Biosynthesizing Kdo₂-lipid A Of Different Structure And Study Of Their Endotoxin Activities

Some Gram-negative bacteria are foodborne pathogen, and their outer membrane consist of plenty lipopolysaccharides(LPS), known as endotoxin, which is closely related to their pathogenicity. Hence, the study of relationship between endotoxin structure and its activity lays a theoretical foundation for the control of foodborne pathogen.Kdo2-lipid A is the minimal LPS structure to sustain bacteria viability and the bioactive centers of LPS, which can activate the mammalian innate immune system. In this study, we constructed five Escherichia coli(E. coli) mutants that producing Kdo2-lipid A with novel structures by deleting the polysaccharide(PS) synthesis-related genes in the chromosome. The immunological and biochemical activities of these strains and their synthetic Kdo2-lipid A samples were investigated. The results are listed below.(1) Based on homologous recombination and site specific recombination, the waaC-waaF genes from the chromosome of laboratory parent strains HW001, HW002, HW003, HW004 and HW005 were deleted, respectively, and the screening markers and auxiliary plasmids were also removed, resulting in five novel waaC-waaF-null mutants BW001, BW002, BW003, BW004 and BW005.(2) The structures of LPS synthesized by mutants BW001, BW002, BW003, BW004 and BW005 were analyzed through SDS-PAGE, TLC, ESI/MS and ESI/MS/MS, and founded that these strains synthesized specific Kdo2-lipid A instead of LPS.(3) Live bacteria cells of BW001, BW002, BW003, BW004 and BW005 mutants wereused to stimulate the HEK-Blue hTLR4 cells,and the TLR4 stimulatory ability of thesemutants were declined compared to wide-type strain W3110 and their parent strains.(4) Kdo2-lipid A samples were purified from mutants BW001, BW002, BW003, BW004 and BW005, and the purified samples were used to stimulate mouse RAW264.7 cells and human THP-1 cells;the release of mouse and human promflammatory cytokines were measured by ELISA. The results showed that all the Kdo2-lipid A samples from the five new mutants were attenuated than LPS from wide-type W3110 to different level and all the five samples were less toxic to both mouse and human cells; besides, their immunological activities were similar to the LPS from their parent strains respectively.(5) Compared to wide-type W3110 and the five parent strains, BW001, BW002, BW003, BW004 and BW005 presented higher outer membrane permeability, higher sensitivity to antibiotics, higher hydrophobicity and auto-aggregation ability, but lower biofilm formation ability and lower MICs.The novel attenuated endotoxin molecules produced by the E. coli mutants in this study have the potential to be further developed as novel vaccine adjuvants, and the premium biochemical phenotypes of the mutants lay the foundation for future industrial application.