Study On The Function Of Zinc Transport System ZnuACB Of F4ac~+ Enterotoxigenic Escherichia Coli

Zinc is the most abundant trace element after iron in all living organisms,which widely exists in both microorganisms and mammalian proteins.Zinc can be acted as a structural composition or catalytic factor of proteins to participate in a variety of physiological activities,such as catalytic reaction,DNA synthesis,gene expression,immune response and so on.The host and pathogenic bacteria establish a competitive relationship at their interaction interface:the host restricts the zinc utilization of pathogenic bacteria through its own zinc transporters and a variety of strategies,affecting the growth and virulence of pathogenic bacteria,resisting pathogenic bacteria infection.At the same time,a variety of intestinal symbiotic microorganisms participate in the competition,making the zinc nutrition in the intestine very scarce.In order to meet their own nutritional needs and establish stable colonization,bacteria have evolved a variety of zinc transport systems to absorb zinc.Therefore,looking for the main zinc uptake system of bacteria to fully understand its main functions can provide a theoretical basis for the study of new antibacterial strategies or antibacterial targets.In this study,enterotoxigenic E.coli reference strain F4ac+ETEC C83902 was used as the main research strain,the main zinc transport system under zinc shortage environment was explored,and the function of zinc transport system in ETEC C83902 was explored.The specific research contents are as follows:1.Screening of major zinc transporters in F4ac+ETEC C83902 under zinc deficiency by RNA-seq techniqueIn order to reveal the coping strategies of F4ac+ETEC C83902 in zinc deficient environment,the differentially expressed genes(DEGs)of F4ac+ETEC C83902 under zinc sufficient and zinc restriction conditions were screened by RNA-seq technique.The results showed that compared with zinc sufficient condition,505 DEGs were significantly up-regulated and 523 DEGs were significantly down-regulated under zinc deficiency condition.GO enrichment and KEGG pathway enrichment analysis showed that these DEGs were involved in a variety of biological processes of bacteria.Among them,genes related to zinc transport were screened out,which are zhuA and znuCB were up-regulated,while zntA,cusA,cusB,cusC and zraP were down-regulated.These data indicate that ETEC C83902 "turn up"the zinc import system and "turn down" the zinc export transporters under zinc shortage to regulate the homeostasis of zinc ions.2.Construction of ETEC C83902 ZnuACB system mutant and the effect of this system on bacterial growth in low-zinc environmentPrevious RNA-seq results showed that ETEC C83902 upregulated the expression of ZnuACB(Zinc uptake)transport system under zinc shortage environment.To clarify the function of ZnuACB in ETEC C83902,we sequenced znuACB genes of ETEC C83902 and constructed the ETEC C83902?znuA,ETEC C83902?znuB,ETEC C83902?znuC and ETEC C83902?znuACB mutants by ?-Red homologous recombination system.The growth of theses mutants under zinc deficient condition,which achieved by adding TPEN,were detected.The results showed that under zinc deficient condition,the loss of ZnuACB transport system resulted in growth perturbation,and the addition of zinc could counteract the growth perturbation of mutants.These results indicated that the ZnuACB transport system maintained the growth of bacteria in zinc deficient environment through zinc uptake.3.Effect of deletion of ZnuACB transport system on F4ac+ETEC biofilm formation and adhesion abilityBiofilm is a complex community of bacteria to adapt to the external environment.In order to explore the effect of ZnuACB transport system on the biofilm formation of ETEC C83902,qualitative and quantitative assays of biofilm were carried out.The results showed that deletion of ZnuACB transport system would lead to the decrease of biofilm formation either in conventional biofilm induction medium or in TEPN-treated zinc deficient medium.In addition,this study explored the effect of ZnuACB transport system on the adhesion of ETEC C83902 to IPEC-J2 cells.The results showed that under the condition of LB culture,the deletion of ZnuACB transport system decreased the adhesion ability of ETEC C83902,but the difference was not significant.In zinc deficient medium,the deletion of full-length gene of ZnuACB system decreased the adhesion ability of ETEC C83902 by 81%(p<0.001),and this reduction was restored after the addition of zinc ion.qPCR was used to detect the effect of deletion of ZnuACB transport system on the transcription level of adhesion-related subunit genes(faeG,fimA,ecpA),enterotoxin gene eltB and biofilm formation related genes(luxS,pfs)under different conditions.The results showed that under the condition of LB medium,the gene transcription level of faeG,ecpA and luxS decreased by 25%,27%and 33%.respectively(p<0.05).The gene transcription level of fimA,eltB and pfs decreased by 16%.23%and 21%,respectively(p>0.05).Under the zinc deficiency condition treated with TPEN,the gene transcription level of faeG,ecpA,eltB,luxS and pfs decreased by 46%,48%,72%,46%and 41%respectively(p<0.01).The.fimA gene transcription was up-regulated by 21%(p>0.05).In summary,this study confirmed that ZnuACB transport system plays an important role in maintaining bacterial biofilm formation,adhesion and virulence factor expression under zinc shortage condition.4.Effects of deletion of ZnuACB transport system on flagella formation and proinflammatory response of intestinal epithelial cells in ETEC C83902Flagella is the main locomotive organelle for bacteria,it is also an important virulence factor involved in the pathogenic process of bacteria.This study explored the effect of the loss of ZnuACB transport system on flagella.The results showed that the loss of ZnuACB transport system resulted in the decrease of bacterial motility.Even wild type(WT)motility ability was lost under zinc deficient conditions.These data suggested that zinc deficiency reduce bacterial motility.After supplementing 0.5 mM Zn2+,the motility ability of WT and ZnuACB transport system mutant were restored,and the recovery of WT motility was more significant than mutant.After adding 1 mM Zn2+,both WT and ZnuACB transport system mutant had no motility,suggesting that excessive zinc could make bacteria lose their motility ability.The expression level of genes related to bacterial flagella synthesis was detected.The results showed that the expression level of flhC,fliA and fliC of ETEC C83902?znuACB decreased by 67%,90%and 92%,respectively(p<0.01)compared to WT.Under TPEN treated zinc shortage condition,the expression level of flhC,fliA and fliC of ETEC C83902 ?nuACB decreased by 75%,75%and 63%(p<0.01)respectively compared to WT.In addition,the expression of inflammatory factors was detected.After infection of IPEC-J2 cells one hour,the expression levels of IL-6,IL-8 and TNF-? in ETEC C83902 ?znuACB infection group decreased by 30%,46%(p<0.01)and 24%(p>0.05)respectively compared with WT infection group;Under zinc deficiency condition,the expression levels of IL-6,IL-8 and TNF-? in ETEC C83902?znuACB infection group decreased by 31%,45%and 22%(p<0.05)respectively compared with WT infection group.After infection of IPEC-J2 cells four hours,IL-6 and IL-8 of ETEC C83902?znuACB infection group increased by 12%and 15%(p>0.05)respectively,and TNF-? had no significant change compared with WT infection group;Under zinc deficiency condition,IL-6 and IL-8 of ETEC C83902?znuA CB infection group decreased by 26%and 46%(p<0.01)respectively,and TNF-? remained unchanged compared with WT group.In summary,zinc deficiency or the deletion of zinc transport system can down-regulate the expression of genes related to bacterial flagellar synthesis at the transcriptional level,resulting in the disturbance of bacterial flagellar synthesis and the decrease of bacterial motility ability.At the same time,the absence of ZnuACB system also reduces the expression of inflammatory factors after bacterial infection under zinc deficiency.5.Effect of deletion of ZnuACB transport system on the pathogenicity of F4ac+ETEC C83902 in vivoPrevious in vitro studies showed that ZnuACB transport system played an important role in maintaining the pathogenicity of ETEC C83902,such as growth,biofilm formation,adhesion and motility,especially in zinc deficeent environment.The purpose of this study is to investigate the effect of ZnuACB transport system on the pathogenicity of ETEC C83902 in piglets.In this study,the challenge experiment of piglets was carried out.The results showed that the deletion of ZnuACB transport system reduced the diarrhea symptoms,fecal bacteria load,intestinal villi damage and host inflammatory response of piglets caused by ETEC C83902,which further confirmed that ZnuACB transport system played an important role in the pathogenicity of ETEC C83902.