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Helicobacter pylori and Porphyromonas gingivalis lipopolysaccharides are poorly transferred to recombinant soluble
M D Cunningham1, C Seachord, K Ratcliffe
1Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Abstract:
Helicobacter pylori and Porphyromonas gingivalis are gram-negative bacteria associated with chronic inflammatory diseases. These bacteria possess lipopolysaccharides (LPSs) that are able to activate human monocytes to produce tumor necrosis factor alpha but fail to activate human endothelial cells to express E-selectin. With Escherichia coli LPS, tumor necrosis factor alpha activation requires membrane-bound CD14 and E-selectin expression requires soluble CD14 (sCD14). Therefore, the ability of H. pylori and P. gingivalis LPSs to transfer to and bind sCD14 was examined by using immobilized recombinant sCD14 and human serum or recombinant LPS-binding protein (LBP). H. pylori and P. gingivalis LPSs were transferred to sCD14 when serum or LBP was present. However, the transfer of these LPSs to CD14 in serum was significantly slower than the transfer of E. coli LPS. Quantitation of the transfer rates by Michaelis-Menten kinetics yielded K(m) values of 6 and 0.1 nM for H. pylori and E. coli LPSs, respectively. The amount of P. gingivalis LPS required to obtain half-maximum binding to CD14 was approximately 10-fold greater than the amount of E. coli LPS required. The slower transfer rates displayed by these LPSs can be explained by the poor binding to LBP observed in direct binding assays. These results are consistent with the proportionately lower ability of these LPSs to activate monocytes compared with E. coli LPS. However, the ability of H. pylori and P. gingivalis LPSs to bind LBP and transfer to sCD14 demonstrates that the lack of endothelial cell CD14-dependent cell activation by these LPSs occurs distal to sCD14 binding.
Insights
Helicobacter pylori and Porphyromonas gingivalis lipopolysaccharides (LPSs) bind soluble CD14 (sCD14) but slower than E. coli LPS. This explains their reduced ability to activate monocytes and endothelial cells.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Gram-negative bacteria like Helicobacter pylori and Porphyromonas gingivalis are linked to chronic inflammatory conditions.
- Their lipopolysaccharides (LPSs) activate human monocytes to produce tumor necrosis factor alpha but not endothelial cells to express E-selectin.
- Escherichia coli LPS activation pathways involve both membrane-bound and soluble CD14 (sCD14).
Purpose of the Study:
- To investigate the binding and transfer of H. pylori and P. gingivalis LPSs to sCD14.
- To understand the role of LPS-binding protein (LBP) in this interaction.
- To elucidate the reasons for differential activation of monocytes and endothelial cells by these LPSs.
Main Methods:
- Utilized immobilized recombinant sCD14 and human serum or recombinant LPS-binding protein (LBP).
- Assessed LPS transfer to sCD14 in the presence of serum or LBP.
- Quantified LPS transfer rates using Michaelis-Menten kinetics and direct binding assays.
Main Results:
- H. pylori and P. gingivalis LPSs transferred to sCD14 with serum or LBP, but at significantly slower rates than E. coli LPS.
- Michaelis-Menten kinetics revealed higher K(m) values for H. pylori LPS (6 nM) compared to E. coli LPS (0.1 nM).
- P. gingivalis LPS required approximately 10-fold more material for half-maximum binding to sCD14 than E. coli LPS, linked to weaker binding to LBP.
Conclusions:
- The slower transfer rates of H. pylori and P. gingivalis LPSs to sCD14 are attributed to their poor binding to LBP.
- These findings correlate with the observed lower capacity of these LPSs to activate monocytes compared to E. coli LPS.
- The ability of H. pylori and P. gingivalis LPSs to bind LBP and transfer to sCD14 indicates that the lack of endothelial cell activation occurs downstream of sCD14 binding.