| 摘要: |
| 【目的】研究模式病原细菌铜绿假单胞菌PA14（Pseudomonas aeruginosa PA14）第三套六型分泌系统（H3-T6SS）的调控机制和在环境适应等方面的功能，为该菌致病机制研究和治疗方案开发奠定基础。【方法】以铜绿假单胞菌PA14为研究对象，利用凝胶迁移阻滞试验（EMSA）验证σs因子（RpoS）与H3-T6SS启动子区的结合能力；构建rpoS敲除菌株（ΔrpoS）及回补菌株（rpoS），用启动子酶活试验研究RpoS对H3-T6SS表达的调控作用；构建H3-T6SS关键组分clpV3敲除菌株（ΔclpV3）及回补菌株（clpV3），测定其在酸、渗透压等不同胁迫下的细胞存活率；检测H3-T6SS对铜绿假单胞菌PA14生物膜形成和运动能力的影响。【结果】RpoS蛋白可以直接结合H3-T6SS启动子区；成功构建了铜绿假单胞菌PA14的rpoS和clpV3敲除菌株及回补菌株，RpoS正调控H3-T6SS的表达，H3-T6SS有助于细菌抵抗酸胁迫，但对渗透压胁迫响应不明显；H3-T6SS可显著增强细菌的生物膜形成和运动能力。【结论】铜绿假单胞菌PA14的H3-T6SS受调控因子RpoS的正调控，在增强细菌抗酸胁迫、生物膜形成和运动能力等方面有重要作用。 |
| 关键词: 铜绿假单胞菌PA14 第三套六型分泌系统 致病机制 生物膜形成 |
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| 基金项目:国家重点研发计划项目（2018YFA0901200）；国家自然科学基金项目（31725003） |
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| Regulation and function of H3-T6SS in Pseudomonas aeruginosa PA14 |
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YU Zonglan,ZHU Kaixiang,ZHANG Chen,et al
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| Abstract: |
| 【Objective】This study investigated the regulation and function of H3-T6SS in Pseudomonas aeruginosa PA14 in environmental adaption to lay foundation for understanding the pathogenicity and development of therapeutic method.【Method】Pseudomonas aeruginosa PA14 was selected for electrophoretic mobility shift assay (EMSA) to determine the binding ability of σs factor (RpoS) on H3-T6SS promoter fragment.The rpoS deletion mutant (ΔrpoS) and the complementary strain (rpoS) of P. aeruginosa PA14 were constructed.The regulation activity of H3-T6SS by RpoS was verified using β-galactosidase assay.The clpV3 deletion mutant and the complementary strains were constructed and the cell survival rates under acid and osmotic stress were measured.Biofilm measurement and motility assay of bacteria were performed to determine the function of H3-T6SS on biofilm formation and motility.【Result】His6-RpoS recombinant protein can bind directly to the H3-T6SS promoter.The deletion mutants and complementary strains of rpoS and clpV3 were constructed.RpoS positively regulated the expression of H3-T6SS.H3-T6SS significantly increased the resistance of bacteria against acidic stress,but not osmotic stress.H3-T6SS ncreased the ability of motility and biofilm formation of P.aeruginosa PA14.【Conclusion】H3-T6SS was positively regulated by RpoS and played important roles in acidic stress resistance,motility and biofilm formation in P. aeruginosa PA14. |
| Key words: Pseudomonas aeruginosa PA14 H3-T6SS pathogenesis biofilm formation |
