Construction of an Escherichia coli mutant producing monophosphoryl lipid A

Chen, Jiuzhou; Tao, Guanjun; Wang, Xiaoyuan

doi:10.1007/s10529-011-0521-z

Cited by 13 publications

(18 citation statements)

References 23 publications

(25 reference statements)

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“…Plasmid pKD-Cre expresses the Cre recombinase, which directly acts on the repeated loxp (Cre recognition target) sites flanking the kan gene and excises the kan gene, resulting in the lacI mutant HW000. Secondly, the DNA fragment lacZ U- lpxE -FRT- kan -FRT- lacZ D was amplified from pWSK29- FnlpxE-Fkan [19] using primers lacZ -F and lacZ -R and transformed into HW000/pKD46. Under the help of Red enzymes expressed by pKD46, the lacZ gene was broken by the inserted DNA fragment lpxE -FRT- kan -FRT.…”

Section: Methodsmentioning

confidence: 99%

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

Han

Chen

et al. 2013

Marine Drugs

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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.

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Section: Methodsmentioning

confidence: 99%

“…For example, deleting the gene lpxM in E. coli can make it synthesize pentaacylated lipid A [17]. Expressing Francisella novicida lpxE in E. coli can remove the phosphate group from the 1-position of lipid A [18,19] (Figure 1). …”

Section: Introductionmentioning

confidence: 99%

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

Han

Chen

et al. 2013

Marine Drugs

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“…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.…”

Section: Structural Modification Of Kdo2-lipid a And Its Regulationmentioning

confidence: 99%

“…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.…”

Section: Structural Modification Of Kdo2-lipid a And Its Regulationmentioning

confidence: 99%

Kdo₂‐lipid A: structural diversity and impact on immunopharmacology

2014

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3-deoxy-d-manno-octulosonic acid-lipid A (Kdo2-lipid A) is the essential component of lipopolysaccharide in most Gram-negative bacteria and the minimal structural component to sustain bacterial viability. It serves as the active component of lipopolysaccharide to stimulate potent host immune responses through the complex of Toll-like-receptor 4 (TLR4) and myeloid differentiation protein 2. The entire biosynthetic pathway of Escherichia coli Kdo2-lipid A has been elucidated and the nine enzymes of the pathway are shared by most Gram-negative bacteria, indicating conserved Kdo2-lipid A structure across different species. Yet many bacteria can modify the structure of their Kdo2-lipid A which serves as a strategy to modulate bacterial virulence and adapt to different growth environments as well as to avoid recognition by the mammalian innate immune systems. Key enzymes and receptors involved in Kdo2-lipid A biosynthesis, structural modification and its interaction with the TLR4 pathway represent a clear opportunity for immunopharmacological exploitation. These include the development of novel antibiotics targeting key biosynthetic enzymes and utilization of structurally modified Kdo2-lipid A or correspondingly engineered live bacteria as vaccines and adjuvants. Kdo2-lipid A/TLR4 antagonists can also be applied in anti-inflammatory interventions. This review summarizes recent knowledge on both the fundamental processes of Kdo2-lipid A biosynthesis, structural modification and immune stimulation, and applied research on pharmacological exploitations of these processes for therapeutic development.

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“…Cronobacter sakazakii is a Gram-negative bacterial pathogen associated with contaminated, temperature-abused, powdered infant formula, which can cause meningitis and enterocolitis in neonates. Cronobacter sakazakii synthesizes two lipid A species, but the biosynthesis of lipid A in C. sakazakii has not been well characterized [5,6]. …”

Section: Introductionmentioning

confidence: 99%

Identification of Three Genes Encoding for the Late Acyltransferases of Lipid A in Cronobacter sakazakii

Cai

Tao

et al. 2013

Marine Drugs

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Lipid A, the hydrophobic anchor of lipopolysaccharide, is an essential component in the outer membrane of most Gram-negative bacteria. Food-borne pathogen Cronobacter sakazakii synthesizes two lipid A species, differing by the length of the secondary acyl chain. In this work, we identified three genes ESA02293, ESA02951 and ESA01386 encoding for the late acyltransferases of lipid A biosynthesis pathway in C. sakazakii. Based on the sequence alignment, proteins YP_001438378.1 encoded by ESA02293, YP_001439016.1 encoded by ESA02951, and YP_001437482.1 encoded by ESA01386 are homologous to E. coli LpxL, LpxP and LpxM, respectively. Functions of the three acyltransferases were confirmed by overexpressing the genes in E. coli, isolating lipid As and analyzing their structures using an ESI/MS. C. sakazakii LpxL and LpxM transfer a C14:0 secondary acyl chain to the 2′- and 3′-position of lipid A, respectively. C. sakazakii LpxP can transfer either a C16:1 or a C14:0 secondary acyl chains to the 2′-position of lipid A.

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Construction of an Escherichia coli mutant producing monophosphoryl lipid A

Cited by 13 publications

References 23 publications

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

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

Kdo₂‐lipid A: structural diversity and impact on immunopharmacology

Identification of Three Genes Encoding for the Late Acyltransferases of Lipid A in Cronobacter sakazakii

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Construction of an Escherichia coli mutant producing monophosphoryl lipid A

Cited by 13 publications

References 23 publications

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

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

Kdo2‐lipid A: structural diversity and impact on immunopharmacology

Identification of Three Genes Encoding for the Late Acyltransferases of Lipid A in Cronobacter sakazakii

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Kdo₂‐lipid A: structural diversity and impact on immunopharmacology