Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions

Abstract

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways

“…In our previous study, we found that sapienic acid treatment of P. gingivalis induces an upregulation of several proteins involved in the biosynthesis of bacterial lipids, metabolism and energy production, degradation of polypeptides, cell adhesion, and virulence (22). Our current study extends those previously reported results, identifies additional proteins in P. gingivalis that are differentially expressed after treatment with sapienic acid, and identifies the primary metabolic pathways affected by sapienic acid treatment.…”

“…Sapienic acid (C 16:1⌬6 ; 254.4 g/mol) kills P. gingivalis within 6 min (MIC, 58.6 g/ml; minimal bactericidal concentration [MBC], 62.5 g/ml), causing disruption of cell membranes that is evident in micrographs (22). Additionally, more than 50% of the sapienic acid used to treat P. gingivalis in our studies stayed associated with the bacterial lipids or was retained intracellularly.…”

“…Fatty acids, especially sapienic acid, appear to be more active against oral species of bacteria than against nonoral bacterial spe-cies (16,22). Sapienic acid (C 16:1⌬6 ; 254.4 g/mol) kills P. gingivalis within 6 min (MIC, 58.6 g/ml; minimal bactericidal concentration [MBC], 62.5 g/ml), causing disruption of cell membranes that is evident in micrographs (22).…”

Itraconazole suppresses the growth of glioblastoma through induction of autophagy

“…[64][65][66] Cells were harvested, pelleted, and fixed in paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, post fixed with 1% OsO 4 for 1.5 h, washed and finally stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water, dehydrated with graded alcohol (50%, 75%, and 95 to 100% alcohol), and embedded in Epon-Araldite resin (Canemco-Marivac, Montreal, PQ, Canada).…”

Phylogenetic and functional gene structure shifts of the oral microbiomes in periodontitis patients

“…As a result, a small group of predominantly Gramnegative anaerobic bacteria and spirochetes, notably Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola, have long been regarded as the main putative pathogens of periodontitis (Holt and Ebersole, 2005;Ertugrul et al, 2013;Fischer et al, 2013). However, more recent studies have shown that, instead of a single pathogen, periodontitis may be a poly-microbial infectious disease caused by potentially pathogenic microbial communities, in which microorganisms interact in a synergistic or cooperative manner, leading to pathogenesis (Darveau, 2010;Hajishengallis and Lamont, 2012;Griffen et al, 2012).…”