Development of a Selective and Stable Antimicrobial Peptide

Abstract

Antimicrobial peptides (AMPs) are presented as potential scaffolds for antibiotic development due to their desirable qualities including broad-spectrum activity, rapid action, and general lack of susceptibility to current resistance mechanisms. However, they often lose antibacterial activity under physiological conditions and/or display mammalian cell toxicity, which limits their potential use. Identification of AMPs that overcome these barriers will help develop rules for how this antibacterial class can be developed to treat infection. Here we describe the development of our novel synthetic AMP, from discovery through in vivo application. Our evolved AMP, DTr18-dab, has broad-spectrum antibacterial activity and is nonhemolytic. It is active against planktonic bacteria and biofilm, is unaffected by colistin resistance, and importantly is active in both human serum and a Galleria mellonella infection model. Several modifications, including the incorporation of noncanonical amino acids, were used to arrive at this robust sequence. We observed that the impact on antibacterial activity with noncanonical amino acids was dependent on assay conditions and therefore not entirely predictable. Overall, our results demonstrate how a relatively weak lead can be developed into a robust AMP with qualities important for potential therapeutic translation.

Keywords: antibiofilm; antimicrobial peptide; serum-stable; synthetic; unnatural amino acids.

Figure 1

Tcp18 and Tr18. (A) The sequences and MICs of Tcp18 (parent) and Tr18 (derivative) are shown. The bold residues show substituted residues. MIC against E. coli ATCC 25922 is shown. The minimum inhibitory concentrations (MICs) were determined by taking the median of all replicates. MICs were repeated for at least biological triplicate in MH Broth. (B) AlphaFold predicted structures of Tcp18 (left) and Tr18 (right). The plDDT confidence score is below with its corresponding color code. (C) Circular dichroism spectra of Tcp18 and Tr18, both have α-helical secondary structure with the addition of TFE. Measurements were taken in technical triplicate. (D) Helical wheel projections for both peptides were generated to visualize residue distribution, they are colored by amino acid type. (E) The fold change in MIC for each alanine derivative in comparison to Tcp18 was graphed with individual points. The black line is set at a 4-fold MIC cutoff where a residue was considered critical. MICs were repeated in biological triplicate.

Figure 2

Biofilm eradication. Established E. coli ATCC 25922 biofilm was treated with DTr18-dab at the indicated concentrations for 24 h at 37 °C. The remaining biofilm was stained with crystal violet, and absorbance at 540 nm was taken. Treated samples were normalized to the no treatment (0 μg/mL) control which was set at 100%. Significance was determined by a one-way ANOVA with Dunnett’s multiple comparison test. Four asterisks (****) represents a p-value of <0.0001.

Figure 3

Assaying DTr18 membrane activity. (A) Measures the uptake of propidium iodide of E. coli ATCC 25922 cells when treated with differing amounts of peptide. PI uptake (fluorescence) was normalized to the PBS control. Melittin is a positive control and kanamycin is a negative control for cell death without membrane disruption. (B) Red blood cells were incubated with 128 μg/mL melittin or DTr18-dab, PBS or 1% TritonX-100 for 3 h at 37 °C. Percent hemolysis was determined by normalizing to the 1% TritonX-100 which was used as the 100% control. Significance was determined by a one-way ANOVA with Dunnett’s multiple comparison test (****) representing a p-value of <0.0001 and ns representing a p-value of >0.9999.

Figure 4

Rescue of G. mellonella with treatment of D-dab. G. mellonella was infected with 10⁶E. coli ATCC 25922 or a 1× PBS control. Thirty minutes postinfection the larvae were treated with DTr18-dab (15 mg/kg) or 1× PBS. Treatment with DTr18-dab (E. coli-DTr18-dab) rescued the larvae from E. coli infection across 48 h as compared to the untreated sample group (E. coli-PBS). Significance was determined by comparing the curves between treatment groups using a log-rank (Mantel–Cox) test. The p-value of PBS–PBS (control) and E. coli-DTr18-dab = 0.02 and is denoted as (*), PBS–PBS and E. coli-PBS = < 0.001 (****), and the E. coli-DTr18-dab and E. coli-PBS = 0.0002 (****).