Abstract
Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.
Highlights
Antimicrobial peptides (AMPs) have been investigated for several decades in an effort to develop alternatives to traditional antibiotics, which face increasing levels of bacterial resistance [1,2,3,4,5]
To determine if the high resistance of growing E. faecalis to L-GL13K could be due to degradation by bacterial proteases, the peptides were tested against the protease deficient strain TX5128
Antimicrobial peptides have been proposed as an alternative to traditional antibiotics [13, 38, 39] and new peptides and derivatives are continuously generated and investigated [40, 41]
Summary
Antimicrobial peptides (AMPs) have been investigated for several decades in an effort to develop alternatives to traditional antibiotics, which face increasing levels of bacterial resistance [1,2,3,4,5]. It has been proposed that the interaction of AMPs with the bacterial cell membrane is associated with relatively low probability of bacterial resistance [9], bacteria that inhabit the host microbiome and some invading bacteria clearly have the ability to co-exist with or overcome host AMPs. experimental resistance to AMPs has been observed [10] and the corresponding bacterial defense mechanisms could protect against therapeutic AMPs [7, 11]. Wide-spread use of therapeutic AMPs could lead to resistance against endogenous host-defense peptides (“arming the enemy”) and render the host unprotected against invading bacteria [12]. To address these concerns, a better understanding of the mechanisms of action and bacterial resistance to AMPs is needed [13]
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