Name: a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis
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Description: Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

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。

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
<ol><li>使用 表1中列出的细菌和质粒。</li>
	<li>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。</li>
	<li>含有温度敏感质粒的培养菌株用于...

<ol>
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H., Romby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNAs+in+bacteria+and+archaea:+who+they+are,+what+they+do,+and+how+they+do+it.">Small RNAs in bacteria and archaea: who they are, what they do, and how they do it.</a> Advances In Genetics. 90, 133-208 (2015).</li><li>Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rough+guide+to+the+non-coding+RNA+world+of+Salmonella.">A rough guide to the non-coding RNA world of Salmonella.</a> Molecular Microbiology. 71 (1), 1-11 (2009).</li><li>Dutta, T., Srivastava, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA-mediated+regulation+in+bacteria:+A+growing+palette+of+diverse+mechanisms.">Small RNA-mediated regulation in bacteria: A growing palette of diverse mechanisms.</a> Gene. 656, 60-72 (2018).</li><li>Waters, L. S., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+RNAs+in+bacteria.">Regulatory RNAs in bacteria.</a> Cell. 136 (4), 615-628 (2009).</li><li>Chen, S., Zhang, A., Blyn, L. B., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicC,+a+second+small-RNA+regulator+of+Omp+protein+expression+in+Escherichia+coli.">MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli.</a> Journal of Bacteriology. 186 (20), 6689-6697 (2004).</li><li>Pfeiffer, V., Papenfort, K., Lucchini, S., Hinton, J. 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M., Masi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Regulation+of+the+Small+RNA+MicC+and+the+Quiescent+Porin+OmpN+in+Response+to+Antibiotic+Stress+in+Escherichia+coli.">Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli.</a> Antibiotics (Basel). 6 (4), 33 (2017).</li><li>Hao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porin+Deficiency+in+Carbapenem-Resistant+Enterobacter+aerogenes+Strains.">Porin Deficiency in Carbapenem-Resistant Enterobacter aerogenes Strains.</a> Microbial Drug Resistance. 24 (9), 1277-1283 (2018).</li><li>Wroblewska, Z., Olejniczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hfq+assists+small+RNAs+in+binding+to+the+coding+sequence+of+ompD+mRNA+and+in+rearranging+its+structure.">Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure.</a> RNA. 22 (7), 979-994 (2016).</li><li>Santiviago, C. 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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<...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>dextrose</td>
			<td>Sangon Biotech</td>
			<td>A610219</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>DNA purification kit</td>
			<td>TIANGEN</td>
			<td>DP214</td>
			<td>for DNA purification</td>
		</tr>
		<tr>
			<td>Ex Taq</td>
			<td>TaKaRa</td>
			<td>RR01A</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017608</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20032116</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>L-Arabinose</td>
			<td>Sangon Biotech</td>
			<td>A610071</td>
			<td>&#955;-Red recombination</td>
		</tr>
		<tr>
			<td>Mini Plasmid Kit</td>
			<td>TIANGEN</td>
			<td>DP106</td>
			<td>plasmid extraction</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10002917</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>PrimeScriptRRT reagent Kit with gDNA Eraser&#160;</td>
			<td>TaKaRa</td>
			<td>RR047</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>SYBRR Premix Ex Taq II</td>
			<td>TaKaRa</td>
			<td>RR820</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202</td>
			<td>Ligation</td>
		</tr>
		<tr>
			<td>TRIzol&#160;</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td>centrifugation</td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>&#160;Electroporation System</td>
			<td>Bio-Rad</td>
			<td>165-2100</td>
			<td>for bacterial transformation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td>Epoch</td>
			<td>Absorbance detection</td>
		</tr>
		<tr>
			<td>Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>7500 system</td>
			<td>qRT-PCR</td>
		</tr>
	</tbody>
</table>

a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis

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。

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...

Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

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。

非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力

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 ...

该协议可用于灭活染色体基因以分离突变体以进行基因功能研究和细菌。它使同源重组过程简单、快速和高效。使用该技术，可以删除病原体的毒力相关基因以获得减毒突变体，该突变体可用作减毒疫苗的候选者。首先扩增含有micC基因血学片段的氯霉素盒。设计正向和反向引物以扩增来自质粒PKD3的氯霉素盒，包括来自micC基因的五个素数和三个素数末端的50个碱基对同源性延伸。按照文本手稿中的说明进行PCR，使用PKD3质粒作为模板扩增氯霉素盒。用琼脂糖凝胶电泳确定PCR产物的大小。然后用DNA凝胶回收试剂盒纯化产物，并通过分光光度法测定DNA的浓度。进行第二次PCR反应以消除PKD3质粒进一步重组的干扰。以1比200的比例稀释第一个PCR产物，并将其用作二级PCR的模板。为了构建第一个重组菌株50336 delta micC点猫，将100微升SE 50336感受态细胞与5微升PKD 46质粒混合，并在冰上孵育30分钟。在42摄氏度下对混合物进行热冲击90秒，然后将其迅速转移回冰上两分钟，将质粒转化为细胞。通过在氨苄西林耐药平板上以30摄氏度培养细胞过夜来筛选阳性菌落。第二天，向SE 50336 PKD 46液体培养物中再加入30毫利尔阿拉伯糖，并在振荡的同时在30摄氏度下孵育一小时诱导重组酶表达。在电击杯中混合 100 纳克纯化的 PCR 产物和 40 微升 SE 50336 PKD 46 感受态细胞。然后进行电击改造。电转化后，将混合物转移到一毫升SOC培养基和150RPM和30摄氏度的振荡培养箱中一小时。将混合物铺在耐氯霉素的LB平板上，并在37摄氏度下培养过夜以筛查阳性菌落。在42摄氏度下培养阳性菌落两个小时。然后在 37 摄氏度下筛选菌落对氨苄西林的敏感性和对氯霉素的耐药性过夜，以获得第一个不含 PKD 46 的重组菌株。使用PCR和凝胶电泳提取50336 Delta micC点猫基因组DNA后，将100纳克质粒PCP 20电镀成40微升50336 Delta micC点猫感受态细胞。在 30 摄氏度的氨苄西林和氯霉素耐药平板上筛查阳性转化体。将正转化体转移到非抗性LB液体培养基中，并在42摄氏度下培养过夜。然后在 37 摄氏度的 LB 平板上分离单个菌落。选择对氨苄西林和氯霉素敏感的菌落。这个突变体是micC缺失突变体SE 50336 Delta micC。按照文本手稿中的说明执行 PCR 验证 50336 Delta micC。该协议用于在补充突变体50336 Delta micC，pmicC中构建缺失突变体50336 Delta micC。通过PCR验证了第一个重组50336 Delta micC点猫，插入氯霉素的预期条带大小约为1200个碱基对PCR产物，而野生型菌株的预期条带大小为279个碱基对。在第二次重组中，氯霉素盒被PCP 20消除。PCR结果结合测序证实，同基因micC突变体构建成功。采用实时定量PCR分析了菌株50336、50336 delta micC和50336 delta micC pmicC中ompA、ompC和ompD基因的表达。与野生型菌株相比，ompA和ompC以及50336 delta micC的转录增加了约2.2倍和3倍。而ompD的转录仅略有增加。LD 50测定显示，在用三种菌株中的每一种感染6至8周龄的BALB / c小鼠后，大多数感染50336 Delta micC的小鼠在感染后48小时表现出倦怠，食欲不振或腹泻，并在96小时后死亡。野生型菌株和50336 delta micC pmicC感染的小鼠在感染后72小时表现出相同的症状，并在120小时后死亡。3株菌株的LD 50表明，突变体50336 Delta micC的毒力比野生型提高了2.5倍。该技术有助于研究人员研究细菌基因的功能，并通过删除靶基因获得所需的工程菌株。

Research

JoVE Journal

Immunology and Infection

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

一个用于标识Allelotyping PCR扩增沙门氏菌肠炎血清型，哈达尔，海德堡和鼠伤寒沙门氏菌

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

科研

免疫与感染

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

一个协议感染<em&gt;秀丽隐杆线虫</em&gt;与<em&gt;鼠伤寒沙门氏菌</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力

非编码小RNA MicC有助于肠炎沙门氏菌外膜蛋白的毒力