The Antimicrobial Peptide lin-SB056-1 and Its Dendrimeric Derivative Prevent Pseudomonas aeruginosa Biofilm Formation in Physiologically Relevant Models of Chronic Infections

Abstract

Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P. aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (lin-SB056-1)2-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)2-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P. aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)2-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P. aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential

“…Differently from artificial sputum medium, patients' sputum contains host/bacterial components such as cell-derived factors, normal flora, inflammatory mediators, proteases, or peptidases that may exert an additional inhibitory effect on the peptide's activity. Nevertheless, herein, we showed that the combination of lin-SB056-1 and EDTA at sub-active and non-cytotoxic concentrations [16,27] determined a significant reduction in P. aeruginosa load in sputa of PCD chronically infected patients, despite certain differences in the level of reduction among different sputum samples being observed. Such differences are not surprising considering that inter-patient variables (e.g., clinical stage and severity of lung infection, bacterial load, sputum sample composition, and consistency) were not standardized in our experiments, in the attempt to mimic conditions found during the actual antimicrobial therapy.…”

“…Similar to CF patients, eradication of chronic P. aeruginosa infection in PCD lungs is hardly obtained, and the reduction of bacterial density during chronic colonization or exacerbations is often the aim of the antimicrobial therapy [3]. In previous studies, we have shown that the combination of lin-SB056-1/EDTA possesses antimicrobial activity against P. aeruginosa in artificial sputum medium and prevents P. aeruginosa biofilm formation in an in vivo-like three-dimensional (3D) lung epithelial cell model [16,27]. Despite resembling the airway mucus, artificial sputum media normally behave like Newtonian fluids lacking many of the intramolecular interactions and covalent cross-links that give respiratory secretions their viscoelastic characteristic [28].…”

The Best Peptidomimetic Strategies to Undercover Antibacterial Peptides

“…Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predicting the clinical potential of novel AMP-based antimicrobials [ 212 ]. The antibiofilm activity of the semisynthetic peptide lin-SB056-1 (KWKIRVRLSA-NH 2 ; Figure 22 ) and its dendrimeric derivative (lin-SB056-1)2-K ([KWKIRVRLSA] 2 -K) against Pseudomonas aeruginosa in an in vivo-like three-dimensional lung epithelial cell model, and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels) gave rewarding results.…”

“…The antibiofilm activity of the semisynthetic peptide lin-SB056-1 (KWKIRVRLSA-NH 2 ; Figure 22 ) and its dendrimeric derivative (lin-SB056-1)2-K ([KWKIRVRLSA] 2 -K) against Pseudomonas aeruginosa in an in vivo-like three-dimensional lung epithelial cell model, and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels) gave rewarding results. [ 212 ]. When alone, Lin-SB056-1 was moderately effective (MIC P.aeruginosa (μM) = 38.5) in reducing P. aeruginosa biofilm formation in 3D lung epithelial cells, but its antibiofilm activity was significantly increased in association with the chelating agent EDTA.…”

“…The combination of lin-SB056-1 at 38.5 mM with EDTA (0.3 to 1.25 mM), know metal chelating agent, resulted in the reduction of the initial bacterial inoculum to the limit of detection (10 CFU/mL). The dimeric derivative (lin-SB056-1) 2 -K demonstrated an enhanced biofilm-inhibitory activity (MIC P.aeruginosa (μM) = 19.25) as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death [ 212 ].…”