Characterization Of InV Autotransporter In Edwardsiella Piscicida

Edwardsiella piscicida is a kind of Gram-negative pathogen causing high mortality edwardsiosis in fish.Type ? and ? secretion systems?T3SS and T6SS?are key virulence factors in E.piscicida.Our lab had previously constructed E.piscicida transposon insertion library based on Mariner Himarl transposon and a total of 20,668 insertion mutant strains were isolated.Based on the auto-aggregation phenotype mediated by T3SS protein EseB,a total of 47 putative T3SS regulatory factors including ETAE₀323 were screened.ETAE₀323?thereafter named InV?encodes a protein with 2,359 amino acid residues and is predicted to be an autotransporter invasin.A transposon insertion mutant of InV was found with significant T3SS deficiency.However,mutant strains with N-terminus or C-terminus deleted in ETAE₀323 as well as their complemented strain showed no effect on the expression of T3SS.Moreover,the localization of InV in T3SS mutant strain was not significantly different from that in WT.Nevertheless,depletion of InV showed dramatic attenuation in internalization in HeLa cell and virulence in zebrafish model,respectively.T3SS was previously found to associate to the fatty acids utilization in E.piscicida.In this project,CRISPR/Cas9 technology was applied in HeLa cells to explore the experimental conditions and efficiency in genome editing.Our data indicated that 50%of the viral solution infecting HeLa cells for 12 h had the highest infection efficiency,and the genes related to fatty acid metabolism?SCD1 and DGAT1?were successfully knocked out in HeLa cells.In addition,the results also indicated that SCD1 and DGAT1 were closely related to the metabolism of lipid and lipid droplet formation in HeLa cells,thus setting up a good foundation for the further experiments in our laboratory.