Mechanism Study Of Extraintestinal Pathogenic Escherichia Coli Disruption Of The Blood-brain Barrier Through ?-Hemolysin

Extraintestinal Pathogenic Escherichia coli(Ex PEC)is a group of pathogens colonized in the host's extraintestinal tissues and cause diseases.The pathogens can lead to pyelonephritis,meningitis,pneumonia,peritonitis,and sepsis,etc.,with a poor prognosis regularly.Besides,the Ex PEC has been reported to obtain serious drug resistance,which was harmful to the livestock and poultry breeding industry,and seriously threatens food safety,public health,and the ecological environment.Bacterial meningitis is one of the diseases caused by Ex PEC.The mortality is relatively high and the survivors are always companied with neurological sequelae of varying degrees,which seriously affects their quality of life,which led to great economic loss.In the animal production industry,the affected animals lose the value for further treatment.Ex PEC causes meningitis depends on its interaction with the blood-brain barrier(BBB).BBB is a complex physiological structure composed of multiple kinds of cells,and the normal communication and regulation between various types of cells in the barrier are of great significance to the structure and function of the barrier itself and even the central nervous system(CNS).Brain microvascular endothelial cells(BMECs),one of the main components of the BBB,are responsible for maintaining the low permeability of the BBB and the characteristics of CNS low immunity activity and determine the homeostasis of the nervous system.For a long time,the interaction between Ex PEC and BMECs has been focused on by the researchers.However,most of the study focused on the interaction between bacteria and monolayer BMECs and ignored the interaction between BMECs and other types of cells in the barrier(such as glial cells),the alterations of cell-to-cell communication upon infection,as well as the effects of such alterations on the function of BBB and CNS.Therefore,this study intends to explore the underly mechanism of cell-to-cell communication in the BBB,and further reveal the changes,significance,and implications of related cell-to-cell communication upon Ex PEC infection,exploring the key targets of pathogens and hosts.The main conclusions are as follows:1.We demonstrated that Astrocytes-derived TGF?1 enhanced the BBB barrier function via activating the non-canonical Hedgehog signaling in BMECs.In this study,we established an in vitro BBB model based on transwell by co-culturing U251 cells together with human BMECs cells.Based on previous transcriptome data of U251,we demonstrated that TGF?1 derived from astrocytes enhanced tight junction proteins ZO-1 expression in BMECs cells,as well as the barrier function of endothelial cells.With a series of in vivo and in vitro studies,we found that TGF?1 modulated the ZO-1 expression via TGFBRI/II-Smad2/3 signals in endothelial cells,while the transcription factor Smad2/3 was unable to bind to the zo-1 gene promoter region directly.Through further exploration,we demonstrated that the Smad2/3 signaling in BMECs activated the intracellular non-canonical HH signal and up-regulated the expression of its transcription factor Gli2.Gli2 was presented to bind to the zo-1 gene promoter to enhance the expression.With luciferase reporter system and Chromatin Immunoprecipitation(Ch IP),we presented the binding of Smad2/3 to the gli2 promoter,as well as Gli2 to the zo-1 promoter.Therefore,we concluded that TGF?1 derived from astrocytes was able to activate the non-classical HH signal in BMECs to promote the expression of tight junction protein ZO-1,and ultimately improved the barrier function of BBB.2.We revealed that ?-hemolysin of meningitic Ex PEC interrupted the TGF?1-based intercellular communication between astrocytes and BMECs and breakdown the BBB.We established in vivo and in vitro infection models with a classic meningitic Ex PEC strain RS218.We found that RS218 infection down-regulated the TGFBRII and Gli2 in the TGFBRI/II-Gli2-ZO-1 axis of BMECs,thereby interfering with the TGF?1-based intercellular communication between astrocytes and BMECs.Through bioinformatics analysis,we found that RS218 inhibits the expression of TGFBRII by down-regulating the positive transcription regulator Sp1 of TGFBRII.When Sp1 overexpressed,the downregulation of TGFBRII due to infection was compensated.Furthermore,we screened the RS218 mutant library,constructed with Tn5-mini transposon,through targeting the gli2 gene promoter.We identified the hly CABD operon in RS218 as a key virulence gene for bacteria decreasing Gli2.By constructing deletion strains and completed strains as well as expressing the corresponding proteins,we proved that the ?-hemolysin Hly A encoded by the operon increased the calcium concentration in BMECs and activated PKA,which was reported to promote Gli2 phosphorylation and degradation.Additionally,we furtherly demonstrated that Hly A was also involved in the decreasing of TGFBRII,in a calcium PKA independent manner.Therefore,we raise the concept that meningitic Ex PEC downregulated TGFBRII and Gli2 in BMECs by secreting ?-hemolysin Hly A,thereby blocking TGF?1-based intercellular communication between astrocytes and BMECs,thus weakening the barrier function.3.We presented that activating the Hedgehog signaling in BMECs repressed the infection-induced endothelial immunity activating via mi R-155.Furtherly,we treated RS218-infected mice with recombinant TGF?1 or the HH signal agonist SAG,and found that SAG protected infected mice,preventing them from death.On the one hand,the activation of the HH signaling directly promoted the expression of tight junction protein ZO-1 and maintained the structural integrity and low permeability of the BBB.On the other hand,the activation of HH signaling up-regulated the mi R-155 in BMECs,thereby inhibiting the activation of the Erk1/2 signal in BMECs due to bacterial infection,and repressed the up-regulating of adhesion molecule E-selectin,inflammatory factor IL-6,and chemokine the expression of MIP-2.Additionally,the activation of microglia in the brain of infected animals and the enhancement of IL-6 and MIP-2 in the brain during infection were also reduced with SAG presence.Recombinant TGF?1 also presented similar protection effects through activating non-canonical HH signaling in BMECs cells.In this work,we demonstrated that astrocytes-derived TGF?1 enhanced BBB barrier function through trigger a non-canonical HH signaling in BMECs.However,the meningitic Ex PEC-secreted hemolysin Hly A interrupted the intercellular communication based on TGF?1 through decreasing TGFBRII and Gli2,which played a key role in the communication between astrocytes and endothelial cells.Furtherly,we also demonstrated that SAG protected the animals upon RS218 infection,which was attributed to the maintaining of BBB integrity and low immunity activity by the induced HH signaling.This work revealed a novel mechanism by which bacterial breakdown BBB,identified Hly A as the key toxin effector,and extended our knowledge to bacterial meningitis.Most of all,this work suggested screening for novel drugs targeting HH signaling for bacterial infection treatment.