Construction Of OmpA Deletion Mutant Of Escherichia Coli NMGCF-19 Strain Associated With Lamb Meningoencephalitis And Its Pathogenesis

Extraintestinal pathogenic Escherichia coli(Ex PEC)is a group of Escherichia coli(E.coli)colonizing other tissues outside the intestinal tract,which can cause extraintestinal infections in humans and animals,such as urogenital tract infections,encephalitis of young livestock and poultry,sepsis and pneumonia,seriously endangering human health and the development of animal husbandry.E.coli strain NMGCF-19 causing lamb encephalitis was clinically isolated from sick and dead lambs characterized by diarrhea and neurological symptoms in our laboratory in 2019.Previous research has found that E.coli is detectable in the mouse brain after infection with the NMGCF-19 strain,proving that NMGCF-19 can disrupt the blood-brain barrier(BBB),and revealed that NMGCF-19 can cause encephalitis by reducing the expression of tight junction proteins(mainly including ZO-1 and occludin)and disrupting the BBB.However,it is not yet clear which virulence genes NMGCF-19 specifically utilizes to disrupt the BBB.Previously,the ompA gene was found to be a potential virulence gene of E.coli strain NMGCF-19 through whole-genome sequencing and analysis and might be involved in the process of NMGCF-19 disrupting the BBB.Outer membrane protein A(ompA)is an important protein in the outer membrane of E.coli,which can maintain the stability of the bacterial structure.In addition,it has been reported that the ompA gene is related to BBB destruction by neonatal meningitis E.coli(NMEC),and it is mainly correlated with bacteremia and bacterial adhesion.To further explore the mechanism of the ompA gene in mediating meningitis,knockout of the ompA gene for E.coli strain NMGCF-19 was performed strain using CRISPR/Cas9 technology based on bioinformatics analysis of the ompA gene from NMGCF-19 strain to constructthe NMGCF-19ΔompAbacteria strains.The growth and biochemical characteristics of NMGCF-19 and NMGCF-19ΔompA,and their pathogenicity to mice were compared.Moreover,NMGCF-19 and NMGCF-19ΔompA strains were used to infect mouse brain microvascular endothelial cells(bend.3)and mice.The m RNA and protein expressions of inflammatory factors and tight junction proteins in cells and mouse brain were detected and compared by quantiatative PCR and western blotting assay.The results showed that the expressions of inflammatory factors in the cell supernatant and mouse brain decreased significantly,while the expressions of tight junction proteins increased significantly after infecting bend.3 cells and mice with NMGCF-19ΔompA in comparison with the NMGCF-19 infection group.These results indicate that the ompA gene plays an important role in BBB invasion by NMGCF-19.Further analysis revealed that the expression of receptor protein gp96 significantly reduced in the supernatant of bent.3 cells and the mouse brain of the NMGCF-19ΔompA infection group compared with the NMGCF-19 infection group,suggesting that the ompA gene may reduce the invasive ability of NMGCF-19 to the BBB by affecting the expression of receptor protein gp96.In summary,the results in this sudy demonstrated that r knockout of the ompA gene in NMGCF-19 strain significantly reduced its ability of disrupting the BBB and the pathogenicity to mice,suggesting that ompA gene is associated with the invasion of NMGCF-19 to the BBB.The findings also demonstrated that knockout of the ompA gene in NMGCF-19 strain affects the invasive ability of NMGCF-19 to the BBB by regulating the expressions of intracellular proinflammatory factors,tight junction proteins,and the expression of receptor protein gp96.