Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes

Abstract

In the questf or new antibiotics, two novel engineered cationic antimicrobialpeptides (eCAPs)h ave been rationallyd esigned. WLBU2 and D8 (all 8v alines are the denantiomer)e fficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxict oe ukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structurea nd pore evidence. We suggest that protein secondary structure is not ac riticald eterminant of bactericidal activity,b ut that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydro-carbonr egion may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model,b oth locate deep into its hydrocarbonr egion where they are primedf or self-promoted uptake into the periplasm.T he partially a-helicals econdarys tructure of WLBU2 in ar ed blood cell (RBC) membrane model containing 50 % cholesterol, could play ar ole in destabilizingt his RBC membrane modelc ausing pore formationt hat is not observed with the D8 randomc oil, which correlates with RBC hemolysis causedb yWLBU2 but not by D8.[a] Prof. Figure 4. CD results of WLBU2i na queouss olution, pH 7and with lipidmembrane models. A) WLBU2 in G(À)I M( black trace) and WLBU2 in water (red trace), B) secondary structure results of WLBU2 in G(À)I Mmodel with decreasing WLBU2:lipid molar ratio in 15 mm PBS, (C) Secondary structure results of 10 mm WLBU2 in 15 mm PBS in six model membranes at 10:1 lipid:peptide molar ratio. R 2 indicates the goodnesso ft he fit to the Brahms and Brahmsd ata set. [49] All CD experiments were carried out at 37 8C. The errors on the percentages are 3-5 %.

The Engineered Antibiotic Peptide PLG0206 Eliminates Biofilms and Is a Potential Treatment for Periprosthetic Joint Infections

“…Several studies have examined how PLG0206 exhibits bactericidal efficacy. Heinrich et al [ 21 ] showed that neither the secondary structure nor pore formation is a critical determinant of bactericidal efficacy for PLG0206. The interaction of PLG0206 with the bacterial cell membrane leads to lipid phase consolidation, resulting in localized stiffening [ 22 ], ordering, and alteration of the thickness of the membranes [ 21 ].…”

“…Heinrich et al [ 21 ] showed that neither the secondary structure nor pore formation is a critical determinant of bactericidal efficacy for PLG0206. The interaction of PLG0206 with the bacterial cell membrane leads to lipid phase consolidation, resulting in localized stiffening [ 22 ], ordering, and alteration of the thickness of the membranes [ 21 ]. Consequently, this disruption of the bacterial cell membrane results in lipid headgroup spacing mismatch and lowering of the energy barrier to ion flow across the membrane.…”

A Novel Lipid-Based MALDI-TOF Assay for the Rapid Detection of Colistin-Resistant Enterobacter Species

“…Colistin, or polymyxin E, is a cationic lipopeptide and “last-resort” antibiotic used for MDR Gram-negative infections, such as those producing extended-spectrum beta-lactamases and those resistant to carbapenems ( 6 ), which is an increasing concern in clinical isolates of Enterobacter species ( 7 ). Targeting the cell membrane, cationic colistin binds with anionic lipopolysaccharide (LPS) through electrostatic interactions ( 8 – 11 ). Colistin resistance is conferred by reduction of the electronegativity of the cell membrane through modification of the terminal phosphate residues of lipid A, the membrane anchor of LPS, thereby decreasing the binding affinity of colistin ( 12 ).…”

Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation

“…13 The detailed secondary structure of WLBU2 in four different lipid model membranes (LMMs) and in aqueous solution was obtained using circular dichroism spectroscopy and was published previously. 16 While WLBU2 in water or 15 mM phosphate-buffered saline (PBS) adopts primarily a random coil or β-sheet structure, the α helical content increases to ∼80% in Gram-negative (G(−)) inner membrane (IM) or Gram-positive (G(+)) LMMs and to ∼40% in lipopolysaccharide (LPS)containing LMMs. Although WLBU2 is not 100% helical by our determination, 16 it is still primarily helical when in contact with the inner membrane of G(−) and G(+) LMMs, partially confirming the locations of R and V on opposite faces in WLBU2′s helical wheel rational design.…”

“…16 While WLBU2 in water or 15 mM phosphate-buffered saline (PBS) adopts primarily a random coil or β-sheet structure, the α helical content increases to ∼80% in Gram-negative (G(−)) inner membrane (IM) or Gram-positive (G(+)) LMMs and to ∼40% in lipopolysaccharide (LPS)containing LMMs. Although WLBU2 is not 100% helical by our determination, 16 it is still primarily helical when in contact with the inner membrane of G(−) and G(+) LMMs, partially confirming the locations of R and V on opposite faces in WLBU2′s helical wheel rational design. When added to a eukaryotic membrane mimic, we found only a low level of αhelicity (∼20%).…”