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

Summary

An λ-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

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 10⁹ CFU, 1.55 x 10⁸ CFU, and 2.54 x 10⁸ CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

Introduction

Non-coding small RNAs (sRNAs) are 40-400 nucleotides in length, which generally do not encode proteins but could be transcribed independently in bacterial chromosomes^1,2,3. Most sRNAs are encoded in the intergenic regions (IGRs) between gene-coding regions and interact with target mRNAs through base-pairing actions, and regulate target genes expression at the post-transcriptional level^4,5. They play important regulation roles in substance metabolism, outer membrane protein synthesis, quorum sensing and virulence gene ....

Protocol

All the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. The animal care and use committee of Yangzhou University approved all experiments and procedures applied on the animals (SYXK2016-0020).

1. Bacterial strains, plasmids, and culture conditions

1. Use the bacteria and plasmids listed in Table 1.
2. Culture bacteria in LB broth or on LB agar plates at 37 °C, in the presence of 50 µg/mL ampicillin (Amp) when appropriate.
3. Culture strains containing temperature sensitive plasmids are used for deletion muta....

Results

Construction of the mutant 50336ΔmicC and complemented strain 50336ΔmicC /pmicC
The micC gene clone result indicated that this gene was composed of 109 bp showing 100% identity with that of S. Typhimurium. Based on the sequence data, the deletion mutant 50336ΔmicC and the complemented mutant 50336ΔmicC/pmicC were constructed succes.......

Discussion

S. Enteritidis is an important facultative intracellular pathogen that can infect young chickens and produces symptoms from enteritis to systemic infection and death^17,18. In addition, S. Enteritidis causes latent infections in adult chickens and chronic carriers contaminate poultry products, resulting in food-borne infections in humans¹⁹. The pathogenic mechanism of S. Enteritidis remains to be further probed. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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