Resisting the resistance: the antimicrobial peptide DGL13K selects for small colony variants of Staphylococcus aureus that show increased resistance to its stereoisomer LGL13K, but not to DGL13K.

Abstract

About 30% of the population are nasal carriers of Staphylococcus aureus, a leading cause of bacteremia, endocarditis, osteomyelitis, and skin and soft tissue infections. Antibiotic-resistant bacteria, in particular, are an increasing problem in both hospital and community settings. In this study, we sought to determine the cellular consequences of long-term exposure of S. aureus to the antimicrobial peptide stereoisomers, DGL13K and LGL13K. Both peptides selected for mutations in the chorismate/menaquinone biosynthetic pathway, which resulted in increased resistance to LGL13K but not DGL13K. DGL13K-selected isolates showed a mutation in aroF, while menA and menH were mutated in LGL13K-selected isolates. The latter also contained a mutation of frsA. The peptide-selected isolates exhibited golden coloration, suggesting increased production of the carotenoid staphyloxanthin, which could enhance resistance to cationic antimicrobial peptides (AMPs). The peptide-selected isolates grew as small colony variants, which have also been associated with resistance to AMPs. Addition of menaquinone to the growth medium reduced the generation time of DGL13K-selected mutants, but not LGL13K-selected mutants. Instead, the latter showed an increased MIC to LGL13K and greatly reduced ATP levels. The peptide-selected isolates showed increased biofilm formation and decreased autolysis, which was further reduced by alkaline shock, consistent with increased Asp23 expression. The mechanisms behind the differential effect of DGL13K and LGL13K on S. aureus resistance remain to be elucidated. The finding that DGL13K induced resistance to the stereoisomer LGL13K but not to DGL13K itself suggests that peptide primary structure is responsible for inducing bacterial defense mechanisms, but the peptide secondary structure determines if the defense mechanisms are effective against each peptide.

Importance: This work examines resistance to stereoisomers of the antimicrobial peptide GL13K in Staphylococcus aureus. Both DGL13K and LGL13K isomers cause mutations in the menaquinone pathway. While LGL13K causes resistance to LGL13K, the bacteria remain susceptible to DGL13K. Conversely, DGL13K also raises resistance to LGL13K, but the cells remain susceptible to DGL13K. The resistant isolates exhibit a small colony variant phenotype and overproduce the pigment staphyloxanthin. Menaquinone supplementation decreases the long generation time of DGL13K-selected isolates and increases the MIC of LGL13K-selected isolates.