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.