Discovery of Homogentisic Acid as a Precursor in Trimethoprim Metabolism and Natural Product Biosynthesis.

Opportunistic infections by Burkholderia cenocepacia are life threatening for patients suffering from cystic fibrosis and chronic granulomatous disease. These infections are often associated with variable clinical outcomes, prompting an interest in molecular investigations of phenotypes associated with disease severity. The production of the pyomelanin pigment is one such phenotype, which was recently linked to the ability of clinical strains to carry out biotransformation of the antibiotic trimethoprim. However, this biotransformation product was not identified, and differences in metabolite production associated with pyomelanin pigmentation are poorly understood. Here, we identify several key metabolites produced exclusively by the pyomelanin-producing strains. To provide insight into the structures and biosynthetic origin of these metabolites, we developed a mass spectrometry-based strategy coupling unsupervised in silico substructure prediction with stable isotope labeling referred to as MAS-SILAC (Metabolite Annotation assisted by Substructure discovery and Stable Isotope Labeling by Amino acids in Cell culture). This approach led to discovery of homogentisic acid as a precursor for biosynthesis of several natural products and for biotransformation of trimethoprim, representing a previously unknown mechanism of antibiotic tolerance. This work presents application of computational methods for analysis of untargeted metabolomic data to link the chemotype of pathogenic microorganisms with a specific phenotype. The observations made in this study provide insights into the clinical significance of the melanated phenotype.

Differences in Cystic Fibrosis-Associated Burkholderia spp. Bacteria Metabolomes after Exposure to the Antibiotic Trimethoprim.

The Burkholderia cepacia complex is a group of closely related bacterial species with large genomes that infect immunocompromised individuals and those living with cystic fibrosis. Some of these species are found more frequently and cause more severe disease than others, yet metabolomic differences between these have not been described. Furthermore, our understanding of how these species respond to antibiotics is limited. We investigated the metabolomics differences between three most prevalent Burkholderia spp. associated with cystic fibrosis: B. cenocepacia, B. multivorans, and B. dolosa in the presence and absence of the antibiotic trimethoprim. Using a combination of supervised and unsupervised metabolomics data visualization and analysis tools, we describe the overall differences between strains of the same species and between species. Specifically, we report, for the first time, the role of the pyomelanin pathway in the metabolism of trimethoprim. We also report differences in the detection of known secondary metabolites such as fragin, ornibactin, and N-acylhomoserine lactones and their analogs in closely related strains. Furthermore, we highlight the potential for the discovery of new secondary metabolites in clinical strains of Burkholderia spp. The metabolomics differences described in this study highlight the personalized nature of closely related Burkholderia strains.