空肠弯曲菌基因组序列及相关致病基因的研究

空肠弯曲菌是1种微需氧的革兰阴性鞭毛螺旋菌,其NCTC11168基因组序列分析显示为1个超变量序列.该菌基因组序列的高变异,尤其是编码膜表面抗原性蛋白的基因序列的高变异可能与其逃逸宿主免疫系统的清除和感染后引起不同疾病有关.结合空肠弯曲菌感染后常见临床表现,本文从基因组水平对该菌趋化性、运动性、黏附力、侵袭力和定植力,以及细胞毒素产生与其感染后诱发格林-巴利综合征等致病作用进行综述.     

空肠弯曲菌fliG基因敲除突变株动力、体外趋化和小鼠定植能力的改变

空肠弯曲菌( Campylobacter jejuni) 是人类细菌性胃肠炎的病原体。趋化是细菌向合适的寄生部位定向运动, 也是空肠弯曲菌在宿主空肠黏膜表面实现定植的关键起始步骤, 该趋化运动由趋化相关二元信号系统( che-TCS) 以MCPs→Ches→Flis/Mots 方式调控。FliG是Fli 蛋白家族成员, 一些病原菌的FliG被证明是鞭毛马达蛋白, 也是细菌鞭毛马达中开关复合体的必需组分, 但空肠弯曲菌FliG在细菌趋化中的作用未明。本研究中, 我们根据同源重组原理, 构建空肠弯曲菌NCTC11168 株fliG基因敲除( fliG^- ) 突变株, 然后对fliG^- 突变株的动力、趋化和定植能力进行测定。动力实验和体外趋化实验结果证实, fliG^- 突变株在半固体琼脂平板上的菌落直径、在硬琼脂平板上对0. 2 mol/L 脱氧胆酸钠( SDC) 的趋化聚集环直径均明显小于野生株( P <0. 05) 。与野生株比较, 黏附于BALB/c-ByJ小鼠空肠黏膜表面以及空肠内容物中的fliG^- 突变株数量也明显减少( P <0. 05) 。实验结果表明, fliG是空肠弯曲菌鞭毛动力以及细菌感染时向空肠黏膜趋化运动的必需基因。     

单增李斯特菌二硫键形成蛋白DsbG介导酸耐受及鞭毛运动性调控北大核心

【目的】本研究旨在通过构建单增李斯特菌(Listeria monocytogenes)二硫键形成蛋白编码基因dsbG缺失株和回补株,探究该蛋白在酸耐受和鞭毛介导的运动性中发挥的生物学功能。【方法】利用同源重组方法构建单增李斯特菌dsbG缺失株和回补株,比较野生株和突变株在不同pH梯度培养基条件下的生长速率和存活率;通过实时荧光定量PCR方法,比较致死酸应激条件下野生株和缺失株酸耐受基因转录水平。通过半固体培养基、荧光定量PCR方法和鞭毛负染色透射电镜观察,比较野生株和突变株的运动能力、鞭毛相关基因转录水平变化和鞭毛形态差异。【结果】与野生株相比,dsbG缺失株的生长能力和速率差异不显著;在pH 3.5(盐酸和柠檬酸)培养条件下,存活率显著降低;精氨酸合成途径基因argD和argF转录水平分别下调2.4和3.7倍。同时,dsbG缺失株的运动能力减弱,且鞭毛形成相关的flaA、flgB和flgD等基因转录水平显著下调(分别为29.7、6.7和6.9倍),鞭毛形成能力减弱。【结论】本研究首次证实了单增李斯特菌二硫键形成蛋白DsbG能感应低pH应激,并形成耐受;证实了DsbG通过调控鞭毛相关基因的转录进而影响细菌的鞭毛形成和运动性。本研究有助于深入了解二硫键形成蛋白家族介导单增李斯特菌环境适应的分子机制,为食源性致病菌的防控提供理论基础。     

空肠弯曲菌生长条件的研究

在国内外,空肠弯曲菌对人的致病性已被反复证明。从腹泻病人已大量分离出该菌。但是由于空肠弯曲菌是一种微嗜氧菌,培养需要特殊条件。因此基层开展培养工作有一定困难。为研制更适合基层使用的方法,对弯曲菌的有关生长因素作进一步探讨是非常必要的,以对该菌特性有进一步的深入了解,从而以便提供研制空肠弯曲菌无血液分离培养基创造     

体外趋化和小鼠定植能力的改变

空肠弯曲菌( Campylobacter jejuni) 是人类细菌性胃肠炎的病原体。趋化是细菌向合适的寄生部位定向运动, 也是空肠弯曲菌在宿主空肠黏膜表面实现定植的关键起始步骤, 该趋化运动由趋化相关二元信号系统( che-TCS) 以MCPs→Ches→Flis/Mots 方式调控。FliG是Fli 蛋白家族成员, 一些病原菌的FliG被证明是鞭毛马达蛋白, 也是细菌鞭毛马达中开关复合体的必需组分, 但空肠弯曲菌FliG在细菌趋化中的作用未明。本研究中, 我们根据同源重组原理, 构建空肠弯曲菌NCTC11168 株fliG基因敲除( fliG^- ) 突变株, 然后对fliG^- 突变株的动力、趋化和定植能力进行测定。动力实验和体外趋化实验结果证实, fliG^- 突变株在半固体琼脂平板上的菌落直径、在硬琼脂平板上对0. 2 mol/L 脱氧胆酸钠( SDC) 的趋化聚集环直径均明显小于野生株( P <0. 05) 。与野生株比较, 黏附于BALB/c-ByJ小鼠空肠黏膜表面以及空肠内容物中的fliG^- 突变株数量也明显减少( P <0. 05) 。实验结果表明, fliG是空肠弯曲菌鞭毛动力以及细菌感染时向空肠黏膜趋化运动的必需基因。     

多位点序列分型分析空肠弯曲菌华东动物源分离株

【目的】研究空肠弯曲菌菌株间的分子特征,对不同宿主来源的空肠弯曲菌进行分子分型研究。【方法】选择空肠弯曲菌的7个看家基因gltA、aspA、glnA、glyA、pgm、tkt和uncA作为目的基因,对2006-2008年间华东地区分离的42株空肠弯曲菌样本进行PCR扩增后测序。将测序结果软件分析并上传到数据库进行比对,将结果制作多位点序列分型(multilocus sequence typing,MLST)遗传进化树并进行分析。【结果】与数据库已有类型比对,发现了24个新的ST型,通过进化树得到其遗传关系。【结论】MLST方法对于研究空肠弯曲菌的菌株群体基因差异与进化趋势具有重要意义。     

空肠弯曲菌CRISPR/Cas系统的研究进展

多数细菌都存在发挥免疫防御机制作用的CRISPR/Cas系统,在不同种属间呈现多态性。空肠弯曲菌是全球范围内重要的食源性病原菌,所致疾病也是典型的自限性疾病,其复杂的致病机制并未得到明确的解析,而空肠弯曲菌CRISPR/Cas系统的结构呈现多态性,研究两者关系仍存在诸多限制。本文从CRISPR/Cas系统在空肠弯曲菌中的结构、机制及技术应用等方面的研究进展进行综述,为探索空肠弯曲菌致病机制提供新思路。     

具有拮抗空肠弯曲杆菌能力鸡源乳酸菌的筛选及特性

【目的】从鸡粪中筛选具有拮抗空肠弯曲杆菌能力的乳酸菌,研究其肠道益生特性,探讨其对空肠弯曲杆菌鞭毛毒力因子的影响。【方法】利用牛津杯法测定40株鸡粪源乳酸菌菌株的抑菌活性以确定抑菌性能好的菌株,利用16S r RNA基因分析进行菌株鉴定,采用HT-29细胞测定菌株的细胞粘附能力,通过模拟胃肠液实验分析菌株对胃肠道环境的耐受性,利用扫描电镜分析乳酸菌无细胞提取物对空肠弯曲杆菌鞭毛毒力因子的影响。【结果】从鸡粪中分离得到40株菌株,进一步筛选得到X13、X14和G20等3株拮抗空肠弯曲杆菌能力较强的菌株,经16S r RNA基因序列分析分别鉴定为罗伊氏乳杆菌、唾液乳杆菌和鸡乳杆菌;HT-29细胞粘附实验表明X13、X14及G20的粘附指数分别为11.5、20.3和14.3个/细胞,均具有良好的粘附能力;3株乳酸菌对人工胃肠液均具有良好的耐受性;扫描电镜观察表明,与对照组相比,3株纯培养乳酸菌无细胞提取物均能抑制空肠弯曲杆菌鞭毛毒力因子的合成。【结论】从鸡粪中筛选得到了3株能有效抑制空肠弯曲杆菌生长并能抑制其鞭毛合成的乳酸菌,有望作为拮抗性饲用益生菌用于控制禽畜的空肠弯曲杆菌感染。     

基于全基因组序列的弯曲菌特征分析

空肠弯曲菌(Campylobacter jejuni)和结肠弯曲菌(Campylobacter coli)是引起人类腹泻的主要致病菌。传统生化方法在鉴定弯曲菌时存在步骤多、耗时长、通量低等问题。本研究通过利用生物信息学方法对弯曲菌全基因组进行序列、基因注释、耐药基因、多位点序列分型以及CRISPR-Cas系统等分析,挖掘能够有效区分空肠弯曲菌和结肠弯曲菌的高分辨力特征。实验结果表明,空肠弯曲菌和结肠弯曲菌在基因组序列长度、GC含量、基因数量、多位点序列分型以及CRISPR-Cas系统等方面存在显著差异。同时,研究还发现了一段在空肠弯曲菌基因组中广泛存在的高分辨力CRISPR重复序列。这些特征可用于构建能够准确鉴别空肠弯曲菌和结肠弯曲菌的生物信息学方法。     

空肠弯曲菌Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(TypeⅥSecretion System,T6SS)是一种倒置于细胞膜上的类噬菌体样结构,能够输送效应蛋白并在定殖和生态位建立中发挥作用。近年来,在空肠弯曲菌(Campylobacter jejuni)中发现了T6SS同源基因且能够表达组装成结构完整的T6SS,但T6SS对空肠弯曲菌的毒力影响尚不清楚。本文就空肠弯曲菌T6SS结构组成、分布特征及效应功能等方面的研究进展进行综述,以期为进一步解析空肠弯曲菌毒力因子的调控机制提供新思路。     

Role of Oxidative Stress in <Emphasis Type="Italic">C. jejuni</Emphasis> Inactivation During Freeze-Thaw Treatment

Campylobacter jejuni represents one of the leading causes of bacterial enteritis throughout the world. Poultry is an important source of C. jejuni. Despite hygiene measures taken in the production chain, C. jejuni is frequently isolated from poultry meat. C. jejuni is a microaerophilic pathogen, affected by oxidative stress. Freeze-thaw treatment induces cell death by several mechanisms, including oxidative stress. In this article, we investigate the role of oxidative stress in C. jejuni sensitivity during and after a freeze-thaw treatment. This treatment results in dead and sublethally injured cells. The latter population might have an increased sensitivity to oxidative stress. To test this, cells were stored for another 24 h at 4°C under aerobic conditions and compared to cells that were not treated. C. jejuni survival was measured in different media (water, BHI broth, chicken juice, and chicken fillets) to test the environment protective effect. Different strains were tested, including sodB (encoding the superoxide dismutase) and cj1371 (encoding a periplasmic protein) mutants. Cell death was particularly important in water but similar in BHI, chicken juice, and chicken fillets. The sodB mutant was more sensitive to freeze-thaw treatment, suggesting that the killing mechanism involves production of superoxide anions. On the contrary, the cj1371 mutant was more sensitive to storage at 4°C, suggesting that it does not play a role in the detoxification of reactive oxygen species. Storage at 4°C after freeze-thaw treatment increases cell death of oxidative stress-sensitive populations. Sensitization to oxidative stress, freeze-thaw treatment, and further storage at 4°C could be a way to reduce C. jejuni populations on carcasses.     

Evaluation of Cytokine Production and Phagocytic Activity in Mice Infected with Campylobacter jejuni

The effect of several Campylobacter jejuni strains on the immune response was analyzed in mice after intraperitoneal inoculation with 10¹⁰ colony forming units (CFU). Three C. jejuni strains were assayed: CCUG 6968 (enterotoxigenic), CCUG 7580 (enterotoxigenic), and CCUG 7440 (non-enterotoxigenic). These C. jejuni strains induced a peritoneal inflammatory response and an important increase in the peritoneal phagocyte oxidative activity measured by chemiluminescence assay, as well as an increase in the number of peritoneal cells. Both interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) production by peritoneal cells were not modified. However, C. jejuni 7440 caused a statistically significant increase in TNFα production. These results have demonstrated that different strains of C. jejuni induce an increase of the inflammatory response without a significant cytokine release. However, these infectious microorganisms may be eliminated efficiently by murine macrophages after phagocytosis. Received: 18 February 1999 / Accepted: 6 May 1999     

Survival of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> Strains from Different Origins Under Oxidative Stress Conditions: Effect of Temperature

Campylobacter jejuni is a microaerophilic pathogen but is able to survive oxidative stress conditions during its transmission to the human host. Strains of different origins (reference, poultry, or human clinical) were tested for survival under oxidative stress conditions. C. jejuni strains were grown in Mueller Hinton broth to obtain late exponential–phase cultures. Then they were exposed to 2 different stresses: (1) cultures were either plated on Columbia agar plates and exposed to atmospheric oxygen or (2) paraquat (a chemical oxidizing agent) was added to liquid cultures to reach a 500-μM concentration. Both of these experimental conditions were realized at 3 different temperatures: 4°C, 25°C, and 42°C. Results obtained with paraquat and atmospheric oxygen were similar. Surprisingly, C. jejuni was found to be very sensitive to oxidative stress at 42°C, which is its optimal growth temperature, whereas it was more resistant at 4°C. A strain effect was observed, but no relationship was found between the origin of the strains and level of resistance. High temperature (42°C) combined with oxidative stress allowed a rapid decrease in the C. jejuni population, whereas low temperature considerably decreased the effect of oxidative stress.     

Pyruvate relieves the necessity of high induction levels of catalase and enables Campylobacter jejuni to grow under fully aerobic conditions

Aims:  Several cases of campylobacteriosis reported worldwide seemingly conflict with the strict growth requirements and sensitivity to environmental stress of Campylobacter jejuni. In this study, the need for a micro‐aerobic environment [dissolved oxygen tension (DOT): 0·1–90%; 100% air saturation)] and the adaptive responses to oxygen stress were studied. Methods and Results:  The growth of C. jejuni in continuous culture was assessed under different DOT in the presence or absence of pyruvate. In a medium without pyruvate, continuous cultures of C. jejuni showed typically micro‐aerobic behaviour and cells were unable to grow under fully aerobic conditions. However in the presence of pyruvate (25 mmol l^?1), continuous cultures of C. jejuni were able to grow in a broad DOT range, varying from 0·1% to at least 90%, and the catalase activity was decreased. Conclusions:  Addition of pyruvate results in the decrease in the concentration of hydrogen peroxide, which enables C. jejuni to grow aerobically. Significance and Impact of the Study:  New information on the oxidative physiology of C. jejuni and its ability to grow aerobically in media supplemented with pyruvate is presented.     

The ALPK1 pathway drives the inflammatory response to Campylobacter jejuni in human intestinal epithelial cells

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.     

Amino‐terminal residues dictate the export efficiency of the Campylobacter jejuni filament proteins via the flagellum

Bacterial flagella play an essential role in the pathogenesis of numerous enteric pathogens. The flagellum is required for motility, colonization, and in some instances, for the secretion of effector proteins. In contrast to the intensively studied flagella of Escherichia coli and Salmonella typhimurium, the flagella of Campylobacter jejuni, Helicobacter pylori and Vibrio cholerae are less well characterized and composed of multiple flagellin subunits. This study was performed to gain a better understanding of flagellin export from the flagellar type III secretion apparatus of C. jejuni. The flagellar filament of C. jejuni is comprised of two flagellins termed FlaA and FlaB. We demonstrate that the amino‐termini of FlaA and FlaB determine the length of the flagellum and motility of C. jejuni. We also demonstrate that protein‐specific residues in the amino‐terminus of FlaA and FlaB dictate export efficiency from the flagellar type III secretion system (T3SS) of Yersinia enterocolitica. These findings demonstrate that key residues within the amino‐termini of two nearly identical proteins influence protein export efficiency, and that the mechanism governing the efficiency of protein export is conserved among two pathogens belonging to distinct bacterial classes. These findings are of additional interest because C. jejuni utilizes the flagellum to export virulence proteins.     

Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

The acid adaptive tolerance response in Campylobacter jejuni induces a global response,as suggested by proteomics and microarrays

Campylobacter jejuni CI 120 is a natural isolate obtained during poultry processing and has the ability to induce an acid tolerance response (ATR) to acid + aerobic conditions in early stationary phase. Other strains tested they did not induce an ATR or they induced it in exponential phase. Campylobacter spp. do not contain the genes that encode the global stationary phase stress response mechanism. Therefore, the aim of this study was to identify genes that are involved in the C. jejuni CI 120 early stationary phase ATR, as it seems to be expressing a novel mechanism of stress tolerance. Two-dimensional gel electrophoresis was used to examine the expression profile of cytosolic proteins during the C. jejuni CI 120 adaptation to acid + aerobic stress and microarrays to determine the genes that participate in the ATR. The results indicate induction of a global response that activated a number of stress responses, including several genes encoding surface components and genes involved with iron uptake. The findings of this study provide new insights into stress tolerance of C. jejuni, contribute to a better knowledge of the physiology of this bacterium and highlight the diversity among different strains.