Sphingolipids and Fatty Acids Alter Porphyromonas gingivalis Protein/Lipid Compositions

Long chain bases: sphingosine, phytosphingosine, and dihydrosphingosine, and fatty acids: lauric acid and sapienic acid, commonly found on oral mucosa and on skin have potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria including Porphyromonas gingivalis.  Treatment with these lipids also induces striking ultrastructural damage to the overall bacterial cell and formation of unique intracellular inclusions in some bacteria.  However, little is known about the mechanisms of antimicrobial action as well as the nature and the origin of the bacterial inclusions.  Objectives: The objective of this study was twofold.  1) We assessed potential effects on whole-cell protein profiles of bacteria following treatment with lipids. 2) We determined if bacteria exhibited cell-association of test lipids following exposure. Methods: P. gingivalis was treated with long chain bases or fatty acids and pelleted by centrifugation. For one treatment, whole-cell protein extracts were reduced and separated by SDS polyacrylamide gel electrophoresis and stained.  For another treatment, lipids were extracted and separated by thin layer chromatography.  Controls consisted of non-treated bacterial cultures and suspended lipids alone.  Standards consisted of purified long chain bases and fatty acids.  Results: Of the long chain bases and fatty acids examined, sapienic acid induced the greatest alterations in the bacterial protein profile and additional high molecular weight proteins were present after SDS-PAGE.  All the long chain bases and fatty acids examined could be identified in cell pellets by TLC.  Conclusions: Lipids, especially sapienic acid, alter whole-cell bacterial protein profiles relative to controls.  Mechanisms for these alterations are not yet known and are being explored.  Long chain bases and fatty acids are present in cell pellets suggesting they may be associating with the bacterial membrane or accumulating in the cell, perhaps in the forming inclusions.  Supported by NIH/NIDCR RO1 DEO18032 and R01 DE014390.