幽门螺杆菌cheA基因在细菌体外趋化和体内定植过程中的作用

目的 了解cheA基因在幽门螺杆菌体外趋化和体内定植中的作用.方法 从幽门螺杆菌NCTC11637株基因组DNA中扩增并克隆全长cheA和cheY基因.构建该两个基因原核表达系统,Ni-NTA亲和层析法提取目的 重组蛋白rCheA和rCheY.rCheA和rCheY免疫家兔制备抗血清,采用硫酸铵沉淀法及DEAE-52柱层析法制备rCheA-IgG和rCheY-IgG.构建cheA基因自杀质粒,根据同源重组交换原理利用该自杀质粒构建cheA基因敲除突变株(cheA-),采用PCR及测序对cheA-突变株进行鉴定.采用rcheA-IgG和rCheY-IgG锚定靶蛋白及蛋白磷酸化检测试剂盒,测定cheA-突变株与野生株CheA和CheY分子磷酸化水平.采用幽门螺杆菌趋化模型及BALB/c小鼠感染模型,比较cheA-突变株与野生株体外趋化及体内定植能力的差异.结果 PCR及测序结果证实cheA-突变株基因组中cheA基因被敲除.0.001～0.1 mol/L盐酸作用10 min,野生株CheA和CheY磷酸化水平分别从(59.6±11.5)和(55.5±10.2)μmol迅速下降至(10.8±2.6)和(5.5±1.2)μmol(P＜0.05),cheA-突变株CheY磷酸化水平均较低且无明显变化(P＞0.05).cheA-突变株对盐酸、硫酸和乙酸趋化聚集环直径[(10～20)±(2～3)mm]明显小于野生株[(16～24)±(2～3)mm],P＜0,05.野生株感染小鼠胃黏膜标本中幽门螺杆菌分离阳性率(90%)明显高于cheA-突变株(40%),P＜0.05;荧光定量PCR结果也显示野生株感染小鼠胃黏膜标本中幽门螺杆菌数量(6.3×103±2.1×103拷贝/mg)也明显高于突变株的(8.3×101±3.1 ×101拷贝/mg),P＜0.05.结论 cheA基因在幽门螺杆菌体外趋化和体内定植中有促进作用.     

幽门螺杆菌cheA和cheY基因表达及其产物与细菌趋化的相关性

目的 克隆幽门螺杆菌趋化相关基因cheA和they并构建其原核表达系统,建立幽门螺杆菌体外趋化模型并确定其趋化诱导物质,了解特异性抗体和氯氰碘柳胺对幽门螺杆菌趋化的抑制作用.方法 PCR扩增幽门螺杆菌NCTC11637株全长cheA和cheY基因片段,T-A克隆后测序.构建上述目的基因原核表达系统,采用SDS-PAGE和Bio-Rad凝胶成像分析系统检查目的重组蛋白rCheA和rCheY的表达情况,Ni-NTA亲和层析法提纯rCheA和rCheY.rCheA和rCheY免疫家兔获得抗血清,用饱和硫酸铵沉淀法和DEAE-32离子交换法提纯IsG,用免疫扩散法检测抗血清及其IsG效价.采用硬琼脂法建立幽门螺杆菌NCTC11637株体外趋化模型并检测11种物质的趋化诱导作用,同时分别观察抗rCheA IgG(rCheA-IgG)和氯氰碘柳胺钠对细菌趋化的抑制作用.结果 PCR扩增获得预期大小的cheA和cheY基因片段,其核苷酸和氨基酸序列与文献报道完全相同.所构建的原核表达系统能有效表达rCheA和rGheY.rCheA和rCheY兔抗血清及其IgG免疫扩散效价均分别为1:4和1:2.盐酸、硫酸和乙酸对该幽门螺杆菌均有趋化诱导作用,一定浓度的rCheA-IgG和氯氰碘柳胺钠均对幽门螺杆菌趋化有抑制作用(P<0.05).结论 本研究成功地构建了幽门螺杆菌NCTC11637株cheA和cheY基因原核表达系统.H~+是诱导幽门螺杆菌趋化的信号物质,rCheA-IgG和氯氰碘柳胺钠均能抑制幽门螺杆菌对H~+的趋化.     

motA基因在空肠弯曲菌致病性相关趋化和定植中的作用

目的 确定鞭毛马达蛋白(flagellar motor protein)MotA编码基因在空肠弯曲菌(Campylobacter jejuni)致病性相关趋化和定植中的作用.方法 采用PCR扩增motA基因以及用于motA基因敲除的Kan~r基因和plus-motA基因片段,目的扩增产物克隆后测序.根据同源重组原理,构建空肠弯曲菌NCTC11168株motA基因自杀质粒(pBlueskrit-Ⅱ-SK~(motA-kan))和motA基因敲除突变株(motA~-).采用半固体平板迁移试验、基于硬琼脂平板(hard agar plus,HAP)的脱氧胆酸钠(SDC)体外趋化试验、小鼠空肠定植试验,了解空肠弯曲菌motA~-突变株和野生株鞭毛动力、SDC趋化和BALB/c-ByJ小鼠空肠内定植能力差异性.结果 所克隆的空肠弯曲菌motA基因核苷酸和氨基酸序列与已报道的相应序列相似性为100%.PCR和测序及含抗生素培养基连续传代培养结果证实,自杀质粒和motA~-突变株构建成功.motA~-突变株在半固体琼脂平板上的菌斑直径、在HAP上对0.2moL/L SDC趋化聚集环直径、黏附于小鼠空肠黏膜表面以及空肠内容物中motA~-突变株数量均明显少于野生株(P<0.05).结论 本研究成功构建了空肠弯曲菌motA基因敲除突变株.motA基因是空肠弯曲菌鞭毛动力以及致病性相关趋化和定植的必需基因.     

空肠弯曲菌mcp1/2/3基因原核表达及其产物与细菌趋化作用的相关性

目的 克隆空肠弯曲菌(Campylobacter jejuni)接受甲基趋化蛋白(MCP)编码基因mcp1、mcp2和mcp3并构建其原核表达系统,建立空肠弯曲菌体外趋化模型并确定趋化诱导物质,了解MCPs与趋化诱导物之间的关系.方法 PCR扩增mcp1、mcp2和mcp3基因片段,T-A克隆后测序.构建上述目的 基因原核表达系统,SDS-PAGE和Bio-Rad凝胶成像分析系统检查目的 重组蛋白rMCP1、rMCP2和rMCP3的表达情况,Ni-NTA亲和层析法提纯rMCPs.rMCPs免疫家兔获得抗血清,免疫扩散法测其效价.用饱和硫酸铵盐析法和DEAE-32离子交换法提纯IgG,经胃蛋白酶酶解和Sephedex G-100层析制备IgGF(ab')2.建立HAP(hard-agar plus)法空肠弯曲菌体外趋化模型并检测8种物质的趋化诱导作用.采用基于IgG F(ab')2封闭的趋化阻断试验,确定不同MCPs的功能及其差异.结果 PCR扩增获得预期大小的mcp1、mcp2和mop3基因片段,其核苷酸和氨基酸序列与文献报道完全相同.所构建的原核表达系统能有效地表达各rMCPs,其产量均约为细菌总蛋白的10%.rMCP1、rMCP2和rMCP3免疫家兔后能产生特异性抗体,其免疫扩散效价均为1∶4.牛胆汁和脱氧胆酸钠(DOC)对空肠弯曲菌趋化有浓度依赖性诱导作用(P<0.05).MCP1和MCP2被其IgG F(ab')2 封闭后,空肠弯曲菌对DOC的趋化能力明显减弱(P<0.05),MCP3被封闭后对空肠弯曲菌趋化能力无影响(P>0.05).结论 本研究成功地表达了空肠弯曲菌MCPs蛋白.牛胆汁和DOC是诱导空肠弯曲菌趋化的信号物质,MCP1和MCP2可能参与该菌对DOC的趋化过程.     

二元信号系统ComD/ComE与肺炎链球菌对β-内酰胺类抗生素耐药的相关性

目的 构建肺炎链球菌comD基因敲除突变株,了解comD基因与细菌β-内酰类抗生素耐药性的相关性,探讨氯氰碘柳胺下调comD、comE和comC基因mRNA的作用.方法 构建用于comD基因敲除的自杀质粒pEVP3comD,通过同源重组及插入失活获得肺炎链球菌ATCC6306株comD基因敲除突变株comD-.采用PCR及免疫荧光法对comD-突变株进行鉴定.采用实时荧光定量RT-PCR检测氯氰碘柳胺处理前后comD-突变株及野生株comD、comE和comC基因mRNA水平变化.采用二倍琼脂稀释法测定comD-突变株及野生株对青霉素G和头孢噻肟的敏感性.结果 测序及免疫荧光试验结果证实,所构建的comD-突变株染色体DNA中comD基因被插入失活.50μmol/L或100 μmol/L的氯氰碘柳胺能明显下调comD、comE和comC基因mRNA水平(P＜0.05),25μmol/L的氯氰碘柳胺则否.comD-突变株对青霉素及头孢噻肟最低抑菌浓度(MIC)值均为32μg/ml,明显高于野生株的0.06 μg/ml和1 μg/ml.结论 本研究成功地构建了肺炎链球菌comD基因敲除突变株.comD基因与细菌对β-内酰类抗生素耐药性密切相关.氯氰碘柳胺可通过下调comD、comE和comC基因的转录水平,从而对细菌感受态形成产生影响.     

Comparison of galE gene of Campylobacter jejuni strains associated Guillain-Barré syndrome

目的 测定3株格林.巴利综合征(Guillain-Barre syndrome,GBS)相关宅肠弯曲菌的gale基因序列,并同GenBank中的空肠弯曲菌菌株相应序列进行比较,了解致GBS的序列特征并分析其遗传进化关系.方法 选取分离自GBS患者粪便并经动物模型证实为致GBS的3株AMAN型空肠弯曲菌菌株进行培养并提取基因组DNA测序.将基因测序结果通过与NCTC11168菌株进行对照比较寻找galE基因突变位点并对gaZE基因片段进行遗传距离计算.结果 3株致GBS空肠弯曲菌菌株的galE基因均由987个碱基构成.与NCTC11168的galE基因序列相比,此3株空肠弯曲菌菌株gale基因核苷酸序列有4个相同碱基突变并导致了4个对应的相同氨基酸突变.遗传距离计算,zhanxing株与qiaoyuntao株距离为1.5%,zhanxing株与lulei株距离为1.6%,qiaoyuntao株与lulei株距离为0.5%.结论 GBS相关空肠弯曲菌中galE基因核苷酸序列的确存在相同变异且发生变异概率较非GBS相关空肠弯曲菌明显增大,遗传距离反映了此3株致GBS的空肠弯曲菌具有一定的区域特征.     

甲型副伤寒沙门氏菌ompW基因功能的初步研究

目的 利用λRed重组系统敲除甲型副伤寒沙门氏菌ompW基因构建突变株,并且制备相应的回复株,进而初步研究该基因的功能.方法 PCR扩增得到上、下游同源臂,与卡那霉素抗性基因片段共同构建打靶片段,将其浓缩后转化含λRed重组系统的甲型副伤寒沙门氏菌(50973株),经PCR鉴定后得到突变株;将重组酶表达质粒pACU184与ompW基因的调控区和编码区序列连接后电转入突变株中,双酶切鉴定得到相应的回复株;SDS-PAGE及Western blot鉴定野生株、突变株与回复株中OmpW蛋白的表达情况;生化鉴定野生株、突变株与回复株,并观察野生株与突变株生长曲线的差异;测定野生株、突变株与回复株活菌半数致死量( LD50),以此来观察ompW基因与细菌毒力的相关性.结果 在甲型副伤寒沙门氏菌50973株中成功敲除了ompW基因,并构建了相应回复株;野生株与回复株均可表达出OmpW蛋白,突变株没有出现该蛋白的表达;野生株、突变株与回复株生化鉴定结果均为甲型副伤寒沙门氏菌,且野生株和突变株生长无明显差异;三者LD50均不存在显著差异.结论 甲型副伤寒沙门氏菌的ompW基因与该菌致病毒力无相关性,但突变株的构建为后续研究该基因具体功能提供了基础.     

Comparison of galE gene of Campylobacter jejuni strains associated Guillain-Barré syndrome

目的 测定3株格林.巴利综合征(Guillain-Barre syndrome,GBS)相关宅肠弯曲菌的gale基因序列,并同GenBank中的空肠弯曲菌菌株相应序列进行比较,了解致GBS的序列特征并分析其遗传进化关系.方法 选取分离自GBS患者粪便并经动物模型证实为致GBS的3株AMAN型空肠弯曲菌菌株进行培养并提取基因组DNA测序.将基因测序结果通过与NCTC11168菌株进行对照比较寻找galE基因突变位点并对gaZE基因片段进行遗传距离计算.结果 3株致GBS空肠弯曲菌菌株的galE基因均由987个碱基构成.与NCTC11168的galE基因序列相比,此3株空肠弯曲菌菌株gale基因核苷酸序列有4个相同碱基突变并导致了4个对应的相同氨基酸突变.遗传距离计算,zhanxing株与qiaoyuntao株距离为1.5%,zhanxing株与lulei株距离为1.6%,qiaoyuntao株与lulei株距离为0.5%.结论 GBS相关空肠弯曲菌中galE基因核苷酸序列的确存在相同变异且发生变异概率较非GBS相关空肠弯曲菌明显增大,遗传距离反映了此3株致GBS的空肠弯曲菌具有一定的区域特征.     

Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa

Infection of the mucous layer of the human stomach by Helicobacter pylori requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylori has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into cheY1, cheY2, and cheA in H. pylori strains N6 and SS1. Video tracking revealed that the wild-type H. pylori strain moves in short runs with frequent direction changes, in contrast to movement of cheY2, cheAY2, and cheAY2 cheY1 mutants, whose motion was more linear. The cheY1 mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1 nor cheAY2 mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1 failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of cheY1, cheY2, and cheA in motility and virulence of H. pylori.     

Colonization and inflammation deficiencies in Mongolian gerbils infected by Helicobacter pylori chemotaxis mutants

Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.     

Campylobacter jejuni colonization of mice with limited enteric flora

We have developed experimental murine Campylobacter infection models which demonstrate efficient establishment and reproducible, high-level colonization. Following oral inoculation, wild-type C3H mice with normal enteric flora were colonized inconsistently and inefficiently by C. jejuni strain 81-176. However, C3H mice with a limited gut flora (LF) were efficiently colonized at high levels (10(8) CFU/g of stool or large intestine tissue) followed by clearance after several weeks. Large intestine tissue showed minimal to mild inflammation at days 7 and 28 postinoculation. In striking contrast, C3H SCID mice with the same limited flora remained persistently colonized at a consistently high level until they were euthanized 8 months postinoculation. Lower gastrointestinal tract tissue from LF-SCID mice showed marked to severe inflammation in the colon and cecum at days 7 and 28 and intense inflammation of the stomach at day 28. These findings indicate that although the innate response alone cannot block colonization persistence, it is sufficient to orchestrate marked gut inflammation. Moreover, the adaptive immune response is critical to mediate C. jejuni clearance from the colonized gut. To validate our LF murine model, we verified that motility and chemotaxis are critical for colonization. Insertion-deletion mutations were generated in motB and fliI, which encode products essential for motility and flagellar assembly, and in the presumptive chemotaxis gene cheA (histidine kinase). All mutants failed to establish colonization in LF mice. Our limited flora murine colonization models serve as tractable, reproducible tools to define host responses to C. jejuni infection and to identify and characterize virulence determinants required for colonization.     

The Campylobacter jejuni Dps Homologue Is Important for In Vitro Biofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination

In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10(5) CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.