Deciphering the role of RND efflux transporters in Burkholderia cenocepacia

Abstract

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4-D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

Figure 1. Differential gene regulation in the B. cenocepacia RND efflux mutants.

The Venn diagram represents the differently expressed genes (down-regulated on the left, up-regulated on the right) in each mutant with respect to the wild-type strain.

Figure 2. Effect of RND-4 and RND-9 mutations on swimming motility.

The average diameter of swimming halos from three different experiments are plotted with standard deviations. Significantly differences with respect to J2315 are indicated by an * (p<0.01). Results are given in percentage, considering B. cenocepacia J2315 (wt) swimming halo as 100%. The panel below the graph shows one representative experiment. J2315, B. cenocepacia wild-type; D4, RND-4 mutant; D9, RND-9 mutant; D4-D9, RND4-RND9 mutant.

Figure 3. Effect of RND-4 and RND-9 mutations on biofilm formation.

(A) Adhesion to polyvinyl chloride mitrotiter plates measured by crystal violet staining. (B) Congo red dye binding ability. In both cases, results are given as a percentage, considering B. cenocepacia J2315 (wild-type) as 100%. The mean of three different experiments with standard deviation is reported. Significantly differences with respect to J2315 are indicated by an * (p<0.01). J2315, B. cenocepacia wild-type; D4, RND-4 mutant; D9, RND-9 mutant; D4–D9, RND4-RND9 mutant.

Figure 4. The Phenotype Microarray profile of B. cenocepacia J2315 and the RND mutants.

Metabolic plates (from PM 11 to PM20) representing the growth of the three B. cenocepacia mutant strains D4, D9 and D4–D9 versus the wild-type strain J2315, in the presence of toxic compounds is shown.

Figure 5. Principal component analysis of phenotype microarrays profiles of B. cenocepacia J2315 and D4, D9, D4–D9 mutants, obtained from an analysis of 960 chemical sensitivity tests (PM11-PM20).

The figure shows the four strains (J2315, D4, D9, D4–D9) and the phenotypical tests plotted in an X-Y diagram corresponding to the first two components.