Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet 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 first-author 1 2 2 2 3 2 3 2 1 corresp corresp Yizhen WANG, yzwang321@zju.edu.cn Yizhen WANG, yzwang321@zju.edu.cn Mingliang JIN, mljin@zju.edu.cn Mingliang JIN, mljin@zju.edu.cn mljin@zju.edu.cn https://orcid.org/0000-0001-6358-5307 1 corresp Yizhen WANG, yzwang321@zju.edu.cn Yizhen WANG, yzwang321@zju.edu.cn yzwang321@zju.edu.cn https://orcid.org/0000-0002-3908-7102 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 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 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 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 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 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 Hunan Provincial Key Laboratory of Animal Nutritional Physiology and Metabolic Process, CAS Key Laboratory of Agro-Ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China Hunan Provincial Key Laboratory of Animal Nutritional Physiology and Metabolic Process, CAS Key Laboratory of Agro-Ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China 中国科学院大学现代农业科学学院， 中国北京市， 100039 中国科学院大学现代农业科学学院， 中国北京市， 100039 College of Advanced Agricultural Science, University of Chinese Academy of Sciences, Beijing 100039, China College of Advanced Agricultural Science, University of Chinese Academy of Sciences, Beijing 100039, China 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, 5×10 10 CFU (CFU: colony forming units) WB800, and 5×10 10 CFU WB800-KR32, respectively, on Days 1‍‒‍14 and all infused with ETEC K88 1×10 10 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、5×10 10 CFU WB800、5×10 10 CFU WB800-KR32，在第15–17天灌喂ETEC K88 1×10 10 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。 工程益生菌 肠道 氧化损伤 断奶仔猪 Nrf2-Keap1途径 Engineered probiotics Intestine Oxidative injury Weaned piglets Nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway 1 Introduction 1 Enterotoxigenic Escherichia coli (ETEC) is a common pathogen of the gut microbiota, causing diarrhea both in humans and animals, especially in the neonatal and suckling periods ( Luise et al., 2019 Luise et al., 2019 ). It has been reported that ETEC can damage the epithelial barrier, increase intestinal permeability ( Tang et al., 2014 Tang et al., 2014 ), induce apoptotic processes ( Xia et al., 2018 Xia et al., 2018 , 2019 2019 ), and disturb the harmony of gut flora ( Xie et al., 2021 Xie et al., 2021 ). As gut microbes will affect the future growth and health of the host ( Bäckhed et al., 2012 Bäckhed et al., 2012 ; Xie et al., 2021 Xie et al., 2021 ), it is essential to maintain their homeostasis. ETEC infection is characterized by the imbalance of intestinal antioxidant homeostasis, with reduced activity of antioxidant enzymes such as glutathione peroxidase (GPx) and superoxide dismutase (SOD), the upregulation of inflammatory responses, and downregulation of antioxidant genes in the intestine ( Yu et al., 2021 Yu et al., 2021 ). It is universally acknowledged that the use of antibiotics constitutes the conventional means to treat pathogen infections. However, due to the food safety reasons and to prevent the development of resistant strains, antibiotics are being phased out from animal feeds ( Wierup, 2001 Wierup, 2001 ; Lekshmi et al., 2017 Lekshmi et al., 2017 ). Consequently, the exploration of new strategies to prevent pathogen infections is extremely urgent. The antimicrobial peptide (AMP) KR32 was designed in our laboratory based on cathelicidin-BF, a natural AMP derived from snake venom ( Hu et al., 2019 Hu et al., 2019 ; Liu et al., 2019 Liu et al., 2019 ). It exhibited high antimicrobial activity with minimal hemolytic activity and cytotoxicity ( Hu et al., 2019 Hu et al., 2019 ). Subsequent studies confirmed that KR32 alleviates diarrhea on post-weaning piglets and decreases the level of inflammatory cytokines ( Hu et al., 2019 Hu et al., 2019 ). However, it is time-consuming and costly to synthesize and purify AMPs ( Luan et al., 2014a Luan et al., 2014a , 2014b 2014b ). Bioengineered probiotics have been demonstrated to be effective against pathogens that commonly infect the gut ( Cruz et al., 2022 Cruz et al., 2022 ). Bacillus subtilis has been considered safe by the United States Food and Drug Administration, which acts as a superb delivery vehicle to produce AMPs ( Luan et al., 2014a Luan et al., 2014a ). Additionally, this species has a naturally large secretory capacity and exports proteins directly into the extracellular medium ( Li et al., 2004 Li et al., 2004 ). Therefore, we constructed a plasmid based on pBE2R, which is a composition-type plasmid for producing AMP KR32 in the B. subtilis expression system. In this study, we used B. subtilis as a delivery vehicle by developing a recombinant strain expressing KR32. The nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway is important in resistance to oxidative stress in the intestine ( Rajput et al., 2021 Rajput et al., 2021 ; Shi et al., 2022 Shi et al., 2022 ; Huang et al., 2023 Huang et al., 2023 ). Nrf2 is an oxidation-reduction-sensitive transcription factor that stimulates antioxidant enzyme expression, thus moderating oxidative injury ( Younis et al., 2020 Younis et al., 2020 ; Wang et al., 2022 Wang et al., 2022 ). Piglets were selected as the model for the similarity of their gastrointestinal tract structure and function to those of human beings ( Guilloteau et al., 2010 Guilloteau et al., 2010 ; Zhang et al., 2013 Zhang et al., 2013 ; Roura et al., 2016 Roura et al., 2016 ). We aimed to validate the hypothesis that recombinant B. subtilis WB800-pBE2R-KR32 could improve intestinal antioxidant capacity induced by ETEC K88 infection via the Nrf2-Keap1 pathway. Based on the findings, the present study provides a scientific basis for treating ETEC-induced impaired intestinal antioxidant capacity in humans and piglets. 2 Materials and methods 1 2.1 Bacterial strains 2 DH5α cells stored in our lab were used for subcloning and plasmid amplification. The B. subtilis WB800 strain (Forhigh Biotech, China) was utilized as the expression host, grown in Luria-Bertani (LB) broth (Sangon, Shanghai, China) at 37 ℃. The shuttle vector pBE2R (MiaoLing Plasmid Platform) was employed for the secretion of target protein (peptide). T4 DNA ligase, Taq DNA polymerase, and all restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA). The KR32 gene was synthesized and inserted into the pBE2R vector by MiaoLing Plasmid Platform (Wuhan, China). The map of the obtained recombinant plasmid pBE2R-KR32 was shown in Fig. S1. This plasmid was then electro-transformed into competent WB800, which was further plated on LB plates (with 50 μg/mL kanamycin) for positive selection to generate B. subtilis WB800-pBE2R-KR32 (WB800-KR32). B. subtilis WB800 transformed with pBE2R plasmid was used as negative control. The E. coli K88 strain used in this study was available from our lab ( Hu et al., 2019 Hu et al., 2019 ) and was cultured in LB broth containing 1% (10 g/L) tryptone, 0.5% (5 g/L) yeast extract, and 1% (10 g/L) NaCl at pH 7.0 for 12 h to reach saturation (≥1.0×10 8 CFU/mL; CFU: colony forming units). The bacterial cells were harvested by centrifugation at 3000 g for 10 min at 4 ℃, washed, and suspended in sterile saline. A solution of ETEC K88 strain containing about 1×10 9 CFU/mL was prepared. 2.2 Animals and experimental design 2 All animal procedures were approved by the Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences (No. ISA-2022-022). Twenty-eight Duroc×Landrace×Yorkshire piglets weaned at 21 d were caged in individual pens. After 3 d of adaption, the piglets ((7.09±0.25) kg) were randomly allocated into four groups ( Fig. 1 Fig. 1 ) ( n =7 per treatment): (1) oral administration of 10 mL 0.9% (9 g/L) NaCl (CON group); (2) oral administration of 10 mL 0.9% NaCl+ETEC challenge (ETEC group); (3) oral administration of 10 mL 5.0×10 9 CFU/mL WB800 bacteria solution+ETEC challenge (ETEC+WB800 group); (4) oral administration of 10 mL 5.0×10 9 CFU/mL WB800-KR32 bacteria solution+ETEC challenge (ETEC+WB800-KR32 group). On Day 15 08:‍00 a.m., all piglets were weighed. ETEC (1×10 9 CFU/mL, 10 mL) was orally administered to animals in the ETEC challenge group once a day for three days from Days 15 to ‍17. The cytokines (interleukin-6 (IL-6) and IL-1β) were increased significantly after three days of treatment with ETEC ( Ren et al., 2019 Ren et al., 2019 ). The doses and durations of ETEC K88 were set according to a previous study ( Ren et al., 2019 Ren et al., 2019 ). Piglets in the CON group were administered with 10 mL 0.9% NaCl. 2.3 Sample collection 2 Fecal samples were collected on Days 1, 14, and 17 from animals in the ETEC+WB800-KR32 group. The collected samples were stored at -80 ℃ until further analysis. On Day 15, prior to ETEC infection, blood samples were taken and centrifuged at 3000 g for 10 min, and then kept at -20 ℃ until further analysis. On Day 17, after 12 h of fasting, all piglets were weighed to evaluate the post-challenge growth performance, and blood samples were collected via vein puncture and then centrifuged at 3000 g for 10 min. The supernatants were collected and stored at -20 ℃ until further analysis. Finally, all piglets were slaughtered via electrical stunning (250 V, 0.5 A, 5–6 s) and bled by the exsanguination of precaval vein. The mucosae of duodenum, jejunum, and ileum were collected immediately, quickly frozen in liquid N 2 , and stored at -80 ℃ until further analysis. 2.4 Analysis of serum and intestinal antioxidant parameters 2 Catalase (CAT), malondialdehyde (MDA), SOD, GPx, and total antioxidant capacity (T-AOC) in the serum and intestinal (duodenum, jejunum, and ileum) mucosa were measured using commercial kits (Hunan AiFang biological, Changsha, China). 2.5 Gene expression analysis 2 Quantitative real-time polymerase chain reaction PCR (qPCR) was performed as described previously ( Li et al., 2015 Li et al., 2015 ; Duan et al., 2017 Duan et al., 2017 ; Wen et al., 2019 Wen et al., 2019 ). Total RNAs of jejunal and ileal mucosae were extracted using the TRIzol reagent (Beyotime Biotechnology, Shanghai, China). The primers for the target genes were listed in Table 2 Table 2 . β‍-actin was used as the housekeeping gene to normalize the expression of target genes. The expression levels of target genes were determined by the 2 <math id="M1"><msup><mrow/><mrow><mo>-</mo><mi mathvariant="normal">Δ</mi><mi mathvariant="normal">Δ</mi><msub><mrow><mi>C</mi></mrow><mrow><mi mathvariant="normal">T</mi></mrow></msub></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=45285884&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=45285837&type= 6.01133299 2.96333337 method. CAT , catalase; SOD1 , superoxide dismutase 1; Keap1 , Kelch-like ECH-associated protein 1; Nrf2 , nuclear factor erythroid 2-related factor 2; GPx , glutathione peroxidase. Gene Sequence (5'→3') Accession No. Size (bp) CAT F: CTCACAGCGAATACCCTC R: TGTTCAACCTCAGCAAAA NC_010444.4 82 SOD1 F: GAGACCTGGGCAATGTGACT R: CCAAACGACTTCCAGCATTT NC_010455.5 189 Keap1 F: TCAACCGTCTGCTCTACG R: CACTCATTCCTCTCTGGG NC_010444.3 465 Nrf2 F: GAAAGCCCAGTCTTCATTGC R: TTGGAACCGTGCTAGTCTCA NC_010457.5 190 GPx F: AGCCCAACTTCATGCTCTTC R: CATTGCGACACACTGGAGAC NC_010455.5 159 β-actin F: TGCGGGACATCAAGGAGAAG R: AGTTGAAGGTGGTCTCGTGG NC_010445.4 216 2.6 Western blotting analysis 2 The relative protein levels for Nrf2 and Keap1 were measured by western blotting according to our previous studies ( Wen et al., 2020a Wen et al., 2020a , 2020b 2020b ). The primary antibodies used in our study included anti-Nrf2, anti-Keap1, and mouse anti-β-actin. All the antibodies were purchased from ABclonal Technology (ABclonal Technology, Wuhan, China). The acquired signals were visualized via ChemiScope 6100 (Clinx, Shanghai, China). Table ‍ 1 ‍ Ingredients and chemical compositions of the experimental diets * All data are expressed in g/100 g dry mass except for digestible energy in MJ/kg. a Supplied in per kg of diet: CuSO 4 ·5H 2 O, 19.8 mg; KI, 0.20 mg; FeSO 4 ·7H 2 O, 400 mg; NaSeO 3 , 0.56 mg; ZnSO 4 ·7H 2 O, 359 mg; MnSO 4 ·H 2 O, 10.2 mg; vitamin K (menadione), 5 mg; vitamin B1, 2 mg; vitamin B2, 15 mg; vitamin B12, 30 μg; vitamin A, 5400 IU; vitamin D3, 110 IU; vitamin E, 18 IU; choline chloride, 80 mg; antioxidants: ethoxyquin, 20 mg. SID: standardized ileal digestible. Composition Proportion (%) * Ingredients Corn meal 59.88 Soybean meal 24.45 Fish meal 4.99 Dried whey 3.99 Soybean oil 2.00 Lysine 0.48 Methionine 0.09 Threonine 0.16 Tryptophan 0.04 NaCl 0.30 CaHPO 4 0.95 Glucose 0.80 Limestone 0.79 Premix a 1.09 Nutrient content Digestible energy (MJ/kg) 14.56 Crude protein 19.31 SID lysine 1.28 SID methionine+cysteine 0.64 SID threonine 0.77 SID tryptophan 0.23 Total calcium 0.85 Total phosphorus 0.63 Digestible phosphorus 0.39 2.7 Analysis of fecal microbiota 2 The microbial community DNA of fecal contents was extracted using the MagPure Stool DNA KF Kit B (Magen, China). The V3–V4 variable region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified with degenerate PCR primers (341F, 5'-ACTCCTACGGGAGGCAGCA-3'; 806R, 5'-GGACTACHVGGGTWTCTAAT-3') and sequenced on the Illumina MiSeq platform (BGI, Shenzhen, China). The assembled MiSeq sequences were uploaded to the Sequence Read Archive of the National Center for Biotechnology Information (NCBI, Sequence Read Archive (SRA) BioProject No. PRJNA894787). The raw reads were filtered and assembled according to described protocols ( Zhou et al., 2019 Zhou et al., 2019 ; Wen et al., 2021 Wen et al., 2021 ). The data were analyzed as previously described ( Jin et al., 2018 Jin et al., 2018 ). Briefly, to investigate bacterial richness and diversity, alpha-diversity was estimated by Mothur (v.1.30.2; https://mothur.org https://mothur.org ), including the Sobs, Chao, Ace, Shannon, and Simpson indices. Beta-diversity was estimated by Quantitative Insights Into Microbial Ecology (QIIME, v.1.8.0; http://qiime. http://qiime. ‍org/1.8.0) at the operational taxonomic unit (OTU) level by calculating Bray-Curtis dissimilarity, and then visualized by principal coordinate analysis (PCoA) and non-metric multi-dimensional scaling (NMDS). Kruskal-Wallis H-test was performed to estimate the significant differences between species. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functions were predicted by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software (v.1.1.0; http://picrust.github.io/picrust http://picrust.github.io/picrust ). 2.8 Statistical analysis 2 All results were expressed as mean±standard error of the mean (SEM). Serum biochemistry index data were analyzed using t -test. Other data were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison test using SAS 8.2 software (SAS Institute, NC, USA). Probability values of < 0.05 were considered statistically significant. 3 Results 1 3.1 Serum biochemical index 2 Prior to ETEC infection, oral WB800-KR32 showed no significant effects on the serum biochemical indices, including albumin (ALB), alkaline phosphatase (ALP), acid phosphatase 2 (ACP2), alanine aminotransferase (ALT), creatine kinase (CK), and immunoglobulin M (IgM). No significant differences were found between before and after infection, and therefore changes were not attributable to ETEC ( Table 3 Table 3 ). The results were expressed as mean±standard error of the mean (SEM), n =7. CON, basal diet infused with sterilized normal saline; ETEC, infected control (piglets receiving a basal diet plus oral dose infused with enterotoxigenic Escherichia coli (ETEC) K88); ETEC+WB800, piglets receiving a basal diet plus oral dose infused with 1×10 10 CFU (CFU: colony forming units) WB800 and ETEC K88; ETEC+WB800-KR32, piglets receiving a basal diet plus oral dose infused with 1×10 10 CFU WB800-KR32 and ETEC K88. ALB, albumin; ALP, alkaline phosphatase; ACP2, acid phosphatase 2; ALT, alanine aminotransferase; CK, creatine kinase; IgM, immunoglobulin M. P a : multivariate analysis of variance in prior to ETEC K88 infection; P b : multivariate analysis of variance in post to ETEC K88 infection; P 1 : prior to ETEC K88 infection (CON) vs. post-ETEC K88 infection (CON); P 2 : prior to ETEC K88 infection (CON) vs. post-ETEC K88 infection (ETEC); P 3 : prior to ETEC K88 infection (WB800) vs. post-ETEC K88 infection (ETEC+WB800); P 4 : prior to ETEC K88 infection (WB800-KR32) vs. post-ETEC K88 infection (ETEC+WB800-KR32). Treatment ALB (U/L) ALP (U/L) ACP2 (U/L) ALT (U/L) CK (U/L) IgM (g/L) 7 Prior to ETEC K88 infection CON 28.20±2.41 341.57±50.65 24.96±4.25 46.34±8.97 585.43±94.59 0.40±0.04 ETEC 32.60±2.05 426.57±30.36 21.86±4.51 43.73±1.89 338.83±57.31 0.42±0.04 WB800 32.11±1.42 351.43±29.30 25.71±5.18 48.09±6.54 607.14±189.98 0.44±0.04 WB800-KR32 30.16±2.37 344.14±30.36 30.48±3.03 50.61±5.40 468.00±85.52 0.38±0.03 7 After ETEC K88 infection CON 30.31±1.37 323.14±49.85 31.10±4.19 49.15±7.45 766.00±240.95 0.55±0.11 ETEC 31.74±1.51 291.29±19.88 22.50±3.85 43.17±1.95 662.50±105.74 0.54±0.06 ETEC+WB800 30.89±0.65 275.86±26.13 26.86±5.17 45.18±4.65 482.83±84.89 0.57±0.06 ETEC+WB800-KR32 28.20±1.83 272.86±29.30 28.46±3.36 49.91±3.58 442.83±31.50 0.46±0.03 7 P -value P a 0.45 0.31 0.57 0.88 0.42 0.73 P b 0.35 0.70 0.54 0.68 0.67 0.28 P 1 0.35 0.08 0.80 0.20 0.32 0.23 P 2 0.70 0.89 0.12 0.07 0.85 0.08 P 3 0.69 0.06 0.08 0.70 0.88 0.07 P 4 0.92 0.96 0.12 0.57 0.66 0.10 3.2 Serum and intestine antioxidant parameters 2 Compared with the CON group, the activity of CAT and T-AOC was significantly decreased in the ETEC and ETEC+WB800 groups ( P < 0.05; Figs. 2 2 a and 2 2 e). However, pretreatment with WB800-KR32 elevated the activity of CAT and T-AOC upon ETEC infection in the serum ( P < 0.05). Relative to the ETEC group, the activity of SOD was significantly increased in the duodenum ( P < 0.05; Fig. 2 Fig. 2 j). Relative to the CON group, ETEC infection decreased both T-AOC and the activity of GPx in the jejunum and ileum ( P < 0.05; Figs. 2 2 g, 2 2 h, 2 2 s, and 2t). Interestingly, these decrements caused by ETEC infection except for T-AOC in ileum were reversed by WB800-KR32 infusion. What is more, the content of MDA was significantly increased in the ETEC group in the jejunum, while the change was alleviated by WB800-KR32 treatment ( P < 0.05; Fig. 2 Fig. 2 o). 3.3 Antioxidant-related gene expression 2 Compared with the CON group, the messenger RNA (mRNA) expression of CAT was not significantly different from the ETEC group ( P > 0.1; Fig. 3 Fig. 3 a). Compared with the CON group, ETEC infection upregulated the mucosal GPx , Nrf2 , and SOD1 mRNA abundance in the ileum; pretreatment with WB800-KR32 could reverse the mRNA expression of GPx , Nrf2 , and SOD1 in ileal mucosae (Figs. 3 3 b, 3 3 d, and 3e). The mRNA expression of Keap1 was inconsistent in the jejunum and ileum of the ETEC group. Compared with the CON group, the mRNA expression of Keap1 was significantly upregulated in the ileum of piglets in the ETEC group; meanwhile, pretreatment with WB800-KR32 could alleviate the increment induced by ETEC infection in the ileum ( P < 0.05; Fig. 3 Fig. 3 c). 3.4 Antioxidant-related protein expression 2 Compared with the CON group, the Nrf2 protein was significantly decreased in the ETEC and ETEC+WB800 groups, and the Keap1 protein was significantly increased in the ETEC and ETEC+WB800 groups; pretreatment with WB800-KR32 could reverse these changes and resulted in no significant difference compared with the CON group ( Fig. 4 Fig. 4 ). 3.5 Fecal microbial community 2 Across all 21 samples, 978 683 high-quality sequences were identified, with an average length of 413.84 bp. Compared with the Day 1 group, the OTUs (Sobs) and richness estimators (Ace and Chao) were significantly increased on Days 14 and 17 ( P < 0.05; Figs. 5 5 a, 5 5 d, and 5e). Besides, no remarkable differences were found in the diversity indices (Shannon and Simpson) (Figs. 5 5 b and 5 5 c). We further investigated the shifts in bacterial taxa that were caused by oral WB800-KR32 (prior to WB800-KR32 and post-WB800-KR32 treatment for 14 d) and ETEC infection (3 d post-ETEC K88 infection). PCoA based on Bray-Curtis distance revealed the distinct clustering of microbiota composition for the three groups ( Fig. 6 Fig. 6 a). The analysis of similarities in Bray-Curtis distance indicated that oral WB800-KR32 and ETEC infection tended to be different ( P =0.001) with an R 2 -value of 0.2315, suggesting that the microbiota of the three groups were different. NMDS ordination plot based on the Bray-Curtis distance metric showed that the fecal bacterial communities in the samples could be differentiated by oral WB800-KR32 and ETEC infection ( Fig. 6 Fig. 6 b). The overall microbial composition of the three groups differed at the class level. The five largest classes represented in each group were Clostridia, Bacteroidia, Bacilli, Spirochaetia, and Negativicutes ( Fig. 6 Fig. 6 c). The abundance of Clostridia gradually increased in response to treatment with WB800-KR32 and ETEC. The abundance of Bacteroidia was significantly decreased after WB800-KR32 treatment for 14 d and significantly increased 3 d after ETEC infection. The abundance of Bacilli was significantly increased after WB800-KR32 treatment and significantly decreased after ETEC infection. At the species level, Eubacterium_rectale_ATCC_33656 was significantly increased in the Day 14 group ( Fig. 6 Fig. 6 d). We found that multiple KEGG (level 3) categories were disturbed (Figs. 6 6 e and 6 6 f). Specifically, the enriched pathways were membrane transport, glycan biosynthesis and metabolism, energy metabolism, and carbohydrate metabolism. Membrane transport and carbohydrate metabolism were significantly upregulated in the Day 14 group compared with Day 1 ( Fig. 6 Fig. 6 e). Replication and repair, as well as “drug resistance: antineoplastic” were significantly upregulated in the Day 14 group compared with Day 17 ( Fig. 6 Fig. 6 f). 4 Discussion 1 The intestinal mucosa is the main tissue of defense against various pathogens in the intestinal lumen ( Smith et al., 2010 Smith et al., 2010 ). However, it is easily attacked by different pathogenic bacteria in the weaning period, as the immune and antioxidant systems are not yet well-developed at this time ( Yin et al., 2014 Yin et al., 2014 ). Intestinal oxidative damage induced by ETEC is regarded as a severe health concern and is a main factor of morbidity and mortality in animals. KR32 is an AMP that has been reported to exert high antimicrobial activity in vitro and decrease the inflammatory response in vivo. The present study revealed that the therapeutic administration of WB800-KR32 improved host defense against ETEC K88 invasion-induced intestinal oxidative damage. The antioxidant and oxidant systems are normally in dynamic balance. Reactive oxygen species (ROS) are produced during cellular metabolism, while perturbations in the balance of antioxidants and the reactive species can lead to alterations in cell composition and the potential damage of cellular activities, collectively named as “oxidative stress.” The effectiveness of the first line of antioxidants including CAT, SOD, and GPx is vital for the whole defense strategy, especially against ROS that are perpetually generated during normal body metabolism. CAT is one of the essential antioxidant enzymes that alleviate oxidative stress to a substantial extent by destroying cellular hydrogen peroxide ( Nandi et al., 2019 Nandi et al., 2019 ), which underlines its importance in the aforementioned physiological processes ( Ighodaro and Akinloye, 2018 Ighodaro and Akinloye, 2018 ). The malfunction of CAT has been postulated to be linked to the pathogenesis of many pathogen infections ( Guan et al., 2019 Guan et al., 2019 ; Yu et al., 2021 Yu et al., 2021 ). In the present study, ETEC challenge significantly reduced the serum activity of CAT and the T-AOC, indicating the disruption of the body’s antioxidant capacity. Furthermore, we detected the antioxidant enzyme and byproduct in the small intestine mucosa. The results showed that ETEC infection had a little effect on duodenum mucosa, which was in agreement with a previous study ( Xie et al., 2021 Xie et al., 2021 ). Therefore, we emphatically examined the antioxidant capacity of jejunum and ileum in subsequent experiments. ETEC challenge did not affect the activity of CAT; however, pretreatment with WB800-KR32 significantly enhanced it in the ileum. It was suggested that our recombinant bacteria exerted a protective effect after ETEC infection. T-AOC was significantly decreased after ETEC challenge, and reached a normal condition with the pretreatment of WB800-KR32 but not WB800, indicating that WB800 had no effect on mucosal antioxidant activity, while the recombinant bacteria WB800-KR32 improved intestinal antioxidant activity through secreting the AMP KR32. The MDA content was significantly increased after ETEC infection in previous studies ( Guan et al., 2019 Guan et al., 2019 ; Xiong et al., 2020 Xiong et al., 2020 ; Wu et al., 2021 Wu et al., 2021 ), whereas it was alleviated by WB800-KR32 in our study. It was suggested that WB800-KR32 could decrease the mucosal lipid peroxide on account of MDA as the main product of lipid peroxidation produced from polyunsaturated fatty acids ( Tsikas, 2017 Tsikas, 2017 ). GPx is well-known for its antioxidant and anti-inflammatory activity, and is highly expressed in the crypt-to-villus axis that secretes microbicidal defensins in response to bacteria ( Esworthy et al., 1998 Esworthy et al., 1998 ; Ayabe et al., 2000 Ayabe et al., 2000 ; Florian et al., 2001 Florian et al., 2001 ). Increased GPx activity inhibits hydroperoxide-mediated apoptosis ( Chu et al., 2004 Chu et al., 2004 ), while insufficient GPx activity induces acute and chronic inflammation in the mucosal epithelium. In our study, the activity of GPx was significantly decreased in response to ETEC infection, which was alleviated by WB800-KR32. This finding indicated that pretreatment with WB800-KR32 could improve mucosal antioxidant level. Previous studies found that ETEC invasion leads to the upregulation of antioxidant genes ( Xie et al., 2021 Xie et al., 2021 ; Yu et al., 2021 Yu et al., 2021 ). Based on the results of antioxidant enzyme activity in the jejunal and ileal mucosae, we examined the mRNA expression of CAT and GPx . The results showed that compared with ETEC group, the relative expression of CAT was significantly decreased in response to pretreatment with WB800-KR32. On the other hand, the expression of GPx was elevated by ETEC infection and reversed by WB800-KR32. GPx1 knockout is more susceptible to oxidative challenge ( Xia et al., 2021 Xia et al., 2021 ), which may explain that WB800-KR32 reduced the ROS while suppressing the expression of antioxidant-related genes. The deficiency and mutation of CAT has been associated with various diseases and abnormalities ( Góth et al., 2004 Góth et al., 2004 ). Members of the SOD family to neutralize the first oxygen-derived ROS are the most powerful antioxidants in the cell, providing protection against bacterial infection ( Wu et al., 2021 Wu et al., 2021 ). An association between SOD deficiency and many pathologies has been found in animals and human beings ( Ighodaro and Akinloye, 2018 Ighodaro and Akinloye, 2018 ); therefore, this enzyme seems indispensable to cellular health, protecting cells from excessive oxygen radicals and free radicals. The expression of SOD was significantly upregulated during oxidative stress ( Wen et al., 2020a Wen et al., 2020a ), which is consistent with our results. In a healthy cell, Nrf2 is anchored to the cytoskeleton of actin in the form of Keap1-Nrf2 complex. In the cytoplasm, the complex is inactivated by ubiquitination to maintain its low concentration and stable state ( Lyakhovich et al., 2006 Lyakhovich et al., 2006 ). The Nrf2 pathway is activated by the specific stimulation of defense mechanism such as oxidative stress. Under oxidative stress, however, the cysteine residues of Keap1 are oxidized and Nrf2 is allowed to dissociate from the inhibitory complex ( Wu et al., 2012 Wu et al., 2012 ). Therefore, we detected the mRNA expression of Nrf2 and Keap1 . The results showed that ETEC infection upregulated the mRNA expression of both Keap1 and Nrf2 , which was reversed by WB800-KR32. These results suggested that ETEC could trigger oxidative stress, and our recombinant bacteria decreased the oxidative response. We further detected the protein expression of Nrf2 and Keap1, which revealed that ETEC infection significantly downregulated the protein expression of Nrf2, while it significantly upregulated the expression of Keap1. Meanwhile, this effect was reversed by pretreatment with WB800-KR32, suggesting that WB800-KR32 exerted a protective effect on intestinal oxidative damage by mediating through the Nrf2-Keap1 pathway. The fecal microbiota analysis showed that WB800-KR32 had a considerable effect on taxonomic composition. The results for the Top 5 significantly different species showed that the abundance of uncultured_bacterium_g_ Clostridium_sensu_stricto_1 and uncultured_ bacterium_g_ Terrisporobacter was significantly increased on Days 14 and 17. Clostridium species possess potential probiotic characteristics important for intestinal homeostasis ( Guo et al., 2020 Guo et al., 2020 ); uncultured_ bacterium_g_ Clostridium_sensu_stricto_1 , referring to the Clostridium cluster I, was increased in colon fed with a low-protein diet in finishing pigs ( Fan et al., 2017 Fan et al., 2017 ). Moreover, it was found that the participation of Akkermansia muciniphila promoted the increments of genera Clostridium_sensu_stricto_1 in the jejunum of broilers, which was associated with necrotic enteritis development ( Yang et al., 2022 Yang et al., 2022 ). Also, a diet enriched with Pediococcus pentosaceus significantly increased the genus level of Clostridium_sensu_stricto_1 in finishing pigs ( Liu et al., 2021 Liu et al., 2021 ). The treatment of chronic diarrhea patients with Lactobaccilus plantarum CCFM1143 (a probiotic) could mitigate the apparent clinical symptoms, accomplished through the enrichment of Terrisporobacter in feces ( Yang et al., 2021 Yang et al., 2021 ). On the contrary, it has been found that flavor supplementation significantly decreased genera Clostridium_ sensu_stricto_1 and Terrisporobacter , while it improved the reproductive performance of sows ( Wang et al., 2021 Wang et al., 2021 ). In addition, with the evolution of gut microbiota, the abundance of Clostridium_sensu_stricto_1 and Terrisporobacter was increased on Days 60 and 75 compared to the younger stage in Ningxiang pigs ( Li et al., 2021 Li et al., 2021 ). Eubacterium_rectale_ATCC_33656 was significantly increased after WB800-KR32 treatment, which is one of the acetate-converting butyrate producers ( Riviere et al., 2015 Riviere et al., 2015 ). Moreover, butyrate was demonstrated to be beneficial on intestinal homeostasis and energy metabolism. It also enhances intestinal barrier function owing to its anti-inflammatory properties ( Liu et al., 2018 Liu et al., 2018 ). These data indicated that WB800-KR32 could protect intestinal health through increasing the relative abundance of butyrate producing bacteria Eubacterium_rectale _ ATCC_33656 . As the complexity of microbial communities makes it challenging to identify which genera or species are beneficial to host health, future studies may use Spearman’s correlation analysis to further estimate the relationship with the apparent symptoms of host. AMPs are crucial for maintaining the balance of microbiota, which has therapeutic implications for infections by enteric pathogens ( Zong et al., 2020 Zong et al., 2020 ). Therefore, further research is needed to investigate the potential benefits of such metabolites. The above findings highlight that WB800-KR32 may play a role in the prevention of ETEC-induced microbiota disruption. 5 Conclusions 1 Pretreatment with WB800-KR32 could alleviate ETEC K88 infection induced by intestinal oxidative injuries in weaned piglets acting through the Nrf2-Keap1 pathway, while microbiota might mediate this effect. 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Anim Nutr , 6 ( 4 ): 389 - 396 . https://doi.org/10.1016/j.aninu.2020.09.002 https://doi.org/10.1016/j.aninu.2020.09.002 Interplay between gut microbiota and antimicrobial peptides 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. 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. 10.1631/jzus.B2200674 1673-1581（2023）06-0496-14 文超越,张红,郭秋平等.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[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(06):496-509. 文超越,张红,郭秋平等.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[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(06):496-509. 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. 2022-12-27 2023-03-01 2023-06-15 2023-07-04 2023-05-16 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 38646254 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). Compliance with ethics guidelines Supplementary information 1 Fig. S1 浙江大学学报（英文版）（B辑：生物医学和生物技术） 6 24