Intestinal drug-resistant pathogens,e.g.,Salmonella enterica subsp.enterica serovar Typhimurium(S.Tm)and enteropathogenic Escherichia coli(E.coli),frequently cause life-threatening infectious enteritis.Probiotic-based...Intestinal drug-resistant pathogens,e.g.,Salmonella enterica subsp.enterica serovar Typhimurium(S.Tm)and enteropathogenic Escherichia coli(E.coli),frequently cause life-threatening infectious enteritis.Probiotic-based therapy is a promising way to eliminate drug-resistant pathogens for treatment of infectious enteritis,but its colonizing and therapeutic efficacy after oral administration are limited.Here,we developed a facile therapeutic agent to treat infectious enteritis by co-assembly of the peptide nanodrug melittin-loaded MSN grafted by polysaccharide-binding protein(MMPB)with the famous probiotic bacteria Lactobacillus plantarum(Lac)and Bifidobacterium animalis subsp.lactis(Bif).The nanodrug was composed of the antimicrobial peptide melittin and mesoporous silica nanoparticles exposing the artificial polysaccharide-binding protein.Owing to presence of the artificial protein on the MMPB surface,the nanodrug strongly bound and cross-linked the probiotic cells,forming the Lac+Bif+MMPB co-assembly.During co-incubation with the kanamycin-resistant E.coli strain(Ecka),the co-assembly strongly reduced the viability of Ecka,leading to the increase in the ratio of probiotic to Ecka from 1.6 to 9.2.After oral administration of the co-assembly to themice pre-colonized by Ecka,Lac+Bif+MMPB almost eliminated the kanamycin-resistant gene in the intestine,and led to 2-3-fold higher levels of the probiotic cells than the nanodrug MMPB or the combined probiotics Lac+Bif.More importantly,in the mice suffering from enteritis caused by drug-resistant S.Tm,the co-assembly remarkably recovered the mouse body weight,reduced intestine colonization of S.Tm cells,and decreased the levels of pro-inflammatory cytokines in both serum and colons.This study realized the synthetic biology technique-mediated abiotic/biotic co-assembly for efficient treating infectious enteritis induced by drug-resistant pathogens.展开更多
由食源性致病菌引发的食品安全问题是威胁人类健康的重要因素。因此,研究食源性致病菌的感染机理对于控制病原菌危害具有重要意义。【目的】以常见的食源性致病菌——鼠伤寒沙门氏菌为研究对象,以其发挥重要致病性的转录调控因子SlyA为...由食源性致病菌引发的食品安全问题是威胁人类健康的重要因素。因此,研究食源性致病菌的感染机理对于控制病原菌危害具有重要意义。【目的】以常见的食源性致病菌——鼠伤寒沙门氏菌为研究对象,以其发挥重要致病性的转录调控因子SlyA为靶标,比较胞嘧啶单碱基编辑技术(CRISPR/Cas9-guided-Cytidine Base Editor,CBE)和λ-Red同源重组技术在构建鼠伤寒沙门氏菌SlyA敲除菌株方面的方法差异,为鼠伤寒沙门氏菌的基因编辑技术应用提供数据。同时,也为其他类型病原菌的基因编辑技术开发提供有力参考。【方法】采用PCR、GoldenGate、Sanger测序等方法完成CBE系统以及λ-Red系统的构建以及敲除结果的验证,采用Editor-R软件分析CBE系统的单碱基编辑效率,采用Western blotting在蛋白表达层面对敲除结果进行验证。此外,本研究还结合了表型鉴定的方法验证了基因敲除结果。【结果】经PCR产物测序鉴定、Western blotting分析及溶血素活性鉴定等结果表明,本研究成功将CBE系统应用于鼠伤寒沙门氏菌slyA的单碱基编辑中,应用前述两种方法构建了鼠伤寒沙门氏菌SlyA敲除菌株。【结论】CBE系统虽然以其操作的简便性在基因编辑中优势明显,但同λ-Red系统相比,该方法需要设立特定的gRNA及PAM位点,在非模式菌株中的普适性较低,且在进行编辑时,CBE系统存在不稳定的问题。尽管如此,但CBE系统在鼠伤寒沙门氏菌中的成功建立,为进一步拓展与完善该菌的基因编辑系统提供了基础。展开更多
基金supported by National Natural Science Foundation of China(32170102)Natural Science Foundation of Tianjin(25JCLMJC00400)the Fundamental Research Funds for the Central Universities(63253191).
文摘Intestinal drug-resistant pathogens,e.g.,Salmonella enterica subsp.enterica serovar Typhimurium(S.Tm)and enteropathogenic Escherichia coli(E.coli),frequently cause life-threatening infectious enteritis.Probiotic-based therapy is a promising way to eliminate drug-resistant pathogens for treatment of infectious enteritis,but its colonizing and therapeutic efficacy after oral administration are limited.Here,we developed a facile therapeutic agent to treat infectious enteritis by co-assembly of the peptide nanodrug melittin-loaded MSN grafted by polysaccharide-binding protein(MMPB)with the famous probiotic bacteria Lactobacillus plantarum(Lac)and Bifidobacterium animalis subsp.lactis(Bif).The nanodrug was composed of the antimicrobial peptide melittin and mesoporous silica nanoparticles exposing the artificial polysaccharide-binding protein.Owing to presence of the artificial protein on the MMPB surface,the nanodrug strongly bound and cross-linked the probiotic cells,forming the Lac+Bif+MMPB co-assembly.During co-incubation with the kanamycin-resistant E.coli strain(Ecka),the co-assembly strongly reduced the viability of Ecka,leading to the increase in the ratio of probiotic to Ecka from 1.6 to 9.2.After oral administration of the co-assembly to themice pre-colonized by Ecka,Lac+Bif+MMPB almost eliminated the kanamycin-resistant gene in the intestine,and led to 2-3-fold higher levels of the probiotic cells than the nanodrug MMPB or the combined probiotics Lac+Bif.More importantly,in the mice suffering from enteritis caused by drug-resistant S.Tm,the co-assembly remarkably recovered the mouse body weight,reduced intestine colonization of S.Tm cells,and decreased the levels of pro-inflammatory cytokines in both serum and colons.This study realized the synthetic biology technique-mediated abiotic/biotic co-assembly for efficient treating infectious enteritis induced by drug-resistant pathogens.
文摘由食源性致病菌引发的食品安全问题是威胁人类健康的重要因素。因此,研究食源性致病菌的感染机理对于控制病原菌危害具有重要意义。【目的】以常见的食源性致病菌——鼠伤寒沙门氏菌为研究对象,以其发挥重要致病性的转录调控因子SlyA为靶标,比较胞嘧啶单碱基编辑技术(CRISPR/Cas9-guided-Cytidine Base Editor,CBE)和λ-Red同源重组技术在构建鼠伤寒沙门氏菌SlyA敲除菌株方面的方法差异,为鼠伤寒沙门氏菌的基因编辑技术应用提供数据。同时,也为其他类型病原菌的基因编辑技术开发提供有力参考。【方法】采用PCR、GoldenGate、Sanger测序等方法完成CBE系统以及λ-Red系统的构建以及敲除结果的验证,采用Editor-R软件分析CBE系统的单碱基编辑效率,采用Western blotting在蛋白表达层面对敲除结果进行验证。此外,本研究还结合了表型鉴定的方法验证了基因敲除结果。【结果】经PCR产物测序鉴定、Western blotting分析及溶血素活性鉴定等结果表明,本研究成功将CBE系统应用于鼠伤寒沙门氏菌slyA的单碱基编辑中,应用前述两种方法构建了鼠伤寒沙门氏菌SlyA敲除菌株。【结论】CBE系统虽然以其操作的简便性在基因编辑中优势明显,但同λ-Red系统相比,该方法需要设立特定的gRNA及PAM位点,在非模式菌株中的普适性较低,且在进行编辑时,CBE系统存在不稳定的问题。尽管如此,但CBE系统在鼠伤寒沙门氏菌中的成功建立,为进一步拓展与完善该菌的基因编辑系统提供了基础。
