【摘要】： 雙歧桿菌屬放線菌科雙歧桿菌屬,革蘭氏陽(yáng)性桿菌,G+C%含量高,專性厭氧。在健康人體腸道內(nèi)存在的多種細(xì)菌中,以雙歧桿菌發(fā)揮的生理功能最為重要。該菌在維護(hù)腸道菌群平衡、抑制病原菌生長(zhǎng)、抗衰老、抗腫瘤以及為人體提供多種營(yíng)養(yǎng)素等方面發(fā)揮了作用。雙歧桿菌能通過(guò)合成乳酸和乙酸等物質(zhì)調(diào)節(jié)腸道pH值,并依靠自身的黏附能力抑制外侵的多種腸道致病菌和條件致病菌的侵襲,預(yù)防和治療胃腸炎、結(jié)腸炎、壞死性小腸結(jié)腸炎以及慢性腸道感染等多種胃腸道疾病。然而,目前報(bào)道中關(guān)于雙歧桿菌與腸道細(xì)胞相互作用的研究很少,尤其是雙歧桿菌在腸道環(huán)境中的適應(yīng)性機(jī)制的研究尚無(wú)報(bào)道。 本研究中所用的長(zhǎng)雙歧桿菌NCC2705分離自健康嬰兒的糞便,是與人體相關(guān)的雙歧桿菌中十分重要的一個(gè)菌株。瑞士雀巢公司的研究人員于2002年完成了該菌的基因組測(cè)序,使其成為第一個(gè)遺傳背景清楚的雙歧桿菌,便于功能基因組學(xué)和蛋白質(zhì)組學(xué)的研究。 為研究長(zhǎng)雙歧桿菌NCC2705在腸道內(nèi)的適應(yīng)性機(jī)制,我們利用家兔作為動(dòng)物模型,將分別在家兔結(jié)腸內(nèi)和家兔體外培養(yǎng)的長(zhǎng)雙歧桿菌NCC2705進(jìn)行了比較蛋白質(zhì)組學(xué)研究。利用雙向凝膠電泳分離蛋白,并用Image Master 2D Elite Platnum軟件對(duì)電泳結(jié)果進(jìn)行分析。研究發(fā)現(xiàn),在經(jīng)過(guò)家兔體內(nèi)培養(yǎng)后,長(zhǎng)雙歧桿菌NCC2705的全基因組蛋白中共有38個(gè)蛋白點(diǎn)表達(dá)豐度發(fā)生了3倍以上的變化,同時(shí)有4對(duì)蛋白點(diǎn)在膠圖上發(fā)生了位置的遷移,可能是蛋白質(zhì)翻譯后修飾的結(jié)果。此后,我們對(duì)這38個(gè)表達(dá)差異的蛋白點(diǎn)及位移的蛋白點(diǎn),用MALDI-TOF-MS和/或ESI-MS/MS進(jìn)行質(zhì)譜鑒定,并利用半定量RT-PCR進(jìn)行了驗(yàn)證。結(jié)果表明,該38個(gè)蛋白點(diǎn)代表了33種蛋白,與體外培養(yǎng)相比,體內(nèi)培養(yǎng)的長(zhǎng)雙歧桿菌有19個(gè)蛋白上調(diào),14個(gè)蛋白下調(diào),4個(gè)蛋白發(fā)生了位置遷移。這些差異蛋白包括應(yīng)激蛋白、能量代謝相關(guān)蛋白以及翻譯相關(guān)蛋白等。 我們對(duì)一些與長(zhǎng)雙歧桿菌NCC2705腸道適應(yīng)性相關(guān)的關(guān)鍵蛋白進(jìn)行了分析:在體內(nèi)環(huán)境中培養(yǎng),膽鹽水解酶比在體外培養(yǎng)表達(dá)豐度提高了5.2倍。膽鹽水解酶能催化水解甘氨酸和牛磺酸共軛結(jié)合膽鹽,釋放氨基酸并使膽鹽失活形成膽酸,使益生菌避免了膽鹽的毒害作用。該酶在益生菌與體內(nèi)環(huán)境交互作用的初期,以及在機(jī)體腸道對(duì)游離氨基酸的利用和腸道抗膽鹽毒害的過(guò)程中發(fā)揮了重要作用。延伸因子EF-Tu在體內(nèi)培養(yǎng)后,表達(dá)豐度增加超過(guò)5倍。它是一種新型表面蛋白,主要在蛋白質(zhì)合成過(guò)程中發(fā)揮作用,具有黏附因子的特征并誘導(dǎo)促炎癥反應(yīng)。其在體內(nèi)培養(yǎng)過(guò)程中的上調(diào)說(shuō)明該蛋白在細(xì)菌的定植和黏附過(guò)程中發(fā)揮了作用。此外,應(yīng)激蛋白包括:與蛋白折疊、聚集和降解相關(guān)的應(yīng)激蛋白(GroEL),觸發(fā)因子的伴侶分子(Tig)和ATP依賴的Clp蛋白酶水解亞單位2蛋白(ClpP2),它們?cè)诩彝皿w內(nèi)培養(yǎng)后表達(dá)豐度均明顯增加,主要作用在于避免長(zhǎng)雙歧桿菌受腸道內(nèi)各種有害因素的影響,得以在腸道內(nèi)苛刻的條件下存活。雙歧桿菌寄居于胃腸道,是通過(guò)其獨(dú)特的Bifid Shunt途徑進(jìn)行糖酵解的。在本研究中,該途徑的相關(guān)酶中有3個(gè)酶在雙歧桿菌經(jīng)體內(nèi)培養(yǎng)后表達(dá)豐度升高,它們分別是醛糖轉(zhuǎn)移酶、酮糖移轉(zhuǎn)酶和核糖5-磷酸異構(gòu)酶,這表明長(zhǎng)雙歧桿菌NCC2705充分利用了這些酶快速生長(zhǎng)來(lái)參與腸道內(nèi)的菌群競(jìng)爭(zhēng)。 本研究中位置發(fā)生遷移的4個(gè)蛋白分別是:GlnA1、PurC、LuxS和Pgk,進(jìn)一步利用Pro-Q Diamond磷酸化蛋白膠染色和Western Blot的方法,對(duì)遷移蛋白點(diǎn)的磷酸化修飾進(jìn)行了驗(yàn)證和分析。結(jié)果顯示,Pgk和LuxS兩個(gè)蛋白發(fā)生了磷酸化作用。長(zhǎng)雙歧桿菌NCC2705中的Pgk被磷酸化并形成磷酸化中間體發(fā)揮作用;LuxS是密度感應(yīng)系統(tǒng)中信號(hào)分子AI-2產(chǎn)生過(guò)程中的關(guān)鍵酶,該蛋白在體內(nèi)環(huán)境中磷酸化修飾作用增強(qiáng)。我們推測(cè)磷酸化形式可能是LuxS的一種活性形式,即LuxS-P,該酶以這種形式在雙歧桿菌的密度感應(yīng)系統(tǒng)中發(fā)揮了關(guān)鍵的作用。 通過(guò)前面蛋白質(zhì)組學(xué)的研究,我們發(fā)現(xiàn)了一些在雙歧桿菌適應(yīng)腸道環(huán)境過(guò)程中發(fā)揮了關(guān)鍵作用的蛋白,這些蛋白增強(qiáng)了雙歧桿菌對(duì)周?chē)焕h(huán)境的適應(yīng)能力。此外,我們還發(fā)現(xiàn)四個(gè)位置發(fā)生遷移的蛋白,其中與細(xì)菌種間交流信號(hào)分子AI-2的產(chǎn)生密切相關(guān)的LuxS蛋白發(fā)生了磷酸化,這一現(xiàn)象表明其在雙歧桿菌適應(yīng)腸道環(huán)境、與其它菌群進(jìn)行信息交流過(guò)程中可能發(fā)揮作用。因此,我們針對(duì)長(zhǎng)雙歧桿菌NCC2705中LuxS蛋白是否具有功能,該菌是否能分泌具有活性的信號(hào)分子AI-2進(jìn)行了研究。我們利用對(duì)AI-2特異的哈氏弧菌BB170報(bào)告系統(tǒng)檢測(cè)長(zhǎng)雙歧桿菌NCC2705和LuxS過(guò)表達(dá)菌中AI-2的分泌及活性。實(shí)驗(yàn)發(fā)現(xiàn),長(zhǎng)雙歧桿菌NCC2705的luxS基因能在大腸桿菌中表達(dá)。通過(guò)對(duì)長(zhǎng)雙歧桿菌NCC2705和LuxS過(guò)表達(dá)重組菌中的AI-2進(jìn)行檢測(cè),發(fā)現(xiàn)長(zhǎng)雙歧桿菌NCC2705和重組菌均能分泌信號(hào)分子AI-2使哈氏弧菌BB170發(fā)出熒光,且AI-2的分泌量在一定范圍內(nèi)與雙歧桿菌菌群密度呈正相關(guān)。這一結(jié)果表明,長(zhǎng)雙歧桿菌NCC2705中LuxS蛋白具有活性,并存在依賴于luxS/AI-2的QS系統(tǒng),能夠分泌群體感應(yīng)信號(hào)分子AI-2。 本研究詮釋了長(zhǎng)雙歧桿菌NCC2705在腸道中的適應(yīng)性機(jī)制,首次利用家兔作為腸道模型研究雙歧桿菌在腸道內(nèi)的生理性變化,并結(jié)合蛋白質(zhì)組學(xué)和磷酸化染色等方法對(duì)該菌在機(jī)體內(nèi)、外條件下的蛋白表達(dá)變化進(jìn)行了有效比較。此外,本研究首次對(duì)長(zhǎng)雙歧桿菌NCC2705的群體感應(yīng)系統(tǒng)信號(hào)分子AI-2的存在以及其產(chǎn)生過(guò)程中的關(guān)鍵酶LuxS的功能活性進(jìn)行了研究和驗(yàn)證。上述這些重要結(jié)論和成果,為雙歧桿菌的分子生物學(xué)研究和以雙歧桿菌為載體的基因工程疫苗等研究提供了重要的理論依據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R371

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