Sandra Alvarez
Cooperative Activity of Human Lysozyme and Fatty Acids Against P. aeruginosa
Abstract
Sandra Alvarez, Michael Waldon, Jose Martinez, Ami Oren, Karl Lohner, and Edith Porter
Background: Innate immunity plays a major role in protecting mucosal membranes from microbial invasion. Antimicrobial peptides with a membrane perturbing activity are well established effector molecules in innate immunity. Lipids, including free fatty acids, are also found in mucosal secretions. However, in spite of reports on altered fatty acid composition in secretions of patients with cystic fibrosis that suffer from chronic lung infection with Pseudomonas aeruginosa and the use of certain lipids as antimicrobials and in food preservation, lipids have not yet been considered as antimicrobial effector molecules. Hypothesis: Selected host-derived fatty acids may contribute to innate host defense alone, or in conjunction with innate antimicrobial peptides, by disrupting bacterial membranes and thereby leading to bacterial cell death. Methods: The potential effects of four free fatty acids; oleic acid (OA), palmitoleic acid (PA), docosahexaenoic acid (DA), and linoleic acid (LA) on lysozyme activity against P. aeruginosa or on model membrane system was assessed using a metabolic assay, electron microscopy (EM), equilibration dialysis (ED), and thermodynamic (TD) studies. Results: Lysozyme-mediated metabolic inhibition and ultra structural damage of PA cell membranes were significantly potentiated by DA. EM observations were consistent with an accumulation of DA in the bacterial cell membranes. ED experiments with radiolabeled fatty acids and PA, and TD studies with mammalian and bacterial model membrane-systems further supported this notion. Conclusion: Our data suggest that DA augments the antibacterial action of lysozyme, whereby lysozyme causes an initial membrane lesion through which DA can gain access into the membrane leading to severe structural and functional damage. These findings support a novel role of lipids as direct effector molecules in innate immunity.
Lipid Signaling at the Floral Transition in Arabidopsis thaliana
Susana Nava
Lipid transfer proteins are a group of plant proteins that have been found to bind and carry lipids between membranes in vitro. In vivo, LTPs have been found to be involved in defense responses and systemic resistance signaling. Microarray studies done in our lab have shown a 9- and 14-fold increase in leaf LTP3 and LTP4 expression, respectively, in Arabidopsis thaliana leaves during the bolting transition. Other results from our lab suggest a lipid signal might be involved in this transition. In order to determine whether LTP3 and LTP4 bind to specific lipids in vivo, transgenic Arabidopsis plants, each expressing either LTP3 or LTP4 tagged with a hemagglutinin epitope, were constructed. Transformant lines were confirmed through PCR with primers specific for the HA-tagged sequence. Expression of the tagged proteins is being verified by RT-PCR and Western blot. The HA-tagged proteins will then be immunoprecipitated from leaf exudates and the chemical structure(s) of the in vivo ligand(s) determined by mass spectrometry. Recombinant LTP3 and LTP4 will also be purified from E.coli for in vitro binding studies.