Bacillus Subtilis And Surfactin Inhibit The Transmissible Gastroenteritis Virus From Entering The Intestinal Epithelial Cells

Intestinal epithelial cells are the targets for transmissible gastroenteritis virus(TGEV)infection.It is urgently to develop a novel candidate against TGEV entry.Bacillus subtilis is a probiotics with excellent anti-microorganism properties,and one of its secretions,surfactin,has been regarded as the versatile weapons for most plant pathogens,especially for the enveloped virus.We demonstrate for the first time that Bacillus subtilis OKB105 and its surfactin can effectively inhibit one animal coronavirus,TGEV,from entering the intestinal porcine epithelial cell line(IPEC-J2).Then,several different experiments were performed to seek for the might mechanisms,in order to provide some basement works for the further study of TGE prevention.1.Isolation and identification of surfactinSurfactin is a cyclic lipopeptide antibiotic and biosurfactant synthesized by Bacillus subtilis.It consists of an anionic seven-membered peptide cyclo and a mixture of several hydrophobic ?-hydroxy fatty acids with chain lengths of 13 to 15 carbon atoms.By this amphiphilic structure surfactin is one of the strongest biosurfactants.Studies on surfactin are focused on properties against phytopathogenic microorganisms,such as anti-bacteria,anti-fungi,inhibition of fibre clot formation,and antiviral ability.But whether surfactin has the activity against TGEV,an animal enveloped virus from coronaviridae family,remains poor understood.In this study,the lipopeptides crude extract was precipitated by 6 mol/L HCl from the supernatant of Bacillus subtilis OKB 105 and dissolved by methanol.And then separated by LH20 chromatographic column.By using MALDI-TOF-MS and hemolytic activity analysis,the identification and bioactivity of lipopeptide were analysed.The results showed the lipopeptide we gathered was standard surfactin isoforms with side C13-to C16-surfactin.And the corresponding mass to charge ratios(m/z)were 1016.473?1030.484?1046.472?1058.636?1060.47?1074.48?1076.439?1082.469[M + Na]+,respectively.And the hemolysis ring of the surfactin was over 8 mm that represent a high bioactivity.These results imply that the surfactin we gathered was pure and had high bioactivity that can be used in subsequent experiments2.Inhibition effects of Bacillus subtilis or surfactinTGEV enters epithelial cells by binding to the cellular receptor and then mediates membrane fusion at the plasma membrane or by endosomal uptake.In order to interrupt TGEV infection in the origination stage,we detected the inhibition effect of Bacillus subtilis OKB105 and the surfactin on TGEV entry process in vitro.Three setups focused on the suppress effect against TGEV entry varying the treatment period.Briefly,monolayers of IPEC-J2 cells were treated with Bacillus subtilis 168,OKB105,and surfactin for 1.5 h,respectively,which was washed away before infection with TGEV for 1.5 h(pre-treatment assay),TGEV was added to the celllayer together with Bacillus subtilis168?OKB105 and surfactin,respectively,during the 1.5 h infection period(co-treatment assay),virus was mixed with Bacillus subtilis 168,OKB 105 and surfactin,respectively,and incubated for 1.5 h at 37?,aliquots were removed and diluted 1:10 with DMEM supplemented with 5%FBS to stop the effect of the surfactin and then sterile filtered through a 0.22 ?m filter.Then the filtrate were added to the cell layer and incubated for 1.5 h(out-treatment assay).Firstly,the toxic effects of the Bacillus subtilis and surfactin on IPEC-J2 cells were determined using the methylthiazolyl-diphenyl-tetrazolium bromide(MTT)viability assay.And then the inhibition effect was detected by RT-qPCRT and western-blot.Results showed that Bacillus subtilis 168 was non-toxic in the used doses,Bacillus subtilis OKB 105 was non-toxic up to 1.00E+09 cfu/ml,and the safe dose of surfactin was up to 0.02 mg/ml.Therefore,the safe dose of Bacillus subtilis(1.00E+ 07 cfu/ml)and surfactin(0.002 mg/ml)were used in the next study.And for the inhibition effect,first,for different 'drugs',our results showed that the relative amounts of viral RNAs in the surfactin-treated IPEC-J2 cells decreased in all treatments.On the other side,Bacillus subtilis OKB 105 reduced the relative amounts of viral RNAs in the pre-treatment and co-treatment,where cells existed.While Bacillus subtilis 168 could only decrease the relative amounts of viral RNAs in the pre-treatment.Second,for different 'drugs' in the same treatment,Bacillus subtilis OKB 105 showed the best suppress activity in the pre-treatment,where it had abundant time of talking to the cells.However,in the out-treatment,where the cells were not on present,Bacillus subtilis did not show significant inhibition,while surfactin did.The similar results were obtained in the western blot analysis.Taken together,these data indicated that there might be a hide association between Bacillus subtilis and IPEC-J2 cells while the surfactin might function on both the virus and cells,and Bacillus subtilis and surfactin might show synergetic effect to some extent.3.The might mechanism of the inhibition effects of Bacillus subtilis OKB105 and surfactinTo explore the possible mechanisms,several studies were carried out.And the effects of Bacillus subtilis OKB 105 and surfactin on viral infectivity as well as the impact on the receptors of TGEV,EGFR and APN,were investigated.Additionally,the toll like receptors(TLRs)and the apoptosis of IPEC-J2 cells were also detected.Our results reveal that Bacillus subtilis 168 and OKB105 could trap most of TGEV on their surface.And the attachment ability of Bacillus subtilis 168 was better than OKB 105.Which might indicate that the surfactin secreted byBacillus subtilis OKB 105 had destroyed the trapped virion so that could not be detected by the Western blot.This hypothesis was confirmed in the plaque assay,and we also confirmed that the inactivity of surfactin was dose-dependent(P<0.01).We also found that after stimulated with Bacillus subtilis 168 and OKB 105 for 1.5 h,the phosphorylation of EGFR and the expression of APN protein were both increased(P<0.01),which indicated that Bacillus subtilis might compete with TGEV for binding to the receptors at the surface of IPEC-J2 cells.After a short time incubation,the expression of TLR-6 protein was increased(P<0.01)and the the percentage of apoptotic cells(P<0.01)was depressed,.which indicates that our 'drugs' were benefit on the state of IPEC-J2 cells.The results of the present study demonstrate that Bacillus subtilis OKB 105 and the surfactin have antiviral activity against TGEV entering IPEC-J2 cells.And that possibly overlapping mechanisms lead to the antiviral activity:might by competing with TGEV in combining to the receptors,adsorptive trapping,inactivation of virus particles of surfactin,improvement of the cell state through activating the innate immunity and induce the apoptosis level.This finding suggest that Bacillus subtilis OKB 105 and its surfactin can antagonize TGEV entry in vitro and may serve as promising new candidates for TGEV prevention.