Mutations in the superoxide-responsive regulator SoxR confer fluoroquinolone resistance in Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia is a multidrug-resistant Gram-negative opportunistic pathogen. Its antimicrobial resistance arises from intrinsic traits and acquired mechanisms. Overexpression of drug efflux transporters is one of the key mechanisms mediating high-level antibiotic resistance. These transporters are regulated by transcriptional factors, including SoxR, a superoxide-responsive regulator containing an iron-sulfur [2Fe-2S] cluster. SoxR controls the multidrug efflux pumps MfsA and SmeVWX, as well as superoxide dismutase SodA1. To examine how soxR mutations affect antimicrobial resistance, random mutagenesis was performed, and mutated genes were ectopically expressed in a ΔsoxR mutant. Transformants were selected for ciprofloxacin resistance, a substrate for both MfsA and SmeVWX. Sequencing of 21 resistant colonies revealed five distinct mutation patterns (A–E), each containing multiple mutations. The ΔsoxR mutant expressing these soxR variants showed increased resistance to multiple antibiotics targeted by the efflux pumps, correlating with elevated MfsA and SmeVWX expression. This indicates constitutive SoxR activation. To assess the role of the [2Fe-2S] cluster, cysteine-to-serine substitutions at three of the four cluster-binding residues abolished SoxR’s ability. Computational structure prediction and molecular dynamics simulations supported these findings, revealing that mutations enhanced solvent exposure of the [2Fe-2S] cluster. This suggests that the mutations shift SoxR into a conformation more prone to activation. Furthermore, a single amino acid substitution at residue R49 was sufficient to activate SoxR and confer resistance. Together, our findings demonstrate that S. maltophilia can enhance antimicrobial resistance through soxR mutations, particularly under antibiotic pressure. This highlights the role of SoxR in the antimicrobial resistance of S. maltophilia.