Cloning And Expression Of Genes Encoding Temperature Sensitive Hemagglutinin And Exploration Of Potential Virulence Factors In Enteropathogenic Escherichia Coli

Extraintestinal pathogenic Escherichia coli(ExPEC)has many types,serotypes and virulence factors.Its potential zoonotic virulence factors have posed a threat to public health to a certain extent.The continuous production and accumulation of antibiotic-resistant bacteria bring severe challenges to the treatment of bacterial infection.The research on the virulence factors of APEC is of great significance for developing specific preventive measures or curative effects against ExPEC.Since autotransporter proteins widely distributed in ExPEC play an essential role in pathogenic mechanisms such as adhesion,invasion,and immune escape.It suggests that these proteins are likely to be a potential target for preventing and controlling ExPEC,which is worth exploring.Among them,temperature sensitive hemagglutinin(Tsh)widely exists in the genome of ExPEC.Some studies have shown that Tsh plays a role in adhesion and invasion as a critical virulence factor of ExPEC infecting livestock and poultry.However,the conservation of the ExPEC tsh gene and the virulence of tsh in human cells are not apparent.In order to clarify the conservation of tsh gene in ExPEC,bioinformatics was used to analyze the tsh gene sequences of E.coli from homo sapiens,swine,and giant pandas,which were downloaded from the NCBI database.Subsequently,taking the tsh gene sequence of APEC IMT5155(CP005930.1)as the reference sequence,we designed specific primers and amplified and sequenced the tsh genes of strains isolated from chicken and dogs which were stored in our laboratory by PCR.At the same time,we analyzed the homology between the sequencing results and the tsh genes sequences of the above three strains downloaded from NCBI.The results showed that the homology rate of tsh gene of these five strains isolated from different hosts and tissues could reach 100%.It indicates that the ExPEC tsh gene is highly conserved in different hosts,indicating that the Tsh protein is a potential prevention and control target of ExPEC.In order to further explore the function of Tsh protein in ExPEC and lay a foundation for exploring its virulence characteristics in HBMEC which is the vitro model of blood-brain barrier,we took APEC TW-XM preserved in our laboratory as a representative and used λRed homologous recombination technique to construct the tsh gene deletion strain APECΔtsh and the complementary strain APECΔtsh/pBR322tsh.The results of bacterial growth curve experiment showed that the deletion of Tsh protein had no effect on the normal growth of ExPEC in vitro.Subsequently,the wild strain APEC TW-XM,the deletion strain APECΔtsh and the complementary strain APECΔtsh/pBR322tsh were co-cultured with HBMEC,respectively.The virulence of Tsh protein in the infected human cell model was further studied through the adhesion and invasion experiment.The results showed that the adhesion ability of the deletion strain APECΔtsh to HBMEC was significantly lower than that of the wild strain APEC TW-XM and the complementary strain APECΔtsh/pBR322tsh(P<0.05).However,there was no significant difference in the invasion experiment.It indicates that Tsh protein mainly plays a role in the adhesion process.In order to understand the mechanism of the effect of tsh deletion on adhesion,we measured the changes of the expression of the previously reported ExPEC virulence factors that are clearly pathogenic to human and animal cells.The results showed that the deletion of tsh gene led to a very significant downregulation of the expression of key adhesion factors fimH,ibeA and OmpA of ExPEC(P<0.01).The results showed that the adhesion mediated by tsh gene deletion was downregulated,including the loss of direct adhesion of Tsh protein and downregulating the expression of other adhesins.From the perspective of host,we measured the expression of HBMEC related pro-inflammatory factors.The results showed that the deletion of Tsh protein led to the transcription level of IL-1β and IL-6 was significantly downregulated(P<0.01).Also,the transcription level of TNF-α was significantly downregulated(P<0.05).These results suggest that Tsh protein promotes the inflammatory response of HBMEC and promotes the process of bacterial meningitis.In order to further explore the pathogenic mechanism of Tsh protein adhesion,we further measured the expression of Tsh binding proteins collagen Ⅵ and fibronectin on human cells by real time PCR.The results showed that the deletion of Tsh protein led to a very significant decrease in the expression of collagen Ⅳ and fibronectin(P<0.01),indicating that the adhesion of Tsh was achieved by promoting the expression of receptor protein on host cells.In conclusion,this study analyzed the virulence mechanism of ExPEC Tsh protein in human cells to a certain extent,suggesting that Tsh can induce the expression of host cell binding protein collagen Ⅳ and fibronectin,promote the adhesion of ExPEC to HBMEC,mediate the upregulation of other virulence factors of ExPEC(fimH,ibeA,OmpA),and then stimulate the upregulation of the pro-inflammatory factor IL-6 and TNF-α of HBMEC.So it promotes the process of bacterial meningitis and directly and indirectly mediates the pathogenic process of ExPEC to the host.In addition,this study proves that Tsh protein has good conservation in different host source ExPEC,and is a potential prevention and control target,which is worthy of further attention and research.