猪链球菌2型SodA、TrxA和TrxC基因的功能研究

doi:10.16431/j.cnki.1671-7236.2025.03.043

摘要/Abstract

摘要： 【目的】探讨猪链球菌2型超氧化物歧化酶A (SodA)、硫氧还蛋白A (TrxA)和TrxC基因在调控氧化应激和参与致病过程中的生物学功能。【方法】比较猪链球菌2型3个突变菌株ΔSodA、ΔTrxA和ΔTrxC对抗应激能力和毒力的调控作用。通过应激存活试验，分别检测SodA、TrxA和TrxC基因对不同氧化应激处理(0.04%过氧化氢(H₂O₂)、10 mol/L百草枯(PQ))的抗氧化应激能力以及不同温度(4、43 ℃)的抗温度应激能力；将不同基因缺失株与猪肠道上皮细胞(IPEC-J2)、小鼠巨噬细胞(RAW264.7)共培养，探究3个基因对细胞黏附和抗吞噬功能的影响；通过小鼠攻毒试验，测定各组织载菌量和血清抗氧化酶与肝功能指标，探明SodA、TrxA和TrxC基因在抗氧化应激以及致病过程中的作用。【结果】抗应激试验结果显示，在不同氧化应激和不同温度应激条件下,ΔSodA和ΔTrxC菌株存活能力均极显著低于野生菌株(P＜0.01)，ΔTrxA菌株在43 ℃高温刺激下存活能力极显著低于野生菌株(P＜0.01)。细胞黏附试验结果显示，SodA和TrxA基因缺失导致细菌黏附能力降低55%～65%，极显著低于野生菌株(P＜0.01)。细胞抗吞噬试验结果显示，ΔSodA、ΔTrxC和ΔTrxA菌株在吞噬细胞中的存活率极显著或显著低于野生菌株(P＜0.01；P＜0.05)。动物攻毒试验结果显示，与野生菌株相比，ΔSodA、ΔTrxA和ΔTrxC菌株毒力均极显著下降(P＜0.01)；ΔSodA、ΔTrxA和ΔTrxC菌株组小鼠死亡率分别为10%、50%和20%，组织载菌量分别为3.6 lg CFU/g～4.4 lg CFU/g、4.2 lg CFU/g～5.1 lg CFU/g和3.1 lg CFU/g～4.1 lg CFU/g；野生菌株组小鼠死亡率为90%，组织载菌量为6.4 lg CFU/g～7.8 lg CFU/g。小鼠感染ΔSodA、ΔTrxC和ΔTrxA菌株后血清中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和总抗氧化能力(T-AOC)极显著高于野生菌株(P＜0.01)，天门冬氨酸转氨酶(AST)、谷丙转氨酶(ALT)和碱性磷酸酶(ALP)活性极显著低于野生菌株(P＜0.01)。【结论】猪链球菌2型SodA和TrxC基因通过中和菌体内超氧根离子和维持蛋白稳态对抗氧化应激引起的损伤，从而介导细菌毒力，而TrxA可能作为一个调控基因参与抗氧化应激反应。

关键词: 猪链球菌2型; 抗氧化应激; SodA基因; TrxA基因; TrxC基因

Abstract: 【Objective】 This study was conducted to investigate the biological functions of superoxide dismutase A (SodA),thioredoxin A (TrxA) and TrxC genes of Streptococcus suis type 2 in regulating oxidative stress and participating in pathogenic processes.【Method】 The regulation of stress resistance and virulence of 3 mutant strains of Streptococcus suis type 2,ΔSodA,ΔTrxA and ΔTrxC,were compared.The anti-oxidative stress ability of SodA,TrxA and TrxC genes to different oxidative stress treatments (0.04% hydrogen peroxide (H₂O₂),10 mol/L paraquat (PQ)) and the anti-temperature stress ability at different temperatures (4 and 43 ℃) were tested by stress survival test.Different gene deletion strains were co-cultured with porcine intestinal epithelial cells (IPEC-J2) and mouse macrophages (RAW264.7) to explore the effects of the 3 genes on cell adhesion and anti-phagocytosis.The effects of SodA,TrxA and TrxC genes on the anti-oxidative stress and the pathogenesis were investigated by testing the bacterial load,serum antioxidant enzymes and liver function in mice.【Result】 The results of anti-stress test showed that under different oxidative stress and different temperature stress conditions,the survival ability of ΔSodA and ΔTrxC strains was extremely significantly lower than that of wild strain (P＜0.01),and the survival ability of ΔTrxA strain was extremely significantly lower than that of wild strain under high temperature stimulation at 43 ℃ (P＜0.01).The results of cell adhesion test showed that the deletion of SodA and TrxA genes reduced the adhesion ability of bacteria by 55%-65%,which was extremely significantly lower than that of wild strain (P＜0.01).The results of anti-phagocytosis test showed that the survival rate of ΔSodA,ΔTrxC and ΔTrxA strains in phagocytosis cells was extremely significantly or significantly lower than that of wild strain (P＜0.01 or P＜0.05).The results of animal challenge test showed that the virulence of ΔSodA,ΔTrxA and ΔTrxC strains decreased extremely significantly compared with wild strain (P＜0.01).The mortality of ΔSodA,ΔTrxA and ΔTrxC strains were 10%,50% and 20%,respectively,and the tissue bacterial load was 3.6 lg CFU/g-4.4 lg CFU/g,4.2 lg CFU/g-5.1 lg CFU/g and 3.1 lg CFU/g-4.1 lg CFU/g,respectively.In the wild strain group,the mortality was 90%,and the tissue load was 6.4 lg CFU/g-7.8 lg CFU/g.The activities of superoxide dismutase (SOD),catalase (CAT) and total antioxidant capacity (T-AOC) in serum of mice infected with ΔSodA,ΔTrxC and ΔTrxA strains were extremely significantly higher than those of wild strain (P＜0.01).The activities of aspartate aminotransferase (AST),alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were extremely significantly lower than those of wild strain (P＜0.01).【Conclusion】 SodA and TrxC genes of Streptococcus suis type 2 counteracted the damage caused by oxidative stress by neutralizing superoxide ions in bacteria and maintaining protein homeostasis,thus mediating bacterial virulence.TrxA might be a regulatory gene involved in antioxidant stress response.

Key words: Streptococcus suis type 2; anti-oxidative stress; SodA gene; TrxA gene; TrxC gene

中图分类号:

S858.28

李熙, 刘剑英, 方丽华, 印遇龙, 唐宇龙. 猪链球菌2型SodA、TrxA和TrxC基因的功能研究[J]. 中国畜牧兽医, 2025, 52(3): 1428-1436.

LI Xi, LIU Jianying, FANG Lihua, YIN Yulong, TANG Yulong. Functional Study on SodA,TrxA and TrxC Genes of Streptococcus suis Type 2[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1428-1436.

参考文献

[1] TANG Y L,WU W，ZHANG X Y, et al. Catabolite control protein A of Streptococcus suis type 2 contributes to sugar metabolism and virulence[J].Journal of Microbiology,2012,50(6):994-1002.
[2] TANG Y L,ZHANG X Y,YIN Y L,et al.Streptococcus suis type 2 SSU0587 protein is a beta-galactosidase that contributes to bacterial adhesion but not to virulence in mice[J].Journal of Veterinary Medical Science, 2014,76(7):1055-1059.
[3] TANG Y L,ZHANG X Y,WU W, et al.Inactivation of the sodA gene of Streptococcus suis type 2 encoding superoxide dismutase leads to reduced virulence to mice[J]. Veterinary Microbiology,2012,158(3-4):360-366.
[4] FANG L H,SHEN H X,TANG Y L, et al.Superoxide dismutase of Streptococcus suis serotype 2 plays a role in anti-autophagic response by scavenging reactive oxygen species in infected macrophages [J].Veterinary Microbiology, 2015,176(3-4):328-336.
[5] LU J,HOLMGREN A.The thioredoxin antioxidant system[J]. Free Radical Biology and Medicine, 2014,66:75-87.
[6] ZHAO C J,JIA X L,PAN Y Y,et al.Thioredoxin A of Streptococcus suis serotype 2 contributes to virulence by inhibiting the expression of pentraxin 3 to promote survival within macrophages[J].Journal of Microbiology,2023,61(4):433-448.
[7] JI C X,PAN Y Y,LIU B C,et al.Thioredoxin C of Streptococcus suis serotype 2 contributes to virulence by inducing antioxidative stress and inhibiting autophagy via the MSR1/PI3K-Akt-mTOR pathway in macrophages[J].Veterinary Microbiology,2024,298:110263.
[8] DENG S M,LIAO J H,LI H J,et al.Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection[J].Virulence,2024,15(1):2301246.
[9] LIU J,WANG J,ZHANG Z,et al.Streptococcus suis deploys multiple ATP-dependent proteases for heat stress adaptation[J].Journal of Basic Microbiology,2024,64(9):e2400030.
[10] ZHANG Y M,LIANG S,PAN Z H, et al.XRE family transcriptional regulator XtrSs modulates Streptococcus suis fitness under hydrogen peroxide stress[J].Archives of Microbiology,2022,204(5):244.
[11] BROXTON C N,CULOTTA V C.SOD enzymes and microbial pathogens:Surviving the oxidative storm of infection [J].PLoS Pathogens, 2016,12(1):e1005295.
[12] GAO Q Q,XIA L,WANG X B,et al.SodA contributes to the virulence of avian pathogenic Escherichia coli O2 strain E058 in experimentally tnfected chickens[J].Journal of Bacteriology,2019,201(6):00625-18.
[13] STAERCK C,GASTEBOIS A,VANDEPUTTE P,et al.Microbial antioxidant defense enzymes[J].Microbial Pathogenesis,2017,110:56-65.
[14] YANY B,LIN Y,HUANG Y,et al.Thioredoxin (Trx):A redox target and modulator of cellular senescence and aging-related diseases[J].Redox Biology，2024,70:103032.
[15] SIES H,BERNDT C,JONES D P.Oxidative stress[J].Annual Review of Biochemistry， 2017,86:715-748.
[16] MAY H C,YU J J,ZHANG H,et al.Thioredoxin-A is a virulence factor and mediator of the type Ⅳ pilus system in Acinetobacter baumannii[J].PLoS One，2019，14(7):e0218505.
[17] HE J J,LIU S,FANG Q J,The thioredoxin system in edwardsiella piscicida contributes to oxidative stress tolerance,motility,and virulence[J].Microorganisms，2023，11(4):827.
[18] CHEN M M,ZHANG J X,XIA J,et al.Listeria monocytogenes GshF contributes to oxidative stress tolerance via regulation of the phosphoenolpyruvate-carbohydrate phosphotransferase system[J].Microbiology Spectrum，2023,11(5):e0236523.
[19] CHENG C Y,DONG Z M,HAN X,et al.Thioredoxin A is essential for motility and contributes to host infection of Listeria monocytogenes via redox interactions[J].Frontiers in Cellular and Infection Microbiology,2017，7:287.
[20] RUHLAND B R,RENIERE M L.YjbH requires its thioredoxin active motif for the nitrosative stress response,cell-to-cell spread,and protein-protein interactions in Listeria monocytogenes [J].Journal of Bacteriology，2020,202(12):e00099-20.
[21] SEGURA M,FITTIPALDI N,CALZAS C,et al.Critical Streptococcus suis virulence factors:Are they all really critical[J] Trends in Microbiology,2017,25(7):585-599.
[22] TAYLOR J P,TSE H M.The role of NADPH oxidases in infectious and inflammatory diseases[J].Redox Biology,2021,48:102159.