Functional Identification Of Neisseria Meningitidis Phosphoethanolamine Transferase And Its Key Sites

Lipid A is the main active component of lipopolysaccharide(LPS).The structural changes of lipid A will affect the extracellular membrane properties of Gram-negative bacteria,resulting in changes in bacterial drug resistance and virulence.Phosphoethanolamine transferase(EptA)is a structural modification enzyme of lipid A.EptA modifies the 1' or 4' phosphate group on lipid A to reduce the negative charge of lipid A,thus neutralizing the electronegativity of the bacterial surface.This modification reduces the binding ability of polypeptide antibiotics to the outer membrane of bacteria,thus reducing the cell autoimmunity to gram-negative bacteria.Escherichia coli is an important model strain,and the construction of phosphate ethanolamine transferase deletion strain of E.coli is convenient for studying phosphate ethanolamine transferase of Neisseria meningitidis and Cronobacter sakazakii.The mechanism of bacterial drug resistance influenced by phosphoethanolamine transferase and the pathogenic mechanism of bacteria were explored through drug resistance experiments and cell experiments.The main research conclusions of this topic are as follows:(1)By comparing the homology of Neisseria meningitidis MC58 and E.coli BL21(DE3)phosphoethanolamine transferase EptA,the EptA coding gene NMB_RS08540 was found in Neisseria meningitidis MC58 and named NmeptA.By constructing E.coli BL21(DE3)?eptA mutation and E.coli BL21(DE3)?eptA/ pET28a-NmeptA,we found that E.coli BL21(DE3)EptA and Neisseria meningitidis MC58 NmEptA were both regulated by weak acid environment.(2)Based on the conservative structure of phosphoethanolamine transferase protein structure,discovered by point mutation,we found Tys150,Trp126,Trp148 and Thr280 are probably the active sites of NmEptA and Thr279 is probably the active site of CsEptA.(3)Modification of lipid A by phosphoethanolamine results in drug resistance of E.coli to polypeptide antibiotics,and eptA mutant strain and wild-strains with mutant active sites are sensitive to polypeptide antibiotics.(4)Through the stimulation experiment of thallus on animal cells,it is found that eptA mutant strain has lower stimulation ability on TLR4 pathway than wild type and expression strain,and its survival ability in THP-1 cells is weakened.(5)By measuring the biological characteristics of bacteria,the absence of eptA has a certain positive effect on the growth of E.coli.The lack of eptA changes the characteristics of the E.coli cell membrane,which increases the permeability of the outer membrane of the cell.