幽门螺杆菌σ~（54）调节稳定期细菌存活及硫代蒜素抗菌的机制研究
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摘要
幽门螺杆菌(Helicobacter pylori, H. pylori)是一种螺杆状、微需氧的革兰氏阴性细菌,世界一半以上的人口都有该菌感染。研究证实幽门螺杆菌感染与慢性胃炎、消化性溃疡、胃癌及胃粘膜相关淋巴组织(MALT)淋巴瘤的发生密切相关。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,幽门螺杆菌必须克服不断变化的体内或体外环境如营养缺乏,氧应激和胃内低pH值等,才能建立有效的感染并在胃内长期定植。因而,幽门螺杆菌在应激环境中存活的调节机制值得研究。
σ因子是原核细胞RNA聚合酶必需的并可解离的组成亚单位,σ因子能够在转录起始位置进行特异的启动子识别。细菌可以利用选择性σ因子引导RNA聚合酶全酶结合到特殊类别的启动子上来调控基因的表达,从而使细菌适应环境的变化。适当的选择性。因子与RNA聚合酶核心酶结合为同时调控大量的原核基因表达提供了一种有效的机制。在革兰氏阴性细菌中,σS被认为是一种普遍性的应激反应性σ因子,它可以有助于细菌在稳定期或其他的应激条件下(如渗透压休克,热应激,低pH值等)存活。然而,由于幽门螺杆菌的基因组比较小,只编码了少数几个转录调节子,仅有3个σ因子σ70(RpoD)、σ54(RpoN)、σ28(FliA),缺少革兰氏阴性菌中典型的与各种应激反应相关的σ因子σS。但是,σ54在所有致病菌中没有一个共同的作用,因为σ54依赖的基因控制着广泛的生物学过程。我们感兴趣的是幽门螺杆菌σ54在环境应激条件下对细菌的存活是否具有适应性调节的功能。
我们的研究发现幽门螺杆菌σ54的表达在营养物质逐渐耗竭、有害代谢产物积累的体外培养稳定期被显著诱导,并且rpoN突变株在稳定期后期的存活率与野生株相比显著下降。这些结果表明σ54对幽门螺杆菌逆境存活起着重要的作用,然而其机制尚不清楚。因此,我们利用比较蛋白质组学的方法分析了幽门螺杆菌野生株和rpoN突变株在稳定期早期的蛋白质表达谱的变化,旨在阐明幽门螺杆菌σ54调节细菌存活的机制。
临床资料表明根除幽门螺杆菌可以使其相关疾病得到显著缓解和恢复。临床上广泛应用的三联疗法,即质子泵抑制剂加上两种抗生素如甲硝唑、阿莫西林或克拉霉素,对幽门螺杆菌有较高的根除率。然而,根除失败也时有发生,这主要是与治疗药物具有副作用、病人依从性差、以及联合用药的费用昂贵等因素相关。更为重要的一点是幽门螺杆菌对抗生素的耐药性日益增加。耐甲硝唑、克拉霉素的幽门螺杆菌菌株已经被报道。因此,寻找一种新的、有效的抗菌制剂来克服以上的临床问题具有重要的意义。我们发现大蒜及其成分很有可能成为一种潜在的抗幽门螺杆菌药物。
大蒜作为一种抗菌药物具有一些显著的优点,首先它来源于自然食物,容易获得,价格低廉,适量摄入没有明显的副作用。而且,近代研究表明大蒜成分对抗生素耐受的细菌仍具有活性。大蒜成分与抗生素联合应用,具有部分或全部的协同作用。另外,大蒜成分还能够抑制某些细菌毒素的产生。
一些体外的研究表明大蒜成分能够有效的抑制幽门螺杆菌的生长。而且,大蒜成分与噢美拉唑联合应用具有一种浓度依赖的协同效应。许多流行病学的研究表明增加摄入葱蒜类蔬菜与胃癌的低发病率正相关,我们认为这可能与大蒜成分的抗幽门螺杆菌作用有关,因为幽门螺杆菌与胃癌的发生密切相关。
由于幽门螺杆菌的高感染率以及世界范围内幽门螺杆菌相关疾病的高发生率,我们关于硫代蒜素的抗幽门螺杆菌效应和抗菌作用机制的研究具有重要的意义。尽管以往的一些研究揭示大蒜成分的抗微生物效应主要是由于其能够与某些含巯基酶(如醇脱氢酶和硫氧环蛋白还原酶)的巯基发生化学反应。然而,这些研究尚缺乏对大蒜成分诱导的细胞内整体分子变化的分析。这就需要利用蛋白质组学的方法从全局上描绘特定条件下细胞在蛋白水平的整体性变化。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σ54调节稳定期细菌的存活及其机制
在幽门螺杆菌感染和传播过程中通常遭遇到不利环境,细菌需要快速的改变基因表达来适应逆境并存活。然而,幽门螺杆菌适应性调控机制尚不清楚。我们以幽门螺杆菌在液体培养环境中的营养物质逐渐耗竭、有害代谢产物积累的稳定期生长状况为模型,研究幽门螺杆菌的抗逆生存机制。实验结果显示幽门螺杆菌σ54的表达在稳定期显著增高,并且rpoN缺失突变株与野生株相比,在稳定期后期的生存力显著下降。以上结果提示σ54对幽门螺杆菌在逆境下存活有相关性。我们利用蛋白质组学方法鉴定出了11种蛋白的表达受σ54的正向调控,还有10种蛋白的表达受σ54的负向调控。我们的结果提示,当幽门螺杆菌逆境生存时,σ54能够通过负向调控参与能量代谢和生物合成过程基因的表达来降低对增殖的需求,同时通过正向调控参与蛋白质命运和氧化还原过程的基因的表达来增强细菌的抗应激能力和存活力,从而延长细菌在逆境中的存活时间。该研究将为幽门螺杆菌在应激条件下的适应性调控机制提供一个新的观点。二：硫代蒜素抗幽门螺杆菌的作用机制
运用常规方法进行的幽门螺杆菌根除治疗在临床上经常失败,因而,寻找一种新的有效的治疗药物势在必行。硫代蒜素是大蒜的一种衍生物,在我国已经成功的被用于治疗系统真菌感染和细菌感染。我们的研究显示硫代蒜素对幽门螺杆菌的生长具有浓度依赖的抑制作用。然而,硫代蒜素的抗菌作用机制至今尚不明了。因此,我们运用了蛋白质组学的方法来分析硫代蒜素所引起的幽门螺杆菌整体蛋白的变化,发现硫代蒜素处理后共有21个蛋白质斑点呈现差异性表达。我们的结果表明硫代蒜素在幽门螺杆菌中的抑菌机制可以归因于硫代蒜素对能量代谢及生物合成(包括氨基酸生物合成、蛋白合成、mRNA合成和脂肪酸合成)过程中的许多靶点具有抑制效应。而且硫代蒜素还能扰乱抗氧化蛋白的表达,以及减少幽门螺杆菌毒力因子的产生。蛋白质印迹结果表明亚抑菌浓度的硫代蒜素能够有效的抑制幽门螺杆菌中两个重要的毒力蛋白CagA和VacA的表达。我们关于硫代蒜素抗菌作用机制的研究,为硫代蒜素作为一种治疗制剂来对抗幽门螺杆菌感染的潜在应用提供了理论基础。
Helicobacter pylori is a spiral-shaped, microaerophilic, Gram-negative bacterium that infects the stomachs of more than 50% of the world's population. It has been demonstrated that Helicobacter pylori infection is strongly associated with some gastrointestinal diseases, such as gastritis, peptic ulcers, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Epidemiological studies show that fecal-oral route is thought to be the primary manner of transmission of H. pylori. In order to establish an infection and to persist in the stomach, H. pylori must overcome the constantly changing environments such as nutrient deficiency, oxygen tension, and low pH in the stomach and so on. The regulatory mechanisms of H. pylori to allow bacterial survival under these environmental stresses are of interest.
The sigma factor is an essential dissociable subunit of prokaryotic RNA polymerase that confers promoter recognition specificity on RNA polymerase in the initiation of transcription. Bacteria can use alternative sigma factors, which direct the RNA polymerase holoenzyme to a specific class of promoters, to adapt to environmental changes. The association of appropriate alternative sigma factors with core RNA polymerase provides an effective mechanism for simultaneously regulating large numbers of prokaryotic genes. In gram-negative bacteria, RpoS (σS) has been recognized as a general stress responsive sigma factor that contributes to survival in stationary-phase and other stress conditions such as osmotic shock, heat, and low pH. However, as the small genome of H. pylori, relatively few transcriptional regulators for gene expression have been annotated, including just three sigma factorsσ70 (RpoD), a54 (RpoN), and a28 (FliA), whereasσs which is typically associated with various stress responses in many gram-negative bacteria is lacked in H. pylori. However, a54 does not share a common role among all pathogens, as a54 dependent genes described to date control a wide diversity of processes. We are interested in whether a54 of H. pylori could regulate bacterial survival when H. pylori encounters environmental stresses.
In the present study, we found that the expression of a54 is induced when H. pylori enters into stationary phase, in which stage the nutrient becomes exhausted and harmful substance is accumulated. Additonally, the rpoN mutant showed a marked decrease in viability in late stationary phase compared with wild-type H.pylori. These results indicatedσ54 played an important role when H.pylori is subjected to unfavorable situations. However, the mechnism by which a54 regulate H. pylori survival in starved environment remains unclear. Thus proteomic analysis was used to compare the protein expression profiles of the H. pylori 26695 and its rpoN null mutant in early stationary phase.
Many clinical evidences show that eradication of H. pylori results in significant remission from these diseases. Widely used triple therapy, consisting of a proton pump inhibitor and two antibiotics such as metronidazole, amoxicillin, or clarithromycin, yields a high eradication rate. However, eradication failure often occurs, which is associated with undesirable side effects of these drugs, poor patient compliance and high cost of combination therapy. An additional reason that should be emphasized is the increasing resistance of H. pylori to antibiotics. For example, strains of H. pylori resistant to metronidazole and clarithromycin have been reported. Thus, it becomes highly necessary to search for an efficacious antibacterial agent to overcome the above clinical problems. Garlic probably has the potential to fulfill these requirements.
Garlic, a natural food in diet, has some extraordinary advantages as an antibacterial agent, including easy accessibility, low cost and negligible side effects with moderate consumption. Garlic is even active against antibiotic-resistant organisms. Garlic extracts in combination with antibiotics can lead to total or partial synergism. Garlic can also suppress toxin production by bacteria.
It has been shown that garlic constituents can inhibit the growth of H. pylori in vitro. Moreover, garlic in combination with omeprazole showed a synergic effect in vitro in a concentration-dependent manner. Several epidemiological studies suggested that a decreased risk of gastric cancer is associated with an increasing consumption of allium vegetables, possibly due to an effect on H. pylori, as this organism is associated with gastric cancer.
Thus, it is very important to study the antibacterial mode of action of garlic constituents because of the high incidences of H. pylori-related diseases throughout the world. Although previous studies have revealed that the antimicrobial effect of garlic is mainly due to its chemical reaction with thiol groups of various enzymes, such as alcohol dehydrogenase and thioredoxin reductase, a detailed analysis is lacking of the global molecular responses induced by garlic and its derivatives, and a proteomic strategy is required to globally profile the cellular responses at the protein level under defined conditions.
The contents of my research and main results are as follows:
1. The regulation ofσ54 on Helicobacter pylori survival of stationary phase
During the process of infection and transmission, Helicobacter pylori should be confronted with unfavorable enviroments and the bacterium requires rapid alterations in gene expression to allow itself survival under stress conditions. However, the mechanism underlining this regulation remains unknown. We set the stationary phase as a model to study the survival regulation of H. pylori in response to adverse enviroments. Our results that the expression ofσ54 is significantly induced in stationary phase and the rpoN mutant showed a significantly lower viability than the wild-type H. pylori in late stationary phase, suggesting that a54 is involved in H. pylori survival under adverse enviroments. Proteomic analysis identified 11 proteins were positively regulated and 10 proteins were negatively regulated by a54 in stationary phase. Our data revealed that when H. pylori enters into stationary phase,σ54 can decrease the need for proliferation through negatively regulating the genes involved in energy metabolism and biosynthesis, and enhance stress-resistant ability through positively regulating the genes involved in protein fate and redox reaction, which would result in prolonged bacterial survival under adverse environment. Our investigations will shed new light on the adaptive regulation of H. pylori under stress conditions.
2. The mode of action of allitridi against Helicobacter pylori Eradication of Helicobacter pylori with traditional therapy often fails in clinical treatment. As a result, a novel efficacious therapeutic agent is strongly needed. Allitridi, a proprietary garlic derivative, has been successfully used to treat both systemic fungal and bacterial infections in China. Our previous study has shown a dose-dependent inhibitory effect of allitridi on H. pylori growth. However, the antibacterial mode of action of allitridi is still unclear. Proteomic analysis was used to study the global protein alterations induced by allitridi. A total of 21 protein spots were identified to be differentially expressed. Our results indicated that the bacteriostatic mechanism of allitridi in H. pylori can be attributed to its multitarget inhibitory effects in energy metabolism and biosynthesis including amino acid biosynthesis, protein synthesis, mRNA synthesis and fatty acid biosynthesis. Allitridi can also disturb the expression of antioxidant proteins and decrease the production of virulence factors. Western blot analysis showed that allitridi at subinhibitory concentrations can potently suppress the production of CagA and VacA. Our investigations on the antibacterial mode of action of allitridi provide an insight into the potential use of allitridi as a therapeutic agent against H. pylori infection.
σ因子是原核细胞RNA聚合酶必需的并可解离的组成亚单位,σ因子能够在转录起始位置进行特异的启动子识别。细菌可以利用选择性σ因子引导RNA聚合酶全酶结合到特殊类别的启动子上来调控基因的表达,从而使细菌适应环境的变化。适当的选择性。因子与RNA聚合酶核心酶结合为同时调控大量的原核基因表达提供了一种有效的机制。在革兰氏阴性细菌中,σS被认为是一种普遍性的应激反应性σ因子,它可以有助于细菌在稳定期或其他的应激条件下(如渗透压休克,热应激,低pH值等)存活。然而,由于幽门螺杆菌的基因组比较小,只编码了少数几个转录调节子,仅有3个σ因子σ70(RpoD)、σ54(RpoN)、σ28(FliA),缺少革兰氏阴性菌中典型的与各种应激反应相关的σ因子σS。但是,σ54在所有致病菌中没有一个共同的作用,因为σ54依赖的基因控制着广泛的生物学过程。我们感兴趣的是幽门螺杆菌σ54在环境应激条件下对细菌的存活是否具有适应性调节的功能。
我们的研究发现幽门螺杆菌σ54的表达在营养物质逐渐耗竭、有害代谢产物积累的体外培养稳定期被显著诱导,并且rpoN突变株在稳定期后期的存活率与野生株相比显著下降。这些结果表明σ54对幽门螺杆菌逆境存活起着重要的作用,然而其机制尚不清楚。因此,我们利用比较蛋白质组学的方法分析了幽门螺杆菌野生株和rpoN突变株在稳定期早期的蛋白质表达谱的变化,旨在阐明幽门螺杆菌σ54调节细菌存活的机制。
临床资料表明根除幽门螺杆菌可以使其相关疾病得到显著缓解和恢复。临床上广泛应用的三联疗法,即质子泵抑制剂加上两种抗生素如甲硝唑、阿莫西林或克拉霉素,对幽门螺杆菌有较高的根除率。然而,根除失败也时有发生,这主要是与治疗药物具有副作用、病人依从性差、以及联合用药的费用昂贵等因素相关。更为重要的一点是幽门螺杆菌对抗生素的耐药性日益增加。耐甲硝唑、克拉霉素的幽门螺杆菌菌株已经被报道。因此,寻找一种新的、有效的抗菌制剂来克服以上的临床问题具有重要的意义。我们发现大蒜及其成分很有可能成为一种潜在的抗幽门螺杆菌药物。
大蒜作为一种抗菌药物具有一些显著的优点,首先它来源于自然食物,容易获得,价格低廉,适量摄入没有明显的副作用。而且,近代研究表明大蒜成分对抗生素耐受的细菌仍具有活性。大蒜成分与抗生素联合应用,具有部分或全部的协同作用。另外,大蒜成分还能够抑制某些细菌毒素的产生。
一些体外的研究表明大蒜成分能够有效的抑制幽门螺杆菌的生长。而且,大蒜成分与噢美拉唑联合应用具有一种浓度依赖的协同效应。许多流行病学的研究表明增加摄入葱蒜类蔬菜与胃癌的低发病率正相关,我们认为这可能与大蒜成分的抗幽门螺杆菌作用有关,因为幽门螺杆菌与胃癌的发生密切相关。
由于幽门螺杆菌的高感染率以及世界范围内幽门螺杆菌相关疾病的高发生率,我们关于硫代蒜素的抗幽门螺杆菌效应和抗菌作用机制的研究具有重要的意义。尽管以往的一些研究揭示大蒜成分的抗微生物效应主要是由于其能够与某些含巯基酶(如醇脱氢酶和硫氧环蛋白还原酶)的巯基发生化学反应。然而,这些研究尚缺乏对大蒜成分诱导的细胞内整体分子变化的分析。这就需要利用蛋白质组学的方法从全局上描绘特定条件下细胞在蛋白水平的整体性变化。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σ54调节稳定期细菌的存活及其机制
在幽门螺杆菌感染和传播过程中通常遭遇到不利环境,细菌需要快速的改变基因表达来适应逆境并存活。然而,幽门螺杆菌适应性调控机制尚不清楚。我们以幽门螺杆菌在液体培养环境中的营养物质逐渐耗竭、有害代谢产物积累的稳定期生长状况为模型,研究幽门螺杆菌的抗逆生存机制。实验结果显示幽门螺杆菌σ54的表达在稳定期显著增高,并且rpoN缺失突变株与野生株相比,在稳定期后期的生存力显著下降。以上结果提示σ54对幽门螺杆菌在逆境下存活有相关性。我们利用蛋白质组学方法鉴定出了11种蛋白的表达受σ54的正向调控,还有10种蛋白的表达受σ54的负向调控。我们的结果提示,当幽门螺杆菌逆境生存时,σ54能够通过负向调控参与能量代谢和生物合成过程基因的表达来降低对增殖的需求,同时通过正向调控参与蛋白质命运和氧化还原过程的基因的表达来增强细菌的抗应激能力和存活力,从而延长细菌在逆境中的存活时间。该研究将为幽门螺杆菌在应激条件下的适应性调控机制提供一个新的观点。二：硫代蒜素抗幽门螺杆菌的作用机制
运用常规方法进行的幽门螺杆菌根除治疗在临床上经常失败,因而,寻找一种新的有效的治疗药物势在必行。硫代蒜素是大蒜的一种衍生物,在我国已经成功的被用于治疗系统真菌感染和细菌感染。我们的研究显示硫代蒜素对幽门螺杆菌的生长具有浓度依赖的抑制作用。然而,硫代蒜素的抗菌作用机制至今尚不明了。因此,我们运用了蛋白质组学的方法来分析硫代蒜素所引起的幽门螺杆菌整体蛋白的变化,发现硫代蒜素处理后共有21个蛋白质斑点呈现差异性表达。我们的结果表明硫代蒜素在幽门螺杆菌中的抑菌机制可以归因于硫代蒜素对能量代谢及生物合成(包括氨基酸生物合成、蛋白合成、mRNA合成和脂肪酸合成)过程中的许多靶点具有抑制效应。而且硫代蒜素还能扰乱抗氧化蛋白的表达,以及减少幽门螺杆菌毒力因子的产生。蛋白质印迹结果表明亚抑菌浓度的硫代蒜素能够有效的抑制幽门螺杆菌中两个重要的毒力蛋白CagA和VacA的表达。我们关于硫代蒜素抗菌作用机制的研究,为硫代蒜素作为一种治疗制剂来对抗幽门螺杆菌感染的潜在应用提供了理论基础。
Helicobacter pylori is a spiral-shaped, microaerophilic, Gram-negative bacterium that infects the stomachs of more than 50% of the world's population. It has been demonstrated that Helicobacter pylori infection is strongly associated with some gastrointestinal diseases, such as gastritis, peptic ulcers, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Epidemiological studies show that fecal-oral route is thought to be the primary manner of transmission of H. pylori. In order to establish an infection and to persist in the stomach, H. pylori must overcome the constantly changing environments such as nutrient deficiency, oxygen tension, and low pH in the stomach and so on. The regulatory mechanisms of H. pylori to allow bacterial survival under these environmental stresses are of interest.
The sigma factor is an essential dissociable subunit of prokaryotic RNA polymerase that confers promoter recognition specificity on RNA polymerase in the initiation of transcription. Bacteria can use alternative sigma factors, which direct the RNA polymerase holoenzyme to a specific class of promoters, to adapt to environmental changes. The association of appropriate alternative sigma factors with core RNA polymerase provides an effective mechanism for simultaneously regulating large numbers of prokaryotic genes. In gram-negative bacteria, RpoS (σS) has been recognized as a general stress responsive sigma factor that contributes to survival in stationary-phase and other stress conditions such as osmotic shock, heat, and low pH. However, as the small genome of H. pylori, relatively few transcriptional regulators for gene expression have been annotated, including just three sigma factorsσ70 (RpoD), a54 (RpoN), and a28 (FliA), whereasσs which is typically associated with various stress responses in many gram-negative bacteria is lacked in H. pylori. However, a54 does not share a common role among all pathogens, as a54 dependent genes described to date control a wide diversity of processes. We are interested in whether a54 of H. pylori could regulate bacterial survival when H. pylori encounters environmental stresses.
In the present study, we found that the expression of a54 is induced when H. pylori enters into stationary phase, in which stage the nutrient becomes exhausted and harmful substance is accumulated. Additonally, the rpoN mutant showed a marked decrease in viability in late stationary phase compared with wild-type H.pylori. These results indicatedσ54 played an important role when H.pylori is subjected to unfavorable situations. However, the mechnism by which a54 regulate H. pylori survival in starved environment remains unclear. Thus proteomic analysis was used to compare the protein expression profiles of the H. pylori 26695 and its rpoN null mutant in early stationary phase.
Many clinical evidences show that eradication of H. pylori results in significant remission from these diseases. Widely used triple therapy, consisting of a proton pump inhibitor and two antibiotics such as metronidazole, amoxicillin, or clarithromycin, yields a high eradication rate. However, eradication failure often occurs, which is associated with undesirable side effects of these drugs, poor patient compliance and high cost of combination therapy. An additional reason that should be emphasized is the increasing resistance of H. pylori to antibiotics. For example, strains of H. pylori resistant to metronidazole and clarithromycin have been reported. Thus, it becomes highly necessary to search for an efficacious antibacterial agent to overcome the above clinical problems. Garlic probably has the potential to fulfill these requirements.
Garlic, a natural food in diet, has some extraordinary advantages as an antibacterial agent, including easy accessibility, low cost and negligible side effects with moderate consumption. Garlic is even active against antibiotic-resistant organisms. Garlic extracts in combination with antibiotics can lead to total or partial synergism. Garlic can also suppress toxin production by bacteria.
It has been shown that garlic constituents can inhibit the growth of H. pylori in vitro. Moreover, garlic in combination with omeprazole showed a synergic effect in vitro in a concentration-dependent manner. Several epidemiological studies suggested that a decreased risk of gastric cancer is associated with an increasing consumption of allium vegetables, possibly due to an effect on H. pylori, as this organism is associated with gastric cancer.
Thus, it is very important to study the antibacterial mode of action of garlic constituents because of the high incidences of H. pylori-related diseases throughout the world. Although previous studies have revealed that the antimicrobial effect of garlic is mainly due to its chemical reaction with thiol groups of various enzymes, such as alcohol dehydrogenase and thioredoxin reductase, a detailed analysis is lacking of the global molecular responses induced by garlic and its derivatives, and a proteomic strategy is required to globally profile the cellular responses at the protein level under defined conditions.
The contents of my research and main results are as follows:
1. The regulation ofσ54 on Helicobacter pylori survival of stationary phase
During the process of infection and transmission, Helicobacter pylori should be confronted with unfavorable enviroments and the bacterium requires rapid alterations in gene expression to allow itself survival under stress conditions. However, the mechanism underlining this regulation remains unknown. We set the stationary phase as a model to study the survival regulation of H. pylori in response to adverse enviroments. Our results that the expression ofσ54 is significantly induced in stationary phase and the rpoN mutant showed a significantly lower viability than the wild-type H. pylori in late stationary phase, suggesting that a54 is involved in H. pylori survival under adverse enviroments. Proteomic analysis identified 11 proteins were positively regulated and 10 proteins were negatively regulated by a54 in stationary phase. Our data revealed that when H. pylori enters into stationary phase,σ54 can decrease the need for proliferation through negatively regulating the genes involved in energy metabolism and biosynthesis, and enhance stress-resistant ability through positively regulating the genes involved in protein fate and redox reaction, which would result in prolonged bacterial survival under adverse environment. Our investigations will shed new light on the adaptive regulation of H. pylori under stress conditions.
2. The mode of action of allitridi against Helicobacter pylori Eradication of Helicobacter pylori with traditional therapy often fails in clinical treatment. As a result, a novel efficacious therapeutic agent is strongly needed. Allitridi, a proprietary garlic derivative, has been successfully used to treat both systemic fungal and bacterial infections in China. Our previous study has shown a dose-dependent inhibitory effect of allitridi on H. pylori growth. However, the antibacterial mode of action of allitridi is still unclear. Proteomic analysis was used to study the global protein alterations induced by allitridi. A total of 21 protein spots were identified to be differentially expressed. Our results indicated that the bacteriostatic mechanism of allitridi in H. pylori can be attributed to its multitarget inhibitory effects in energy metabolism and biosynthesis including amino acid biosynthesis, protein synthesis, mRNA synthesis and fatty acid biosynthesis. Allitridi can also disturb the expression of antioxidant proteins and decrease the production of virulence factors. Western blot analysis showed that allitridi at subinhibitory concentrations can potently suppress the production of CagA and VacA. Our investigations on the antibacterial mode of action of allitridi provide an insight into the potential use of allitridi as a therapeutic agent against H. pylori infection.
引文
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mRNACagA、VacA、 NapA
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2. Magalhaes Queiroz DM & Luzza F (2006) Epidemiology of Helicobacter pylori infection. Helicobacter 11:1-5.
3. Delport W & van der Merwe SW (2007) The transmission of Helicobacter pylori: the effects of analysis method and study population on inference. Best Pract Res Clin Gastroenterol 21:215-236.
4. Jishage M, Iwata A, Ueda S & Ishihama A (1996) Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli:intracellular levels of four species of sigma subunit under various growth conditions. J Bacteriol 178:5447-5451.
5. Gross CA, Chan C, Dombroski A, Gruber T, Sharp M, Tupy J & Young B (1998) The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb Symp Quant Biol 63:141-155.
6. Kazmierczak MJ, Wiedmann M & Boor KJ (2005) Alternative sigma factors and their roles in bacterial virulence. Microbiol Mol Biol Rev 69:527-543.
7. Tomb JF, White O & Kerlavage AR et al. (1997) The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547.
8. Beier D, Spohn G, Rappuoli R & Scarlato V (1998) Functional analysis of the Helicobacter pylori principal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription. Mol Microbiol 30:121-134.
9. Shirai M, Fujinaga R, Akada JK & Nakazawa T (1999) Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H. pylori rpoD gene in E. coli. Gene 239:351-359.
10. Helmann JD (1991) Alternative sigma factors and the regulation of flagellar gene expression. Mol Microbiol 5:2875-2882.
11. Ishimoto KS & Lory S (1989) Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci USA 86:1954-1957.
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