Short and Robust Anti-Infective Lipopeptides Engineered Based on the Minimal Antimicrobial Peptide KR12 of Human LL-37

Abstract

This study aims to push the frontier of the engineering of human cathelicidin LL-37, a critical antimicrobial innate immune peptide that wards off invading pathogens. By sequential truncation of the smallest antibacterial peptide (KR12) of LL-37 and conjugation with fatty acids, with varying chain lengths, a library of lipopeptides is generated. These peptides are subjected to antibacterial activity and hemolytic assays. Candidates (including both forms made of l- and d-amino acids) with the optimal cell selectivity are subsequently fed to the second layer of in vitro filters, including salts, pH, serum, and media. These practices lead to the identification of a miniature LL-37 like peptide (d-form) with selectivity, stability, and robust antimicrobial activity in vitro against both Gram-positive and negative bacteria. Proteomic studies reveal far fewer serum proteins that bind to the d-form than the l-form peptide. C10-KR8d targets bacterial membranes to become helical, making it difficult for bacteria to develop resistance in a multiple passage experiment. In vivo, C10-KR8d is able to reduce bacterial burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC in neutropenic mice. In addition, this designer peptide prevents bacterial biofilm formation in a catheter-associated mouse model. Meanwhile, C10-KR8d also recruits cytokines to the vicinity of catheters to clear infection. Thus, based on the antimicrobial region of LL-37, this study succeeds in identifying the smallest anti-infective peptide C10-KR8d with both robust antimicrobial, antibiofilm, and immune modulation activities.

Structure and Activity of a Selective Antibiofilm Peptide SK-24 Derived from the NMR Structure of Human Cathelicidin LL-37

“…SK-24 was superior to LL-37, which displayed higher MIC values in most of the cases even in 15% tryptic soy broth (TSB). We recently found similar MIC values for a short lipoLL-37 peptide in 100% TSB and Mueller Hinton Broth (MHB) [39]. It appeared that 17BIPHE2, engineered based on GF-17 [32], was most potent in killing these pathogens with MIC in the range of 2-8 µM.…”

“…During measurement, the high-tension signal applied to the detector was also recorded and was subsequently converted to absorbance. Data represent the average of five individual scans with a corresponding reference measurement on pure solvent subtracted [21,39]. The peptide concentration was fixed at 0.5 mM in 10 mM PBS (pH 7) with or without 60 mM of sodium dodecyl sulfate (SDS).…”

“…Membrane permeabilization was evaluated as described [39]. In brief, bacteria were inoculated and grown overnight.…”

“…The experiment was conducted as described [39]. In brief, an overnight culture of S. aureus USA300 LAC was subcultured in a fresh TSB medium and grown to the exponential phase.…”

Antimicrobial Tear Lipids in the Ocular Surface Defense

“…Lipidated analogs of peptides generated by conjugation of human peptide with fatty acids provide antimicrobial activity against ESKAPE bacteria and biofilms of S. aureus ( Kamysz et al., 2020 ). An engineered short lipopeptide generated by conjugating human cathelicidin LL-37, an innate immune antimicrobial peptide, with fatty acids shows robust antimicrobial activity against Gram-positive and Gram-negative bacteria in vitro and reduces bacterial burden of MRSA in mice in vivo ( Lakshmaiah Narayana et al., 2021 ). It targets bacterial membranes and makes it helical so that bacteria find it difficult to develop resistance.…”