| In this study,Salmonella was cultured with xylose,xylobiose,xylotriose,xylotetraose,xylopentaose and xylohexaosetoresearch its effect on the proliferation of Salmonella and meanwhile the cultured Salmonella was used to incubate IPEC-J2,measuring the ability of Salmonella to adhere to IPEC-J2.In order to investigate the mechanism of xylose and xylo-oligosaccharides effect on the Salmonella proliferation and adhesion to IPEC-J2,we were sequencing transcriptomics of the cultured Salmonella.In addition,Mixing Salmonella cultures with xylose and xylo-oligosaccharides incubated IPEC-J2 to assess the ability of Salmonella to adhere or invade to IPEC-J2,cell damage and the expression of mucin gene,which to find out how homogeneous component of xylo-oligosaccharides protected cell in vitro.The results were as follows:(1)Salmonella was cultured with xylose and xylo-oligosaccharides at a concentration of 5 mg/mL,and the same volume of PBS was added to the control group.The OD value of Salmonella cultures was measured at 3h,6h,9h,14h,24h and 36h and calculated bacterial concentration.The results showed that xylose had a significant inhibitory effect on Salmonella growth(P<0.05)but xylobiose,xylotetraose had no effect(P>0.05).Xylotriose promoted Salmonella growth from 9h to 24h(P<0.05).Xylopentaose inhibited Salmonella proliferation from 0h to 9h(P<0.05)but had no effect after that.Xylohexaose on the proliferation of Salmonella have a certain inhibitory effect but did not reach a significant level(P>0.05).(2)Salmonella was cultured with xylose and xylo-oligosaccharides at a concentration of 5 mg/mL,and the same volume of PBS was added to the control group.At 9h,washing Salmonella with PBS then incubated IPEC-J2 for 45min to determine the bacterial adhesion rate to cells.The results showed that culturing Salmonella with xylose,xylobiose,xylotriose,xylotetraose,xylopentaose and xylohexaose significantly reduced the adhesion rate(P<0.05),the reductions were 49.35%,27.13%,37.04%,17.48%,21.91%and 41.20%respectively.(3)Salmonella was cultured with xylose and xylo-oligosaccharides at a concentration of 5 mg/mL,and the same volume of PBS was added to the control group.At 9h,total bacterial RNA was extracted for transcriptomics.The results showed that:(1)The addition of xylose caused altered expression of 87 genes,of which 63 genes were up-regulated and 24 were down-regulated.The significantly xylose up-regulated genes galK,spe A,smp B are related to growth,adhesion colonization capacity.Down-regulated gene wzzE is associated with LPS assembly.(2)The addition of xylobiose caused altered expression of 45 genes,of which 25 genes were up-regulated and 20 were down-regulated.The significantly xylobiose up-regulated genes araB?ara E both involved in arabinose metabolism and galK is related to adhesion colonization capacity.(3)The addition of xylotriose caused altered expression of 61 genes,of which 39 genes were up-regulated and 22 were down-regulated.The differentially expressed genes screening of xylotriose groupswere not related to growth and adhesion.(4)The addition of xylotetraose caused altered expression of 24 genes,of which 9genes were up-regulated and 15 were down-regulated.The differentially expressed genes screening of xylotetraose groupswere not related to growth and adhesion.(5)The addition of xylopentaose caused altered expression of 209 genes,of which 114genes were up-regulated and 95 were down-regulated.The significantly xylopentaoseup-regulated genes sic A,lip A,lonH are related to bacterial adhesion ability.AroA encoding 5-enolpyruvylshikimate-3-phosphate synthase,involved in flagellum formation and dps,bioF is related to biofilm formation.Bacterial proliferation(speE,graK,osmY,yfdQ,rne),intracellular replication and survival(pur G,metC)were also significantly up-regulated in xylopentaose group.Down-regulated genes yidE,nar Z are related to bacterial adhesion ability and mgt A is associated with bacterial motility.Biofilm formation fad L and gene nanA related to the ability to use carbon in the human intestinal mucus were also significantly down-regulated in xylopentaose group.(6)The addition of xylohexaose caused altered expression of 263 genes,of which 212genes were up-regulated and 51 were down-regulated.The significantly xylohexaose up-regulated genes are involved in bacterial proliferation and metabolic activity(dgt,oad A,yjgB,tktA,pgm,spe E,spe A,rne),intestinal colonization and intracellular survival(htp G),biofilm and outer membrane formation(artI,cyaA,yae T,bioF).The significantly xylohexaose down-regulated genes are involved in bacterial proliferation and metabolic activity(aspA),related to the ability to use carbon in the human intestinal mucus(nanA).(4)Mixing Salmonella culture(10~8CFU/mL)with xylose and xylo-oligosaccharides(the concentration was 3 mg/mL)in a 2:1 ratio incubated IPEC-J2 for 45min to determine bacterial adhesion rate,invasion rate and LDH release from cell.The results showed that xylose,xylobiose,xylotetraose,xylopentaose and xylohexaose all could reduce the ability of Salmonella to adhere to IPEC-J2(P<0.05),and the reduction rates were 60.81%,45.95%,41.89%,59.46%and 27.03%.Xylotriose has a tendency to reduce Salmonella adhesion(P=0.068)that decreased by 24.32%comparing with the control group;xylose,xylobiose,xylotriose,xylotetraose,xylopentaose and xylohexaose could significantly reduce the invasion rate of Salmonella to IPEC-J2(P<0.05);The experiment of LDH release,except for xylobiose group was significantly higher than control group(P<0.05),there was no significant difference between the other groups and control group(P>0.05).(5)Mixing Salmonella culture(10~8CFU/mL)with xylose and xylo-oligosaccharides(the concentration was 3 mg/mL)in a 2:1 ratio incubated IPEC-J2 for 45min,extract cell RNA to measure the expression of mucin gene MUC1,MUC2,MUC12,MUC13 and MUC20.The results showed that the expression of MUC1,MUC2,MUC12 and MUC20were not significantly different(P>0.05).The expression of MUC13 in the xylotriose group had no significantly difference with control group(P>0.05)and xylose,xylobiose,xylotetraose,xylopentaose and xylohexaose group were significantly lower than control group(P<0.05).The above results showed that xylo-oligosaccharides affected Salmonella proliferationand the abilityto adhere cells,and its effect on the adhesion of Salmonellawas a more important way to interfere with damaged cells. |
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