Comparative antimicrobial activity and mechanism of action of bovine lactoferricin-derived synthetic peptides

Abstract

Lactoferricin B (LfcinB), a 25 residue peptide derived from the N-terminal of bovine lactoferrin (bLF), causes depolarization of the cytoplasmic membrane in susceptible bacteria. Its mechanism of action, however, still needs to be elucidated. In the present study, synthetic LfcinB (without a disulfide bridge) and LfcinB (C-C; with a disulfide bridge) as well as three derivatives with 15-, 11- and 9-residue peptides were prepared to investigate their antimicrobial nature and mechanisms. The antimicrobial properties were measured via minimum inhibitory concentration (MIC) determinations, killing kinetics assays and synergy testing, and hemolytic activities were assessed by hemoglobin release. Finally, the morphology of peptide-treated bacteria was determined by atomic force microscopy (AFM). We found that there was no difference in MICs between LfcinB and LfcinB (C-C). Among the derivatives, only LfcinB15 maintained nearly the same level as LfcinB, in the MIC range of 16-128 μg/ml, and the MICs of LfcinB11 (64-256 μg/ml) were 4 times more than LfcinB, while LfcinB9 exhibited the lowest antimicrobial activity. When treated at MIC for 1 h, many blebs were formed and holes of various sizes appeared on the cell surface, but the cell still maintained its integrity. This suggested that LfcinB had a major permeability effect on the cytoplasmic membrane of both Gram-positive and Gram-negative bacteria, which also indicated it may be a possible intracellular target. Among the tested antibiotics, aureomycin increased the bactericidal activity of LfcinB against E. coli, S. aureus and P. aeruginosa, but neomycin did not have such an effect. We also found that the combination of cecropin A and LfcinB had synergistic effects against E. coli.

Antimicrobial Activity of Lactoferrin-Related Peptides and Applications in Human and Veterinary Medicine

“…No antagonism was observed between the antibacterial agents used and bLfcin against E. coli, S. aureus and P. aeruginosa. The MICs for the antibiotics markedly decreased when the peptide was added, as expected, indicating that bLfcin might act in combination with these antimicrobial agents against E. coli, S. aureus and P. aeruginosa [74].…”

“…Studies have indicated that bLfcin leads to depolarization of the cell membrane without causing the lysis of the cells [70], that it exerts its bactericidal effect initially by acting on the bacterial cell surface, and subsequently on the cytoplasmic contents [74].…”

“…The role of disulfide is not yet fully understood, and Liu et al [74] recently failed to find a significant difference in MICs between the disulfide-bridged peptide and its linear counterpart, while another study showed three-times-higher activity for the disulfide-bridged peptide [63]. Intracellular targets, such as phosphoenolpyruvate carboxylase, were detected using E. coli proteome chips, indicating that one of Lfcin's mechanisms of action may be associated with pyruvate metabolism [75].…”

“…In regard to its toxicity on eukaryotic cells, measured as haemolytic activity, bLfcin displayed slight hemolytic activity at a concentration of 64 µg/mL, exhibiting a marked antimicrobial activity against most of the test bacteria [74]. However, a study of the relationship between structure and activity found that the undecapeptide structure is essentially non-haemolytic, but that undecapeptides containing more than three tryptophan residues produced 50% haemolysis of human red blood cells at concentrations above 400 µg/mL (>230 µM) [72].…”

Killing of Mycobacterium avium by Lactoferricin Peptides: Improved Activity of Arginine- and d -Amino-Acid-Containing Molecules

“…Furthermore, the possibility of an internal target for the action of these peptides against M. avium cannot be discarded. In fact, there are numerous evidences in the literature pointing to the probable existence of an internal target for lactoferricins (5,(47)(48)(49).…”