Analysis of the oxidative stress regulon identifiessoxSas a genetic target for resistance reversal in multi-drug resistantKlebsiella pneumoniae

Abstract

AbstractIn bacteria, the defense system deployd to counter oxidative stress is orchestrated by three transcriptional factors – SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data is not available forKlebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced inK. pneumoniaeMGH 78578 using paraquat and the corresponding oxidative stress regulon recorded using RNA-seq. ThesoxSgene was significantly induced during oxidative stress and a knock-out mutant was constructed, to explore its functionality. The wild-type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate thesoxSregulon inK. pneumoniaeMGH78578. Genes that are commonly regulated both in the oxidative stress regulon andsoxSregulon were identified and denoted as the ‘oxidative SoxS regulon’ – these included a stringent group of genes specifically regulated by SoxS. Efflux pump encoding genes such asacrAB-tolC, acrE, and global regulators such asmarRABwere identified as part of this regulon. Consequently, the isogenicsoxSmutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. ThesoxSmutant was also susceptible to tetracyclinein vivo, in a zebrafish embryo model. We conclude that thesoxSgene could be considered as a genetic target against which an inhibitor could be developed in the future and used in combinatorial therapy with tetracycline to combat infections associated with multi-drug resistantK. pneumoniae.