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Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Published on: May 28, 2012
Identification and characterization of a lipopolysaccharide binding, anti-inflammatory protein from Parabacteroides
Weimin Guo1, Laura Carretero Santos2,3, Gar Yee Koh4
1Department of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine, Boston, MA, USA.
Abstract:
Gut bacteria are well known to impact a diverse array of acute and chronic diseases. One mechanism by which gut bacteria can impact health is by modulating inflammation. We and others previously found that membrane components of the gram-negative bacterium Parabacteroides distasonis (Pd) can suppress proinflammatory signaling in vitro and reduce intestinal inflammation in mouse models. Here, we sought to identify anti-inflammatory proteins from the membrane fraction of Pd. Using size exclusion chromatography, cell-based assays, fluorescence anisotropy, pull-down assays, and proteomics, we identified a putative anti-inflammatory protein named DUF4925 domain-containing protein (Protein ID: A6LA28, Gene ID: BDI_0772). Recombinant A6LA28 dose-dependently suppressed LPS induced IL-8 production from HT-29 and SW-480 cells, IL-6 and TNFα from splenocytes and blocked LPS binding to HT-29 and HEK reporter cells. Deletion of the C-terminal 201 amino acids, which are predicted to form a LPS binding pocket, destroyed its function, whereas deletion of the N-terminal 133 amino acids had little impact. Although its native function in Pd remains unclear, these data indicate that A6LA28 can bind to LPS, thereby preventing TLR4-mediated cytokine production. Further work is required to determine whether A6LA28 contributes to the anti-inflammatory capacity of Pd in vivo and to evaluate the therapeutic potential of this protein.
Insights
Researchers identified a novel anti-inflammatory protein, A6LA28, from gut bacteria Parabacteroides distasonis. This protein binds to lipopolysaccharide (LPS), suppressing inflammatory responses and offering potential therapeutic applications.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Gut bacteria significantly influence health and disease, often by modulating inflammation.
- Membrane components of Parabacteroides distasonis (Pd) are known to suppress pro-inflammatory signaling and reduce intestinal inflammation.
Purpose of the Study:
- To identify specific anti-inflammatory proteins within the Pd membrane fraction.
- To characterize the function and mechanism of action of a newly identified protein.
Main Methods:
- Size exclusion chromatography, cell-based assays, fluorescence anisotropy, pull-down assays, and proteomics were employed.
- Recombinant protein A6LA28 was tested for its ability to suppress LPS-induced cytokine production and block LPS binding.
- Site-directed mutagenesis (N-terminal and C-terminal deletions) was used to determine functional domains.
Main Results:
- A DUF4925 domain-containing protein (A6LA28) was identified as a putative anti-inflammatory agent.
- Recombinant A6LA28 dose-dependently inhibited LPS-induced IL-8, IL-6, and TNFα production.
- A6LA28 blocked LPS binding to cells, indicating interference with Toll-like receptor 4 (TLR4) activation.
- The C-terminal region of A6LA28 is crucial for its LPS-binding and anti-inflammatory function.
Conclusions:
- A6LA28 binds to lipopolysaccharide (LPS), thereby inhibiting TLR4-mediated cytokine production.
- The protein A6LA28 shows therapeutic potential for inflammatory conditions.
- Further research is needed to confirm A6LA28's in vivo role and therapeutic efficacy.
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