The Effect of 2-Thiocyanatopyridine Derivative 11026103 on Burkholderia Cenocepacia: Resistance Mechanisms and Systemic Impact

Abstract

Bacteria of the Burkholderia cepacia complex (Bcc) are associated with significant decline of lung functions in cystic fibrosis patients. Bcc infections are virtually impossible to eradicate due to their irresponsiveness to antibiotics. The 2-thiocyanatopyridine derivative 11026103 is a novel, synthetic compound active against Burkholderia cenocepacia. To characterize mechanisms of resistance to 11026103, B. cenocepacia was subjected to chemical mutagenesis, followed by whole genome sequencing. Parallel mutations in resistant isolates were localized in a regulatory protein of the efflux system Resistance-Nodulation-Division (RND)-9 (BCAM1948), RNA polymerase sigma factor (BCAL2462) and its cognate putative anti-sigma factor (BCAL2461). Transcriptomic analysis identified positive regulation of a major facilitator superfamily (MFS) efflux system BCAL1510-1512 by BCAL2462. Artificial overexpression of both efflux systems increased resistance to the compound. The effect of 11026103 on B. cenocepacia was analyzed by RNA-Seq and a competitive fitness assay utilizing an essential gene knockdown mutant library. 11026103 exerted a pleiotropic effect on transcription including profound downregulation of cluster of orthologous groups (COG) category "Translation, ribosomal structure, and biogenesis". The competitive fitness assay identified many genes which modulated susceptibility to 11026103. In summary, 11026103 exerts a pleiotropic cellular response in B. cenocepacia which can be prevented by efflux system-mediated export.

Keywords: Burkholderia cenocepacia; RNA-Seq; Tn-Seq; chemical genetics; chemical mutagenesis; whole-genome sequencing.

Figure 1

Mutations in B. cenocepacia isolates resistant to 11026103. (A) All mutations, mapped to chromosomes 1 and 2, which resulted from chemical mutagenesis with ethyl methanesulfonate (EMS) (five isolates; outer circles) or 5-azaC (four isolates; inner circles). The genes/operons which displayed convergent mutations are denoted by asterisks. (B) Detailed view of parallel mutations. MICs of corresponding 11026103-resistant mutants are denoted. The visualizations were carried out using the BRIG software [12].

Figure 2

The effect of artificial expression of BCAL2462 (wild-type (WT) or 51P→S allele) in B. cenocepacia J2315 on (A) expression of major facilitator superfamily (MFS) transporter BCAL1510; (B) MIC of 11026103. Expression changes were calculated in comparison with empty vector control experiment (J2315 + pSCrhaB2 with or without rhamnose).

Figure 3

The effect of artificial expression of efflux systems BCAL1510-1512 (MFS) and BCAM1945-1947 (RND-9) in B. cenocepacia J2315 on MIC of 11026103. MICs were determined in media containing increasing concentrations of expression inducer (L-rhamnose) up to 0.0005% (w/v). Rhamnose concentrations of 0.001% (w/v) and higher impaired viability of bacteria overexpressing both efflux systems, regardless of the presence of 11026103.

Figure 4

Effect of 11026103 on essential gene knockdown mutants. (A) The bimodal distribution of genes affected by 11026103. Normal distribution, resulting from chloramphenicol treatment, is shown for comparison. Abundance of each mutant relative to the no antibiotic control was calculated by taking the log₂ of the normalized read count for the control divided by the normalized read count for the 11026103- or chloramphenicol-treated sample. Positive values indicate susceptibility. (B) Cluster of orthologous groups (COG) categories affected by 11026103. The values for individual genes per COG category were averaged. The total number of genes recovered after the growth with 11026103 is shown for each COG category.

Figure 5

Effect of 11026103 on transcriptome of B. cenocepacia J2315. The differentially expressed genes are color-coded according to the concentration of 11026103 used (sub-inhibitory: 5 µg mL⁻¹; inhibitory: 10 µg mL⁻¹ and 15 µg mL⁻¹). The extent of differential expression (fold-change (FC) of expression with respect to untreated control experiment) is denoted by the intensity of corresponding colors. Genes whose differential expression did not reach statistical significance (p > 0.2) were discarded from the calculations. (A) Total numbers of differentially expressed genes. (B) Representation of COG categories among differentially expressed genes. Total numbers of B. cenocepacia J2315 genes in each COG category are denoted.