The last decades have witnessed an exponential increase in bacterial antimicrobial resistance and a dramatic decline in the development of novel antibiotics. Recent WHO reports have urged the need for new therapeutic strategies to combat the immediate threat posed by multidrug resistant bacterial infections. Antivirulence drugs are considered a promising avenue in the race to develop new antimicrobial therapeutics. However, their evolutionary robustness often quoted as an advantage of this strategy over traditional antibiotics, warrants further investigation.
Inhibition of the DiSulfide Bond (DSB) oxidative protein folding machinery, a major facilitator of virulence in Gram‐negative bacteria, represents a promising antivirulence strategy. We previously developed small molecule inhibitors of DsbA from Escherichia coli K‐12 (EcDsbA) and showed that they attenuate virulence of Gram‐negative pathogens by directly inhibiting multiple diverse DsbA homologues.
In this work we use an experimental evolution approach to test the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials against Salmonella enterica serovar Typhimurium under pathophysiological in vitro conditions. We show that phenylthiophene DsbA inhibitors can slow S. Typhimurium growth in minimal media, phenocopying S. Typhimurium isogenic dsbA null mutants. We establish the hierarchical contribution of different DsbA enzymes encoded by Salmonella for growth under these conditions. Through passaging experiments, we demonstrate that DsbA inhibitor resistance is not induced under conditions that rapidly induce resistance to ciprofloxacin, an antibiotic commonly used to treat Salmonella infections. Furthermore, no drug-specific adaptations are detected in inhibitor‐treated S. Typhimurium, while pathogen virulence remains susceptible to DsbA inhibition.
Our work demonstrates that under in vitro pathophysiological conditions, DsbA inhibitors can have both antivirulence and antibiotic activity, supporting the proposition that different antivirulence drugs will have different fitness cost for bacteria. Importantly, our finding that inhibition of DsbA attenuates pathogen growth and virulence without detectable resistance development, supports the case for their further development as novel and possibly “evolution proof” antimicrobials against critical-priority Gram-negative pathogens.