非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力
Summary
使用λ-Red介导的重组系统创建小的非编码RNA micC的缺失突变体。
Abstract
非编码小RNA（sRNA）是在转录后水平调控基因表达的新因子。一种在大肠杆菌和鼠伤寒沙门氏菌中已知的sRNA MicC可以抑制外膜蛋白的表达。为了进一步研究micC在肠炎沙门氏菌中的调控功能，我们在肠炎沙门氏菌菌株50336中克隆了micC基因，然后通过λ Red基重组系统和互补突变体50336Δ micC/p micC携带重组质粒pBR322表达micC构建了突变体50336Δ micC。 qRT-PCR结果表明，50336Δ micC的ompD转录率是野生型菌株的1.3倍，而50336ΔmicC中ompA和ompC的转录量分别是野生型菌株的2.2倍和3倍。这些表明micC抑制了ompA和ompC的表达。在随后的研究中，通过感染6周龄Balb / c小鼠和1日龄鸡来检测50336ΔmicC的致病性。结果表明，野生型50336株的LD50、突变体50336Δ micC和50336Δ micC/pmicC分别为12.59 CFU、5.01 CFU和19.95 CFU。1日龄鸡的LD50品系分别为1.13 x 109 CFU、1.55 x 10 8 CFU和2.54 x 108 CFU。结果表明，micC的缺失增强了S的毒力。 通过调节外膜蛋白的表达在小鼠和鸡中的肠炎。
Introduction
非编码小RNA（sRNA）的长度为40-400个核苷酸，通常不编码蛋白质，但可以在细菌染色体1，2，3中独立转录。大多数sRNA编码在基因编码区之间的基因间区（IGR）中，并通过碱基配对作用与靶mRNA相互作用，并在转录后水平调节靶基因表达4，5。它们在物质代谢、外膜蛋白合成、群体感应和毒力基因表达中起重要的调节作用5。
MicC是存在于大肠杆菌和鼠伤寒沙门氏菌中的109核苷酸小RNA转录本，可调节多种外膜蛋白表达，如OmpC、OmpD、OmpN、Omp35和Omp366，7，8，9。MicC通过在体外抑制核糖体与ompC mRNA领导者的结合来调节OmpC的表达，并且它需要Hfq RNA伴侣才能在大肠杆菌6中发挥作用。在鼠伤寒沙门氏菌中，MicC 通过编码序列（密码子 23-26）中的 ≤bp RNA 双链体沉默 ompD mRNA，然后破坏核内裂解 mRNA7 的稳定性。该调节过程由伴侣蛋白Hfq10辅助。OmpC是一种丰富的外膜蛋白，被认为在营养和毒素浓度高的环境中很重要，例如在肠道中6。OmpD孔蛋白是鼠伤寒沙门氏菌中含量最高的外膜蛋白，约占总细胞蛋白的11%。OmpD参与人巨噬细胞和肠上皮细胞的粘附12。MicC还抑制OmpC和OmpD孔蛋白的表达。人们认为MicC可以调节毒力。为探索MicC调控的新靶基因，研究micC的毒力调控功能，我们在肠炎沙门氏菌（SE）菌株50336中克隆了micC基因，构建了突变体50336ΔmicC和互补突变体50336ΔmicC/p micC。采用qRT-PCR筛选新型靶基因。50336ΔmicC的毒力由小鼠和鸡感染检测到。
Protocol
所有实验均按照国家研究委员会的《实验动物护理和使用指南》进行。扬州大学动物护理和使用委员会批准了所有应用于动物的实验和程序（SYXK2016-0020）。 1. 细菌菌株、质粒和培养条件 使用 表1中列出的细菌和质粒。 在LB肉汤或LB琼脂平板上培养细菌，在37°C下，适当时在50μg/ mL氨苄青霉素（Amp）存在下。 含有温度敏感质粒的培养菌株用于…
Representative Results
突变体50336Δ micC和互补菌株50336Δ micC /pmicC的构建 micC基因克隆结果表明，该基因由109 bp组成，与S基因100%相同。 鼠伤寒。基于序列数据，成功构建缺失突变体50336ΔmicC和互补突变体50336ΔmicC/pmicC。详细地，测序结果表明，扩增1.1 kb Cm电阻盒并用于构建<sup…
Discussion
S. 肠炎是一种重要的兼性细胞内病原体，可感染幼鸡并产生从肠炎到全身感染和死亡的症状17，18。此外，肠炎链球菌在成年鸡中引起潜伏感染，慢性携带者污染家禽产品，导致人类食源性感染19。肠炎链球菌的致病机制仍有待进一步探讨。迄今为止，已经发现一些sRNA，如IsrJ，SroA和IsrM会影响沙门氏菌的毒力<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本研究得到了中国国家自然科学基金（31972651号和31101826号）、江苏省高等学校科学基金（No.14KJB230002）、兽医生物技术国家重点实验室（No.SKLVBF201509）、扬州市自然科学基金（No.YZ2014019）的资助，该项目由江苏省高等学校优先学术项目发展（PAPD）资助。
Materials
| dextrose | Sangon Biotech | A610219 | for broth preparation |
| DNA purification kit | TIANGEN | DP214 | for DNA purification |
| Ex Taq | TaKaRa | RR01A | PCR |
| KH2PO4 | Sinopharm Chemical Reagent | 10017608 | for broth preparation |
| K2HPO4 | Sinopharm Chemical Reagent | 20032116 | for broth preparation |
| L-Arabinose | Sangon Biotech | A610071 | λ-Red recombination |
| Mini Plasmid Kit | TIANGEN | DP106 | plasmid extraction |
| NaCl | Sinopharm Chemical Reagent | 10019308 | for broth preparation |
| (NH4)2SO4 | Sinopharm Chemical Reagent | 10002917 | for broth preparation |
| PrimeScriptRRT reagent Kit with gDNA Eraser | TaKaRa | RR047 | qRT-PCR |
| SYBRR Premix Ex Taq II | TaKaRa | RR820 | qRT-PCR |
| T4 DNA Ligase | NEB | M0202 | Ligation |
| TRIzol | Invitrogen | 15596018 | RNA isolation |
| Tryptone | Oxoid | LP0042 | for broth preparation |
| Yeast extract | Oxoid | LP0021 | for broth preparation |
| centrifuge | Eppendorf | 5418 | centrifugation |
| Electrophoresis apparatus | Bio-Rad | 164-5050 | Electrophoresis |
| Electroporation System | Bio-Rad | 165-2100 | for bacterial transformation |
| Spectrophotometer | BioTek | Epoch | Absorbance detection |
| Real-Time PCR system | Applied Biosystems | 7500 system | qRT-PCR |
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Citazione di questo articolo
Meng, X., Cui, W., Meng, X., Wang, J., Wang, J., Zhu, G. A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis. J. Vis. Exp. (167), e61808, doi:10.3791/61808 (2021).