Characterization Of The IcmF Gene And Identification Of Genes That Contribute To Invasion Of Brain Microvascular Endothelial Cells In Extraintestinal Pathogenic Escherichia Coli

Extraintestinal pathogenic Escherichia coli(ExPEC) can cause opportunistic infections in extraintestinal organs, like lung infections, urinary tract infections, and meningitis. Although many virulence factors have been identified, the pathogenesis is still not clear. Many kinds of pathogenic bacteria have been identified having the type VI secretion system including Ex PEC. And T6 SS is found to play a role in pathogenic processes. In this study, two T6 SS gene deletion mutantsΔhcp1-3 and ΔicmFwere used to investigate the function of T6 SS in an EXPEC strain PCN033. The majorresults are as follows:(1) Construction of the icmF gene detection mutant ΔicmFby anegative and positive selection system, and improve the selection process.(2) Compared with the wild type, Δhcp1-3 and ΔicmF showed no change in motility, drug tolerance, but were increased in biofilm formation on plastic surfaces.(3) When interacting with eukaryocyte, T6 SS mutants were increased in adherence to and invasion of HBMEC, and decreased in intra-macrophage survival, but no changed in macrophage toxicity. These results suggest that icmF in PCN033 may play a role in virulence through the function of T6 SS.In order to get more understanding of the pathogenesis of E.coli meningitis, a Tn5 insertion mutant library was built to identify the virulence factors in RS218. A total of 764 insertion mutants of RS218 were screened, and 33 mutants were find out to be decreased invasion of HBMEC distinctly. The growth rates of these 33 mutants were determined,and 2 mutants which had defect in growth were removed from the library. Then 31 potential HBMEC invasion mutants were used for testing invasion frequence. The mutants were all found to exhibit significantly decreased invasion of HBMEC. TAIL-PCR was used to get the genome DNA flanking Tn5. Sequencing data showed that 29 out of 31 insertions were located in genes, and 2 were intergenic. In addition, fimH, a gene previously shown to play a role in bacterial adhesion and invasion, was identified. The results of this study laid the foundation of the future research of E.coli meningitis.