Cell-Penetrating Antimicrobial Peptides Derived from an Atypical Staphylococcal δ-Toxin

Revisiting underutilized classes of antibiotics is a pragmatic approach to the identification of alternative therapies for antimicrobial-resistant pathogens. To this end, we designed and screened a set of seven staphylococcal δ-toxin-inspired peptides (STIPs) for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, a pathogen-specific protease was leveraged to generate shorter peptides from these δ-toxin derivatives to expand the screen of putative antimicrobial peptides (AMPs) and to counterscreen against AMP inactivation. Remarkably, a 17-amino acid peptide based on the atypical δ-toxin sequence of Staphylococcus auricularis was discovered to possess an ability to kill MRSA and related pathogens. An alanine scan and series of rational substitutions improved AMP activity, and phenotypic assays characterized the STIPs’ ability to rapidly interact with and permeabilize the staphylococcal membrane without causing lysis on a commensurate timescale. Instead of rapid lysis, both l- and d-enantiomers of STIP3-29, an AMP with low micromolar activity, were observed to penetrate and accumulate within cells. Finally, we observed that STIP3-29 was capable of controlling MRSA infection in a three-dimensional skin infection model. Overall, the results suggest that this unconventional source of AMPs can provide promising candidates for further development as therapeutic agents. IMPORTANCE The continued emergence and global distribution of infections caused by antimicrobial-resistant pathogens fuel our perpetual need for new or alternative therapies. Here, we present the discovery and initial characterization of bacterial cell-penetrating AMPs that were based on a family of virulence factors. In contrast to the multitude of AMPs that are sourced from animals, these potential therapeutic molecules have not undergone extensive selection for their antimicrobial properties and have proven to be amenable to activity-optimizing modifications. The staphylococcal toxin-inspired peptides described here represent a source of AMPs that can kill common opportunistic pathogens, such as MRSA, and have the potential to be improved for application in medicine.