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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 17, 2013
Reactivity of murine and human recombinant LPS-binding protein (LBP) within LPS and gram negative bacteria
S Lengacher1, C V Jongeneel, D Le Roy
1Division of Infectious Diseases, CHUV, Laussane, Switzerland.
Insights
Serum lipopolysaccharide (LPS) binding protein (LBP) enhances cellular responses to LPS. Recombinant LBP binds avidly to free LPS and heat-killed bacteria, but weakly to live Gram-negative bacteria.
Area of Science:
- Immunology
- Microbiology
- Biochemistry
Background:
- Serum lipopolysaccharide (LPS) binding protein (LBP) significantly amplifies cellular reactions to low LPS concentrations.
- LBP facilitates LPS transfer to CD14, initiating signaling cascades in susceptible cells.
Purpose of the Study:
- To clone and sequence murine LBP cDNA.
- To produce recombinant murine LBP using a baculovirus expression system.
- To investigate the binding characteristics of recombinant LBP to LPS and bacteria.
Main Methods:
- Cloning and sequencing of murine LBP cDNA.
- Production of recombinant murine LBP via baculovirus expression.
- Solid-phase and cytofluorometric assays to assess LBP binding to LPS and bacteria.
Main Results:
- Recombinant murine and human LBP exhibit avid binding to free LPS.
- LBP binds weakly to live Gram-negative bacteria, with binding loosely correlating to O chain length and composition.
- Recombinant LBP demonstrates strong binding to heat-killed Gram-negative bacterial preparations.
Conclusions:
- LBP's binding affinity differs significantly between live and killed Gram-negative bacteria.
- Findings suggest LBP's primary role in immune response may be directed towards killed, rather than live, Gram-negative bacteria.
Abstract:
The serum lipopolysaccharide (LPS) binding protein, LBP, has been shown to greatly enhance cellular responses to low concentrations of LPS. Purified LBP facilitates the transfer of LPS to membrane-bound or soluble CD14; the CD14/LPS complex then triggers a signal in responsive cells. We have cloned and sequenced a cDNA encoding murine LBP, and produced recombinant murine LBP using a baculovirus expression system. Using either a solid-phase or a cytofluorometric assay, recombinant murine and human LBP were found to bind avidly to free LPS, but only weakly to live bacteria from most LPS-containing Gram negative strains. Binding correlated loosely with the length and composition of the polysaccharide O chains. However, recombinant LBP did bind well to all heat-killed bacterial preparations. These findings suggest that LBP could be implicated in the response to killed but not live Gram negative bacteria.
