长双歧杆菌NCC2705肠道适应性机制研究

发布时间：2018-08-24 10:03

【摘要】： 双歧杆菌属放线菌科双歧杆菌属,革兰氏阳性杆菌,G+C%含量高,专性厌氧。在健康人体肠道内存在的多种细菌中,以双歧杆菌发挥的生理功能最为重要。该菌在维护肠道菌群平衡、抑制病原菌生长、抗衰老、抗肿瘤以及为人体提供多种营养素等方面发挥了作用。双歧杆菌能通过合成乳酸和乙酸等物质调节肠道pH值,并依靠自身的黏附能力抑制外侵的多种肠道致病菌和条件致病菌的侵袭,预防和治疗胃肠炎、结肠炎、坏死性小肠结肠炎以及慢性肠道感染等多种胃肠道疾病。然而,目前报道中关于双歧杆菌与肠道细胞相互作用的研究很少,尤其是双歧杆菌在肠道环境中的适应性机制的研究尚无报道。本研究中所用的长双歧杆菌NCC2705分离自健康婴儿的粪便,是与人体相关的双歧杆菌中十分重要的一个菌株。瑞士雀巢公司的研究人员于2002年完成了该菌的基因组测序,使其成为第一个遗传背景清楚的双歧杆菌,便于功能基因组学和蛋白质组学的研究。为研究长双歧杆菌NCC2705在肠道内的适应性机制,我们利用家兔作为动物模型,将分别在家兔结肠内和家兔体外培养的长双歧杆菌NCC2705进行了比较蛋白质组学研究。利用双向凝胶电泳分离蛋白,并用Image Master 2D Elite Platnum软件对电泳结果进行分析。研究发现,在经过家兔体内培养后,长双歧杆菌NCC2705的全基因组蛋白中共有38个蛋白点表达丰度发生了3倍以上的变化,同时有4对蛋白点在胶图上发生了位置的迁移,可能是蛋白质翻译后修饰的结果。此后,我们对这38个表达差异的蛋白点及位移的蛋白点,用MALDI-TOF-MS和/或ESI-MS/MS进行质谱鉴定,并利用半定量RT-PCR进行了验证。结果表明,该38个蛋白点代表了33种蛋白,与体外培养相比,体内培养的长双歧杆菌有19个蛋白上调,14个蛋白下调,4个蛋白发生了位置迁移。这些差异蛋白包括应激蛋白、能量代谢相关蛋白以及翻译相关蛋白等。我们对一些与长双歧杆菌NCC2705肠道适应性相关的关键蛋白进行了分析:在体内环境中培养,胆盐水解酶比在体外培养表达丰度提高了5.2倍。胆盐水解酶能催化水解甘氨酸和牛磺酸共轭结合胆盐,释放氨基酸并使胆盐失活形成胆酸,使益生菌避免了胆盐的毒害作用。该酶在益生菌与体内环境交互作用的初期,以及在机体肠道对游离氨基酸的利用和肠道抗胆盐毒害的过程中发挥了重要作用。延伸因子EF-Tu在体内培养后,表达丰度增加超过5倍。它是一种新型表面蛋白,主要在蛋白质合成过程中发挥作用,具有黏附因子的特征并诱导促炎症反应。其在体内培养过程中的上调说明该蛋白在细菌的定植和黏附过程中发挥了作用。此外,应激蛋白包括:与蛋白折叠、聚集和降解相关的应激蛋白(GroEL),触发因子的伴侣分子(Tig)和ATP依赖的Clp蛋白酶水解亚单位2蛋白(ClpP2),它们在家兔体内培养后表达丰度均明显增加,主要作用在于避免长双歧杆菌受肠道内各种有害因素的影响,得以在肠道内苛刻的条件下存活。双歧杆菌寄居于胃肠道,是通过其独特的Bifid Shunt途径进行糖酵解的。在本研究中,该途径的相关酶中有3个酶在双歧杆菌经体内培养后表达丰度升高,它们分别是醛糖转移酶、酮糖移转酶和核糖5-磷酸异构酶,这表明长双歧杆菌NCC2705充分利用了这些酶快速生长来参与肠道内的菌群竞争。本研究中位置发生迁移的4个蛋白分别是:GlnA1、PurC、LuxS和Pgk,进一步利用Pro-Q Diamond磷酸化蛋白胶染色和Western Blot的方法,对迁移蛋白点的磷酸化修饰进行了验证和分析。结果显示,Pgk和LuxS两个蛋白发生了磷酸化作用。长双歧杆菌NCC2705中的Pgk被磷酸化并形成磷酸化中间体发挥作用;LuxS是密度感应系统中信号分子AI-2产生过程中的关键酶,该蛋白在体内环境中磷酸化修饰作用增强。我们推测磷酸化形式可能是LuxS的一种活性形式,即LuxS-P,该酶以这种形式在双歧杆菌的密度感应系统中发挥了关键的作用。通过前面蛋白质组学的研究,我们发现了一些在双歧杆菌适应肠道环境过程中发挥了关键作用的蛋白,这些蛋白增强了双歧杆菌对周围不利环境的适应能力。此外,我们还发现四个位置发生迁移的蛋白,其中与细菌种间交流信号分子AI-2的产生密切相关的LuxS蛋白发生了磷酸化,这一现象表明其在双歧杆菌适应肠道环境、与其它菌群进行信息交流过程中可能发挥作用。因此,我们针对长双歧杆菌NCC2705中LuxS蛋白是否具有功能,该菌是否能分泌具有活性的信号分子AI-2进行了研究。我们利用对AI-2特异的哈氏弧菌BB170报告系统检测长双歧杆菌NCC2705和LuxS过表达菌中AI-2的分泌及活性。实验发现,长双歧杆菌NCC2705的luxS基因能在大肠杆菌中表达。通过对长双歧杆菌NCC2705和LuxS过表达重组菌中的AI-2进行检测,发现长双歧杆菌NCC2705和重组菌均能分泌信号分子AI-2使哈氏弧菌BB170发出荧光,且AI-2的分泌量在一定范围内与双歧杆菌菌群密度呈正相关。这一结果表明,长双歧杆菌NCC2705中LuxS蛋白具有活性,并存在依赖于luxS/AI-2的QS系统,能够分泌群体感应信号分子AI-2。本研究诠释了长双歧杆菌NCC2705在肠道中的适应性机制,首次利用家兔作为肠道模型研究双歧杆菌在肠道内的生理性变化,并结合蛋白质组学和磷酸化染色等方法对该菌在机体内、外条件下的蛋白表达变化进行了有效比较。此外,本研究首次对长双歧杆菌NCC2705的群体感应系统信号分子AI-2的存在以及其产生过程中的关键酶LuxS的功能活性进行了研究和验证。上述这些重要结论和成果,为双歧杆菌的分子生物学研究和以双歧杆菌为载体的基因工程疫苗等研究提供了重要的理论依据。
[Abstract]:Bifidobacterium is an actinomycete bifidobacterium. Gram-positive bacilli contain high G+C% and are anaerobic. Among the various bacteria in the intestine of healthy people, the physiological function of Bifidobacterium is the most important. Bifidobacterium can regulate intestinal pH by synthesizing lactic acid and acetic acid, and inhibit the invasion of invasive intestinal pathogens and conditional pathogens by its own adhesion ability. Bifidobacterium can prevent and treat gastroenteritis, colitis, necrotizing enterocolitis and chronic intestinal infection. However, few studies have been reported on the interaction between Bifidobacterium and intestinal cells, especially on the adaptive mechanism of Bifidobacterium in intestinal environment.
The strain NCC2705 used in this study, isolated from the feces of healthy infants, is an important human-related strain of Bifidobacterium longum. Researchers at Nestle Switzerland completed the genome sequencing of the bacterium in 2002, making it the first Bifidobacterium with a clear genetic background to facilitate functional genomics and genomics. Proteomics research.
In order to study the adaptive mechanism of Bifidobacterium longum NCC2705 in intestinal tract, we used rabbits as animal models to study the comparative proteomics of Bifidobacterium longum NCC2705 cultured in rabbit colon and rabbit colon respectively. The results showed that 38 protein spots in the whole genome of Bifidobacterium longum NCC2705 were more than three times as abundant as those in rabbits, and 4 pairs of protein spots migrated on the map, which may be the result of post-translational modification. MALDI-TOF-MS and/or ESI-MS/MS were used to identify the different protein spots and displaced protein spots, and semi-quantitative RT-PCR was used to verify them. The results showed that the 38 protein spots represented 33 proteins. Compared with in vitro culture, 19 proteins were up-regulated, 14 proteins were down-regulated and 4 proteins were down-regulated. These differential proteins include stress proteins, energy metabolism related proteins, and translation related proteins.
We analyzed some key proteins related to intestinal adaptation of Bifidobacterium longum NCC2705. The expression abundance of biliary saline hydrolase was 5.2 times higher in vitro than in vivo. Bile saline hydrolase can catalyze the hydrolysis of glycine and taurine conjugated bile salts, release amino acids and deactivate bile salts to form cholic acid. Probiotics avoid the toxicity of bile salts. The enzyme plays an important role in the initial interaction between probiotics and the environment in vivo, in the utilization of free amino acids in the intestinal tract and in the intestinal anti-bile salt toxicity. Its up-regulation in vivo suggests that the protein plays a role in bacterial colonization and adhesion. In addition, stress proteins include: stress proteins related to protein folding, aggregation and degradation (GroEL), triggers. Bifidobacterium longum can survive in the intestinal tract under harsh conditions. Bifidobacterium bifidum resides in the gastrointestinal tract by means of gastrointestinal tract. In this study, three of the enzymes involved in the Bifid Shunt pathway, aldose-transferase, ketose-transferase and ribose-5-phosphate isomerase, were highly expressed in Bifidobacterium longum after incubation in vivo, suggesting that the rapid growth of these enzymes was fully utilized by Bifid longum NCC2705. Bacterial competition in the gut.
In this study, GlnA1, PurC, LuxS and Pgk were the four proteins that migrated to their sites. The phosphorylation modification of the migrating protein sites was further verified and analyzed by Pro-Q Diamond phosphorylated protein glue staining and Western Blot method. The results showed that Pgk and LuxS were phosphorylated. Bifidobacterium longum NCC2705 P GK is phosphorylated to form phosphorylated intermediates; LuxS is a key enzyme in the production of AI-2, a signal molecule in density sensing systems, and the phosphorylation of this protein is enhanced in vivo. We speculate that phosphorylation may be an active form of LuxS, LuxS-P, which is densely present in Bifidobacteria. The degree induction system plays a key role.
Through previous proteomic studies, we have identified proteins that play a key role in the adaptation of Bifidobacterium to the intestinal environment. These proteins enhance the ability of Bifidobacterium to adapt to adverse environments. In addition, we have also identified four proteins that migrate, including the intercellular signaling molecule AI- The phosphorylation of LuxS protein, which is closely related to the production of Bifidobacterium longum NCC2705, suggests that LuxS protein may play a role in the intestinal adaptation of Bifidobacterium longum to other flora. We detected the secretion and activity of AI-2 in the over-expressed strains NCC2705 and LuxS of Bifidobacterium longum using the AI-2-specific Vibrio harringtonii BB170 reporting system.It was found that the luxS gene of Bifidobacterium longum NCC2705 could be expressed in E.coli.The AI-2 in the over-expressed recombinant strains NCC2705 and LuxS of Bifidobacterium longum was detected. It was found that both NCC2705 and recombinant bacterium could secrete signal molecules AI-2 to make Vibrio harringiensis BB170 fluorescent, and the secretion of AI-2 was positively correlated with the density of Bifidobacterium flora in a certain range. Body sensing signal molecule AI-2.
In this study, the adaptive mechanism of Bifidobacterium longum NCC2705 in intestine was elucidated. Rabbits were used as intestinal model to study the physiological changes of Bifidobacterium longum in intestine for the first time. The existence of the quorum-sensing system signal molecule AI-2 of Bifidobacterium longum NCC2705 and the functional activity of the key enzyme LuxS during its production were studied and verified for the first time. These important conclusions and achievements provide the basis for the molecular biology of Bifidobacterium longum and the genetic engineering vaccine with Bifidobacterium longum as vector. Important theoretical basis.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R371

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