Screening Of Bacillus Subtilis And Its Mechanism Of Inducing The Innate Immune Response To Anti-infection With Escherichia Coli In Rabbit

Probiotics are a safe alternative to antibiotics and are supplemented in the diet to prevent diseases and promote growth.Due to the spore's resistance,survival in extreme environments and long-term storage,spore-forming Bacillus spp.are considered to be suitable probiotics.They not only produce antibacterial substances,certain essential nutrients,such as amino acids,and vitamins K and B12 to promote growth performance,but they also promote the proliferation of beneficial anaerobic bacteria by consuming the free oxygen in the intestinal tract.The innate immune system is used as the first line of defense to various pathogens.Pattern recognition receptors?PRRs?have been implicated in pathogen control and elimination and have been recently identified as key mediators of inflammatory and immune responses.Nucleotide-binding oligomerization domain-like receptors?NLRs?are a kind of PRRs.NLRs detect and sense microbial components,such as Bacillus subtilis?B.subtilis?,and then lead to the activation of an inflammatory response and promote the secretion of cytokines.The mechanism of B.subtilis to immune response has not been studied experimentally.In this study,In order to find probiotics instead of antibiotics,we separated B.subtilis strains from the soil,screened for the strain with the best performance in an in vitro probiotic potential evaluation.In order to confirm the viability of the strain in the gastrointestinal tract,artificial gastric juice,intestinal juice,and bile salt test were carried out.In order to prove its antimicrobial activities,gram positive and negative bacterial inhibition tests were carried out,and the infection resistance of B.subtilis was clarified.In order to verify the anti-infection effect of B.subtilis in vivo,a feeding experiment was carried out with rabbits as experimental animals.The results showed that dairy with B.subtilis effects on improved growth performance,serum immunoglobulin,immune organ index,intestinal homeostasis and immune response of rabbits,as well as its antibacterial infection.It confirmed that dairy with B.subtilis could replace antibiotics and showed good industrial prospect.The mechanism of B.subtilis to resist bacterial infection is further revealed.By detecting the expression of NLRs,we find that B.subtilis resist to Escherichia coli?E.coli?infection through rabbit NOD1?rNOD1?.We cloned rNOD1 and investigated rNOD1 downstream signaling pathways and antibacterial activity to clarify its role during bacterial infections.This study is divided into four parts:1.Isolation and probiotic potential evaluation of B.subtilis in vitroIn this research,according to colony morphology and the results of microscopic examination,Gram-positive Bacillus strains with the typical volcano shape were selected as subjects for biochemical experiments.In total,180 B.subtilis were isolated from the soil and confirmed by 16S rRNA sequencing.All B.subtilis were selected for artificial gastric juice,intestinal juice and bile salt test.Total 68 B.subtilis which were tolerant to acidic conditions,intestinal juice,and bile salt were performed functional screening,including antimicrobial activities?enteropathogenic Escherichia coli,Salmonella,Staphylococcus aureus,and Clostridium perfringens?and enzyme production test.BYS2,BQ3,and BD17 showed good antimicrobial activities.B.subtilis has the strong inhibitory effect on Gram positive bacteria,such as Staphylococcus aureus and Clostridium perfringens.Especially for Clostridium perfringens,the diameter of inhibition zone can reach 20-30mm.For BYS2,BQ3 and BD17,the inhibitory diameter for Staphylococcus aureus is larger than 20mm,and the inhibition to Salmonella is slightly weaker.The inhibition zone of BQ3 reaches the maximum of18.1±1.7mm.The inhibition ability on enteropathogenic Escherichia coli is the weakest and the inhibition zone diameter is 12-14mm.It also has the similar bacteriostasis for ESBL-producing E.coli.Finally,antibiotic sensitivity test was carried out to determine that it did not carry drug-resistant genes.Finally,we selected BG5 and BGY12 with the most enzyme producing ability,and BYS2,BQ3,and BD17 with the strongest antimicrobial activities.The five B.subtilis were mixed to investigate their effect on rabbits.2.The effect of B.subtilis on growth performance,immunomodulation,and intestinal florawas evaluated in vivo.Rabbits with a diet of 10⁶CFU g^-1mixed B.subtilis exhibited the best growth performance,and higher serum IgG and IgA than controls?P<0.05?.The relative weights of the thymus and spleen were significantly increased by diet with B.subtilis after 5 weeks of feeding?P<0.05?.High-throughput sequence analysis of bacterial 16S rRNA V3-V4 region was conducted on cecal contents of rabbits after 5 and 7 weeks of feeding.According to the analysis of?diversity studies,the species diversity in B.subtilis group is higher than that of control.The95.0%-96.5%readsbelongtoFirmicutes,Bacteroidetes,Verrucomicrobia,and Proteobacteria at the phylum level.The relative abundance of the phyla Firmicutes was increased after feeding B.subtilis.The relative abundance of the phyla Bacteroidetes was decreased after feeding B.subtilis?P<0.05?.The shifts in bacterial composition at the phylum level were caused by the changes at the genus level.The dominant bacteria of the genus level in control group are Ruminococcus 8.13%,Oscillospira 4.62%,Bacteroides5.20%,Akkermansia 3.46%,Coprococcus 1.28%,Clostridium 1.12%,Faecalibacterium0.59%,Desulfovibrio 0.23%,Parabacteroides 0.38%,Dorea 0.39%.After 5 weeks of feeding,the dominant bacteria of the genus level in rabbits fed with B.subtilis are Ruminococcus12.00%,Oscillospira 4.19%,Bacteroides 0.69%,Akkermansia 1.18%,Coprococcus 0.76%,Clostridium 1.05%,Faecalibacterium 0.49%,Desulfovibrio 0.20%,Parabacteroides 0.10%,Dorea 0.17%.After 7 weeks of feeding,the dominant bacteria of the genus level in rabbits fed with B.subtilis are Ruminococcus 9.61%,Oscillospira 7.50%,Bacteroides 1.57%,Akkermansia 0.65%,Coprococcus 2.54%,Clostridium 0.24%,Faecalibacterium 0.23%,Desulfovibrio 0.38%,Parabacteroides 0.21%,Dorea 0.05%.Dairy with B.subtilis can promote intestinal flora balance and intestinal digestion function,and maintain intestinal health.In particular,the Ruminococcus contributed to the digestion and absorption of fiber in herbivores.The relative abundance of Ruminococcus was increased after 5 weeks of feeding?P<0.05?.The percentage of Bacteroides and Clostridium were significantly decreased after5 and 7 weeks of feeding?P<0.05?.It suggested that B.subtilis could promote the growth of beneficial bacteria and inhibit pathogenic bacteria in intestine.These findings indicated that dairy with B.subtilis can improve growth performance and immune organ index,as well as maintain intestinal health.3.Innate immune response to B.subtilisThis research is the first report on the innate immune response to B.subtilis in rabbits.To determine the induction of innate immunity in rabbits fed with a diet of B.subtilis,the expressions of innate immune-related genes were examined in the spleen and jejunum of rabbits fed with the probiotic compared to the controls after 5 and 7 weeks of feeding.The dietary administration of B.subtilis increased the expression of rNOD1,rNLRC3 and rNLRX1.The major pro-inflammatory factor IL-1?,IL-8,and IFN-?,as well as?-defensin were up-regulated compared with the controls.After 7 weeks of feeding,the survival rate of the rabbits fed with B.subtilis was significantly higher than those in the control groups post-infected with E.coli?P<0.05?.At the same time,this study detected reduced bacteria contents of the heart and cecal contents from the diet mixed with B.subtilis compared with the control groups.Moreover,The NLRs and?-defensin were up-regulated in the B.subtilis feeding group post-infection with E.coli,especially DEFB114,DEFB134,and DEFB135.The increased expression of?-defensin can directly kill pathogenic microorganisms and enhance the ability of anti-infection.The similar results were observed in RK-13 cells co-cultured with B.subtilis in vitro.The expression of rNOD1 was significantly increased in rabbit kidney cells?RK-13?after stimulated with B.subtilis.However,after knockdown of rNOD1,B.subtilis can not induce the expression of pro-inflammatory cytokines and defensins.These findings showed that B.subtilis stimulates and activates rNOD1 to induce the production of pro-inflammatory cytokine and defensins and improve disease resistance.4.Characterization of rNOD1 and the role of rNOD1 signaling pathway during bacterialinfectionNOD1 is the most prominent of all NOD-like receptors,which in the mammalian innate immune system,serve as intracellular receptors for pathogens and endogenous molecules during tissue injury.From RK-13 cells,we cloned rNOD1 and identified an N-terminal caspase activation and recruitment domain,a central NACHT domain,and C-terminal leucine-rich repeat domains.rNOD1 was expressed in all tested tissues;infection with E.coli induced significantly higher expression in the spleen,liver,and kidney compared to other tissues.It indicates that the organism can quickly identify the external pathogens and induce the immune response to anti-infection.The overexpression of rNOD1 induced the expression of pro-inflammatory cytokines IL-1?,IL-6,IL-8,IFN-?,and TNF-?and defensins,including DEFB124,DEFB125,DEFB128,DEFB135,and NP5 via activation of the nuclear factor?NF?-?B pathway.Overexpression of rNOD1 inhibited the growth of E.coli,whereas knockdown of rNOD1 or inhibition of the NF-?B pathway promoted the growth of E.coli.Such results suggest that the antibacterial activity for rNOD1 is mediated through NF-?B signaling.In addition,our results showed that rNOD1 induce the production of pro-inflammatory cytokine?IL-1?,IL-6,IL-8,IFN-?,and TNF-??and defensins?DEFB124,DEFB125,and DEFB128?in E.coli-infected cells.After knockdown of rNOD1 or inhibition of the NF-?B pathway,overexpression of rNOD1 decreased the expression of pro-inflammatory cytokines and defensins induced by E.coli.It is suggested that rNOD1activated NF-?B signaling to mediate the immune response to anti-infection of E.coli.Moreover,rNOD1 colocalized with LC3,upregulated autophagy pathway protein LC3-?,and increased autolysosome formation in RK-13 cells infected with E.coli.rNOD1 induced autophagy and played an important role in the inhibition of E.coli.rNOD1 and it's ligand i E-DAP may represent as new targets for vaccine adjuvant and drug development.