Adriana Di Stasi

Background/Objectives: Developing novel strategies to combat respiratory infections caused by multidrug-resistant “priority pathogens” like the ESKAPEE Pseudomonas aeruginosa and Staphylococcus aureus is an urgent priority. Methods: We investigated two shortened variants of the proline-rich antimicrobial peptide (PrAMP) B7-005, B7-006 (15-mer) and B7-007 (13-mer). Evaluation included MIC assays against laboratory and clinical multidrug-resistant isolates, mechanistic studies of membrane permeabilization, cytotoxicity testing on BEAS-2B bronchial epithelial cells, and proteolytic stability assays in human elastase and sputum. Results: Despite their reduced size, lower positive charge, and decreased proline content, both variants retained full antimicrobial activity against clinical pathogens with consistent MIC values ≤ 25 µM. These variants exhibit membrane permeabilization in P. aeruginosa but may also relay on a hybrid mode of action involving also intracellular targets. Notably, B7-006 and B7-007 displayed low cytotoxicity compared to the lytic peptide BMAP-18. While B7-007 showed greater susceptibility to proteolytic degradation than its parent B7-005, it preserved partial integrity during the initial hours of exposure. Conclusions: Overall, these findings demonstrate that the B7 scaffold tolerates substantial truncation while preserving potency and selectivity, identifying a minimal 13-amino-acid active core. This work provides critical insights into structure–activity relationships and supports the development of compact, mechanistically versatile antimicrobial peptides to address the growing threat of multidrug-resistant respiratory pathogens.

Proline-rich antimicrobial peptides (PrAMPs) primarily exert their antimicrobial effects intracellularly, inhibiting protein synthesis. B7-005, a synthetic 16-amino acid PrAMP, has a broader antimicrobial spectrum compared to native counterparts, despite shorter PrAMPs typically exhibiting reduced activity. This study aimed to enhance B7-005’s potency by extending it with 6 or 11 amino acids derived from the C-terminal sequences of cetacean Tur1A and Lip1 PrAMPs, as well as bovine Bac7(1-35). Six chimeric derivatives were evaluated for antimicrobial and bactericidal potency, cytotoxicity, bacterial membrane permeabilization, and in vitro inhibition of protein synthesis. Extending B7-005 with sequences from other PrAMPs increased its activity against most ESKAPE+E pathogens, reducing minimum inhibitory concentration (MIC) values by 2- to 8-fold, with notable differences among bacterial species, without increasing cytotoxicity toward the A549 cell line. All chimeras retained the ability to inhibit protein synthesis in Escherichia coli and to modestly perturb the E. coli membranes like B7-005. These novel chimeric PrAMPs, particularly the 22-mer derivatives, hold promise for developing new antimicrobial agents. The study also highlights variability in bacterial responses to PrAMPs and underscores how minor sequence differences can significantly impact efficacy against specific microorganisms. PrAMPs thus represent a valuable scaffold to rationally design derivatives targeting high-priority pathogens.

Proline-rich antimicrobial peptides (PrAMPs) have gained attention due to their antimicrobial properties and low cytotoxicity. B7-005, a small optimized PrAMP, exhibits a broader spectrum of activity than native PrAMPs, due to an antimicrobial mechanism based on inhibiting prokaryotic protein synthesis and destabilizing bacterial membranes. However, the toxicity and the in vivo efficacy of B7-005 remain poorly understood, so in vitro and in vivo microbiology and toxicology experiments were used to assess its suitability as an anti-infective agent. The incidence of resistance towards B7-005 by E. coli was lower than for other PrAMPs and antibiotics; moreover, it maintained antimicrobial activity in the presence of human serum. B7-005 exerted its antimicrobial effect at a much lower concentration than those causing harmful effects on four different cell types, such as membrane permeabilization or non-lytic depolarization of mitochondria. The latter effect may be related to the inhibition of eukaryotic protein synthesis by B7-005 observed in vitro . In a zebrafish embryo model, B7-005 was well tolerated and reduced mortality from pre-existing E. coli bacteraemia. Overall, B7-005 was safe for human cells and effective against systemic infection in vivo , making it a promising lead for developing new antibiotics.