Construction Of Escherichia Coli Mutants That Produce Lipid A With Different Structures

Lipid A is an important integrant of LPS, and plays an important role in membranepermeability. Lipid A is also responsible for the bioactivity of LPS. It can be recognized bythe TLR4/MD-2complex of mammalian immune cells, leading to activation of in vivosignaling pathways and release of proinflammatory cytokines. The level of cytokines isclosely related to its precise chemical structures. Lipid A with right chemical structure couldbe used as vaccine adjuvants to enhance the immune response. A series of E.coli mutants thatsynthesize lipid A of different structures were constructed in this study by chromosomal genedeletion and integration. The main achievements are listed below：(1) By chromosomal gene deletion of lacI in W3110, an original strain for chromosomalconstitutive expression of integrated heterologous genes in E.coli was constructed, namedHW000. lpxE from Francisella was integrated into lacZ of HW000, resulting in HW001,which could synthesize pure MPLA confirmed by TLC and ESI/MS. HW002was constructedby further deletion of lpxM in HW001, which could synthesize pure M-MPLA confirmed byTLC and ESI/MS.(2) HW003, HW004and HW005were constructed by integrating gene clusters ofpagL-lpxE, lpxE-SepagP, pagL-lpxE-SepagP respectively into lacZ of HW000. pagL andSepagP were derived from Salmonella. The structures of lipid A in HW003, HW004andHW005were changed according to their genetic modifications confirmed by TLC andESI/MS, which could synthesize L-MPLA, P-MPLA and LP-MPLA respectively.(3) waaC and waaF, which encode heptosyltransferase, were deleted respectively inHW001and HW002to construct HW006and HW007. The two strains could synthesizeKdo2-lipid A confirmed by SDS-PAGE.(4) The changes in the structure of lipid A had no influences in either growth rate orsurface hydrophobicity of E. coli cells. However, HW001, HW002, HW004and HW005showed an enhanced membrane permeability; HW006and HW007showed a distinct slowergrowth rate comparing to W3110.(5) Purified LPSs of HW001, HW002, HW003, HW004and HW005were testedrespectively for immunological competence. There is a clear decline in generation of IL-6andTNF-α when the five LPSs were used to stimulate RAW264.7cells, with that of HW002LPSreduce the most. This suggests that the immunological competence of LPS relies largely onthe precise structure of its lipid A.