Abstract
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.
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.
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References
Ayabe T, Satchell DP, Wilson CL, et al., 2000. Secretion of microbicidal α-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol, 1(2): 113–118. https://doi.org/10.1038/77783
Bäckhed F, Fraser CM, Ringel Y, et al., 2012. Defining a healthy human gut microbiome: current concepts, future directions, and clinical applications. Cell Host Microbe, 12(5):611–622. https://doi.org/10.1016/j.chom.2012.10.012
Chu FF, Esworthy RS, Doroshow JH, 2004. Role of Sedependent glutathione peroxidases in gastrointestinal inflammation and cancer. Free Radic Biol Med, 36(12):1481–1495. https://doi.org/10.1016/j.freeradbiomed.2004.04.010
Cruz KCP, Enekegho LO, Stuart DT, 2022. Bioengineered probiotics: synthetic biology can provide live cell therapeutics for the treatment of foodborne diseases. Front Bioeng Biotechnol, 10:890479. https://doi.org/10.3389/fbioe.2022.890479
Duan YH, Zeng LM, Li FN, 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. https://doi.org/10.1016/j.jiut.2016.10.016
Esworthy RS, Swiderek KM, Ho YS, 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. https://doi.org/10.1016/S0304-4165(98)00032-4
Fan PX, Liu P, Song PX, 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. https://doi.org/10.1038/srep43412
Florian S, Wingler K, Schmehl K, 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. https://doi.org/10.1080/10715760100301181
Góth L, Rass P, Páy A, 2004. Catalase enzyme mutations and their association with diseases. Mol Diagn, 8(3):141–149. https://doi.org/10.1007/BF03260057
Guan GP, Ding SJ, Yin YL, 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. https://doi.org/10.1007/s11427-018-9494-6
Guilloteau P, Zabielski R, Hammon HM, et al., 2010. Nutritional programming of gastrointestinal tract development. Is the pig a good model for man? Nutr Res Rev, 23(1):4–22. https://doi.org/10.1017/S0954422410000077
Guo PT, Zhang K, Ma X, et al., 2020. Clostridium species as probiotics: potentials and challenges. J Anim Sci Biotechnol, 11:24. https://doi.org/10.1186/s40104-019-0402-1
Hu WY, Yang YY, Li Z, 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. https://doi.org/10.1007/s10989-019-09898-0
Huang JJ, Bai YM, Xie WT, 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. https://doi.org/10.1631/jzus.B2200213
Ighodaro OM, Akinloye OA, 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. https://doi.org/10.1016/j.ajme.2017.09.001
Jin ML, Zhang H, Zhao K, et al., 2018. Responses of intestinal mucosal barrier functions of rats to simulated weightlessness. Front Physiol, 9:729. https://doi.org/10.3389/fphys.2018.00729
Lekshmi M, Ammini P, Kumar S, et al., 2017. The food production environment and the development of antimicrobial resistance in human pathogens of animal origin. Microorganisms, 5(1):11. https://doi.org/10.3390/microorganisms5010011
Li FN, Duan YH, Li YH, 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. https://doi.org/10.1017/S0007114514004346
Li H, Ma LT, Li ZQ, et al., 2021. Evolution of the gut micro-biota and its fermentation characteristics of Ningxiang pigs at the young stage. Animals (Basel), 11(3):638. https://doi.org/10.3390/ani11030638
Li WF, Zhou XX, Lu P, 2004. Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol, 155(8):605–610. https://doi.org/10.1016/j.resmic.2004.05.002
Liu H, Wang J, He T, et al., 2018. Butyrate: a double-edged sword for health? Adv Nutr, 9(1):21–29. https://doi.org/10.1093/advances/nmx009
Liu HY, Cao XX, Wang H, 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. https://doi.org/10.1093/jas/skz110
Liu SN, Zhang B, Xiang DC, 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). https://doi.org/10.13344/j.microbiol.china.200898
Luan C, Zhang HW, Song DG, et al., 2014a. Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol, 98(8):3651–3658. https://doi.org/10.1007/s00253-013-5246-6
Luan C, Xie YG, Pu YT, 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. https://doi.org/10.1139/cjm-2013-0652
Luise D, Lauridsen C, Bosi P, 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. https://doi.org/10.1186/s40104-019-0352-7
Lyakhovich VV, Vavilin VA, Zenkov NK, et al., 2006. Active defense under oxidative stress. The antioxidant responsive element. Biochemistry (Mosc), 71(9):962–974. https://doi.org/10.1134/S0006297906090033
Nandi A, Yan LJ, Jana CK, et al., 2019. Role of catalase in oxidative stress- and age-associated degenerative diseases. Oxid Med Cell Longev, 2019:9613090. https://doi.org/10.1155/2019/9613090
National Research Council, 2012. Nutrients Requirements of Swine, 11th Ed. National Academy Press, Washington, USA, p.20–26.
Rajput SA, Liang SJ, Wang XQ, et al., 2021. Lycopene protects intestinal epithelium from deoxynivalenol-induced oxidative damage via regulating Keap1/Nrf2 signaling. Antioxidants, 10(9):1493. https://doi.org/10.3390/antiox10091493
Ren M, Cai S, Zhou T, 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. https://doi.org/10.1039/C9FO00218A
Riviere A, Gagnon M, Weckx S, 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. https://doi.org/10.1128/AEM.02089-15
Roura E, Koopmans SJ, Lalles JP, et al., 2016. Critical review evaluating the pig as a model for human nutritional physiology. Nutr Res Rev, 29(1):60–90. https://doi.org/10.1017/S0954422416000020
Shi Y, Hu Y, Wang ZQ, 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. https://doi.org/10.3390/antiox10111690
Smith F, Clark JE, Overman BL, 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. https://doi.org/10.1152/ajpgi.00081.2009
Tang YL, Li FN, Tan B, et al., 2014. Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy. Vet Microbiol, 171(1-2):160–164. https://doi.org/10.1016/j.vetmic.2014.03.025
Tsikas D, 2017. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: analytical and biological challenges. Anal Biochem, 524:13–30. https://doi.org/10.1016/j.ab.2016.10.021
Wang J, Su LQ, Zhang L, 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. https://doi.org/10.1631/jzus.B2100988
Wang RJ, Liu N, Yang YC, 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. https://doi.org/10.1016/j.aninu.2021.01.007
Wen CY, Li FN, Duan YH, 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. https://doi.org/10.1016/j.jff.2019.103569
Wen CY, Li FN, Guo QP, et al., 2020a. Protective effects of taurine against muscle damage induced by diquat in 35 days weaned piglets. J Anim Sci Biotechnol, 11:56. https://doi.org/10.1186/s40104-020-00463-0
Wen CY, Guo QP, Wang WL, et al., 2020b. Taurine alleviates intestinal injury by mediating tight junction barriers in diquat-challenged piglet models. Front Physiol, 11:449. https://doi.org/10.3389/fphys.2020.00449
Wen CY, Li SY, Wang JJ, et al., 2021. Heat stress alters the intestinal microbiota and metabolomic profiles in mice. Front Microbiol, 12:706772. https://doi.org/10.3389/fmicb.2021.706772
Wierup M, 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. https://doi.org/10.1089/10766290152045066
Wu T, Shi YT, Zhang YY, 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. https://doi.org/10.3389/fcimb.2021.724401
Wu X, Zhang Y, Liu Z, 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. https://doi.org/10.2527/jas.53752
Xia XJ, Zhang XL, Liu MC, et al., 2021. Toward improved human health: efficacy of dietary selenium on immunity at the cellular level. Food Funct, 12(3):976–989. https://doi.org/10.1039/D0FO03067H
Xia YY, Bin P, Liu SJ, et al., 2018. Enterotoxigenic Escherichia coli infection promotes apoptosis in piglets. Microb Pathog, 125:290–294. https://doi.org/10.1016/j.micpath.2018.09.032
Xia YY, Chen SY, Zhao YY, 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. https://doi.org/10.1039/C9FO01863H
Xie WC, Song LY, Wang X, 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. https://doi.org/10.1080/19490976.2021.1956281
Xiong W, Huang J, Li XY, 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. https://doi.org/10.1096/fj.201902265R
Yang B, Yue Y, Chen Y, 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. https://doi.org/10.3389/fimmu.2021.746585
Yang WY, Chou CH, Wang C, 2022. The effects of feed supplementing Akkemansia muciniphila on incidence, severity, and gut microbiota of necrotic enteritis in chickens. Poult Sci, 101(4):101751. https://doi.org/10.1016/j.psj.2022.101751
Yin J, Wu MM, Xiao H, et al., 2014. Development of an antioxidant system after early weaning in piglets. J Anim Sci, 92(2):612–619. https://doi.org/10.2527/jas.2013-6986
Younis NS, Abduldaium MS, Mohamed ME, 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. https://doi.org/10.3390/antiox9100977
Yu E, Chen DW, Yu B, et al., 2021. Amelioration of enterotoxigenic Escherichia coli-induced disruption of intestinal epithelium by manno-oligosaccharide in weaned pigs. J Funct Foods, 82:104492. https://doi.org/10.1016/j.jff.2021.104492
Zhang QS, Widmer G, Tzipori S, 2013. A pig model of the human gastrointestinal tract. Gut Microbes, 4(3):193–200. https://doi.org/10.4161/gmic.23867
Zhou J, Xiong X, Yin J, et al., 2019. Dietary lysozyme alters sow’s gut microbiota, serum immunity and milk metabolite profile. Front Microbiol, 10:177. https://doi.org/10.3389/fmicb.2019.00177
Zong X, Fu J, Xu BC, et al., 2020. Interplay between gut microbiota and antimicrobial peptides. Anim Nutr, 6(4):389–396. https://doi.org/10.1016/j.aninu.2020.09.002
Acknowledgments
This work was supported by the Zhejiang Provincial Key R&D Program of China (No. 2021C02008), the China Agriculture Research System of MOF and MARA (No. CARS-35), the National Natural Science Foundation of China (No. 32022079), the Fundamental Research Funds for the Central Universities (No. 2022QZJH46), and the Taishan Industrial Leading Talents Project.
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Chaoyue WEN performed the experimental research and data analysis, and wrote and edited the manuscript. Hong ZHANG, Qiuping GUO, Yehui DUAN, Sisi CHEN, Mengmeng HAN, and Fengna LI contributed to the study design, data analysis, and writing and editing of the manuscript. Mingliang JIN designed the experiment, and wrote and revised the manuscript. Yizhen WANG contributed to the study design and editing of the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Chaoyue WEN, Hong ZHANG, Qiuping GUO, Yehui DUAN, Sisi CHEN, Mengmeng HAN, Fengna LI, Mingliang JIN, and Yizhen WANG declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed. All animal procedures were approved by the Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences (No. ISA-2022-022).
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Wen, C., Zhang, H., Guo, Q. et al. Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet. J. Zhejiang Univ. Sci. B 24, 496–509 (2023). https://doi.org/10.1631/jzus.B2200674
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DOI: https://doi.org/10.1631/jzus.B2200674
Key words
- Engineered probiotics
- Intestine
- Oxidative injury
- Weaned piglets
- Nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway