Primate cathelicidin orthologues display different structures and membrane interactions

Abstract

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.

Broad-Spectrum Activity against Bacterial Mastitis Pathogens and Activation of Mammary Epithelial Cells Support a Protective Role of Neutrophil Cathelicidins in Bovine Mastitis

“…The ability of BMAPs to retain activity in serum is common to those helical AMPs that acquire this conformation only in the presence of an anisotropic environment, such as at the bacterial membrane surface, whereas they remain largely unstructured in aqueous medium at physiological salt concentrations (38,49). We have previously termed this type of helical peptide F form, with reference to some helical primate cathelicidins that behave similarly (26,45,56). In rarer cases, such as the human LL-37, a helical conformation can also be induced by physiological salts in the absence of biological membranes (Aform helical peptides), and we have shown these to be considerably more susceptible to serum or medium components (45).…”

Lipid Segregation Explains Selective Toxicity of a Series of Fragments Derived from the Human Cathelicidin LL-37

“…One of the factors contributing to the resistance of some strains of S. aureus to LL-37 is the presence of proteolytic enzymes (25). One of these enzymes has been identified as a glutamylendopeptidase that cleaves LL-37 between Glu 16 and Phe 17 . This enzyme thus cleaves LL-37 but not GF-17.…”

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

“…Minimum inhibitory concentration (MIC) assays, which determine the lowest concentration of peptide that inhibits bacterial growth, show that LL-37 is effective against a broad spectrum of bacteria that is both Gram-positive and -negative (12). In vitro assays using lipid bilayers indicate that LL-37 permeabilizes model membranes by a carpeting or toroidal pore mechanism (13)(14)(15)(16)(17).…”

“…Our current understanding of how this AMP attacks bacteria has been shaped by bulk assays (11,12), model membrane assays (13)(14)(15)(16)(17)(18), and assumptions based on studies of other AMPs. Minimum inhibitory concentration (MIC) assays, which determine the lowest concentration of peptide that inhibits bacterial growth, show that LL-37 is effective against a broad spectrum of bacteria that is both Gram-positive and -negative (12).…”