Mechanistic Insights into Outer Membrane Permeability and Synergistic Activity of Temporins

The increasing emergence of multidrug-resistant microbes has prompted the search for new antibiotics with a new mode of action. Naturally occurring antimicrobial peptides (AMPs), that are produced by almost all forms of life, represent attractive candidates for the development of a new generation of anti-infective agents [1]. There is compelling evidence that unlike conventional antibiotics, most AMPs do act by altering the permeability of the target cell membrane. However, before reaching it, they need to cross the microbial cell wall that, in Gram-negative bacteria, is surrounded by the lipopolysaccharide (LPS)-outer membrane, which forms an efficient barrier against a variety of molecules. In Amphibia, temporins are among the shortest (10 to 14 residues) AMPs, with up to ten isoforms within the same specimen [2]. We have shown that some of them (temporin-1Tb, TB, and temporin-1Tl, TL) have a synergistic effect in killing Gram-negative bacteria [3]. This suggests an important strategy to overcome bacterial resistance due to the LPS layer. More precisely, we have found that LPS induces oligomerization of TB. This would prevent its translocation across the outer membrane making the peptide inactive on Gram-negative bacteria. Differently, TL is highly active on these bacteria, presumably due to the lack of such oligomerization. To gain insight into interactions of TL and TB within LPS, we investigated the peptides’ structures and interactions in LPS micelles using NMR spectroscopy. Interestingly, TL assumes a novel anti-parallel dimeric helical structure stabilized by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, TB demonstrates populations of helical and aggregated conformations in LPS. Note that the LPS-induced aggregated states of TB are largely destabilized in the presence of TL. STD (saturation transfer difference)-NMR studies have delineated residues of TL and TB in close contacts with LPS and enhanced interactions of these two peptides with LPS, when combined together. Importantly, these results provide the first structural insights into the mechanism of action and synergism of AMPs at the level of the LPS-outer membrane. 1 Zasloff M (2002) Nature 415: 389-95 2 Mangoni ML (2006) Cell Mol Life Sci 63: 1060-69 3 Mangoni ML et al. (2008)J. Biol Chem 283:22907-17