Metabolomic profiling of Burkholderia cenocepacia in synthetic cystic fibrosis sputum medium reveals nutrient environment-specific production of virulence factors

Abstract

Infections by Burkholderia cenocepacia lead to life-threatening disease in immunocompromised individuals, including those living with cystic fibrosis (CF). While genetic variation in various B. cenocepacia strains has been reported, it remains unclear how the chemical environment of CF lung influences the production of small molecule virulence factors by these strains. Here we compare metabolomes of three clinical B. cenocepacia strains in synthetic CF sputum medium (SCFM2) and in a routine laboratory medium (LB), in the presence and absence of the antibiotic trimethoprim. Using a mass spectrometry-based untargeted metabolomics approach, we identify several compound classes which are differentially produced in SCFM2 compared to LB media, including siderophores, antimicrobials, quorum sensing signals, and various lipids. Furthermore, we describe that specific metabolites are induced in the presence of the antibiotic trimethoprim only in SCFM2 when compared to LB. Herein, C13-acyl-homoserine lactone, a quorum sensing signal previously not known to be produced by B. cenocepacia as well as pyochelin-type siderophores were exclusively detected during growth in SCFM2 in the presence of trimethoprim. The comparative metabolomics approach described in this study provides insight into environment-dependent production of secondary metabolites by B. cenocepacia strains and suggests future work which could identify personalized strain-specific regulatory mechanisms involved in production of secondary metabolites. Investigations into whether antibiotics with different mechanisms of action induce similar metabolic alterations will inform development of combination treatments aimed at effective clearance of Burkholderia spp. pathogens.

Discovery of homogentisic acid as a precursor in trimethoprim metabolism and natural product biosynthesis

“…Our lab has previously shown that sublethal concentrations of trimethoprim induce a strain-specific response in the K56-2 strain not seen in other B. cenocepacia strains, resulting in increased production of secondary metabolites such as fragin and pyrazine- N -oxides. 12, 13 Plotting PC1 against PC3 revealed an additional trend where samples from pigmented strains show separation from those of non-pigmented strains along PC3 (10.9% of the total variance explained) (Fig. 3B).…”

“…Culturing was performed in the presence of antibiotic trimethoprim since it is a known inducer of secondary metabolites produced by B. cenocepacia K56-2 and also undergoes biochemical modification by pigmented B. cenocepacia , while also being the first line of defense again B. cenocepacia infections. 12, 13 To enhance extraction of chemically diverse metabolites, liquid-liquid and solid-phase extractions were performed. These extracts were pooled together prior to UHPLC-HRMS/MS analysis in positive and negative ionization modes.…”

“…The chemical structure of this motif was previously annotated as trimethoprim (Fig S3A,B). 12, 13 Mass spectral annotation of additional nodes in the network revealed that this motif was present in metabolites containing adenine and anthranilic acid as substructures (Figs. S4,S5).…”

“…We had previously linked the biotransformation of trimethoprim to an increase in the minimal inhibitory concentration of this antibiotic against clinical B. cenocepacia strains. 12, 13 The direct link between pyomelanin biosynthesis and biochemical modification of the antibiotic trimethoprim suggests that inhibition of pyomelanin production in conjunction with antibiotic treatment can also be investigated as a treatment strategy for infections by pyomelanin-producing B. cenocepacia . For example, 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC) is an FDA-approved drug which inhibits the 4-hydroxyphenylpyruvate dioxygenase (Hppd, Figure 1) enzyme responsible for producing homogentisic acid.…”

“…10, 11 We recently showed that pigmented strains can metabolize the antibiotic trimethoprim, increasing the minimal inhibitory concentration of this antibiotic. 12, 13 Trimethoprim in combination with sulfamethoxazole (co-trimoxazole) is used as one of the first lines of treatment in the clinic. 14 However, the details of how trimethoprim undergoes metabolic transformation by pigmented strains are not known.…”