A Synergism between Temporins toward Gram-negative Bacteria Overcomes Resistance Imposed by the Lipopolysaccharide Protective Layer

Abstract

Temporins are short and homologous antimicrobial peptides (AMPs) isolated from the frog skin of Rana genus. To date, very little is known about the biological significance of the presence of closely related AMPs in single living organisms. Here we addressed this question using temporins A, B, and L isolated from Rana temporaria. We found that temporins A and B are only weakly active toward Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L. To shed light on the underlying mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the effect of the peptides' interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction with lipopolysaccharides (LPS) and the effect of LPS on the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution and when bound to LPS, by using circular dichroism and ATR-FTIR spectroscopies. Our data reveal that temporin L synergizes with A and B by preventing their oligomerization in LPS. This should promote their translocation across the outer membrane into the cytoplasmic membrane. To the best of our knowledge, this is the first study that explains how a combination of native AMPs from the same species can overcome bacterial resistance imposed by the LPS leaflet.

NMR Structure of Pardaxin, a Pore-forming Antimicrobial Peptide, in Lipopolysaccharide Micelles

“…In particular, a higher positive charge content has been thought to be necessary to overcome the LPS-mediated barrier in Gramnegative organisms (11)(12)(13). Recent studies with amphibian AMPs temporins demonstrated the role of LPS in preventing the outer membrane permeabilization by low cationic (ϩ1 to ϩ2) antimicrobial peptides (20,21). Temporins A and B are largely inactive against Gram-negative bacteria as a result of the lack of their outer membrane permeabilization.…”

“…Temporins A and B are largely inactive against Gram-negative bacteria as a result of the lack of their outer membrane permeabilization. However, an interesting synergistic effect has been demonstrated among these temporins to disrupt outer membranes (20,21). The structural basis of the synergistic mechanism is not known.…”

“…The structural basis of the synergistic mechanism is not known. Although it has been demonstrated that single temporins might aggregate in the LPS layer, thus preventing transversal through the outer membrane (20,21). In contrast to these, Pa4 contains a net positive charge of ϩ1, disregarding terminal charges, and is found to be independently active.…”

Lipopolysaccharide, a Key Molecule Involved in the Synergism between Temporins in Inhibiting Bacterial Growth and in Endotoxin Neutralization

“…The majority of them act by increasing the permeability of the microbial membrane (34 -36). Recently, the physiological significance of the existence of multiple forms of temporins has been addressed and has revealed that temporins A and B strongly synergize in killing Gramnegative bacteria, when each is combined with temporin L (15). Temporin L has also been studied for its potential to suppress the endotoxic effect of LPS (37).…”

“…The LPS barrier is believed to be stabilized by LPS-associated cations (Mg 2ϩ ) through salt bridges that neutralize the repulsive forces of adjacent LPS molecules. This leads to the formation of an oriented and tightly crosslinked leaflet that protects bacteria from a variety of host-defense hydrophobic molecules (13,14), including some AMPs (15)(16)(17). Note that for all enterobacterial and most nonenterobacterial strains, LPS is composed of three parts: (i) a hydrophobic moiety, also referred to as lipid A, consisting of fatty acid chains linked to two phosphorylated glucosamine residues; (ii) an oligosaccharide core, covalently bound to the lipid A via ketodeoxyoctonic acid (KDO); and (iii) a hydrophilic O-antigenic domain, composed of repeating saccharide units, which protrudes into the surrounding medium.…”