Structure Modification Of Lipid A In Escherichia Coli

Lipid A is the major component of lipopolysaccharide, which forms the outer monolayer of the outer membrance in most Gram-negative bacteria. Lipid A is responsible for the bioactivity of endotoxin. After entering the host, lipid A can be recognized by immune cells, causing a series of reactions. The biosynthetic pathway of lipid A in bacteria is relatively conservative, but structure modifications can occur during its transport to the bacterial surface. These modifications differ in bacteria in order to adapt to different external environment. They are not necessary for survival, but are tightly regulated in the cell and closely related to the virulence of bacteria. This work focused on the structure modification of lipid A in Escherichia coli. The main results are listed below:(1) Recombination vectors pWSK29-PagL and pWSK29-PagP have been constructed. The expression of PagL and PagP in E. coli and the corresponding modification of lipid A were studied. The results showed that the modification of PagL and PagP on the structure of lipid A are different. In the strain expressing PagL, almost all the lipid As lack the fatty acyl chain at 3 position; the modification of lipid A by expressing PagP was significantly weaker in E. coli. These suggest that the function of PagP was not only regulated by PhoP-PhoQ in transcription, but also influenced by other external conditions.(2) The co-expression vector of pWSK29-pagL-lpxE-pagP was constructed and MPL could be produced in vivo, though other three lipid A species were also produced.(3) A rapid and efficient method was constructed to integrate heterologous genes into the chromosome of E. coli. The cat gene was used as a reporter to test the generality of this method. The results indicate that the reporter gene cat has been inserted and the kan gene has been removed in the chromosome of W3110-cat. This method provides a novel and vector independent method for the structure modification of lipid A in E. coli.(4) Three mutant strains, CW001, CW002 and CW004 were constructed, using the above method. CW001 carries an lpxE gene in the chromosome, and predominantly produces MPLA in vivo. CW001 provides a solid foundation for the development of the MPL vaccine adjuvant strains and the attenuated living vaccine strains. CW002 carries lpxE gene and pagL gene in the chromosome. CW004 is a papP deletion mutant strain and carries pagL, lpxE, pagP gene under the control of kan promoter in the chromosome. CW004 can produce the same level of MPL to the strain containing the corresponding expression vector.