Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria

Abstract

We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1-5 min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1 μM. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections.

Escherichia coli biofilm: development and therapeutic strategies

“…It was proposed that these peptides could traverse inside the cell and eventually bind the DNA for its antimicrobial action (Anunthawan et al . ). Bacteriocin isolated from Citrobacter freundii showed antimicrobial activity against a wide range of bacteria including E. coli in both planktonic as well as in biofilm form (Shanks et al .…”

Anti-biofilm peptides as a new weapon in antimicrobial warfare

“…For example, Anunthawan et al . demonstrated that the two tryptophan-rich cationic antimicrobial peptides KT2 and RT2 showed anti-biofilm activity at sub-MIC levels against the multidrug-resistant, enterohemorrhagic E. coli O157:H7 strain and were able to prevent biofilm formation and eradicate mature biofilms at a concentration of 1 μM [13]. Both peptides interacted with and bound to negatively-charged LPS molecules to enable self-promoted uptake (without forming pores or aggregates) across the outer membrane and subsequently interacted with cytoplasmic membrane phospholipids [13].…”

Antimicrobial Peptides: Mechanisms of Action and Resistance

“…On one hand, the cell wall of gram-positive bacteria represents a porous 40-to 80-nm-thick mesh that many AMPs seem to pass with relative ease (Malanovic and Lohner 2016). On the other, gram-negative bacteria present an outer membrane that some AMPs can quickly cross by a charge-exchange mechanism where the cationic peptides compete with Ca 2+ and Mg 2+ bound to lipopolysaccharide, possibly promoted by binding to outer membrane proteins (self-promoted uptake hypothesis; e.g., Anunthawan et al 2015). These peptides then have access to the cell wall in gram-negative bacteria as well as the cell membrane and intracellular targets.…”