INTRODUCTION

Porphyromonas gingivalis (Pg), an etiologic agent for periodontitis, causes a highly unusual innate host response. Two different LPSs are available with differing biologic activity. One fraction, designated Pg₁₆₉₀, purified using a phenol method, yields a negative mass ion of 1690 which represents a penta-acyl lipid A. This fraction is an agonist for human monocytes and HUVEC. Another fraction, designated Pg_1435/1449, obtained through the cold magnesium chloride purification method, yields lipid A species with mass ions of 1435/1450 representing tetra-acyl forms. This preparation is a weak agonist for macrophages, does not stimulate HUVEC and is an antagonist of Pg₁₆₉₀ and E.coli LPS stimulation of these cells.

BIOCHEMICAL ANALYSIS   

Gas chromatographic/mass spectroscopy (GC/MS) of LPS fatty acids.

Pg₁₆₉₀ and Pg_1435/1450 LPS samples were analyzed by their trimethylsilyl ethers after transmethylation with N,O,-bis(trimethylsilyl) trifluoroacetimide (BSTFA) containing 1% trimethylchlorosilane (TMCS). The fatty acids are accounted for along with trace amounts of C14:0 and C18:0. No other fatty acids nor phospholipid, glycolipid, or lipoprotein were detected.

Mass Spectrometry

The deduced structure of the Lipid A moiety of Pg₁₆₉₀ and Pg_1435/1449 was analyzed by matrix assisted laser desorption ionization time of flight (MALDI-TOF). Pg 1690 contains a negative mass ion of 1690, which represents a penta-acyl Lipid A. Pg_1435/1449 LPS contains 2 species of lipid A with mass ions of 1435/1449, representing tetra-acyl forms. A minor species of tria-acyl is also present with a negative mass of 1195.

BIOLOGIC ACTIVITY

The biologic response of the purified LPS was tested for E-Selectin expression on HUVEC cells (Astarte HUVEC/E-Selectin Kit #2016). These cells normally produce E-selectin in response to E.coli LPS. Purified Pg₁₆₉₀ LPS is a potent agonist for E-selectin expression while Pg1435/1449 is a weak agonist. In addition, Pg_1435/1449 is able to antagonize Pg₁₆₉₀ stimulation of E-Selectin at 1:1 and E.coli LPS at 100:1. With respect to myeloid cells, Pg₁₆₉₀ is a more potent agonist of TNF-α and IL-8 expression by macrophages cells compared to Pg_1435/1449.

REFERENCES

• Somerville JE Jr, Cassiano L, Bainbridge B, Cunningham MD, Darveau RP. A novel Escherichia coli lipid A mutant that produces an anti-inflammatory lipopolysaccharide. J Clin Invest 1996;97:359-365.
• Darveau RP, Cunningham MD, Bailey T, et al. Ability of bacteria associated with chronic inflammatory disease to stimulate E-selectin expression and promote neutrophil adhesion. Infect Immun 1995;63:1311-1317.
• Reife, RA, et al. Porphyromonas gingivalis lipopolysaccharide lipid A heterogeneity: differential activities of tetra- and penta-acylated lipid A structures on E-selectin expression and TLR4 recognition. Cellular Microbiology (2006)8 (5), 857–868.
• Coats, S.R., Reife, R.A., Bainbridge, B.W., Pham, T.T.T., Darveau, R.P. P. gingivalis LPS antagonizes E.coli LPS at TLR 4 in human endothelial cells. Infect Immun 71(12):6799-6807 (2003).

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