The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms

Abstract

Objectives To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms. Methods Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths. Results Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified. Conclusions Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties

“…E. coli ATCC 25922 (“smooth” LPS) and S. aureus ATCC 25923 are common control strains for antimicrobial susceptibility testing [50,51]. E. coli DC2 CGSC 7139 is hypersensitive to antibacterial agents and more permeable to dyes [52], while S. aureus ATCC 6538 is known to form biofilms [53]. Tachyplesin peptides and their cyclic analogues were tested against planktonic cultures of all strains and against S. aureus ATCC 6538 in the biofilm form for direct comparison of their antimicrobial activity.…”

“…Against the S. aureus biofilm (see Figure 3 and Table 4), the tachyplesin peptides were >5-fold less active compared to the planktonic cells, likely due to the reduced accessibility of the peptides to the bacterial cell in biofilm form or the low ability of the peptides to penetrate the extracellular matrix of exopolysaccharides and access the cells [64]. The activities of the tachyplesin peptides against biofilms are comparable to other tested antimicrobial peptides [53]. The efficacy against the biofilms could be increased by using higher concentrations of the tachyplesin peptides or by using the peptide in combination with other antibiofilm or extracellular matrix disrupting agents [65].…”

“…Two-fold serial dilutions of each peptide (highest concentration tested was 32 µM and lowest was 0.25 µM) were added to the biofilms for 4 h. Untreated 24 h preformed biofilms were used as a control. The metabolic activity of biofilm-embedded cells was determined using a resazurin reduction fluorometric assay as previously described [53].…”

The Two Weapons against Bacterial Biofilms: Detection and Treatment

“…This increased resistance can be attributed to different factors, including decreased diffusion of antimicrobial agents through the self-produced extracellular matrix (made of polysaccharides, nucleic acids, and proteins matrix) [ 8 , 9 , 10 ], altered metabolic activity, and formation of persister cells [ 11 ]. Moreover, a relevant characteristic of biofilms is the fact that bacteria are able to inter-communicate and collaborate to survive in even the hardest conditions.…”

Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant Pseudomonas aeruginosa and Staphylococcus aureus Planktonic Cells and Biofilm

“…CAMA, a hybrid peptide containing an N-terminus of cecropin A and melittin, effectively inhibited methicillin-resistant S. aureus (MRSA) biofilm formation [23]. Several anti-biofilm mechanisms have been discovered and include the membrane potential disruption of biofilm-embedded bacterial cells [24], the interruption of quorum-sensing systems [20], EPS reduction [25,26], alarmone system inhibition [27], and downregulation of genes related to biofilm formation and binding protein transportation [28]. Although many anti-biofilm peptides have been designed and investigated, further studies identifying and defining key amino acids and/or structural features responsible for biofilm prevention and eradication are needed.…”