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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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