Fatty Acid Conjugation Leads to Length-Dependent Antimicrobial Activity of a Synthetic Antibacterial Peptide (Pep19-4LF)

Abstract

The increasing number of infections caused by multidrug-resistant bacteria requires an intensified search for new antibiotics. Pep19-4LF is a synthetic antimicrobial peptide (GKKYRRFRWKFKGKLFLFG) that was previously designed with the main focus on high antimicrobial activity. The hydrophobic motif, LFLFG, was found to be essential for antimicrobial activity. However, this motif shows several limitations such as aggregation in biological media, low solubility, and small yields in peptide synthesis. In order to obtain more appropriate peptide characteristics, the hydrophobic motif was replaced with fatty acids. For this purpose, a shortened variant of Pep19-4LF (Pep19-short; GKKYRRFRWKFKGK) was synthesized and covalently linked to saturated fatty acids of different chain lengths. The peptide conjugates were tested with respect to their antibacterial activity by microdilution experiments on different bacterial strains. The length of the fatty acid was found to be directly correlated to the antimicrobial activity up to an ideal chain length (undecanoic acid, C11:0). This conjugate showed high antimicrobial activity in absence of toxicity. Time-kill studies revealed a fast and bactericidal mode of action. Furthermore, the first in vivo experiments of the conjugate in rodents demonstrated pharmacokinetics appropriate for application as a drug. These results clearly indicate that the hydrophobic motif of the peptide can be replaced by a single fatty acid of medium length, simplifying the design of this antimicrobial peptide while retaining high antimicrobial activity in the absence of toxicity.

Keywords: Enterococcus faecium; Staphylococcus aureus; antimicrobial activity; fatty acid conjugation; synthetic antimicrobial peptides.

Figure 1

Schematic illustration of peptides synthesized and tested for antimicrobial activity by microdilution experiments: (A) Pep19-short, the peptide without a hydrophobic moiety, (B) Pep19-short conjugated to fatty acids including C6:0 (caproic acid), C8:0 (caprylic acid), C10:0 (capric acid), C11:0 (undecanoic acid), C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid), and C18:0 (stearic acid), (C) Pep19-2.5 (original peptide), (D) Pep19-4LF (parent peptide), and (E) Pep19-4LF conjugated to undecanoic acid (C11:0) for an additional hydrophobic moiety.

Figure 2

MIC values of Pep19-short conjugated to different fatty acids on (A) S. aureus (ATCC 25923), (B) E. faecium (UL602570*, clinical isolate, vanA-resistant), and (C) A. bohemicus (DSM 100419) and time–kill studies of the most potent fatty acid conjugate (C11-Pep19-short) on Rothia kristinae (DSM 20032) for (D) 4 × MIC and (E) 0.5 × MIC (n = 3). The length of the conjugated fatty acid is crucial for the antimicrobial activity of the peptide conjugates against all bacterial strains. Short fatty acids (C6:0, C8:0) did not show antimicrobial activity on S. aureus (A) or E. faecium (B) and only moderate antimicrobial activity on A. bohemicus (C). In contrast, fatty acids of medium length showed the highest antimicrobial activity with minimal inhibitory concentrations (MICs) at least comparable to or even lower than those of the control peptide, Pep19-4LF. Longer fatty acids showed lower antimicrobial activities resulting in higher MICs. Time–kill studies showed a bactericidal activity of C11-Pep19-short, Pep19-4LF and bacitracin at a concentration of 4 × MIC (D). As expected, a concentration of 0.5 × MIC was not high enough for an effective antimicrobial activity (E). For statistical analysis, a one-way ANOVA followed by a Turkey’s comparison test was applied. The significance is given by **** p < 0.0001.

Figure 3

(A) Hemolysis study for Pep19-4LF, Pep19-2.5, C6-Pep19-short, C11-Pep19-short, and C18-Pep19-short as well as cytotoxicity assay for Pep19-4LF and C11-Pep19-short on HEK 293 cells (B) and HepG2 cells (C) (n = 3). C6-Pep19-short showed no hemolytic activity at all concentrations measured. C11-Pep19-short showed only low hemolytic activity at a concentration higher than 75 µg/mL. However, this was lower compared to Pep19-4LF, Pep19-2.5, and C18-Pep19-short. Pep19-4LF and C11-Pep19-short had no cytotoxic effects on both cell lines at MIC-relevant concentrations.

Figure 4

Scintigraphical images of (A) Pep19-4LF, (B) Pep19-short, and (C) C11-Pep19-short. The peptides were labeled with ¹²⁵I and injected intravenously into the tail vein of female Wistar rats, the images were recorded 0–24 h post administration. All peptides showed comparable pharmakokinetics with fast liver enrichment and subsequent hepatobiliary excretion.