Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans.The previous found that a combination of kojic acid and tea polyphenols exhibited better activity ag...Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans.The previous found that a combination of kojic acid and tea polyphenols exhibited better activity against E.coli O157:H7 than using either alone.This study aimed to explore responses underlying the antibacterial mechanisms of kojic acid and tea polyphenols from the gene level.The functional enrichment analysis by comparing kojic acid and tea polyphenols individually or synergistically against E.coli O157:H7 found that acid resistance systems in kojic acid were activated,and the cell membrane and genomic DNA were destructed in the cells,resulting in“oxygen starvation”.The oxidative stress response triggered by tea polyphenols inhibited both sulfur uptake and the synthesis of ATP,which affected the bacteria's life metabolic process.Interestingly,we found that kojic acid combined with tea polyphenols hindered the uptake of iron that played an essential role in the synthesis of DNA,respiration,tricarboxylic acid cycle.The results suggested that the iron uptake pathways may represent a novel approach for kojic acid and tea polyphenols synergistically against E.coli O157:H7 and provided a theoretical basis for bacterial pathogen control in the food industry.展开更多
目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞...目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞株,观察Caco-2单层细胞中的微菌落数量和m in i-Tn5Km2插入位点。结果:经诱变,共分离到3组粘附缺陷突变株,1组完全丧失了粘附力,2组粘附力减弱,3组的粘附力更弱。1组和3组拥有LEE岛上多基因的转座子插入,而2组m in i-Tn5Km2的插入位点在LEE岛之外。结论:O157Sakai的肠道粘附能力可能与LEE岛的III型分泌系统有关。展开更多
To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into ...To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.展开更多
基金supported by National Natural Science Foundation of China(31972021)R&D Projects in Key Areas of Guangdong Province(2019B020212003)+4 种基金the Science and Technology Program of Guangzhou,China(202206010177)Guangdong key research and development program(2021B0202060001)Foshan and agricultural academy cooperation projectGuangdong Modern Agriculture project(2022KJ117)Aquatic Products Center Project of GAAS。
文摘Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans.The previous found that a combination of kojic acid and tea polyphenols exhibited better activity against E.coli O157:H7 than using either alone.This study aimed to explore responses underlying the antibacterial mechanisms of kojic acid and tea polyphenols from the gene level.The functional enrichment analysis by comparing kojic acid and tea polyphenols individually or synergistically against E.coli O157:H7 found that acid resistance systems in kojic acid were activated,and the cell membrane and genomic DNA were destructed in the cells,resulting in“oxygen starvation”.The oxidative stress response triggered by tea polyphenols inhibited both sulfur uptake and the synthesis of ATP,which affected the bacteria's life metabolic process.Interestingly,we found that kojic acid combined with tea polyphenols hindered the uptake of iron that played an essential role in the synthesis of DNA,respiration,tricarboxylic acid cycle.The results suggested that the iron uptake pathways may represent a novel approach for kojic acid and tea polyphenols synergistically against E.coli O157:H7 and provided a theoretical basis for bacterial pathogen control in the food industry.
文摘目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞株,观察Caco-2单层细胞中的微菌落数量和m in i-Tn5Km2插入位点。结果:经诱变,共分离到3组粘附缺陷突变株,1组完全丧失了粘附力,2组粘附力减弱,3组的粘附力更弱。1组和3组拥有LEE岛上多基因的转座子插入,而2组m in i-Tn5Km2的插入位点在LEE岛之外。结论:O157Sakai的肠道粘附能力可能与LEE岛的III型分泌系统有关。
文摘To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.
