Antimicrobial Agent Trimethoprim Influences Chemical Interactions in Cystic Fibrosis Pathogens via the ham Gene Cluster

Abstract

The fungus Aspergillus fumigatus and the bacterium Burkholderia cenocepacia cause fatal respiratory infections in immunocompromised humans and patients with lung disease, such as cystic fibrosis (CF). In dual infections, antagonistic interactions contribute to increased mortality. These interactions are further altered by the presence of antimicrobial and antifungal agents. However, studies performed to date on chemical interactions between clinical B. cenocepacia and A. fumigatus have focused on pathogens in isolation and do not include the most abundant chemical signal, i.e., clinically administered therapeutics, present in the lung. Here, we characterize small molecule-mediated interactions between B. cenocepacia and A. fumigatus and their shift in response to trimethoprim exposure by using metabolomics and mass spectrometry imaging. Using these methods, we report that the production of several small-molecule natural products of both the bacteria and the fungus is affected by cocultivation and exposure to trimethoprim. By systematic analysis of metabolomics data, we hypothesize that the B. cenocepacia-encoded ham gene cluster plays a role in the trimethoprim-mediated alteration of bacterial-fungal interactions. We support our findings by generating a genetically modified strain lacking the ham gene cluster and querying its interaction with A. fumigatus. Using comparative analyses of the extracts of wild-type and knockout strains, we report the inactivation of a bacterially produced antifungal compound, fragin, by A. fumigatus, which was verified by the addition of purified fragin to the A. fumigatus culture. Furthermore, we report that trimethoprim does not inhibit fungal growth, but affects the biochemical pathway for DHN-melanin biosynthesis, an important antifungal drug target, altering the pigmentation of the fungal conidia and is associated with modification of ergosterol to ergosteryl-3β-O-l-valine in coculture. This study demonstrates the impact of therapeutics on shaping microbial and fungal metabolomes, which influence interkingdom interactions and the expression of virulence factors. Our findings enhance the understanding of the complexity of chemical interactions between therapeutic compounds, bacteria, and fungi and may contribute to the development of selective treatments.

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Overview of study design, sample processing, and metabolomics data acquisition and analyses. (a) Schematic representation of the workflow for culturing, extractions of samples, and UHPLC-MS/MS-based untargeted metabolomics data acquisition. (b) Data analysis workflow for untargeted metabolomics. (c) Schematic representation of the MALDI-ToF-MSI workflow. (Created with permission from BioRender.com).

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Metabolome profiling. (a) Principal component analysis of untargeted metabolomics data acquired on extracts of B. cenocepacia K56-2 (Bc) and A. fumigatus (Af) monocultures and B. cenocepacia K56-2 + A. fumigatus (Bc + Af) cocultures treated without (−) and with (+) trimethoprim. (b) Heatmap showing differences in relative abundances of the top 50 significant features identified via hierarchical clustering analysis. (c) UpSet plot analysis displaying the number of features detected across different growth conditions of antibiotic exposure and mono- and cocultures. For the sake of clarity, only the top 20 combinations of conditions in which the largest number of unique features were detected are shown.

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Feature-based molecular network of metabolomics data. UHLPC-MS/MS-based untargeted metabolomics data were processed and organized into 3539 nodes. By MS² spectral matching with the GNPS library spectra, 210 nodes (6%) returned with putative annotations. Singletons were removed for the sake of clarity.

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Fungal siderophore triacetylfusarinin C detected upon cocultivation of A. fumigatus and B. cenocepacia. (a) Molecular network of TAFC analogues connected via FBMN or MS2LDA. (b) Mass2Motif 559 corresponding to the TAFC substructure. (c) Boxplots of the relative abundance of TAFC and TAFC-iron complex in extracts derived from A. fumigatus monocultures and A. fumigatus + B. cenocepacia cocultures in the presence and absence of trimethoprim. Asterisks indicate the significant differences between the compared groups, as determined by the Kruskal–Wallis test with Dunn’s post-test (adjusted p-value <0.05). (d) MALDI-MSI-derived images for TAFC in mono- and cocultures. The visualization of the spatial distribution of TAFC is shown as a heatmap in false color (cyan, m/z 875.401, [M + Na]⁺). A white rectangle represents the measurement area used for data acquisition.

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Shift in chemical interactions between B. cenocepacia and A. fumigatus in the absence of the functional ham gene cluster. Boxplots of the relative abundance of (a) triacetylfusarinin C, (b) the ergosterol-related compound with m/z 496.415, (c) the unknown compound 1 with m/z 318.202, and (d) the unknown compound 2 with m/z 304.223 in the extracts derived from A. fumigatus monocultures and cocultures with either B. cenocepacia WT or ΔhamC strains in the presence and absence of sublethal trimethoprim.

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MAS-SILAC-guided annotation of fragin-related compounds. (a) Molecular network of features extracted using query language MassQL (see Methods), containing NL values of 76.027 Da (left) and 29.998 Da (right). Nodes with the same color represent fragin analogues with the same acyl chain. (b) Annotated MS² spectrum of unlabeled O ²-Me-fragin and labeled O ²-Me-fragin (using the incorporation of ¹³C₅,¹⁵N,D₂-valine). (c) EICs of fragin and O ²-Me-fragin detected in culture extracts of A. fumigatus supplemented with purified fragin and extracts of a media control without A. fumigatus, but supplemented with purified fragin. ND refers to not detected.

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MS² spectral analysis of labeled and unlabeled ergosterol-related compounds and incorporation of ¹³C₅,¹⁵N,D₂-valine. (a) Annotated MS² spectrum of unlabeled and labeled ergosterol-related compounds with m/z 496.415 and (b) annotated MS² spectrum of unlabeled and labeled ergosterol-related compounds with m/z 509.410.

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Phenotypic changes of A. fumigatus upon exposure to trimethoprim at a sublethal concentration. (a) Assessment of the antifungal activity of B. cenocepacia against A. fumigatus in the absence and presence of sublethal trimethoprim. Supplementing the culture media with trimethoprim at sublethal concentrations led to a significantly increased inhibition of A. fumigatus by B. cenocepacia. (b) Loss of antifungal activity of B. cenocepacia lacking the hamC gene against A. fumigatus A1160+. (c) Dysregulation of fungal pigment production in the presence of trimethoprim. Images of A. fumigatus A1160+ in the monoculture on solid agar supplemented with and without trimethoprim are shown. (d) Dysregulation of the fungal DHN-melanin biosynthesis pathway in the presence of trimethoprim. Biosynthetic pathway of DHN (dihydroxynaphthalene)-melanin is shown in the top panel. Images of the A. fumigatus B-5233 WT strain and six single-gene deletions (Δalb1, Δayg1, Δarp2, Δarp1, Δabr1, Δabr2) cultured on solid agar supplemented with (bottom row) and without (top row) trimethoprim are shown. T4HN: tetrahydroxynaphthalene and T3HN: triydroxynaphthalene.