Analysis of the oxidative stress regulon identifies soxS as a genetic target for resistance reversal in multi-drug resistant Klebsiella pneumoniae

In bacteria the defence system to counter oxidative stress is orchestrated by three transcriptional factors – SoxS, SoxR and OxyR. Although the transcriptional regulon of these factors are known in many bacteria, similar data is not available for K. pneumoniae. To address this data gap, oxidative stress was induced in K. pneumoniae MGH 78578 using paraquat and the corresponding regulon was identified using RNA-seq. Since soxS was significantly induced , a soxS mutant was constructed to decipher this regulon in K. pneumoniae MGH75878. The 'oxidative SoxS regulon', comprising common genes differentially regulated genes in oxidative and soxS regulon was identified from both regulons – characterised a stringent group of genes which were regulated by SoxS during oxidative stress. Efflux pump encoding genes like acrAB-tolC, acrE along with marRABwere identified in the oxidative SoxS regulon. The phenotypic effect of the observed efflux pump regulation was confirmed in the soxS mutant that exhibited an 2 fold reduction in the minimum bactericidal concentration (MBC) against tetracycline compared to that of the isogenic wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further confirmed using tetraphenylphosphonium (TPP+) ion accumulation assay. The susceptibility of the soxS mutant against tetracycline was also apparent in vivo, in the zebrafish embryo model. We conclude that the soxS gene could be considered as a genetic target against which an inhibitor could be developed and be used in combinatorial therapy with tetracycline to combat infections associated with multi-drug resistant K. pneumoniae. Overall design: Bacterial transcriptomic profile generated by inducing oxidative stress by exposing the culture to Paraquat, deep sequencing, in duplicates, using Illumina HiSeq2500.