Multi-Omic Analyses Provide Links between Low-Dose Antibiotic Treatment and Induction of Secondary Metabolism in Burkholderia thailandensis

Abstract

Low doses of antibiotics can trigger secondary metabolite biosynthesis in bacteria, but the underlying mechanisms are generally unknown. We sought to better understand this phenomenon by studying how the antibiotic trimethoprim activates the synthesis of the virulence factor malleilactone in Burkholderia thailandensis. Using transcriptomics, quantitative multiplexed proteomics, and primary metabolomics, we systematically mapped the changes induced by trimethoprim. Surprisingly, even subinhibitory doses of the antibiotic resulted in broad transcriptional and translational alterations, with ∼8.5% of the transcriptome and ∼5% of the proteome up- or downregulated >4-fold. Follow-up studies with genetic-biochemical experiments showed that the induction of malleilactone synthesis can be sufficiently explained by the accumulation of methionine biosynthetic precursors, notably homoserine, as a result of inhibition of the folate pathway. Homoserine activated the malleilactone gene cluster via the transcriptional regulator MalR and gave rise to a secondary metabolome which was very similar to that generated by trimethoprim. Our work highlights the expansive changes that low-dose trimethoprim induces on bacterial physiology and provides insights into its stimulatory effect on secondary metabolism. IMPORTANCE The discovery of antibiotics ranks among the most significant accomplishments of the last century. Although the targets of nearly all clinical antibiotics are known, our understanding regarding their natural functions and the effects of subinhibitory concentrations is in its infancy. Stimulatory rather than inhibitory functions have been attributed to low-dose antibiotics. Among these, we previously found that antibiotics activate silent biosynthetic genes and thereby enhance the metabolic output of bacteria. The regulatory circuits underlying this phenomenon are unknown. We take a first step toward elucidating these circuits and show that low doses of trimethoprim (Tmp) have cell-wide effects on the saprophyte Burkholderia thailandensis. Most importantly, inhibition of one-carbon metabolic processes by Tmp leads to an accumulation of homoserine, which induces the production of an otherwise silent cytotoxin via a LuxR-type transcriptional regulator. These results provide a starting point for uncovering the molecular basis of the hormetic effects of antibiotics.

A rapid screening method for the detection of specialised metabolites from bacteria: Induction and suppression of metabolites from Burkholderia species

“…Previously, exposure to trimethoprim at subinhibitory concentrations has been reported as a global activator for Burkholderia thailandensis specialised metabolites, able to induce previously uncharacterised BGCs ( Seyedsayamdost 2014 ; Okada et al 2016 ; Li et al 2020 ). To evaluate the trimethoprim induction phenomenon on different Burkholderia species, four strains of B. gladioli (BCC1665, BCC1678, BCC1686 and BCC1701) were grown on BSMG agar plates with a range of trimethoprim concentrations (0–10 μg ml −1 ) and metabolites were analysed as above.…”

“…Low doses of rifampicin and erythromycin have been shown to change the expression of up to 5% of the transcripts in Salmonella enterica , with many of them being upregulated ( Goh et al 2002 ). Similarly, the addition of subinhibitory concentrations of trimethoprim to B. thailandensis resulted in both transcriptional and translational alterations, with 8.5% of the transcriptome and 5% of the proteome up or downregulated by more than 4-fold ( Li et al 2020 ). It was proposed that the low concentrations of trimethoprim inhibit one‑carbon metabolic processes, which leads to an accumulation of homoserine, that subsequently induces silent BGCs by a LuxR-type transcriptional regulator ( Li et al 2020 ).…”

“…Similarly, the addition of subinhibitory concentrations of trimethoprim to B. thailandensis resulted in both transcriptional and translational alterations, with 8.5% of the transcriptome and 5% of the proteome up or downregulated by more than 4-fold ( Li et al 2020 ). It was proposed that the low concentrations of trimethoprim inhibit one‑carbon metabolic processes, which leads to an accumulation of homoserine, that subsequently induces silent BGCs by a LuxR-type transcriptional regulator ( Li et al 2020 ). Understanding the mechanisms of antibiotic-based induction and/or suppression of B. gladioli and B. ambifaria metabolites seen in the current study would be interesting to address by global transcriptomic analysis.…”

The Antibiotic Andrimid Produced by Vibrio coralliilyticus Increases Expression of Biosynthetic Gene Clusters and Antibiotic Production in Photobacterium galatheae

“…These data could therefore indicate that P. galatheae uses its stress response to increase production of holomycin, but more work is needed to address the mechanism. It was recently shown that the trimethoprim-induced production of malleilactone in B. thailandensis occurs via the accumulation of metabolic intermediates rather than via stress response (Li et al, 2020). However, malleilactone is a virulence factor necessary for infection of the nematode Caenorhabditis elegans by B. thailandensis (Biggins et al, 2012), and therefore competition sensing via stress response may not be an ecological cue for its production.…”

“…This response was mediated by a newly identified two-component regulatory system. In B. thailandensis, sub-MICs of trimethoprim change intracellular metabolite concentrations, which leads to the overproduction of malleilactone (Li et al, 2020). These examples add to our knowledge on how, on a mechanistic and molecular level, sub-MICs of antibiotics induce physiological and transcriptional responses in microbes (Andersson and Hughes, 2014).…”