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一种非编码的小 RNA MicC 有助于肠炎沙门氏菌外膜蛋白的毒力。

A non-coding small RNA MicC contributes to virulence in outer membrane proteins in Salmonella Enteritidis.

机构信息

College of Veterinary Medicine, Yangzhou University; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses;

College of Veterinary Medicine, Yangzhou University.

出版信息

J Vis Exp. 2021 Jan 27(167). doi: 10.3791/61808.

Abstract

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336ΔmicC by the λ Red-based recombination system and the complemented mutant 50336ΔmicC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336ΔmicC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336ΔmicC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336ΔmicC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336ΔmicC and 50336ΔmicC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 10 CFU, 1.55 x 10 CFU, and 2.54 x 10 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

摘要

一种非编码的小 RNA (sRNA) 是一种新的调节转录后基因表达的因子。在大肠杆菌和鼠伤寒沙门氏菌中发现的一种 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作用,我们在肠炎沙门氏菌 50336 株中克隆了 micC 基因,然后利用 λ Red 重组系统构建了突变株 50336ΔmicC,并通过携带表达 micC 的重组质粒 pBR322 构建了互补突变株 50336ΔmicC/pmicC。qRT-PCR 结果表明,50336ΔmicC 中 ompD 的转录水平比野生型菌株高 1.3 倍,而 50336ΔmicC 中 ompA 和 ompC 的转录水平比野生型菌株高 2.2 倍和 3 倍。这表明 micC 抑制了 ompA 和 ompC 的表达。在接下来的研究中,我们通过感染 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、突变株 50336ΔmicC 和 50336ΔmicC/pmicC 对 6 周龄 Balb/c 小鼠的 LD50 分别为 12.59 CFU、5.01 CFU 和 19.95 CFU。对 1 日龄鸡的 LD50 分别为 1.13x10 CFU、1.55x10 CFU 和 2.54x10 CFU。这表明通过调节外膜蛋白的表达,micC 的缺失增强了肠炎沙门氏菌在小鼠和鸡中的毒力。

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