Construction of Monophosphoryl Lipid A Producing Escherichia coli Mutants and Comparison of Immuno-Stimulatory Activities of Their Lipopolysaccharides

Abstract

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine phosphorylated at the 1- and 4′-positions. It can be recognized by the TLR4/MD-2 complex of mammalian immune cells, leading to release of proinflammatory cytokines. The toxicity of lipid A depends on its structure. In this study, two E. coli mutants, HW001 and HW002, were constructed by deleting or integrating key genes related to lipid A biosynthesis in the chromosome of E. coli W3110. HW001 was constructed by deleting lacI and replacing lacZ with the Francisella novicida lpxE gene in the chromosome and only synthesizes monophosphoryl lipid A. HW002 was constructed by deleting lpxM in HW001 and synthesizes only the pentaacylated monophosphoryl lipid A. The structures of lipid A made in HW001 and HW002 were confirmed by thin layer chromatography and electrospray ionization mass spectrometry. HW001 and HW002 grew as well as the wild-type W3110. LPS purified from HW001 or HW002 was used to stimulate murine macrophage RAW264.7 cells, and less TNF-α were released. This study provides a feasible way to produce interesting lipid A species in E. coli.

Mucosal Prevalence and Interactions with the Epithelium Indicate Commensalism of Sutterella spp.

“…and E. coli and, thus, speculate that this could be due to their varying LPS structures. LPS on the surface of Gram-negative bacteria is composed of Kdo 2 -Lipid A (endotoxin), core-oligosaccharide and O-antigen repeats and recognized by the host TLR4–MD2 complex (Miller et al, 2005; Han et al, 2013; Polissi and Sperandeo, 2014). We visualized the differences in the LPS structure between the species by silver staining of the SDS-PAGE separated LPS, which revealed differences in O-antigen repeats, and by immunoblotting with an antiserum against E. coli LPS, which did not recognize LPS from Sutterella spp.…”

“…We visualized the differences in the LPS structure between the species by silver staining of the SDS-PAGE separated LPS, which revealed differences in O-antigen repeats, and by immunoblotting with an antiserum against E. coli LPS, which did not recognize LPS from Sutterella spp. However, the endotoxicity of LPS is more determined by the lipid A structure, such as phosphorylation degree as well as the number and length of acyl chains, which affects its ligand affinity to TLR4–MD2 (Kobayashi et al, 2006; Park et al, 2009; Han et al, 2013). The E. coli lipid A is hexa-acylated with two phosphate groups and therefore can activate the TLR4–Mal–MyD88 pathway making it highly virulent, whereas the penta-acylated or monophosphorylated forms of lipid A are 100-fold less toxic by signaling through TLR4–TRAM–TRIF (Park et al, 2009; Herath et al, 2011; Han et al, 2013).…”

Construction and Characterization of an Escherichia coli Mutant Producing Kdo2-Lipid A

“…Samples were analyzed by TLC [42,43] and electrospray ionization mass spectrometry (ESI/MS) [42]. Typically, 400-mL cultures were grown to an OD 600 of 1.5.…”

Kdo2‐lipid A: structural diversity and impact on immunopharmacology

“…The absence of the phosphate group would greatly decrease the surface negative charge of these bacteria. Two genes lpxE and lpxF encoding phosphatases have been identified in F. novicida (Wang et al , 2004, 2007; Chen, Tao & Wang, 2011; Han et al , 2013). LpxE selectively removes the phosphate group at the 1-position of Kdo 2 -lipid A, while LpxF selectively removes the phosphate group at the 4′-position.…”

“…LpxE from Francisella tularensis has been used to construct recombinant, plasmid-free strains of E. coli and Salmonella Typhimurium that produce predominantly 1-dephosphorylated lipid A (Chen et al , 2011; Han et al , 2013). LpxE expression in Salmonella Typhimurium reduced its virulence in mice.…”