Vandenbussche, I. | Search bioRxiv

bioRxiv2020

DNA methylation epigenetically regulates gene expression in Burkholderia cenocepacia

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation has shown to be an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2, and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant {Delta}BCAL3494 showed changes in biofilm structure and cell aggregation, {Delta}BCAM0992 was less motile. B. cenocepacia wild type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-Molecule Real-Time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified and qPCR experiments showed an upregulation of several genes, including biofilm and motility related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation and motility. ImportanceCF patients diagnosed with B. cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multi-replicon genome and a lot remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to lead to new insights in how to tackle B. cenocepacia infections in CF patients.

microbiology↗

bioRxiv2018

Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant and -tolerant bacteria. As these potentiators typically do not interfere with essential processes of bacteria, it has been hypothesized that they are less likely to induce resistance than conventional antibiotics. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop resistance towards one such adjuvant, baicalin hydrate (BH), a quorum sensing inhibitor known to increase antibiotic-induced oxidative stress. Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After each cycle of treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to treatments with TOB and TOB+BH, compared to the start population, and the potentiating effect of BH towards TOB was lost. Whole genome sequencing was performed to probe which changes were involved in the reduced effect of BH and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter), as well as a partial deletion of two larger regions. No changes in the minimal inhibitory or minimal bactericidal concentration of TOB or changes in the number of persister cells were observed in the evolved populations. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that resistance towards antibiotic-potentiating activity can develop rapidly in B. cenocepacia J2315 biofilms and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as potential mechanisms.\n\nImportanceBacteria show a markedly reduced susceptibility to antibiotics when growing in a biofilm, which hampers effective treatment of biofilm-related infections. The use of potentiators that increase the activity of antibiotics against biofilms has been proposed as a solution to this problem, but it is unclear whether resistance to these potentiators could develop. Using an experimental evolution approach, we convincingly demonstrate that Burkholderia cenocepacia biofilms rapidly develop resistance towards the tobramycin-potentiating activity of baicalin hydrate. Whole genome sequencing revealed that there are different mechanisms that lead to this resistance, including mutations resulting in metabolic changes, changes in production of intracellular levels of reactive oxygen species, and differences in transporter-mediated tobramycin uptake. Our study suggests that this form of combination therapy is not evolution-proof and highlights the usefulness of experimental evolution to identify mechanisms of resistance and tolerance in biofilm-grown bacteria.

microbiology↗