The Construction Of Engineered Strain WB800＿KR32 And Its Regulation Mechanism On Intestinal Health In ETEC Infected Piglets

The health of piglets has important value on swine production.However,due to it’s immunodeficiency in pre and post weaning period,piglets are susceptible to the pathogens infection,such as Escherichia coli(E.coli),which would induce diarrhea and threate the health breeding of piglets.Antimicrobial peptides(AMPs)play roles in immunomodulation and microbiome modulation,and become a research focus of intestinal health of piglets.Previous study in our lab have found that antimicrobial peptides KR32 has a strong antimicrobial capacity,low cytotoxicity,and no obvious killing effect on probiotics,it has a well effect on the treatment of diarrheal piglets.However,chemosynthetic KR32 have difficulty in applying industry for high cost.Applying engineered bacteria to produce recombinanted AMPs could reduce production costs.In this study,we build the engineered bacteria(WB800/p BE2R/KR32,WB800＿KR32)by applying genetic engineering technology,and invested the antimicrobial(Enterotoxigenic Escherichia coli K88,ETEC K88)activity of the fermented WB800＿KR32 supernatant using oxford cup method in vitro,and invested the effects of WB800＿KR32 on intestinal antioxidant and barrier function in ETEC K88 infected piglets in vivo,and final invested the effects of fermented WB800＿KR32 supernatant on antioxidant and barrier function of ETEC K88 infected IPEC-J2.The main methods and results as follows:1.The construction and antimicrobial activity detection of engineered bacteria WB800＿KR32We build the recombinant bacteria WB800＿KR32 by genetic engineering technology and compared the antibicrobial activity of supernatant of WB800＿KR32 at varied fermented time(24～39 h)by oxford cup method.The results showed that fermented supernatant reached highest anti-bacteria(E.coli,1×10~6CFU/m L)activity at fermentation 33 h,and the ati-bacteria activity is obvious strong than the fermented 33h supernatant of WB800 and WB800/p BE2R.LC/MS/MS analyses were performed to detect the concentration of WB800＿KR32 supernatant fermented at 33h,the calculation showed that the expression level of WB800＿KR32 fermented 33 h reached 2.24μg/L.2.The effect of engineered bacteria WB800＿KR32 on intestinal antioxidant and barrier function in ETEC K88 infected pigletsTwenty eight Duroc×(Landrace×Yorkshire)piglets(7.09±0.25 kg)weaned at 21days were caged in individual pens and randomly allocated into 4 groups(n=7/treatment).The treatments were as follows(1)CON group:oral administration of10 m L 0.9%Na Cl,once a day(d1-d17).(2)ETEC group:oral administration of 10 m L0.9%Na Cl once a day at d1 to d14.(3)ETEC+WB800 group:oral administration of 10m L 5.0×10~9 CFU/m L WB800 bacteria solution once a day at d1 to d14.(4)ETEC+WB800＿KR32 group:oral administration of 10 m L 5.0×10~9 CFU/m L WB800＿KR32 at d1 to d14.Except for CON group,the other three group were chanllenged 10 m L 1×10~9 CFU/m L ETEC K88 once a day at d15 to d17.The samples were collected at d18 for further analyisis.The effect of engineered bacteria WB800＿KR32 on growth performance of ETEC K88 infected piglets:Compared with ETEC group and ETEC+WB800 group,oral WB800＿KR32 could significantly improve the growth performance and decrease the diarrhea score(P<0.05),and showed no significantly difference compared with CON group(P>0.05).The effect of engineered bacteria WB800＿KR32 on serum and intestinal antioxidant function in ETEC K88 infected piglets:Compared with ETEC group,oral WB800＿KR32 could significantly increase total antioxidant capacity(T-AOC)in serum(P<0.05),significantly increase the activity of glutathione peroxidase(GPx)in jejunnal and ileal mocosa(P<0.05),significantly decrease the relative expression of antioxidant related genes(GPx,Nuclear factor erythroid 2-related factor 2(Nrf2)and superoxide dismutase 1(SOD1))in jejunal and ileal mucosa(P<0.05),significantly increase the relative protein expression of Nrf2 while decrease the relative protein expression of kelch like ECH associated protein 1(Keap1)in ileal mucosa(P<0.05)and showed no significantly difference compared with CON group(P>0.05).The effect of engineered bacteria WB800＿KR32 on intestinal barrier function in ETEC K88 infected piglets:(1)The effect of engineered bacteria WB800＿KR32on intestinal physical barrier in ETEC K88 infected piglets:Compared with ETEC group,oral WB800＿KR32 significantly increased the villus height and villus height/crypt depth in jejunum and ileum(P<0.05),significantly decrease the activity of diamine oxidase(DAO)and the concentration of D-lactic acid(D-LA)in serum and have the tendency to increase the relative gene(P=0.086)and protein(P=0.093)expression of occludin in ileal mucosa,upregulated the relative protein expression of ZO-1(P<0.05),.(2)The effect of engineered bacteria WB800＿KR32 on immunological barrier in ETEC K88 infected piglets:Compared with ETEC group,oral WB800＿KR32could significantly decrease the concentration of proinflammatory cytokine IL-6,while increase the concentration of anti-inflammatory cytokine IL-10 in serum and small intestinal mucosa(P<0.05),significantly decrease the phosphorylation level of NF-κB p65 in ileal mucosa,and showed no significantly difference compared with CON group(P>0.05).(3)The effect of engineered bacteria WB800＿KR32 on ileal microbial barrier in ETEC K88 infected piglets:Compared with CON group,the relative abundance of Lactobacillus＿paralimentarius was significantly decreased(P<0.05),while the relative abundance of Romboutsia＿sedimentorum was significantly increased(P<0.05)in ETEC group.Compared with ETEC group,the relative abudance of Clostridium＿chartatabidum was significantly increased(P<0.05),while the relative abundance of Escherichia was significantly decreased(P<0.05)in ETEC+WB800＿KR32 group.The effect of engineered bacteria WB800＿KR32 on serum metabolites in ETEC K88 infected piglets:The GC/LC-MS were performed to analysis the serum metabolites,the main differential metabolites were organic nitrogen compounds and organic acids,such as D-asparegine,organoheterocycllic compounds and fatty acyls,such as margaric acid.The analysis of KEGG pathway of differtial metabolites were enriched in the pathway of glutathione metabolism,selenocompound metabolism and pyruvate metabolism.The effect of engineered bacteria WB800＿KR32 on ileal content mi RNAs in ETEC K88 infected piglets:The total RNA was extracted from ileal content,and mi RNA library were constructed and the final ligation PCR products were sequenced using the BGISEQ-500 platform.We performed RT-q PCR methods to verifiy the differential expression of mi RNAs and found that compared with CON and ETEC+WB800＿KR32 group,the relative expression level of novel-ssc-mi R1250-5p(mi R1250-5p)was significantly increased in ETEC group(P<0.05).The KEGG analysis of mi R1250-5p targeted genes showed that the pathway were enriched in tight junction,NF-κB signal pathway,and Escheria infection pathway.We performed RT-q PCR methods to verifiy relative expression of the mi R1250-5p targeted genes(NF-κB p65),and found that compared with ETEC group,the relative gene expression of NF-κB p65 was significantly decreased in ETEC+WB800＿KR32 group(P<0.05),and showed no significantly difference compared with CON group(P>0.05).3.The effect of engineered bacteria WB800＿KR32 fermented supernatant on antioxidant capacity and barrier function in ETEC K88 infected IPEC-J2 cellsThe 6.2×10~6 CFU/m L ETEC K88 treated IPEC-J2 2 h was used as the condition for ETEC K88 infected IPEC-J2,and the cell viability reached 62.53%.Compared with ETEC group,40%WB800＿KR32 fermented supernatant treated IPEC-J2 2.5 h could significantly increase the cell viability of IPEC-J2 cells(P<0.05).The engineered bacteria WB800＿KR32 fermented supernatant improve antioxidant function of ETEC K88 infected IPEC-J2 cells:Compared with ETEC group,treated with WB800＿KR32 fermented supernatant could significantly increase the capacity of GPx and CAT,and T-AOC of IPEC-J2 cells(P<0.05),significantly increase the relative gene expression of Keap1 while significantly decrease the relative gene expression of Nrf2 in IPEC-J2 cells(P<0.05).The engineered bacteria WB800＿KR32 fermented supernatant imrove barrier function of ETEC K88 infected IPEC-J2 cells:Compared with ETEC group,treated with WB800＿KR32 could significantly improve the relative protein expression of occludin and ZO-1(P<0.05),significantly decrease the concentration of IL-6,IL-10,IL-17,and s Ig A(P<0.05)in the cell medium.Compared with WB800 fermented supernatant,treated with WB800＿KR32 fermented supernatant could significantly downregulate the relative gene expression of IL-6,IL-8,and NF-κB p65(P<0.05).Compared with ETEC group,treated with WB800＿KR32 fermented supernatant could significantly downregulated the relative protein phosphorylate level of NF-κB p65(P<0.05).Conclusion:We have built an engineered bacteria WB800＿KR32 which could secrete antimicrobial peptide KR32 independent.Engineered bacteria WB800＿KR32and its fermented supernatant could improve the antioxidant and barrier function in ETEC K88 infected piglets and IPEC-J2 cells,respectively.It may by inhibiting the relative expression of mi R1250-5p,and then downregulate it’s targeted gene NF-κB p65 and then reduce the relative protein phosphorylate level of NF-κB p65,and final decrease the inflammatory response induced by ETEC K88 infection.This study provided a new perspective for applying engineered bacteria WB800＿KR32 to regulate the disturbance of intestinal oxidative damage and barrier function induced by ETEC K88 infection.