The Molecular Mechanism Study Of The Induction Of EGR-1 By Meningitic Escherichia Coli Mediates Disruption Of The Blood-brain Barrier

Bacterial meningitis is a huge challenge to human health and livestock breeding.In human medicine,bacterial meningitis is one of the top ten infectious diseases causing high mortality,and most survivors will develop different degrees of neurological sequelae.In veterinary medicine,a large number of farm animals lost their economic value due to meningitis,which caused huge economic losses to the animal husbandry industry.Extraintestinal pathogenic Escherichia coli?ExPEC?has attracted wide attention in recent years;it can infect multiple organs of the host,leading to urinary tract infection and meningitis.Meanwhile,ExPEC can also infect pets and transmit diseases,contaminating food that causes food hygiene and safety panics.Therefore,ExPEC-induced epidemics in different hosts have become an important zoonotic infectious disease that seriously affecting human health,food safety and the development of animal husbandry.Up to now,research on E.coli meningitis mainly focuses on the screening of target genes involved in how meningitic E.coli invades into the central nervous system?CNS?.However,it is rarely reported on the function of host genes that involved in the process of meningitic E.coli when it destroys the blood brain barrier?BBB?permeability in meningitis.This study was based on clinically isolated ExPEC from different species,combined with meningitis E.coli infection of human brain microvascular endothelial cells?hBMECs?differentially coding RNA and non-coding RNA sequencing results,to explore the molecular mechanism of BBB permeability increase in meningitic E.coli-induced central inflammation.In this study,7 ExPEC strains from patients,20 ExPEC strains from diseased pigs and 3 ExPEC strains from avian stored in our lab were selected to testify their invasion abilities to the hBMECs and the mouse brain.8 strains were observed to possess strong invasion abilities to hBMECs,including PCN033,Xiantao 10,ACN005,PCN079,HCN10618,PCN080,A764 and B958.Among them PCN033 and A764 have strong viability in mouse blood and colonization ability in brain tissue.Considering that PCN033 was originally isolated from the brain of a diseased pig,we subsequently used PCN033 as our experimental meningitic strain.Subsequently,we found that meningitic E.coli increased BBB permeability by downregulating the expression of tight junctions?TJs?in hBMECs,and altering the distribution of TJs.The high-throughput sequencing was used to analyze the transcriptional profiles of host coding RNA and non-coding RNA in hBMECs in response to meningitic E.coli.A total of 366 mRNAs,895 lncRNAs,32 miRNAs and 308 circRNAs were significantly altered.In addition,regulatory network diagrams of significant changed circRNAs,miRNAs and mRNAs were drawn.By analyzing the whole transcriptome data of meningitis E.coli infected or uninfected hBMECs,we have explored the biological function of host factor EGR-1,which plays an important regulatory role in the process of meningitic E.coli destroying host BBB integrity.Via hBMECs EGR-1 gene-deleted cell lines and EGR-1 gene-deleted mice,we demonstrated that EGR-1 was involved in the regulation of CNS inflammatory response and BBB permeability.We found that transcription regulator EGR-1 was significantly upregulated in the early stage of infection,and can bind to the promoter of VEGFA,PDGFB,ICAM-1 and TNF?.Through further in vivo and in vitro experiments,we found that meningitis E.coli infection induced the expression of EGR-1 to promote host secreting VEGFA and PDGF-BB,which acted on BMECs decreasing TJs expression and altering the distribution of TJs.On the other hand,Snail-1 also significantly upregulated in BMECs in response to meningitic E.coli,and it can directly inhibit the transcription of TJs such as ZO-1 and Occludin by binding to their promoter region.Meanwhile,we demonstrated that meningitis E.coli infection induced the expression of EGR-1 to cause the high expression level of ICAM-1 on BMECs which contributed to inflammatory cells crossing the BBB to mediate CNS inflammation,and then enhancing the permeability of BBB.In addition,this study was the first time to report the function of circRNAs in bacterial meningitis.We found that the expression of hsa_circ₂858 was significantly upregulated in meningitic E.coli infected-hBMECs,which can upregulate the expression of VEGFA by sponging hsa-miR-93-5p.This study demonstrated the roles of EGR-1 in mediating meningitic E.coli-induced BBB damage as well as neuroinflammation,providing new concepts and potential targets for future prevention and treatment of bacterial meningitis.