Membrane Activity of LL-37 Derived Antimicrobial Peptides against Enterococcus hirae: Superiority of SAAP-148 over OP-145

Abstract

The development of antimicrobial agents against multidrug-resistant bacteria is an important medical challenge. Antimicrobial peptides (AMPs), human cathelicidin LL-37 and its derivative OP-145, possess a potent antimicrobial activity and were under consideration for clinical trials. In order to overcome some of the challenges to their therapeutic potential, a very promising AMP, SAAP-148 was designed. Here, we studied the mode of action of highly cationic SAAP-148 in comparison with OP-145 on membranes of Enterococcus hirae at both cellular and molecular levels using model membranes composed of major constituents of enterococcal membranes, that is, anionic phosphatidylglycerol (PG) and cardiolipin (CL). In all assays used, SAAP-148 was consistently more efficient than OP-145, but both peptides displayed pronounced time and concentration dependences in killing bacteria and performing at the membrane. At cellular level, Nile Red-staining of enterococcal membranes showed abnormalities and cell shrinkage, which is also reflected in depolarization and permeabilization of E. hirae membranes. At the molecular level, both peptides abolished the thermotropic phase transition and induced disruption of PG/CL. Interestingly, the membrane was disrupted before the peptides neutralized the negative surface charge of PG/CL. Our results demonstrate that SAAP-148, which kills bacteria at a significantly lower concentration than OP-145, shows stronger effects on membranes at the cellular and molecular levels.

The membrane activity of the amphibian Temporin B peptide analog TB_KKG6K sheds light on the mechanism that kills Candida albicans

“…The ANTS/DPX containing LUVs were separated from free ANTS/DPX by exclusion chromatography using a column filled with Sephadex™ G-75 (Amersham Biosciences, Amersham, United Kingdom) fine gel swollen in an isosmotic buffer (10 mM HEPES, 140 mM NaCl, pH 7.4). After the void volume, fractions were collected and the phospholipid concentration was determined by phosphate analysis as described by Piller et al (76). The LUVs containing ANTS/DPX were stored in the dark at room temperature.…”

“…For the latter, amino acids 13-37, marked in bold, were considered for analysis. In silico helical wheel projection, bilayer partitioning free energy (ΔG), and hydrophobic moment (µ) were obtained as described by Piller P et al (76).…”

“…Unilamellarityand size were confirmed by dynamic light scattering (DLS) using a Zetasizer Nano (ZSP, Malvern Panalytical, Prager Electronics, Wolkersdorf, Austria).The ANTS/DPX containing LUVs were separated from free ANTS/DPX by exclusion chromatography using a column filled with Sephadex™ G-75 (Amersham Biosciences, Amersham, United Kingdom) fine gel swollen in an isosmotic buffer (10 mM HEPES, 140 mM NaCl, pH 7.4). After the void volume, fractions were collected and the phospholipid concentration was determined by phosphate analysis as described by Piller et al(76). The LUVs containing ANTS/DPX were stored in the dark at room temperature.The leakage of aqueous contents from the prepared LUVs induced by the TB analog was determined using the GloMax® Discover Microplate Reader (Promega Corporation, Madison, WI, USA).…”

Where Electrostatics Matter: Bacterial Surface Neutralization and Membrane Disruption by Antimicrobial Peptides SAAP-148 and OP-145

“…Due to high levels of hydrophobic and cationic residues in AMPs [ 13 ], positive charges are believed to be responsible for the initial binding of the AMP to the negatively charged bacterial membrane, while hydrophobicity is proposed to dominate the insertion into and perturbation of the membrane [ 13 , 15 , 16 , 17 ]. These features ( Figure 1 ) have also been studied in OP-145 [ 8 , 18 , 19 , 20 ], an AMP derived from the human cathelicidin LL-37 [ 21 , 22 , 23 , 24 ]. Like its parent peptide, OP-145 belongs to a class of linear cationic peptides, which adopts nearly an ideal alpha helix in presence of the membrane [ 20 ].…”

“…In further developments, all negatively charged amino acids in the OP-145 sequence were replaced and the number of positively charged amino acids was nearly doubled resulting in a peptide named SAAP-148 [ 8 ]. The overall hydrophobicity was not changed, but the hydrophobic character in the hydrophobic region was increased, while the predicted alpha-helical structure and total amphipathicity were kept almost constant [ 19 ]. Indeed, in our recent studies [ 8 , 19 ] we confirmed more efficient membrane disruption and an improved antimicrobial profile for SAAP-148.…”