Adapting antibacterial display to identify serum-active macrocyclic peptide antibiotics

Abstract

The lack of available treatments for many antimicrobial-resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity toward human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of Gram-negative bacteria without strong inner-membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality.

Keywords: antibiotic discovery; antimicrobial peptide; bacterial display; macrocycle; serum.

Fig. 1.

Synthetic discovery of serum-active AMPs. A) Diagram of the potential residues at each position of a peptide library based on natural β-AMP sequence frequencies. Potential disulfide bonds are indicated with a dotted line. Octagons potentiate early stop codons. B) Diagram of how SLAY functions. C) Uninduced versus induced mean reads for peptides in the library with a significant log₂-fold change (L2FC) less than −0.5 or less than −1. D) Distribution of log₂-fold change, charge, length, and number of potential S–S bonds for select BHS peptides examined in vitro.

Fig. 2.

SAP-26 functions differently from traditional cationic AMPs. A) Table describing the structure and activity of various antibiotics. NPN B) and PI C) fluorescence of E. coli cells treated with various concentrations of antibiotic for 30 min. D) Kill curve of E. coli cells treated with antibiotic at 2× their listed MIC. E) Growth curve of E. coli cells treated with antibiotic at 8× their listed MIC. Listed MICs are the median of three replicates data points are an average of three replicates with error bars representing 1 SD.

Fig. 3.

SAP-26 causes cell elongation, is nontoxic, and functions in vivo. A) Fluorescent microscopy images of E. coli 25922 cells expressing cytoplasmic GFP with and without treatment with SAP-26 (Scale = 5 µm). A scatter plot shows individual cell length and mean from both groups (n ≥ 148, ****P < 0.0001, Welch's t-test). B) Table showing % hemolysis caused by 128 µg/ml AMP, IC₂₅ for HEK293T tissue cultured cell, and LD₅₀ for G. mellonella larvae. Percentage of hemolysis error is 1 SD of triplicate samples. c) Survival of G. mellonella larvae infected with E. coli 25922 +/− treatment with indicated concentrations of PG-1 or SAP-26. Significance was determined in relation to the untreated group (n ≥ 13, ****P < 0.0001, ***P = 0.0003, ns = not significant, log-rank test on Kaplan–Meier curves).