Structures and biological activity of phosphorylated dihydroceramides of Porphyromonas gingivalis

Frank C. Nichols, Birgit Riep, JiYoung Mun, Martha D. Morton, Mike T. Bojarski, Floyd E. Dewhirst, and Michael B. Smith

Department of Periodontology, University of Connecticut, Farmington, CT 06030

Corresponding Author: nichols{at}nso.uchc.edu

Porphyromonas gingivalis, a recognized periodontal pathogen, synthesizes free ceramides as well as other phosphorylated complex lipids that potentiate interleukin-1b-mediated secretory responses in gingival fibroblasts. The purpose of this study was to separate complex lipids of P. gingivalis by HPLC, determine the structures of the major ceramides recovered in HPLC fractions and assess biological activity of the purified ceramide lipids. P. gingivalis was grown in pure culture and complex lipids extracted. HPLC fractionation revealed specific dihydroceramide lipid fractions that were pooled and refractionated using the same HPLC system. The repurified dihydroceramide lipids were then subjected to detailed structural analysis using electrospray MS/MS and NMR approaches as well as hydrolytic approaches combined with GC-MS analysis. Three major ceramide lipid fractions were recovered. The earliest eluting dihydroceramide class (HPLC fractions 7-8) contained free dihydroceramides consisting of 3-OH isobranched (iso) C17:0 in amide linkage to saturated long chain bases of 17, 18 or 19 carbons. This fraction revealed little capacity to potentiate IL-1b-mediated prostaglandin secretion. The second major dihydroceramide fraction (HPLC fraction 20) contained the base dihydroceramide structures of HPLC fractions 7-8 but was substituted with phosphoglycerol and isoC15:0 linked to the hydroxyl of 3-OH isoC17:0. This fraction demonstrated significant potentiation of IL-1b-mediated prostaglandin secretion from gingival fibroblasts. The third major dihydroceramide fraction (HPLC fraction 28) contained the dihydroceramide base structures of HPLC fractions 7-8 but was substituted with phosphoethanolamine. HPLC fraction 28 demonstrated minimal potentiation of prostaglandin secretion. The novel phosphoceramides produced by P. gingivalis demonstrate varying capacities to promote inflammatory mediator secretion or cell morphology changes based on the phosphorylated head group substitution and/or the addition of esterified fatty acid. These results demonstrate the capacity of phosphoglycerol dihydroceramides of P. gingivalis to act as a strong virulence factor by promoting inflammatory factor secretion from fibroblasts.

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