Full Text:   <427>

Summary:  <158>

Suppl. Mater.: 

CLC number: 

On-line Access: 2023-06-13

Received: 2022-12-27

Revision Accepted: 2023-03-01

Crosschecked: 2023-07-21

Cited: 0

Clicked: 602

Citations:  Bibtex RefMan EndNote GB/T7714


Mingliang JIN


Yizhen WANG


-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2023 Vol.24 No.6 P.496-509


Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet

Author(s):  Chaoyue WEN, Hong ZHANG, Qiuping GUO, Yehui DUAN, Sisi CHEN, Mengmeng HAN, Fengna LI, Mingliang JIN, Yizhen WANG

Affiliation(s):  Institute of Feed Science, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Key Laboratory of Animal Nutrition and Feed Science in Eastern China, Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; more

Corresponding email(s):   yzwang321@zju.edu.cn, mljin@zju.edu.cn

Key Words:  Engineered probiotics, Intestine, Oxidative injury, Weaned piglets, Nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway

Chaoyue WEN, Hong ZHANG, Qiuping GUO, Yehui DUAN, Sisi CHEN, Mengmeng HAN, Fengna LI, Mingliang JIN, Yizhen WANG. Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet[J]. Journal of Zhejiang University Science B, 2023, 24(6): 496-509.

@article{title="Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet",
author="Chaoyue WEN, Hong ZHANG, Qiuping GUO, Yehui DUAN, Sisi CHEN, Mengmeng HAN, Fengna LI, Mingliang JIN, Yizhen WANG",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet
%A Chaoyue WEN
%A Qiuping GUO
%A Yehui DUAN
%A Sisi CHEN
%A Mengmeng HAN
%A Fengna LI
%A Mingliang JIN
%A Yizhen WANG
%J Journal of Zhejiang University SCIENCE B
%V 24
%N 6
%P 496-509
%@ 1673-1581
%D 2023
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2200674

T1 - Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet
A1 - Chaoyue WEN
A1 - Hong ZHANG
A1 - Qiuping GUO
A1 - Yehui DUAN
A1 - Sisi CHEN
A1 - Mengmeng HAN
A1 - Fengna LI
A1 - Mingliang JIN
A1 - Yizhen WANG
J0 - Journal of Zhejiang University Science B
VL - 24
IS - 6
SP - 496
EP - 509
%@ 1673-1581
Y1 - 2023
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2200674

engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)‍-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‍‒‍14 and all infused with ETEC K88 1×1010 CFU on Days 15‍‒‍17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

工程枯草芽孢杆菌通过Nrf2-Keap1途径缓解ETEC K88感染仔猪导致的肠道氧化损伤

1饲料科学研究所, 浙江省动物饲料与营养重点实验室,教育部分子动物营养重点实验室, 农业部华东动物营养与饲料重点实验室, 浙江大学动物科学学院,中国杭州市,310058
2动物营养生理与代谢过程湖南省重点实验室, 中国科学院亚热带农业生态过程重点实验室, 畜禽养殖污染控制与资源化利用国家工程实验室, 中国科学院亚热带农业生态研究所,中国长沙市,410125
3中国科学院大学现代农业科学学院, 中国北京市,100039
摘要: 工程益生菌具有产生重组免疫刺激物质的特性,可以作为一种治疗药物。本研究使用基因工程技术构建了表达抗菌肽KR32的重组枯草芽孢杆菌(WB800-KR32),并且探究了其在通过激活Nrf2-Keap1途径对产肠毒素大肠埃希氏菌(ETEC) K88感染断奶仔猪导致的肠道氧化态紊乱的保护作用。我们将28头断奶仔猪随机分成4组,每组7个重复,均饲喂基础日粮。对照组灌喂灭菌生理盐水;ETEC组、ETEC+WB800组和ETEC+WB800-KR32组分别在第1~14天灌喂灭菌生理盐水、5×1010 CFU WB800、5×1010 CFU WB800-KR32,在第15-17天灌喂ETEC K88 1×1010 CFU。结果表明,WB800-KR32预处理能够缓解ETEC K88导致的肠道紊乱,提高肠道粘膜抗氧化酶活性(过氧化物酶、超氧化物歧化酶和谷胱甘肽过氧化物酶),降低丙二醛含量。更重要的是,WB800-KR32预处理可上调回肠粘膜Nrf2的蛋白表达量,同时下调Keap1的蛋白表达量。此外,WB800-KR32预处理还显著改变了粪便微生物的丰度(Ace和Chao指数),并增加了Eubacterium_rectale_ ATCC_33656在粪便中的丰度。综上,WB800-KR32可能通过Nrf2-Keap1途径缓解ETEC K88导致的肠道氧化损伤,这为将WB800-KR32作为调节ETEC K88感染导致的肠道氧化失调的潜在治疗手段提供了一个新的视角。


Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]AyabeT, SatchellDP, WilsonCL, et al., 2000. Secretion of microbicidal α‍-‍defensins by intestinal Paneth cells in response to bacteria. Nat Immunol, 1(2):‍113-118.

[2]BäckhedF, FraserCM, RingelY, et al., 2012. Defining a healthy human gut microbiome: current concepts, future directions, and clinical applications. Cell Host Microbe, 12(5):611-622.

[3]ChuFF, EsworthyRS, DoroshowJH, 2004. Role of Se-dependent glutathione peroxidases in gastrointestinal inflammation and cancer. Free Radic Biol Med, 36(12):1481-1495.

[4]CruzKCP, EnekeghoLO, StuartDT, 2022. Bioengineered probiotics: synthetic biology can provide live cell therapeutics for the treatment of foodborne diseases. Front Bioeng Biotechnol, 10:890479.

[5]DuanYH, ZengLM, LiFN, et al., 2017. Effect of branched-chain amino acid ratio on the proliferation, differentiation, and expression levels of key regulators involved in protein metabolism of myocytes. Nutrition, 36:8-16.

[6]EsworthyRS, SwiderekKM, HoYS, et al., 1998. Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine. Biochim Biophys Acta, 1381(2):213-226.

[7]FanPX, LiuP, SongPX, et al., 2017. Moderate dietary protein restriction alters the composition of gut microbiota and improves ileal barrier function in adult pig model. Sci Rep, 7:43412.

[8]FlorianS, WinglerK, SchmehlK, et al., 2001. Cellular and subcellular localization of gastrointestinal glutathione peroxidase in normal and malignant human intestinal tissue. Free Radic Res, 35(6):655-663.

[9]GóthL, RassP, PáyA, 2004. Catalase enzyme mutations and their association with diseases. Mol Diagn, 8(3):‍141-149.

[10]GuanGP, DingSJ, YinYL, et al., 2019. Macleaya cordata extract alleviated oxidative stress and altered innate immune response in mice challenged with enterotoxigenic Escherichia coli. Sci China Life Sci, 62(8):1019-1027.

[11]GuilloteauP, ZabielskiR, HammonHM, et al., 2010. Nutritional programming of gastrointestinal tract development. Is the pig a good model for man? Nutr Res Rev, 23(1):4-22.

[12]GuoPT, ZhangK, MaX, et al., 2020. Clostridium species as probiotics: potentials and challenges. J Anim Sci Biotechnol, 11:24.

[13]HuWY, YangYY, LiZ, et al., 2019. Antibacterial, cytotoxicity and mechanism of the antimicrobial peptide KR-32 in weaning piglets. Int J Pept Res Ther, 26(2):943-953.

[14]HuangJJ, BaiYM, XieWT, et al., 2023. Lycium barbarum polysaccharides ameliorate canine acute liver injury by reducing oxidative stress, protecting mitochondrial function, and regulating metabolic pathways. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 24:157-171.

[15]IghodaroOM, AkinloyeOA, 2018. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alex J Med, 54(4):287-293.

[16]JinML, ZhangH, ZhaoK, et al., 2018. Responses of intestinal mucosal barrier functions of rats to simulated weightlessness. Front Physiol, 9:729.

[17]LekshmiM, AmminiP, KumarS, et al., 2017. The food production environment and the development of antimicrobial resistance in human pathogens of animal origin. Microorganisms, 5(1):11.

[18]LiFN, DuanYH, LiYH, et al., 2015. Effects of dietary n-6:n-3 PUFA ratio on fatty acid composition, free amino acid profile and gene expression of transporters in finishing pigs. Br J Nutr, 113(5):739-748.

[19]LiH, MaLT, LiZQ, et al., 2021. Evolution of the gut microbiota and its fermentation characteristics of Ningxiang pigs at the young stage. Animals (Basel), 11(3):638.

[20]LiWF, ZhouXX, LuP, 2004. Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol, 155(8):605-610.

[21]LiuH, WangJ, HeT, et al., 2018. Butyrate: a double-edged sword for health? Adv Nutr, 9(1):21-29.

[22]LiuHY, CaoXX, WangH, et al., 2019. Antimicrobial peptide KR-32 alleviates Escherichia coli K88-induced fatty acid malabsorption by improving expression of fatty acid transporter protein 4 (FATP4). J Anim Sci, 97(6):2342-2356.

[23]LiuSN, ZhangB, XiangDC, et al., 2021. Effect of Pediococcus pentosaceus 368 on grow performance, fecal microbiota and metabolite in pigs. Microbiol China, 48(6):2035-2048 (in Chinese).

[24]LuanC, ZhangHW, SongDG, et al., 2014a. Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol, 98(8):3651-3658.

[25]LuanC, XieYG, PuYT, et al., 2014b. Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli. Can J Microbiol, 60(3):113-120.

[26]LuiseD, LauridsenC, BosiP, et al., 2019. Methodology and application of Escherichia coli F4 and F18 encoding infection models in post-weaning pigs. J Anim Sci Biotechnol, 10:53.

[27]LyakhovichVV, VavilinVA, ZenkovNK, et al., 2006. Active defense under oxidative stress. The antioxidant responsive element. Biochemistry (Mosc), 71(9):962-974.

[28]NandiA, YanLJ, JanaCK, et al., 2019. Role of catalase in oxidative stress- and age-associated degenerative diseases. Oxid Med Cell Longev, 2019:9613090.

[29]National Research Council, 2012. Nutrients Requirements of Swine, 11th Ed. National Academy Press, Washington, USA, p.20-26.

[30]RajputSA, LiangSJ, WangXQ, et al., 2021. Lycopene protects intestinal epithelium from deoxynivalenol-induced oxidative damage via regulating Keap1/Nrf2 signaling. Antioxidants, 10(9):1493.

[31]RenM, CaiS, ZhouT, et al., 2019. Isoleucine attenuates infection induced by E. coli challenge through the modulation of intestinal endogenous antimicrobial peptide expression and the inhibition of the increase in plasma endotoxin and IL-6 in weaned pigs. Food Funct, 10(6):3535-3542.

[32]RiviereA, GagnonM, WeckxS, et al., 2015. Mutual cross-feeding interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 explain the bifidogenic and butyrogenic effects of arabinoxylan oligosaccharides. Appl Environ Microbiol, 81(22):7767-7781.

[33]RouraE, KoopmansSJ, LallesJP, et al., 2016. Critical review evaluating the pig as a model for human nutritional physiology. Nutr Res Rev, 29(1):60-90.

[34]ShiY, HuY, WangZQ, et al., 2022. The Protective effect of taurine on oxidized fish-oil-induced liver oxidative stress and intestinal barrier-function impairment in juvenile Ictalurus punctatus. Antioxidants, 10(11):1690.

[35]SmithF, ClarkJE, OvermanBL, et al., 2010. Early weaning stress impairs development of mucosal barrier function in the porcine intestine. Am J Physiol Gastrointest Liver Physiol, 298(3):G352-G363.

[36]TangYL, LiFN, TanB, et al., 2014. Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy. Vet Microbiol, 171(1-2):160-164.

[37]TsikasD, 2017. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: analytical and biological challenges. Anal Biochem, 524:13-30.

[38]WangJ, SuLQ, ZhangL, et al., 2022. Spirulina platensis aqueous extracts ameliorate colonic mucosal damage and modulate gut microbiota disorder in mice with ulcerative colitis by inhibiting inflammation and oxidative stress. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 23(6):481-501.

[39]WangRJ, LiuN, YangYC, et al., 2021. Flavor supplementation during late gestation and lactation periods increases the reproductive performance and alters fecal microbiota of the sows. Anim Nutr, 7(3):679-687.

[40]WenCY, LiFN, DuanYH, et al., 2019. Dietary taurine regulates free amino acid profiles and taurine metabolism in piglets with diquat-induced oxidative stress. J Funct Foods, 62:103569.

[41]WenCY, LiFN, GuoQP, et al., 2020a. Protective effects of taurine against muscle damage induced by diquat in 35 days weaned piglets. J Anim Sci Biotechnol, 11:56.

[42]WenCY, GuoQP, WangWL, et al., 2020b. Taurine alleviates intestinal injury by mediating tight junction barriers in diquat-challenged piglet models. Front Physiol, 11:449.

[43]WenCY, LiSY, WangJJ, et al., 2021. Heat stress alters the intestinal microbiota and metabolomic profiles in mice. Front Microbiol, 12:706772.

[44]WierupM, 2001. The Swedish experience of the 1986 year ban of antimicrobial growth promoters, with special reference to animal health, disease prevention, productivity, and usage of antimicrobials. Microb Drug Resist, 7(2):183-190.

[45]WuT, ShiYT, ZhangYY, et al., 2021. Lactobacillus rhamnosus LB1 alleviates enterotoxigenic Escherichia coli-induced adverse effects in piglets by improving host immune response and anti-oxidation stress and restoring intestinal integrity. Front Cell Infect Microbiol, 11:724401.

[46]WuX, ZhangY, LiuZ, et al., 2012. Effects of oral supplementation with glutamate or combination of glutamate and N-carbamylglutamate on intestinal mucosa morphology and epithelium cell proliferation in weanling piglets. J Anim Sci, 90(S4):337-339.

[47]XiaXJ, ZhangXL, LiuMC, et al., 2021. Toward improved human health: efficacy of dietary selenium on immunity at the cellular level. Food Funct, 12(3):976-989.

[48]XiaYY, BinP, LiuSJ, et al., 2018. Enterotoxigenic Escherichia coli infection promotes apoptosis in piglets. Microb Pathog, 125:290-294.

[49]XiaYY, ChenSY, ZhaoYY, et al., 2019. GABA attenuates ETEC-induced intestinal epithelial cell apoptosis involving GABAAR signaling and the AMPK-autophagy pathway. Food Funct, 10(11):7509-7522.

[50]XieWC, SongLY, WangX, et al., 2021. A bovine lactoferricin-lactoferrampin-encoding Lactobacillus reuteri CO21 regulates the intestinal mucosal immunity and enhances the protection of piglets against enterotoxigenic Escherichia coli K88 challenge. Gut Microbes, 13(1):1956281.

[51]XiongW, HuangJ, LiXY, et al., 2020. Icariin and its phosphorylated derivatives alleviate intestinal epithelial barrier disruption caused by enterotoxigenic Escherichia coli through modulate p38 MAPK in vivo and in vitro. FASEB J, 34(1):1783-1801.

[52]YangB, YueY, ChenY, et al., 2021. Lactobacillus plantarum CCFM1143 alleviates chronic diarrhea via inflammation regulation and gut microbiota modulation: a double-blind, randomized, placebo-controlled study. Front Immunol, 12:746585.

[53]YangWY, ChouCH, WangC, 2022. The effects of feed supplementing Akkemansia muciniphila on incidence, severity, and gut microbiota of necrotic enteritis in chickens. Poult Sci, 101(4):101751.

[54]YinJ, WuMM, XiaoH, et al., 2014. Development of an antioxidant system after early weaning in piglets. J Anim Sci, 92(2):612-619.

[55]YounisNS, AbduldaiumMS, MohamedME, 2020. Protective effect of geraniol on oxidative, inflammatory and apoptotic alterations in isoproterenol-induced cardiotoxicity: role of the Keap1/Nrf2/HO-1 and PI3K/Akt/mTOR pathways. Antioxidants, 9(10):977.

[56]YuE, ChenDW, YuB, et al., 2021. Amelioration of enterotoxigenic Escherichia coli-induced disruption of intestinal epithelium by manno-oligosaccharide in weaned pigs. J Funct Foods, 82:104492.

[57]ZhangQS, WidmerG, TziporiS, 2013. A pig model of the human gastrointestinal tract. Gut Microbes, 4(3):193-200.

[58]ZhouJ, XiongX, YinJ, et al., 2019. Dietary lysozyme alters sow’s gut microbiota, serum immunity and milk metabolite profile. Front Microbiol, 10:177.

[59]ZongX, FuJ, XuBC, et al., 2020. Interplay between gut microbiota and antimicrobial peptides. Anim Nutr, 6(4):389-396.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2024 Journal of Zhejiang University-SCIENCE