Lipid A is the active center of lipopolysaccharide which also known as endotoxin. Monophosphoryl-lipid A (MPLA) has less toxicity but retains potent immunoadjuvant activity; therefore, it can be developed as adjuvant for improving the strength and duration of the immune response to antigens. However, MPLA cannot be chemically synthesized and can only be obtained by hydrolyzing lipopolysaccharide (LPS) purified from Gram-negative bacteria. Purifying LPS is difficult and time-consuming and can damage the structure of MPLA. In this study, Escherichia coli mutant strains HWB01 and HWB02 were constructed by deleting several genes and integrating Francisella novicida gene lpxE into the chromosome of E. coli wild type strain W3110. Compared with W3110, HWB01 and HWB02 synthesized very short LPS, Kdo2-monophosphoryl-lipid A (Kdo2-MPLA) and Kdo2-pentaacyl-monophosphoryl-lipid A (Kdo2-pentaacyl-MPLA), respectively. Structural changes of LPS in the outer membranes of HWB01 and HWB02 increased their membrane permeability, surface hydrophobicity, auto-aggregation ability and sensitivity to some antibiotics, but the abilities of these strains to activate the TLR4/MD-2 receptor of HKE-Blue hTLR4 cells were deceased. Importantly, purified Kdo2-MPLA and Kdo2-pentaacyl-MPLA differed from wild type LPS in their ability to stimulate the mammalian cell lines THP-1 and RAW264.7. The purification of Kdo2-MPLA and Kdo2-pentaacyl-MPLA from HWB01 and HWB02, respectively, is much easier than the purification of LPS from W3110, and these lipid A derivatives could be important tools for developing future vaccine adjuvants.
Pseudomonas putida KT2442 is an important bacterium for producing various types of polyhydroxyalkanoate polymers. Phospholipids and lipid A in membranes of P. putida play important roles in stress responses, but detailed structural information of these lipids is not known. In this study, phospholipids and lipid A were isolated from P. putida KT2442, and their structures were analyzed using thin layer chromatography, high performance liquid chromatography, and electrospray ionization/mass spectrometry. Major phospholipids in P. putida KT2442 were phosphatidylethanolamine (79.9%), phosphatidylglycero1 (12.7%), and cardiolipin (7.4%), with C16:1 and/or C18:1 acyl chains. Four lipid A species were found in P. putida KT2442: two are hexa-acylated, and the other two are penta-acylated. Compared with lipid A of P. aeruginosa, P. putida lipid A has less hydroxylation on the secondary acyl chains and less modification. Therefore, P. putida lipid A could be used as a base structure to investigate lipid A modification of P. aeruginosa for understanding its pathogenesis.
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