Screening And Identification Of Biofilm Genes And The Pathogenic Mechanism Of YbgD Fimbriae In Avian Pathogenic Escherichia Coli

Avian Pathogenic Escherichia coli(APEC)is the main pathogenic bacteria in poultry farming.As an extra-intestinal pathogen,APEC has a broad host spectrum,which not only seriously harms the poultry industry but also poses a potential threat to human health.When APEC infects the host,the biofilm can resist the high concentrations of antibiotics to achieve its continuous colonization of the host.In addition,when bacteria reside in biofilms,the pathogen-associated molecular patterns of bacteria are less exposed,which is conducive to their escape from host pattern recognition receptor recognition and immune evasion.Therefore,identification of key genes involved in APEC biofilm and analysis of its regulatory mechanism can provide a theoretical basis for the prevention and control of APEC from the perspective of biofilm.To identify the virulence factors involved in the formation and pathogenicity of APEC biofilm.To screen and identify genes affecting APEC biofilm,transposon random mutation libraries were constructed using APEC81.Mutation libraries were used to identify genes affecting APEC biofilms.A total of 30 genes affecting biofilm formation were identified,and 13 genes were adhesion genes.Adhesin is a key factor for bacteria to adhere to the surface of biological and abiotic media to form biofilm.According to their characteristics and functions,they are divided into fimbrial adhesins and afimbrial adhesins.Among the screened adhesin genes,type 1 fimbriae genes had the greatest influence on the biofilm,followed by flagella genes and curli fimbriae genes.At 25 °C,the absence of YbgD fimbrial adhesin resulted in a 66.06% reduction in the biofilm.It is speculated that YbgD fimbrial adhesin enhances the ability to resist the external environment by participating in biofilms at 25 °C.At 37°C,the absence of YbgD fimbrial adhesin resulted in a 22.39% enhancement of its biofilm formation.It is speculated that it interferes with host immunity through biofilm and participates in systemic infection of the host.In the early stage of infection,adhesins initially interact with host cells,recognize cell surface-specific receptors,and trigger signaling pathways for colonization and invasion.The type III secretion system 2(ETT2),as an important virulence island in APEC,mainly regulates bacterial surface adhesins globally.The roles of YbgD fimbriae as adhesins in APEC biofilm formation and host infection and the role of ETT2 as an important regulatory system for YbgD fimbriae remain unclear.Therefore,this study first analyzed the role of YbgD fimbriae in pathogenicity from the perspective of aetiology,then applied transcriptome analysis and verified that YbgD fimbriae were involved in biofilm formation and pathogenicity by mediating the expression of the adhesin Fde C,and Yqe I can regulate YbgD fimbriae to participate in the pathogenic process.Secondly,from the perspective of pathogen-host interaction,the signal pathway activated by YbgD fimbriae in the process of infecting host cells was screened by transcriptome analysis,which further confirmed that the YbgD fimbrial adhesin mediates inflammatory responses by regulating the innate immune system,thereby realizing the anti-inflammatory response to the host.pathogenicity.This project can provide a theoretical basis for the prevention and control of APEC from the perspective of biofilm regulation by researching the regulation of APEC biofilm formation and the pathogenic mechanism by YbgD fimbrial adhesin.The specific research content and results are as follows:1.Construction of a transposon random mutation library to screen and identify genes involved in APEC biofilm formationFirst,the transposon Tn5 was randomly inserted into the APEC81 genome by conjugative transduction,and a random mutation library containing 6318 mutants was successfully constructed through resistance screening and PCR identification.Crystal violet staining was used to screen 45 mutants with significantly reduced biofilm from3000 mutants,and 31 insertion site genes were further identified by thermal asymmetric interlaced PCR(TAIL-PCR).The deletion of 31 genes in APEC81 was completed by the red-homologous recombination method.Since 25°C is the ambient temperature suitable for E.coli to form biofilms to resist environmental stress,while 37°C is suitable for E.coli growth,it is beneficial to synthesize virulence factors to infect the host.Therefore,the biofilmforming ability of the deletion strains was tested at 25°C and 37°C.The results showed that deletion of 30 genes significantly affected biofilm formation ability,including flagella genes(fli S,fli D,fli R),Curli fimbriae genes(csg D,csg A,csg F,csg G),fimbrial adhesin genes(fim A,fim D,fim C,fim H,ybg D and yeh E),coenzyme genes(glt A,bgl X,mlt F,glx K,suf B,ydc U,ydc V,mdo C,ydi F,pde H,evg S,pfs),toxin-antitoxin gene rat A,putative protein genes(07045,11735,11255,and 03110).Among these genes,type 1 fimbriae genes had the greatest effect on biofilm,followed by flagella genes and Curli fimbriae genes.Interestingly,however,under the condition of25°C,the biofilms without the fimbriae gene ybg D decreased by 66.06% respectively,and the difference was extremely significant(P < 0.01);it indicated that ybg D was involved in the formation of biofilms against the external environment at 25°C.At 37°C,ybg D deletion enhanced the biofilm by 22.39%,a very significant difference(P < 0.01).The biofilm is more conducive to infecting the host.2.Analysis of the role of YbgD fimbriae mediating Fde C adhesin in biofilm and pathogenicityYbgD fimbriae are molecular chaperones or lead protein fimbriae.In order to explore the pathogenic mechanism of YbgD fimbriae,the adhesion and invasion assays showed that ybg D deletion significantly enhanced the adhesion and invasion ability of APEC to host cells.And the survival rate of the deletion strain APEC81Δybg D in host cells was significantly higher than that of the wild strain.However,the enhanced adhesion of APEC81Δybg D to the host could not be inhibited by mannose,indicating that the enhanced adhesion of ybg D to host cells was not mediated by type 1 fimbriae,indicating that other adhesins were involved.To screen the target genes regulated by YbgD,the m RNA level of the adhesin gene fde C gene in the deletion strain APEC81Δybg D was significantly increased by transcriptome and real-time quantitative PCR analysis.Therefore,the adhesin gene fde C was selected as the target gene.To verify the regulatory relationship between YbgD and Fde C,the fde C gene was further deleted based on the ybg D gene deletion to construct a double gene deletion strain APEC81Δybg DΔfde C,and the pathogenicity analysis and biofilm detection.The results showed that compared with the APEC81Δybg D deletion strain,the ybg D and fde C double gene deletion strain APEC81Δybg DΔfde C had significantly lower biofilm formation ability and host cell adhesion and invasion ability(P < 0.05).In addition,it was proved by hemagglutination assay that ybg D deletion is beneficial to the expression of type 1 fimbriae,and the enhanced expression of type 1fimbriae is beneficial to biofilm formation,although it does not mediate the enhancement of adhesion ability.This study demonstrates that YbgD fimbriae can participate in biofilm formation and pathogenic processes by regulating the expression of the adhesin Fde C and type 1 fimbriae.3.ETT2 transcription factor Yqe I regulates Ybg fimbriae and participates in pathogenic researchThe ETT2 virulence island mainly has a global effect on bacterial membrane surface proteins.The laboratory previously demonstrated that the transcriptional regulator Yqe I in ETT2 can regulate the down-regulation of type 1 fimbriae genes,resulting in a significant decrease in the adhesion ability and biofilm formation ability.To prove that Yqe I can regulate YbgD fimbriae on the bacterial surface to participate in pathogenicity.It was found by real-time quantitative PCR that the deletion of yqe I could significantly increase the transcription levels of Ybg fimbriae genes ybg D,ybg Q and ybg P(P < 0.05),indicating that Yqe I is involved in the regulation of the ybg operon.On the basis of the deletion strain APEC81Δyqe I,the double gene deletion strains APEC81Δyqe IΔybg D,APEC81Δyqe IΔybg Q and APEC81Δyqe IΔybg P were further constructed.The pathogenicity analysis of the deletion strain showed that compared with APEC81Δybg D,the adhesion ability of the double gene deletion strain APEC81Δyqe IΔybg D was significantly enhanced(P < 0.05),and was significantly inhibited by the addition of mannose.Compared with Δybg P,the adhesion ability of APEC81Δyqe IΔybg P in the double gene deletion strain was significantly enhanced(P <0.05),which was also significantly inhibited by adding mannose,indicating that APEC81Δyqe IΔybg D and APEC81Δyqe IΔybg P enhanced type 1 Fimbriae expression results in enhanced adhesion.Furthermore,hemagglutination tests proved that the expression levels of type 1fimbriae in APEC81Δyqe IΔybg P and APEC81Δyqe IΔybg D were significantly higher than those of wild APEC81 and APEC81Δyqe I(P < 0.05).At the same time,real-time quantitative PCR analysis showed that compared with the wild strain APEC81,the transcription level of the type 1 fimbriae gene fim A in the deletion strain Δyqe I was significantly down-regulated(P < 0.05),while the deletion strain APEC81Δyqe IΔybg P and APEC81Δyqe I,the transcription level of type 1 fimbriae gene fim A was significantly up-regulated in Δybg D(P < 0.05).It indicated that the transcriptional regulator Yqe I affects the expression of type 1 fimbriae by negatively regulating Ybg fimbriae and then participating in the pathogenic process.4.Study on the interaction mechanism between YbgD fimbriae and the hostFimbriae can recognize specific receptors and trigger signal transduction pathways or cytoskeletal rearrangements for colonization and invasion.It has been demonstrated that YbgD fimbriae deletion leads to an enhanced ability of APEC to adhere to and invade host cells.To explore the YbgD fimbriae-mediated signaling pathway of the host.In this study,RNA-Seq was used to analyze the transcriptomes of wild APEC81 and APEC81Δybg D cells infected with RAW264.7 cells.Through GO function analysis and KEGG enrichment analysis,it was found that most of the differential genes were enriched in immune system processes,and the NF-κB signaling pathway,Toll-like receptor pathway,and MAPK signaling pathway were involved.The transcription of pathwayrelated genes was detected by real-time fluorescence quantitative PCR.The host cell’s Toll-like receptors(TLR3 and TRL9),chemokine CCL2 and inflammatory cytokines(IL-1β,TNF-α),IFN-β and IL-6)genes were significantly up-regulated after APEC81Δybg D deletion,but the gene transcription levels of key proteins(My D88,NF-κB1,NF-κB2,JNK and ERK)in the NF-κB signaling pathway and MAPK signaling pathway were not significantly different.Further detection of cytokine levels found that APEC81Δybg D infection induced higher levels of the inflammatory cytokines IL-6 and IL-1β(P < 0.05).By infecting mice with the deletion strain APEC81Δybg D,it was found that the bacterial load in the liver,spleen,kidney,and lungs of the mice was significantly increased(P <0.05).It causes spleen enlargement in mice,indicating that mice induced a stronger inflammatory response.The results of this study indicate that YbgD fimbriae mediate APEC to inhibit the activation of TLR3 and TLR9 receptors in the innate immune system during host infection,thereby inhibiting the release of inflammatory cytokines IL-6 and IL-1β,and avoiding the occurrence of inflammatory responses,thereby preventing premature clearance of APEC by the host immune system.In conclusion,this study identified multiple biofilm-forming genes by constructing a transposon random mutation library of APEC81.It is confirmed that YbgD fimbriae can prevent APEC from enhancing the adhesion and invasion of host cells by inhibiting the expression of type 1 fimbriae and adhesin Fde C,thereby avoiding the activation of intracellular TLR3 and TLR9 receptors,thereby inhibiting the occurrence of inflammatory responses and facilitating escape.Colonization is achieved by the host’s innate immunity.And ETT2 virulence island can regulate YbgD fimbriae to participate in this pathogenic process.It indicates that ETT2 virulence island and YbgD fimbriae play an important role in escaping the recognition process of the host’s innate immune system,and this study provides a theoretical basis for the pathogenic mechanism of APEC.