Abstract
Mono- and multispecies microbial populations alter the chemistry of their surrounding environments during colony development thereby influencing multicellular behavior and interspecies interactions of neighboring microbes. Here we present a methodology that enables the creation of three-dimensional (3D) models of a microbial chemotype that can be correlated to the colony phenotype through multimodal imaging analysis. These models are generated by performing matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS) on serial cross-sections of microbial colonies grown on 8 mm deep agar, registering data sets of each serial section in MATLAB to create a model, and then superimposing the model with a photograph of the colonies themselves. As proof-of-principle, 3D models were used to visualize metabolic exchange during microbial interactions between Bacillus subtilis and Streptomyces coelicolor, as well as, Candida albicans and Pseudomonas aeruginosa. The resulting models were able to capture the depth profile of secreted metabolites within the agar medium and revealed properties of certain mass signals that were previously not observable using two-dimensional MALDI-TOF IMS. Most significantly, the 3D models were capable of mapping previously unobserved chemical distributions within the array of sub-surface hyphae of C. albicans and how this chemistry is altered by the presence of P. aeruginosa, an opportunistic pathogen known to alter virulence of C. albicans. It was determined that the presence of C. albicans triggered increased rhamnolipid production by P. aeruginosa, which in turn was capable of inhibiting embedded hyphal growth produced beneath the C. albicans colony at ambient temperature.
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Acknowledgements
This work was supported by the Keck Foundation and National Institute of General Medical Sciences Grant NIH GM086283, GM094802 (PCD), NIH Molecular Biophysics Training Grant NIH GM08326 (JDW), and European Union 7th Framework Programme grant 305259. We thank Suzanne Noble, University of California San Francisco, for kindly providing us with C. albicans ySN250.
Author contributions
JDW, TA and PCD designed the research plan; JDW, VVP and C-CH performed the experiments; TA performed 3D modeling of the MALDI-TOF IMS data; C-CH, WJM and BMD performed NMR analysis; JDW, VVP, C-CH, WJM and PCD analyzed MS data; JDW, VVP, C-CH, TA and PCD wrote the paper.
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Watrous, J., Phelan, V., Hsu, CC. et al. Microbial metabolic exchange in 3D. ISME J 7, 770–780 (2013). https://doi.org/10.1038/ismej.2012.155
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DOI: https://doi.org/10.1038/ismej.2012.155
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