, and proteins are the executors of all biological functions. Due to the influence of post-transcriptional regulation and translation, changes in the level of mRNA can not be achieved. Proteomics is a high-throughput system for studying specific proteins that function in different time and space at an overall level. It has been proved to be a powerful tool for studying the adaptive responses of bacteria to various environmental stresses. Two strains of Helicobacter pylori (H. pylori 26) Genome-wide sequences of 695 and H. pylori J99 have been sequenced. In order to make full use of these bioinformatics resources, biotin conversion and high-throughput comparative proteomics methods were used to analyze the sulfhydryl nitroso modification of Helicobacter pylori protein by NO and the protein expression of Helicobacter pylori under NO pressure. The main research contents and experimental results are as follows:1.
Nitric oxide on thiol nitrosation of Helicobacter pylori and its bacteriostatic effect
When Helicobacter pylori infects humans, it can activate the innate and secondary immune responses of the host. NO is an important non-specific immune effector molecule. Up to now, no studies have been done to analyze the antibacterial activity of reactive nitrogen substances (RNS) such as NO against Helicobacter pylori. One of the main mechanisms of RNS against pathogenic microorganisms is that However, the target protein of RNS to Helicobacter pylori is still unclear. In this experiment, the strong bacteriostatic and bactericidal activity of NO to Helicobacter pylori was detected in vitro for the first time, and the effect of GSNO on Helicobacter pylori was analyzed. After incubation of Helicobacter pylori bacterial proteins with GSNO, the proteins labeled with biotin conversion method (BSM) were purified and separated by gel electrophoresis. Finally, the proteins were identified by MALDI-TOF-TOF mass spectrometry. Sulfhydryl nitrosogenic proteins are chaperone molecule and heat shock protein (GroEL), urease alpha subunit (UreA), alkyl hydroperoxide reductase (TsaA), HP0721 and HP0129, which preliminarily reveal the possible bacteriostatic mechanism of RNS to Helicobacter pylori. Defense function, adhesion and colonization ability.
Two, the proteome reaction characteristics of Helicobacter pylori under no pressure.
Helicobacter pylori has a variety of strategies to escape innate immune responses. Active nitrogen compounds, such as NO, synthesized via NOS pathway, are important effectors in innate immune responses. However, the mechanism of how Helicobacter pylori survives under NO pressure has not been fully elucidated. Proteomic analysis of Helicobacter pylori has been carried out in this study. Characteristics of bacterial protein reactions under nitrosation pressures. 38 proteins regulated by nitric oxide donors (sodium nitroprusside) were screened by comparative proteomics. These proteins are involved in protein processing, antioxidation, general stress response, virulence factors, and some unknown functions, respectively. Some of the selected proteins are involved in iron metabolism, and the results of the literature suggest that they may be under the control of iron-carrying regulator (Fur). Although Fur proteins may not be captured in comparative proteomics due to low protein abundance, the results of real-time quantitative PCR show that nitroso In addition, thioredoxin reductase (TrxR) was analyzed in 38 stress proteins. To confirm the role of Fur or TrxR in the tolerance of H. pylori to NO stress, we constructed mutants with deletion of Fur and TrxR genes, which were detected in pylorus after deletion of fur or trxR genes. Helicobacter spp. is more sensitive to nitrosation pressure, suggesting that Fur regulator and TrxR play an important role in NO pressure regulation.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

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