An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes.

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due to their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Interaction with lipid II induces conformational changes in bovicin HC5 structure.

Bovicin HC5 is a lantibiotic produced by Streptococcus bovis HC5 that targets the cell wall precursor lipid II. An understanding of the modes of action against target bacteria can help broadening the clinical applicability of lantibiotics in human and veterinary medicine. In this study, the interaction of bovicin HC5 with lipid II was examined using tryptophan fluorescence and circular dichroism spectroscopy with model membrane systems that do or do not allow pore formation by bovicin HC5. In the presence of lipid II, a blue-shift of 12 nm could be observed for the fluorescence emission maximum of the tryptophan residue for all of the membrane systems tested. This change in fluorescence emission was paralleled by a decrease in accessibility toward acrylamide and phospholipids carrying a spin-label at the acyl chain; the tryptophan residue of bovicin HC5 was located near the twelfth position of the membrane phospholipid acyl chains. Moreover, the binding of lipid II by bovicin HC5 induced remarkable conformational changes in the bovicin HC5 structure. The interaction of bovicin HC5 with lipid II was highly stable even at pH 2.0. These results indicate that bovicin HC5 interacts directly with lipid II and that the topology of this interaction changes under different conditions, which is relevant for the biological activity of the peptide. To our knowledge, bovicin HC5 is the only bacteriocin described thus far that is able to interact with its target in extreme pH values, and this fact might be related to its unique structure and stability.

Role of lipid II and membrane thickness in the mechanism of action of the lantibiotic bovicin HC5.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria, nisin being the most well-known member. Nisin inhibits peptidoglycan synthesis and forms pores at sensitive membranes upon interaction with lipid II, the essential bacterial cell wall precursor. Bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5, has the putative N-terminal lipid II binding motif, and we investigated the mode of action of bovicin HC5 using both living bacteria and model membranes, with special emphasis on the role of lipid II. Bovicin HC5 showed activity against Staphylococcus cohnii and Staphylococcus warneri, but bovicin HC5 hardly interfered with the membrane potential of S. cohnii. In model membranes, bovicin HC5 was not able to cause carboxyfluorescein release or proton influx from DOPC vesicles containing lipid II. Bovicin HC5 blocked lipid II-dependent pore formation activity of nisin, and a high-affinity interaction with lipid II was observed (apparent binding constant [K(a)] = 3.1 × 10(6) M(-1)), with a 1:1 stoichiometry. In DOPC vesicles containing lipid II, bovicin HC5 was able to assemble with lipid II into a prepore-like structure. Furthermore, we observed pore formation activity of bovicin HC5, which was stimulated by the presence of lipid II, in thin membranes. Moreover, bovicin HC5 induced the segregation of lipid II into domains in giant model membrane vesicles. In conclusion, bovicin HC5 has a primary mode of action similar to that of nisin, but some differences regarding the pore-forming capacity were demonstrated.