Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae

Abstract

In bacteria, the defense system deployed 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 are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout 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 the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant 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. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance.

Keywords: AMR; Klebsiella pneumoniae; mechanisms of resistance; oxidative stress; soxS.

FIG 1

Oxidative, soxS, and oxidative soxS regulon of K. pneumoniae MGH78578. (A) The number of statistically significant genes identified in the oxidative regulon of K. pneumoniae MGH78578. These are categorized according to their expression pattern and depicted in a color code based on the color key given below. (B) The number of statistically significant genes identified in the soxS regulon and categorized according to their expression pattern. (C) Differentially regulated genes common in the soxS regulon of both K. pneumoniae MGH78578 and E. coli K-12 (3). The genes that are in green font and highlighted in yellow are those identified in the oxidative soxS regulon. (D) The number of genes in the oxidative soxS regulon of K. pneumoniae MGH78578 expressed as a percentage. The most significant soxS-induced and -repressed genes are indicated.

FIG 2

Expression of soxS gene under paraquat stress (A) and tetracycline stress (B). In both cases, the compounds were added to bacteria growing at mid-exponential phase for 30 min. Error bars represent standard deviations (SD) calculated from three biological replicates with three technical replicates each.

FIG 3

TPP⁺ accumulation in K. pneumoniae MGH78578 and its isogenic mutant MGH78578 ΔsoxS. All measurements were performed in 100 mM NaPi buffer containing 0.1% glucose, pH 8.0. Concentrated cell suspensions were added to obtain OD₆₀₀ of 1. Final concentrations of NMP (μg/ml) are indicated in panels B and C. The final concentrations of polymixin B (PMB) are indicated in the figure: 3, 6 and 9 μg/ml (A), 50 μg/ml (B), or 6 and 4 μg/ml for wt and ΔsoxS cells, respectively (C).

FIG 4

Survival of larvae in a zebrafish infection model. The survival of larvae infected with the isogenic wild-type K. pneumoniae MGH78578 reduced as the days postinfection increased (red line). The survival of larvae infected with the K. pneumoniae MGH78578 ΔsoxS strain (green line) was maintained at 100% throughout the experiment similar to the negative (avirulent E. coli XI1 blue, uninfected and DPBS) (black line) controls. Log-rank (Mantel-Cox) test, P < 0.0001.

FIG 5

Survival of bacteria in a zebrafish larvae infection model where the larvae are treated with tetracycline. Tetracycline induces ROS generation in the zebrafish larvae. The bar charts show the survival of the different bacterial cultures in the larval blood at 0, 6, 18, and 24 h postinfection and at different concentrations of tetracycline. Error bars represent standard deviations (SD) calculated from 3 independent reads. Significance was determined by two-way ANOVA and Sidak's multiple-comparison test, comparing the different tetracycline concentrations with the control group for each time point.