Study Of Function And Application Of N-acyltransferase LpxD In F.novicida

Lipopolysaccharide(LPS,endotoxin)is the significant component of outer membrane,which can be recognized by Toll-like receptor4(TLR4)in the innate immune responses.Lipid A is the key and biologically active component.Modification of lipid A can alter the virulence of the bacteria and biological activity of LPS.Some specific lipid A have been used as vaccine adjuvants.LpxD is N-acyltransferase that catalyzes the third step transferring acyl chains to the 2 and 2' positions in the lipid A biosynthesis.Francisella is a gram-negative bactria with extreme virulence.Two kinds of N-acyltransferasewere identified in F.novicida in our previous study.Mutational analysis showed the lpxD1-null mutant was attenuated.Effects of lipid A acyltransferases on the pathogenesis of F.novicida were analyzed.LpxD1 and Lpx D2 were expressed in serious E.coli.The detail results are listed below:(1)ESI/MS were used to determine the lipid A structure of F.novicida,lpxD1-null and lpxD2-null mutants.The effects of lipid A acyltransferases on the pathogenesis of F.novicida were analyzed by compariation of environmental resistance,level of stimulation of TLR4,intracellular survival and replication,toxicity to cells of F.novicida,lpxD1-null and lpxD2-null mutants.The lpxD1-null mutant showed sensitive to environmental stress,loss of replication ability and lower toxicity.(2)LpxD1 and lpxD2 were expressed in E.coli RL25.LpxD1 can transfer 3-OH-C18 and 3-OH-C16 to 2 and 2' position of lipid A.Lpx D2 can transfer 3-OH-C16 to the position of lipid A.Compared to RL25/pUC,RL25/pUC-lpxD1 and RL25/pUC-lpxD2 presented higher outer membrane permeability,higher auto-aggregation and lower hydrophobicity.Cells were used to stimulate HEK-Blue hTLR4 cells.LPS of the three strains were used to stimulate RAW267.4 cells and THP-1 cells.RL25/pUC-lpx D1,RL25/pUC-lpxD2 and their LPS showed declined immune activity compared with RL25/pUC.(3)LpxD1 were expressed in W3110,HW001,HW002,HW003 and HW004.The structures of them were determinged by MS;Cells and LPS were used to stimulate HEK-Blue hTLR4,RAW267.4 cells and THP-1 cells.Immune activity of the strains with expression of LpxD1 showed different degrees of decline.