Resistance to the Cyclotide Cycloviolacin O2 in Salmonella enterica Caused by Different Mutations That Often Confer Cross-Resistance or Collateral Sensitivity to Other Antimicrobial Peptides

Abstract

Antimicrobial peptides (AMPs) are essential components of innate immunity in all living organisms, and these potent broad-spectrum antimicrobials have inspired several antibacterial development programs in the past 2 decades. In this study, the development of resistance to the Gram-negative bacterium-specific peptide cycloviolacin O2 (cyO2), a member of the cyclotide family of plant miniproteins, was characterized in Salmonella enterica serovar Typhimurium LT2. Mutants isolated from serial passaging experiments in increasing concentrations of cyO2 were characterized by whole-genome sequencing. The identified mutations were genetically reconstituted in a wild-type background. The additive effect of mutations was studied in double mutants. Fitness costs, levels of resistance, and cross-resistance to another cyclotide, other peptide and nonpeptide antibiotics, and AMPs were determined. A variety of resistance mutations were identified. Some of these reduced fitness and others had no effect on fitness in vitro, in the absence of cyO2. In mouse competition experiments, four of the cyO2-resistant mutants showed a significant fitness advantage, whereas the effects of the mutations in the others appeared to be neutral. The level of resistance was increased by combining several individual resistance mutations. Several cases of cross-resistance and collateral sensitivity between cyclotides, other AMPs, and antibiotics were identified. These results show that resistance to cyclotides can evolve via several different types of mutations with only minor fitness costs and that these mutations often affect resistance to other AMPs.

Antimicrobial Peptide Exposure Selects for Resistant and Fit Stenotrophomonas maltophilia Mutants That Show Cross-Resistance to Antibiotics

“…One potential problem with using AMPs clinically is the possibility that resistance to these compounds might be associated with cross-resistance to other classes of antimicrobials ( 25 , 63 – 65 ). We hypothesize that the genetic changes altering the membrane composition or membrane permeability, such as in lptB , ftsW , or hydG , generates low susceptibility not only to AMPs, but also to antibiotics.…”

Harnessing cyclotides to design and develop novel peptide GPCR ligands

“…40 All four cyclotides tested against nine strains of microbes showed activity against Gram-positive and negative bacteria, moderate activity against two fungi strains, and no activity against Candida albicans. 40 Antimicrobial activity of cyclotides has been confirmed by other studies [41][42][43] but it remains unclear whether this activity is biologically relevant as often the tested conditions did not include physiologically relevant media or bacteria relevant to plants. 44 Recently, Slazak et al examined the activity and mode of action of cycloviolacin cyclotides isolated from V. odorata and V. uliginosa against phytopathogens, thereby emphasizing their primary role in plant defense mechanisms.…”

Structural Characterization of the PawL-Derived Peptide Family, an Ancient Subfamily of Orbitides

“…Cyclotides and cyclic knottins adopt the cyclic cystine knot motif, an arrangement of disulfide bonds in which a ring like shape is formed by two disulfides, which brace the backbone of the peptide, with the knot 'tied' by the third disulfide bond threading through this ring. 15 Numerous bioactivities are reported for members of these families like uterotonic, 16 insecticidal, 17 and antimicrobial 18 activity as well as protease inhibition. 19 While most cyclotides are produced from dedicated precursor proteins, 20 some examples are encoded within precursor proteins for seed storage albumins.…”