Gene Expression Profiling of Pseudomonas aeruginosa Upon Exposure to Colistin and Tobramycin

Abstract

Pseudomonas aeruginosa (Pae) is notorious for its high-level resistance toward clinically used antibiotics. In fact, Pae has rendered most antimicrobials ineffective, leaving polymyxins and aminoglycosides as last resort antibiotics. Although several resistance mechanisms of Pae are known toward these drugs, a profounder knowledge of hitherto unidentified factors and pathways appears crucial to develop novel strategies to increase their efficacy. Here, we have performed for the first time transcriptome analyses and ribosome profiling in parallel with strain PA14 grown in synthetic cystic fibrosis medium upon exposure to polymyxin E (colistin) and tobramycin. This approach did not only confirm known mechanisms involved in colistin and tobramycin susceptibility but revealed also as yet unknown functions/pathways. Colistin treatment resulted primarily in an anti-oxidative stress response and in the de-regulation of the MexT and AlgU regulons, whereas exposure to tobramycin led predominantly to a rewiring of the expression of multiple amino acid catabolic genes, lower tricarboxylic acid (TCA) cycle genes, type II and VI secretion system genes and genes involved in bacterial motility and attachment, which could potentially lead to a decrease in drug uptake. Moreover, we report that the adverse effects of tobramycin on translation are countered with enhanced expression of genes involved in stalled ribosome rescue, tRNA methylation and type II toxin-antitoxin (TA) systems.

Keywords: Pseudomonas aeruginosa; RNA-Seq; Ribo-seq; colistin; ribosome profiling; tobramycin.

FIGURE 1

Scatter plots showing the correlation between normalized RNA-seq and Ribo-seq sequencing reads (BaseMean) obtained from two biological replicates of (A) control, (B) colistin, and (C) tobramycin treated samples. ρ – Spearman correlation value.

FIGURE 2

Venn diagram showing the number of transcripts with increased (I), decreased (D) or opposite (O) abundance in RNA-seq and Ribo-seq data obtained after (A) colistin treatment and (B) tobramycin treatment. For significance, only transcripts with a fold-change ≥ 2 or ≤ –2 and a multiple testing adjusted p-value ≤ 0.05 were considered. The corresponding transcripts and ribosomal footprints with increased, decreased and opposite abundance are listed in Supplementary Table 1.

FIGURE 3

Scatter plots showing the correlation between gene expression fold-changes in RNA-seq and Ribo-seq data obtained after (A) colistin treatment and (B) tobramycin treatment. The X-axis corresponds to the RNA-seq data, or transcriptome, and the Y-axis to the Ribo-seq data, or translatome.

FIGURE 4

PseudoCAP functional class distribution of annotated genes with altered expression in response to colistin or tobramycin. (A) Up-regulated and (B) down-regulated genes in colistin treated samples. Blue and pink bars indicate the number of de-regulated genes based on the RNA-seq and Ribo-seq data, respectively. (C) Up-regulated and (D) down-regulated genes in tobramycin treated samples. Brown and orange bars indicate the number of de-regulated genes based on the RNA-seq and Ribo-seq data, respectively.

FIGURE 5

Depiction of novel functions/pathways revealed in this study that are de-regulated upon (A) colistin and (B) tobramycin treatment. Major genes/pathways that are down-regulated and up-regulated based on the RNA-seq and/or Ribo-seq data are highlighted in rose and green, respectively. Positive- and negative regulation of gene expression is denoted by arrows and blocked lines, respectively. RIP - regulated intramembrane proteolysis.