Immuno-Stimulatory Activity of Escherichia coli Mutants Producing Kdo2-Monophosphoryl-Lipid A or Kdo2-Pentaacyl-Monophosphoryl-Lipid A

Abstract

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.

A phosphoethanolamine transferase specific for the 4′-phosphate residue of Cronobacter sakazakii lipid A

“…; Wang et al . ). Briefly, bacterial cells were harvested at an OD 600 of 1·5 and washed twice with phosphate‐buffered saline (PBS).…”

Insights into the structure of Escherichia coli outer membrane as the target for engineering microbial cell factories

“…E. coli K-12 W3110 mutants WBB06 [ 55 ] and WJW00 [ 56 ] could be used to produce Kdo 2 -lipid A. Furthermore, the least toxic structure Kdo 2 -P-MPLA could be synthesized by the mutant HWB02 [ 37 ].

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“…Modifications of both PG and PE are well-known in resistance mechanisms, especially in response to aminoglycosides and cationic antimicrobial peptides [ 35 ]. Kdo 2 -lipid A, as the bioactive center of LPS, is known to be responsible for the toxic effects of infections, and is recognized by the Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2) complex [ 12 , 36 , 37 ]. Kdo 2 -lipid A also represents a significant obstacle for the effective delivery of numerous antimicrobial agents [ 12 ].…”

“…3 c) [ 36 ]. The MPLA had been developed as adjuvant [ 37 ], and the PS-MPLA showed decreased ability to activate the TLR4/MD-2 receptor of HKE-Blue hTLR4. Therefore, the attenuated modifications of lipid A in E. coli might be helpful to improve application safety for microbial cell factories; and better understanding phospholipids and Kdo 2 -lipid A contributes to design metabolic engineering strategies to improve robustness and security of bacterial producers.…”

The Characterization of Two-Component System PmrA/PmrB in Cronobacter sakazakii

“…Lipid A is the biologically active component of LPS, which can be recognized by the innate immune system through TLR4 (Wang et al, 2010). It triggers an inflammatory response with the production of a large number of cytokines and even leads to septic shock or death (Wang B. et al, 2015). In C. sakazakii, the backbone of the lipid A general structure is a hexa-acylated β-1 , 6linked disaccharide of glucosamine, which has phosphate groups at the 1 and 4 positions, 3-hydroxymyristate (3-OH C14:0) at the 2 and 3 positions, and secondary fatty acid derivatives at the 2 b (C14:0) and 3 b (C12:0 or C14:0) positions Jia et al, 2018).…”