Identification Of Lipid A Phosphoethanolamine Transferase Related Genes In Cronobacter Sakazakii

Cronobacter sakazakii is a Gram-negative opportunistic pathogen contaminated in powdered infant formula, which can cause meningitis, sepsis, bacteremia and necrotizing enterocolitis. Lipid A is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, which plays an important role in cell membrane stability, permeability and pathogenesis. Previous research indicated that an encoding phosphoethanolamine ept A gene exists in Escherichia coli and Salmonella typhimurium, by which to modify the lipid A structure to further change the virulence of bacteria.In this study, we constructed Cronobacter sakazakii mutants by using chromosomal gene expression and gene knockout technology to identify the function of ESA_RS09200 and ESA_RS16425. The immunological and biochemical activities of these strains were further investigated, several major findings were shown as follows:(1) Lipid A structure isolated from C. sakazakii BAA894 under pH 5.0 with modification of phosphoethanolamine was identified by analytical chemistry. Two genes ESA_RS09200 and ESA_RS16425 in BAA894 were found to be highly homologous to eptA gene in S. typhimurium and E. coli by the BLAST.(2) ESA_RS9200 and ESA_RS16425 were expressed respectively in E. coli W3110 and C. sakazakii BAA894. Lipid A were isolated from strains under pH 5.0 and the structure was identified by ESI/MS. The results showed that ESA_RS09200 encoded enzyme is a phosphoethanolamine aminotransferase.(3) WLL001 and WLL002 were constructed by deleting ESA_RS09200 and ESA_RS16425 from the genome of C. sakazakii BAA894. The lipid A structures isolated from WLL001 and WLL002 were identified, which further prove that ESA_RS09200 encoded enzyme is phosphoethanolamine aminotransferase, while ESA_RS16425 encoded enzyme is not lipid A modification related enzyme. Biological characteristics in WLL001 mutant were determined and analysised, including growth curve, cell surface hydrophobicity, membrane permeability and antibiotic minimal inhibitory concentration. The biochemical phenotype of the mutants with the modified lipid A structure were studied as compared to the wild type strain.(4) BAA894/pWSK29-lpxE, WLL001/pWSK29-lpxE, WLL003/pWSK29-lpxE and WLL003/pWSK29-lpxF were constructed to identify phosphoethanolamine modification occurs in 4' position, but not 1 position.(5) To further analyze the impact of structural changes in the lipid A immune recognition, live bacteria cells of WLL001 mutant was used to stimulate the HEK-Blue hTLR4 cells and lipopolysaccharide purified from mutant strains were used to stimulate RAW264.7 cells. The cell infection experiment showed that the WLL001 mutant decreased the infection ability of Caco-2 cells, and also the survival rate in THP-1 cells was decreased.