Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite

Abstract

Background: Burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis (CF). As treatment of infected CF patients is problematic, multiple preventive measures are taken to reduce the infection risk. Besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. However, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of B. cepacia complex bacteria have been reported. In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of reactive oxygen species (ROS), the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2 or NaOCl.

Results: The exposure to H2O2 and NaOCl resulted in an upregulation of the transcription of 315 (4.4%) and 386 (5.4%) genes, respectively. Transcription of 185 (2.6%) and 331 (4.6%) genes was decreased in response to the respective treatments. Many of the upregulated genes in the NaOCl- and H2O2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ROS. In addition, multiple upregulated genes encode proteins that are necessary to repair ROS-induced cellular damage. Unexpectedly, a prolonged treatment with H2O2 also resulted in an increased transcription of multiple phage-related genes. A closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6S RNA.

Conclusion: Our results reveal that the transcription of a large fraction of B. cenocepacia J2315 genes is altered upon exposure of sessile cells to ROS. These observations have highlighted that B. cenocepacia may alter several pathways in response to exposure to ROS and they have led to the identification of many genes not previously implicated in the stress response of this pathogen.

Figure 1

Effect of treatments with H₂O₂ or NaOCl on the number of viable sessile B. cenocepacia J2315 cells. The average relative fluorescence signals (%) show the fraction of viable B. cenocepacia J2315 sessile cells in untreated biofilms and in biofilms treated with H₂O₂ (0.3%, 30 min; dark grey bars) or NaOCl (0.02%, 5 min; pale grey bars). Error bars represent standard deviations.

Figure 2

Putative OxyR-binding sites upstream of BCAL2297, BCAL3299 (katB), BCAM0931 and ahpCF (BCAM1217-BCAM1216). The four tetranucleotide sequences in the E. coli OxyR-binding consensus sequence are underlined and the nucleotides matching the consensus sequence are indicated by grey shading [74].

Figure 3

Effect of treatment with H₂O₂ on the number of viable sessile B. cenocepacia cells. The average relative fluorescence signals (%) show the fraction of viable sessile cells in untreated biofilms and in biofilms treated with H₂O₂ (0.3%) for 15, 30 or 60 min. Data were obtained for biofilms of B. cenocepacia J2315 (black bars), C5424 (dark grey bars), MDL1 (katA mutant; pale grey bars) and MDL2 (katB mutant; white bars). Error bars represent standard deviations.

Figure 4

The protein encoded by BCAL1766 belongs to the Ohr family. Amino acid sequences of three upregulated Ohr proteins in B. cenocepacia J2315 (encoded by BCAS0085, BCAM0896 and BCAL1766) and of P. aeruginosa PAO1 Ohr (OhrPAO1) and OsmC (OsmCPAO1) and E. coli K-12 MG1655 OsmC (OsmCK12) were aligned using the CLUSTAL W program using standard settings [75]. The regions of black shading and white lettering show the conserved regions found in either the Ohr homologues or the OsmC homologues. Grey shading indicates that identical amino acid sequences are found in both Ohr and OsmC homologues. The triangles point to the highly conserved C residues in both families.

Figure 5

Increased lipase activity in the supernatant of H₂O₂-treated B. cenocepacia J2315 biofilms. Lipase activity was determined in the supernatant of B. cenocepacia J2315 biofilms that were either untreated (diamonds) or that were treated with 0.3% H₂O₂ for 15 (rectangles), 30 (triangles) or 60 min (circles). The graph shows a representative example of the obtained curves using 4-MU oleate as fluorogenic substrate. Signals are expressed as normalized fluorescence units (RFU). Error bars represent standard deviations.

Figure 6

Effect of treatments with H₂O₂ on the expression of the IGs IG1_2935724 and IG1_3008003 and their adjacent genes. The expression of the genes BCAL2667 and BCAL2737 (grey bars) and BCAL2668 and BCAL2738 (black bars) and of the IGs (IG1_2935724 and IG1_3008003 [white bars]) in the treated biofilms is compared to the expression observed in the untreated biofilms. Error bars represent SEM. *: significant upregulation in treated biofilms compared to untreated biofilms (p < 0.05).

Figure 7

Secondary structure of the putative 6S RNA in IG1_2935724 and the 6S RNA consensus structure. A. Secondary structure obtained for the intergenic region spanning nucleotides 2935805 to 2935985 (B. cenocepacia J2315; chromosome 1) as predicted by mfold using standard settings [76]. B. Secondary structure of the 6S RNA consensus structure (RFAM database [53]).

Figure 8

Effect of treatments with H₂O₂ on the expression of three selected BcepMu prophage genes. The expression of BCAS0543 (BcepMu12; grey bars), BCAS0546 (BcepMu9; white bars) and BCAS0547 (BcepMu8; black bars) in the H₂O₂-treated biofilms is compared with the expression observed in the untreated biofilms. Error bars represent SEM. *: significant upregulation in treated biofilms compared to untreated biofilms (p < 0.05).