Molecular mechanisms of sphingosine-mediated killing of bacteria

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We have shown that micromolar or even nanomolar concentrations of sphingosine are sufficient to kill many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus (even methicillin-resistant S. aureus [MRSA]), Acinetobacter baumannii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia species, which are important pathogens causing pneumonia. In vivo studies showed that sphingosine also kills P. aeruginosa and S. aureus in the lungs of mice with cystic fibrosis (CF), with no measurable adverse effects on the mammalian cells. Additional studies of the inhalation of sphingosine in mice and mini-pigs in concentrations as high as 1 mM in the inhalation solution and also of locally administered sphingosine confirmed that sphingosine has no adverse effects on the mammalian epithelial cells of the trachea, bronchi, and nose. Thus, sphingosine seems to have a relatively selective antibacterial function. Sphingosine contains an NH2 group and an OH group. At neutral or slightly acidic pH, as found in airways and on many epithelial cell surfaces, the NH2 group will be protonated and, thus, positively charged. We hypothesize that sphingosine, depending on its positive charge, binds to negatively charged phospholipids, mainly cardiolipin. Preliminary studies confirm this hypothesis. Cardiolipin is expressed in bacterial plasma membranes and has very important functions in respiration. In mammalian cells cardiolipin is expressed only in mitochondria, but it is absent from the cell plasma membrane and, thus, is much less accessible to sphingosine applied to cells in the form of micelles. The proposed studies will investigate the hypothesis that sphingosine/sphingosine micelles bind to cardiolipin and induce a clustering of cardiolipin in the bacterial plasma membrane, with the subsequent formation of large gel- or even crystal-like structures that disturb the biophysical properties of the membrane, rapidly resulting in leakiness of the plasma membrane and bacterial cell death.