Effects of sRNA STnc1220 Gene Deletion Strain on Biological Characteristics and Pathogenicity of Salmonella typhimurium

doi:10.7668/hbnxb.20192539

Abstract

Abstract:

In order to explore the molecular characteristics,biological functions and effects of sRNA STnc1220 of Salmonella typhimurium on bacterial virulence,the sRNA STnc1220 gene of Salmonella typhimurium was amplified by PCR,and its molecular characteristics were analyzed.Use the TargetRNA2 website to predicted the target genes that might be regulated by the STnc1220 gene;the λ-Red homologous recombination technology was used to construct STM(Salmonella)-The gene deletion mutant strain,and compared with the parent SL1344,its growth characteristics,biochemical characteristics,genetic stability,biofilm(BF)formation ability,and its effects in different stress environments(pH,H₂O₂,high salt and ethanol stress)adaptability,through adhesion and infection of macrophages and mouse infection tests to study pathogenicity.The results showed that the full length of STnc1220 gene was 73 bp,and its upstream 5'-UTR had-10 and-35 promoter binding sites.It was predicted that its potential target gene was barA gene;The test successfully constructed the deletion strain STM-ΔSTnc1220,and it had good performance genetic stability;compared with the SL-1344 parent strain,the growth rate,biochemical characteristics and biofilm formation ability of the missing strain did not change;the growth rate was not significantly different under the conditions of pH=4.8,pH=9 and H₂O₂.However, in the 4% NaCl environment, the growth rate of the deletion strain was significantly higher than that of the parent strain after 7 h, and the growth rate of the deletion strain in the logarithmic phase was significantly increased in the 3.8% ethanol environment(P<0.05);It significantly reduces the ability of adhesion and invasion in macrophages(P<0.01), and the LD₅₀ of mice was significantly elevated,the pathological damage to the liver and spleen of mice was weakened.It showed that the STnc1220 gene played an important role in environmental adaptation and virulence regulation.

Key words: Salmonella typhimurium, sRNA, STnc1220, Biological characteristics, Virulence

LI Na, NING Chengcheng, GUO Yun, JI Chunhui, WANG Lixia, ZHANG Yaping, MENG Qingling, QIAO Jun, CAI Xuepeng. Effects of sRNA STnc1220 Gene Deletion Strain on Biological Characteristics and Pathogenicity of Salmonella typhimurium[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 188-194. doi: 10.7668/hbnxb.20192539.

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Figures/Tables 10

Tab.1 Primers required for PCR amplification

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	扩增片段大小/bp Amplified fragment size
P1	TTGAGCAGCAACAAGAACTA	275
P2	CAGGATGAATACGCAAATAA
P3	AGCGTGCTTGCGTAAAAATACGCTGGAAAAAATGGAAGTGGAATGGCTGGGCGATTGTGTAGGCTGGAG	1 159
P4	CCGACTGTTGGTCGAACCAGGCCAACAGAACCTGTTCTATACTATTTTTAAGGAACACTTAACGGCTGACAT

Fig.1 PCR amplification of STM STnc1220 gene,analysis of gene promoter region and sRNA STnc1220 and mRNA barA complementary pairing position
A.STM STnc1220 gene clone:M.DNA Marker DL2000;1.Negative control;2.sRNA STnc1220 gene.
B.Sequencing analysis of STM STnc1220 gene.C.Complementary pairing position of sRNA STnc1220 and target gene barA.

Fig.2 Identification of deletion strains ΔSTnc1220 by PCR
A.STnc1220 targeting fragment:M.Marker DL2000;1.Negative control;2.Targeting fragment.B.Identification of STM ΔSTnc1220:M.Marker DL2000; 1.Negative control;2.sRNA STnc1220 gene;3. ΔSTnc1220∷cat amplification product;4.ΔSTnc1220 gene amplification product.

Fig.3 Identification genetically stable of ΔSTnc1220 by PCR

Fig.4 Growth curve measurement

Fig.5 Growth curves of strains under different stress environments
Different lowercase letters indicate significant differences(P<0.05).

Fig.6 Adhesion and invasion of SL1344, ΔSTnc1200 to macrophage RAW264.7
Different capital letters in the same tissue showed significant differences(P<0.01);There was no significantly different analysis between different tissues.

Tab.2 Determination of LD₅₀ after intraperitoneal injection of SL1344 and ΔSTnc1220 strains in 6-week-old mice

菌株 Strains	菌落总数/ (cfu/mL) Total number of bacterial colonies	小鼠 Mice	小鼠死亡数 Mouse death	半数致死量/ (cfu/mL) LD₅₀
SL1344	1.0×10³	6	0	6.46×10⁴
	1.0×10⁴	6	1
	1.0×10⁵	6	3
	1.0×10⁶	6	6
	1.0×10⁷	6	6
ΔSTnc1220	1.0×10³	6	0	1.48×10⁵
	1.0×10⁴	6	1
	1.0×10⁵	6	3
	1.0×10⁶	6	4
	1.0×10⁷	6	6
对照 Control	PBS	6	0	—

Fig.7 Death and survival curve of mice by intraperitoneal infected with different strains

Fig.8 Pathological changes of organs in mice infected with SL1344 and ΔSTnc1220

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