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

Biwen Wang,Ye Li,Yaning Han,Xiaoyuan Wang,Yanyan Li,Eliane Namie Miyaji

doi:10.1371/journal.pone.0144714

Biwen Wang, Ye Li + Show 4 more

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https://doi.org/10.1371/journal.pone.0144714

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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.

Highlights

Lipopolysaccharide (LPS), the major component of the outer layer of outer membrane in most Gram-negative bacteria, is known as endotoxin [1,2] and is important for membrane stability [3]
The lipid A moiety of LPS can be recognized by the Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2) complex that activates the mammalian innate immune system and results in the production of potent inflammation mediators, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) [4,5,6]
Mutant strains HWB01 and HWB02 were constructed by deletion and integration of certain key genes related to LPS biosynthesis in the chromosome of E. coli W3110

Summary

Introduction

Lipopolysaccharide (LPS), the major component of the outer layer of outer membrane in most Gram-negative bacteria, is known as endotoxin [1,2] and is important for membrane stability [3]. The lipid A moiety of LPS can be recognized by the Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2) complex that activates the mammalian innate immune system and results in the production of potent inflammation mediators, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) [4,5,6]. Native lipid A is hexaacylated and bisphosphorylated, it can efficiently activate TLR4/MD-2 to induce leukocyte activation, cytokine production, and inflammation. This is beneficial for host because local bacterial infections can be cleared, but if these mediators are overproduced, they can damage small blood vessels and cause shock. Native lipid A cannot be used as an immunomodulator because it can cause pronounced inflammatory responses, but certain lipid A analogs such as monophosphoryl lipid A retain some immunomodulatory properties while possessing attenuated proinflammatory activity [8,9]

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