【作者】 邵春红；

【导师】 贾继辉；

【作者基本信息】 山东大学 ， 病原生物学， 2008， 博士

【摘要】 幽门螺杆菌（Helicobacter pylori,H.pylori）主要定植于人胃粘膜上皮细胞,是慢性胃炎和消化性溃疡的主要致病因素,与胃癌及胃粘膜相关淋巴组织（MALT）淋巴瘤发生密切相关,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。流行病学调查显示,粪—口途径是幽门螺杆菌的重要传播途径之一,该菌在体内存活及体外传播过程中必然会遭受到多种环境压力的胁迫,细菌通过启动一系列的适应性调节机制来抵抗这些胁迫因素以达到保种生存并导致疾病的目的。幽门螺杆菌的一个显著特征就是能够在人胃的极端酸性环境中生存,人胃腔内的pH值在饥饿时可低达1.0,在自酸性的胃腔向胃粘膜层移动过程中,幽门螺杆菌不可避免的应对pH值的不断波动。现今研究较清楚的幽门螺杆菌抗酸机制就是其脲素酶系统,脲素酶可分解脲素产生氨和二氧化碳来提高细菌微环境的pH值,然而,胃液内的脲素浓度往往约为1mM,仅通过该系统不能完全保证幽门螺杆菌在酸性胃环境的存活。因此,推测在幽门螺杆菌的酸性应答过程中还存在其它的pH平衡系统。餐后胆汁返流入胃是一种正常的生理现象,患有胃肠道疾病者此现象更为严重;许多研究证明粪-口途径是幽门螺杆菌进入人体消化道的一种重要途径,那么在其传播过程中,幽门螺杆菌必定经过富含胆汁的肠道,耐受并抵抗胆汁对它的胁迫作用;另外,人们利用聚合酶链反应和DNA印迹等方法在胆汁,胆囊和肝组织中检测到幽门螺杆菌的存在。以上均说明幽门螺杆菌对胆汁的耐受性是其在人类胃肠道定植和生存重要机制,幽门螺杆菌通过何种机制在富含胆汁的环境中生存还不清楚。形态学改变是细菌抗逆生存的重要调节机制之一,在O₂压力,低pH值等不利的培养条件下或抗菌治疗后,幽门螺杆菌可转变为活的非可培养状态的球形体,这种形态学转变是一种积极的生物学应答过程,是幽门螺杆菌的一种保护性抗逆生存机制。当细菌受到胁迫时也可进入非分裂状态,这在医学研究上具有重要意义。已有研究证明当细菌的分裂被抑制而它们的生长仍在继续时细菌可转变成丝状体形态。研究发现幽门螺杆菌在高盐培养基中于静止早期可形成丝状体结构,胺曲南也可以诱导幽门螺杆菌丝状体的形成。因此,对其丝状体的研究有可能发现幽门螺杆菌的细菌分裂检查点。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。蛋白质是生命活动的直接执行者,由于翻译和转录后修饰的影响,mRNA水平的变化不能完全反映蛋白质丰度的变化。蛋白质组学研究是在整体水平上高通量的解析基因组所表达的体现生命活动特征的蛋白质的变化规律,并且蛋白质组学技术已被证实是研究细菌应对各种不同环境压力的生理性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,本研究通过高通量的比较蛋白质组学分析了幽门螺杆菌在酸,胆汁等压力条件下及幽门螺杆菌非分裂状态丝状体的蛋白质表达变化,旨在为阐明幽门螺杆菌在体内外的生存机制提供有价值的理论基础。本学位论文的主要研究内容及实验结果如下:一.幽门螺杆菌在无脲素存在时对酸性胁迫的蛋白质反应酸性胁迫是幽门螺杆菌在人胃内面临的最大挑战,当幽门螺杆菌暴露于酸性环境时,脲素酶系统是其保持胞内及胞质pH接近于中性的基本调节机制,然而,胃液内的脲素浓度往往在1mM左右,仅仅通过分解脲素不足以有效保证幽门螺杆菌应对酸的胁迫,因此推测幽门螺杆菌还存在其它的酸性调节机制。为了鉴定除脲素酶以外幽门螺杆菌存在的抗酸相关的蛋白分子,我们利用双向凝胶电泳技术比较了该细菌在pH7.4,6.0,5.0,4.0,3.0及2.0条件下暴露30分钟后的蛋白质表达谱,差异性的蛋白进行MALDI-TOF-TOF质谱分析,共得到36个具有不同功能的蛋白质分子的信息。这些蛋白参与产氨,分子伴侣,能量代谢,细胞外膜,信号调节等多种生物学功能,还有一些蛋白质的功能至今仍不清楚,然后我们对差异性的蛋白进行了SOM分析,结果显示幽门螺杆菌通过多种蛋白的改变响应酸性胁迫。二.幽门螺杆菌对人胆汁胁迫的蛋白质反应幽门螺杆菌对胆汁的耐受能力是其在人胃肠道的定植和存活的重要机制,由于胆汁返流入低pH的胃内可被酸化,因此本研究首先检测了胆汁酸化前后对幽门螺杆菌抑制能力的改变,结果显示胆汁的酸化后对幽门螺杆菌的抑制活性降低。然后,通过双向凝胶电泳得到幽门螺杆菌在正常条件下及在胆汁和酸化胆汁胁迫下的蛋白质表达谱,差异性蛋白进行MALDL-TOF-TOF质谱分析,结果发现与正常条件下的菌体蛋白相比,胆汁及酸化胆汁胁迫下的蛋白质表达谱的变化规律相似。胆汁和酸化胆汁的存在使幽门螺杆菌28个菌体蛋白质的表达发生变化,并且大多数蛋白是诱导表达。这些蛋白包括分子伴侣蛋白,参与铁离子储存的蛋白,趋化性蛋白,与能量代谢相关的酶类及编码鞭毛装置的蛋白等,这说明幽门螺杆菌通过对多种信号分子的调节应对胆汁和酸化胆汁的胁迫。为了证实某些蛋白在幽门螺杆菌胆汁耐受中的作用,我们构建了Hp0721和鞭毛马达开关蛋白的基因缺失突变株,经检测发现两个突变株对胆汁胁迫的抵抗力有所降低,说明这两个蛋白在幽门螺杆菌的胆汁调节反应中起一定作用。三.胺曲南诱导的非分裂状态幽门螺杆菌丝状体的蛋白质组学分析处于压力环境中,细菌可进入非分裂状态,这在医学上具有重要意义。本研究利用胺曲南诱导了非分裂状态的幽门螺杆菌丝状体,细胞分裂时FtsI基因编码细胞壁上参与肽聚糖交联的一种转肽酶,是一种青霉素结合蛋白,胺曲南可抑制该蛋白的功能从而阻止细胞分裂。我们运用实时荧光定量PCR检测了FtsI和其它几个青霉素结合蛋白（Hp0597,Hp1565,Hp1372,Hp1373）在幽门螺杆菌丝状体中的表达情况,结果显示FtsI在幽门螺杆菌丝状体中低表达,而其它几个青霉素结合蛋白的表达水平与螺杆状细菌相比并无明显差异。为了寻找幽门螺杆菌可能存在的其它细胞分裂检查点,我们利用双向凝胶电泳技术比较了能够正常分裂的螺杆状和处于非分裂状态的幽门螺杆菌丝状体的蛋白质表达谱,在丝状体中具有差异表达的蛋白质斑点通过MALDI-TOF-TOF质谱分析,得到21个蛋白质的信息。其中有一个与细胞分裂相关的蛋白在幽门螺杆菌丝状体中被诱导表达,该蛋白是由MinD基因编码的一种细胞分裂抑制因子,通过序列比较发现该蛋白与大肠杆菌的相应蛋白具有50%的同源性。我们构建了MinD基因缺失的幽门螺杆菌26695突变株,形态学观察发现该基因的缺失使得部分菌体转变为小细胞型（mini cell）,幽门螺杆菌的生长率和存活率降低,这可能是由于这种小细胞型细菌无细胞核,没有增殖能力所致。更多还原

【Abstract】 As one of the most important human pathogenic bacteria, Helicobacter pylori（H.pylori） colonize mainly in human stomach. It has been recognized as the aetiological agent of gastritis and peptic ulcer disease. Moreover, long-term infection with this bacterium is closely related to the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. In 1994 international cancer institute had determined it being type I carcinogen. As shown by epidemiology, the mechanism of transmission of H.pylori is thought to be primarily by the fecal-oral route. It is necessarily inhibited by kinds of adverse conditions during growth in vivo and transmission in vitro. Only by starting a series of adaptive regulatory mechanisms, H. pylori can resist these stressing factors to survive and induce diseases.H.pylori has a unique feature which is its ability to survive in the extremely acidic environment of human stomach. The pH of human stomach content ranges from 1.0 during starvation to 5.0 in the digestive phase. When migrating from acidic lumen into gastric mucus layer, H.pylori invariably faces pH fluctuations. One of the best-studied processes in H pylori to resist acid stress is the urease system. However, the urea in gastric juice is approximately 1 mM. which sometimes may be insufficient to ensure the survival of H.pylori. Thus it is assumed that additional mechanisms of pH homeostasis may be required for the acid adaptation in H.pylori.The reflux of bile into the stomach post-prandially is a normal occurrence, especially in patients with gastroduodenal disease. Many studies support that faecal-oral is one important route of H.pylori entering the digestive tract. During transmission, H.pylori must tolerate and resist bile stress when traversing the bile-rich intestinal tract. Furthermore, H.pylori has been detected in bile, gall-bladder tissue and liver samples through polymerase chain reaction and Southern blot hybridization. All these points indicate that the ability of H.pylori to tolerate bile is likely to be very important for their colonization and survival in the gastrointestinal tract of humans. Although it has been established that H. pylori is bile-sensitive, the mechanism allowing it to colonize a bile-containing environment is unclear.Morphologic change is an important adaptive ways for bacteria to resist various unfavourable conditions. The viable but nonculturable coccoid form of H.pylori can be induced by many environmental stresses such as O₂ stress, pH alteration or exposure to antibiotics. Transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism. When stressed, bacteria can enter various nondividing states, which are medically important. It has been shown that the filaments of bacteria are formed when cell division is inhibited while their growth continues. Previous studies found that H.pylori changes into filamentous form in a high-salt level medium at early stationary phase, aztreonam can also induce pronounced filamentation in H.pylori. The study on filamentous H.pylori is helpful to identify possible cell division checkpoints in this bacterium.Genomic and proteomic technologies are both integrative and high-throughput technologies whose rapid development led to a burst across all branches of the life sciences. Proteins are the direct executors of vital activities and mRNA level is not consistently reliable to predict protein abundance due to the posttranslational and posttranscriptional modification. Proteomics is to analyze the structure and function of the whole proteins expressed by genome in a cell, tissue or organism at global level. Furthermore, proteomic technologies have been proven to be particularly useful to study the physiological responses of bacteria to various environmental stresses. The genomic sequencing of two H.pylori strains （H.pylori26695 and H.pyloriJ99） has been completed. In order to efficiently exploit this information, we analyzed the protein expression changes of H.pylori under acid and bile stress and filamentous H.pylori using comparative proteomics. The aim is to reveal the possible survival mechanisms underlying in this important human pathogen. The research contents and main results are as follows:1. The changes of proteomes components of Helicobacter pylori in response to acid stress without urea.Acid stress is the most obvious challenge Helicobacter pylori （H. pylori） encounters in human stomach. The urease system is the basic process to maintain periplasmic and cytoplasmic pH near neutrality when H.pylori is exposed to acidity. However, since the urea concentration in gastric juice is approximately 1 mM, which may be insufficient to ensure the survival of H. pylori, it is postulated that additional mechanisms of pH homeostasis seem to contribute to the acid adaptation in H.pylori. To identify the acid-related proteins other than the urease system we have compared the proteome profiles of H.pylori strain 26695 exposed to different levels of external pH （7.4, 6.0, 5.0, 4.0, 3.0 and 2.0） for 30min in the absence of urea using 2-DE. Differentially expressed proteins were identified by MALDI-TOF-TOF-MS analysis, which turned out to be 36 different proteins with diverse functions, including ammonia production, molecular chaperones, energy metabolism, cell envelope, response regulator and some proteins with unknown function. SOM analysis indicated that H.pylori responds to acid stress through multi-mechanisms involving many proteins, which depend on the levels of acidity the cells encounter.2. Helicobacter pylori proteins response to human bile stress.The ability of H.pylori to tolerate bile is likely to be very important for its colonization and survival in the gastrointestinal tract of humans. Since bile can be acidified after reflux into the low pH of human stomach, we first tested the inhibitory effect of fresh normal-appearing human bile on H.pylori before and after acidification. The result showed that the acidification of bile attenuated its inhibitory activity on H.pylori. Next, the protein profiles of H.pylori under human bile and acidified bile stress were obtained by two-dimensional electrophoresis. Protein spots with differential expression were identified using tandem matrix assisted laser desorption ionzation time of flight mass spectrometry （MALDI-TOF-TOF）. The results showed that the changes of proteomic profiles under bile and acidified bile are similar when compared with the normal H.pylori. The expression of 28 proteins was found to be modulated with the majority being induced both duing bile or acidified bile exposure. These proteins include molecular chaperones, proteins involved in iron storage, chemotaxis protein, enzymes related to energy metabolism and flagellar protein. It indicates that H.pylori responds to bile and acidified bile stress through multi-mechanisms involving many signal pathways. And furthermore, to confirm the function of some proteins in H.pylori response to bile stress, we constructed the deficiency mutant of H pylori predicted coding region Hp0721 and flagellar motor switch protein and found they have lower resistant ability to bile stress. This suggests that these two proteins are likely to play a role in bile resistant response of H.pylori.3. Analysis of aztreonam-inducing proteome changes in nondividing filamentous Helicobacter pyloriWhen stressed, bacteria can enter various nondividing states, which are medically important. In the present study, nondividing filamentous form in H.pylori was induced by aztreonam, which can block cell division by inhibiting FtsI, a transpeptidase required for cross-linking of the peptidoglycan cell wall during division. Realtime-PCR results showed that FtsI has a lower expression level in filamentous H.pylori while the expression of other several penicillin-binding proteins （Hp0597, Hp1372, Hp1373 and Hp1565） did not display obvious difference when H.pylori becomes to nondividing filamentous form. In order to find other possible cell division checkpoints in H.pylori, 2-DE was used to compare the proteomic profile of nondividing filamentous H.pylori with its spiral form. Differentially expressed protein spots in 2-DE map of filamentous H.pylori were identified by MALDI-TOF-TOF analysis. These turned out to be twenty-one different proteins that are involved in various cellular processes. Out of them there is one protein, cell division inhibitor （MinD）, related to cell division was induced by aztreonam. Sequence comparison showed that MinD of H.pylori and that of E.coli share 50% identical residues. We then constructed the depletion mutant of MinD in H.pylori26695. Scanning electron microscope observation showed that the depletion of this protein provoked some bacteria change into minicell-shaped and the growth and viability of the mutant is lower than that of the wild type. This may be due to the minicells induced by depletion of MinD are almostly anucleated cells and do not give rise to viable colony forming units.更多还原