Optimization of host cell-compatible, antimicrobial peptides effective against biofilms and clinical isolates of drug-resistant bacteria

Abstract

Here, we describe the synthetic molecular evolution of a family of host-compatible antimicrobial peptides (AMP) designed for the treatment of wounds infected with drug-resistant, biofilm forming bacteria. The peptides tested are variants of an evolved AMP called D-CONGA, which has excellent antimicrobial activities in vitro and in vivo. In this generation of rational D-CONGA variants, we tested multiple sequence-function hypotheses that had not been tested in previous generations. Many of the peptide variants tested have lower antibacterial activity against Gram-positive and Gram-negative pathogens, especially those that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, D-CONGA is generally well tuned for good antimicrobial activity. However, we identified one variant, D-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria and lower cytotoxicity against human fibroblasts. Against clinical isolates of K. pneumoniae, innate resistance to D-CONGA was surprisingly common despite a lack of inducible resistance in P. aeruginosa reported in our previous work. Yet, these same isolates are susceptible to D-CONGA-Q7. Additional mechanistic work will be required to understand why D-CONGA-Q7 is less prone to innate resistance in Gram-negative bacteria. In the spirit of synthetic molecular evolution, the discovery of D-CONGA-Q7 achieved a critical gain-of-function, and has provided a significantly better template sequence for the next generation of synthetic evolution.

Mutational signature analysis predicts bacterial hypermutation and multidrug resistance

“…MMR-pro cient MPAO1 eventually displayed elevated MICs to both AZ and COL, but it did so more slowly indicating that MMR-de ciency catalyzed resistance acquisition. Despite lack of resistance acquisition previously observed in PAO1 37,38 , MPAO1-ΔmutS rapidly developed resistance to both D-CONGA and D-CONGA-Q7 (Fig. 1f).…”

“…AZ and COL are common treatments for P. aeruginosa infections used as a ' rst line' or 'last-resort' option, respectively 35,36 . D-CONGA and D-CONGA-Q7 are cationic antimicrobial peptides developed via synthetic molecular evolution and are e cacious in vitro and in vivo against drug-resistant clinical isolates of Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and P. aeruginosa, including P. aeruginosa CF isolates 37 . Notably, induction of resistance to D-CONGA and D-CONGA-Q7 has not been observed for WT P. aeruginosa (PAO1) 37,38 .…”

“…D-CONGA and D-CONGA-Q7 are cationic antimicrobial peptides developed via synthetic molecular evolution and are e cacious in vitro and in vivo against drug-resistant clinical isolates of Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and P. aeruginosa, including P. aeruginosa CF isolates 37 . Notably, induction of resistance to D-CONGA and D-CONGA-Q7 has not been observed for WT P. aeruginosa (PAO1) 37,38 . Prior to experimentation, MPAO1-DmutS was validated as a hypermutator via rifampicin reversion frequency (Supplementary Fig.…”