Study On The Related Functions Of Avian Meningitis-Induced E.coli Tsh Protein In The Destruction Of The Blood-brain Barrier

Avian Pathogenic Escherichia coli(APEC)APEC TW-XM and Neonatal Meningitis Escherichia coli(Neonatal Meningitis Escherichia coli,NMEC)play important roles in neonatal and avian meningitis,and become one of the challenges for the development of livestock and poultry breeding and human health.Using bioinformatics to compare the genomes of APEC TW-XM and NMEC,it is found that they belong to the B2 system group and the 95 multi-locus sequence typing complex(ST95).They are highly homologous at the level of their genomic DNA,amino acid sequence and protein.There are many similar virulence factors and the same animal infection model,which indicates that APEC TW-XM has potential public health significance.Temperature-sensitive hemagglutinin(Tsh)is one of the important components of avian pathogenic Escherichia coli.As an important adhesive and key virulence factor,it can mediate the interaction between bacteria and host cells.Tsh is highly conserved in the evolution of Gram-negative bacteria.In the past few decades,the structure,function and expression of Tsh in APEC have been extensively studied for an important virulence factor of avian pathogenic Escherichia coli.Studies have shown that it can promote the adhesion of bacteria to epithelial cells such as avian respiratory tract host cells,play a key role in the adhesion and invasion of APEC.Multilocus sequence typing and virulence plasmid detection found that Tsh is the common virulence target of the two kind of NMEC and APEC TW-XM and the amino acid sequence is highly homologous.Therefore,exploring the function of Tsh in APEC TW-XM is of great significance for early warning of the potential risk of zoonotic diseases.Tsh as ainitialing point in this study was explored its pathogenic mechanism in the process of APEC TW-XM pathogen infection for the host.This study used the meningitis-causing Escherichia coli APEC TW-XM strain to detect the pathogenic effect of the virulence factor Tsh on meningitis,laying the foundation for subsequent research on the potential risks of preventing zoonotic diseases.First,this study compared the tsh gene sequence of related meningitis-causing E.coli strains on NCBI GenBank,designed a pair of primers for PCR amplification of the tsh gene in APEC TW-XM,and then cloned it into the pBR322 expression vector.Sequencially this study transformed recombinant pBR322 expressing tsh into E.coli DH5a to express Tsh protein.Then this study verified the agglutination activity of Tsh on red blood cells by hemagglutination test,and confirmed that Tsh exists in APEC TW-XM.Then by using the blood-brain barrier(Blood-Brain Barrier,BBB)in vitro model mouse brain microvascular endothelial cells(mouse brain microvascular endothelial cells,bEnd.3)to detect APEC TW-XM adhesion to bEnd.3 cells at low temperature(26?),mammalian body temperature(37?)and avian body temperature(42?).The adhesion results showed that the APEC TW-XM strain had the strongest adhesion ability to bEnd.3 cells under the condition of avian body temperature(42?),providing theoretical support for subsequent in vitro experiments.In order to explore the effect of Tsh on APEC TW-XM crossing bEnd.3 cells,based on the tsh gene sequence,the deletion strain and complementary strain of the coding gene tsh in the APEC TW-XM strain were constructed using the ?-Red homologous recombination system and gene cloning technology.The results of the growth curve experiment show that the absence of Tsh does not affect the growth pattern of APEC TW-XM,and it also excludes the influence of different growth patterns on the results of subsequent experiments.The adhesion and invasion results of bEnd.3 cells showed that compared with the wild strain,APEC TW-XM?tsh deletion strain had a significant decrease in the adhesion and invasion ability of bEnd.3 cells at 30,37,and 42?(0.005<P<0.01),the effection was the most significant at 42?,and the adhesion ability of the complementary infection group was not statistically different from that of the wild group(P>0.05).As an inflammatory factor that affects the destruction of BBB function,it is often used as a parameter for detecting pathogen adhesion and invasion of BBB.Therefore,this study used qRT-PCR to detect the expression of inflammatory factors IL-1?,IL-6 and TNF-? after infection of bEnd.3 cells at three different temperatures.The experimental results showed that,compared with the wild strain infection group,the expression of IL-1?,IL-6,TNF-? inflammatory factors in the bEnd.3 cells of the tsh gene-deletion strain infection group decreased significantly at the transcription level,and the most significant of decrease was at 42?,There was no significant difference between the invasive ability of the complementary infection group and the wild group(P>0.05).Collagen ? and fibronectin are binding proteins of Tsh protein,which can mediate the adhesion of Tsh to respiratory epithelial cells.In order to further explore how Tsh protein promotes the adhesion of APEC TW-XM to bEnd.3 cells,this study used qRT-PCR to detect the expression of collagen ?and fibronectin after infection of bEnd.3 cells at three different temperatures.The results showed that compared with the wild strain infection group,the expression of collagen ? and fibronectin decreased significantly in the bEnd.3 cells of the deletion strain infection group.The transcription levels of collagen ? and fibronectin of Tsh-deletion strains decreased most significantly at 42?.There was no statistical difference between the complementary group and the wild group(P>0.05).The above in vitro experimental results show that Tsh can promote APEC TW-XM adhesion and invasion of bEnd.3 cells by inducing the expression of collagen ? and fibronectin,and induce the expression of pro-inflammatory factors in the cells,thereby affecting the invasion of BBB by bacteria.In order to further explore the adverse effect of Tsh on APEC TW-XM-induced meningitis,this study established an animal model of APEC TW-XM-induced meningitis and compared the neurological symptomsmortality,BBB integrity,brain pathology,collagen ? and fibronectin protein expression after infection of mouse with APEC TW-XM strain,APEC TW-XM?tsh deletion strain and APEC TW-XM?tsh/ptsh complementary strains.By observing and scoring the clinical neurological symptoms of meningitis,this study successfully established a mouse model of APEC TW-XM-induced meningitis.The neurological symptoms of the mouse in the deletion strain infection group were reduced.The survival experiment of mouse found that the mortality of mouse infected with deletion strain decreased significantly.The mortality rate of the wild strain,deletion strain and complementary strains infection group were 100%(10/10),66%(8/12),and 92%(11/12),respectively.The results of bacterial colonization showed that the number of bacteria in the blood,brain,heart,liver,spleen,and lungs of the mouse in the deletion strain infection group was significantly reduced compared with the mouse in the wild strain infection group(P<0.01).Evans Blue(EB)dye was injected into the tail vein to determine the degree of damage to the BBB of mouse.The results showed that the degree of brain staining in the deletion strain infection group was significantly lower than that of the wild strain and complementary strain infection group.The pathological section results showed that compared with the deletion group,there were obvious histopathological lesions such as meningeal thickening and neutrophil infiltration in the brains of the wild group and the complementary strains group.qRT-PCR was used to detect the expression of inflammatory factors and Tsh-binding protein collagen ? and fibronectin in the brain tissue after APEC TW-XM,APEC TW-XM?tsh and APEC TW-XM?tsh/ptsh infection.The results showed that compared with the wild strain infection group,when the mouse were infected with the deletion strain,the levels of IL-1?,IL-6 and TNF-?in the brain tissue were significantly reduced by 81.2%,78.5%,and 82.1%(P<0.01);the expression of collagen ? and fibronectin decreased by 83.3%and 66.7%,(P<0.01).Further immunohistochemical experiments were carried out to explore the expression and distribution of collagen ? and fibronectin on blood vessels in the brain tissue of mouse after infection.The results showed that,compared with the wild group,the expression of collagen ? and fibronectin on the vascular endothelial cells of the brain tissue of the deletion group were significantly reduced.The above results are consistent with the results of in vitro experiments,proving that Tsh affects APEC TW-XM's ability to break through BBB and cause meningitis.The above in vitro and in vivo experimental results show that Tsh can up-regulate the expression of collagen ? and fibronectin to promote the colonization of APEC TW-XM in the brain and the adhesion and invasion of BBB.After invading the central nervous system,it induces the production of pro-inflammatory factors,and finally causes bacterial meningitis.