Metabolome-Informed Proteome Imaging of Lignocellulose Decomposition by a Naturally Evolved Fungal Garden Microbial Consortium
Marija Velickovic1, Ruonan Wu1, Margaret W. Thairu2, Yuqian Gao1, Dusan Velickovic1, Carrie D. Nicora1, Jennifer E. Kyle1, Nathalie Munoz1, Chaevien S. Clendinen1, Aivett Bibao1, Priscila M. Lalli1, Kevin Zemaitis1, Rosalie K. Chu1, Daniel Orton1, Sarai Williams1, Ying Zhu1, Rui Zhao1, Matthew E. Monroe1, Ronald J. Moore1, Bobbie-Jo M. Webb-Robertson1, Lisa M. Bramer1, Cameron R. Currie2, Paul D. Piehowski1, and Kristin E. Burnum-Johnson1* (email@example.com)
1Pacific Northwest National Laboratory; and 2University of Wisconsin–Madison
The objective of this Early Career Research project is to gain transformative molecular-level insights into microbial lignocellulose deconstruction through comprehensive and informative review of underlying biological pathway data yielded by the integration of spatiotemporal multiomic measurements (i.e., proteomics, metabolomics, and lipidomics). One of this project’s goals is to uncover the mechanisms that drive cooperative fungal-bacterial interactions that result in the degradation of lignocellulosic plant material in the leafcutter ant fungal garden ecosystem. The project’s approach will enrich the current knowledge base needed for a predictive systems-level understanding of the fungal-bacterial metabolic and signaling interactions that occur during cellulose deconstruction in an efficient, natural ecosystem.
The leafcutter ant fungal garden is known as a natural model system for efficient plant matter degradation. The degradation processes are largely mediated by the symbiotic fungal and bacterial members within the complex microbial consortium. These symbiotic microbes with unique metabolic capabilities, however, are heterogeneously spatially organized in the sample. Previous mass spectrometry (MS) studies profiled molecules from bulk fungal garden samples; thereby, averaging the biological processes across the ecosystem and masking their spatial localization, biological origin, and molecular dynamics (Khadempour et al. 2021). To overcome this limitation, researchers, hereby, performed microscale imaging across 12 µm-thick fungal garden serial sections by applying a metabolome-informed proteome imaging (MIPI) approach. This approach combines two spatial multiomics MS modalities that enable obtaining comprehensive molecular characterization across and through the fungal garden. Matrix-assisted laser desorption/ionization (MALDI) imaging profiled metabolites with a spatial resolution of 50 µm and correlated morphologically unique features with metabolome profiles of interest (i.e., lignocellulose degradation). The identified regions of interest (ROIs) were selected for subsequent microdissection and microscale proteomic imaging using microPOTS (microdroplet processing in one pot for trace samples) with an integrated metaproteomic approach to detect metabolic activities and identify microbial community members.
Untargeted MALDI-Fourier-transform ion cyclotron resonance–MS imaging analysis revealed heterogeneous spatial distribution of various molecular features across the fungal garden sections. Researchers leveraged the METASPACE annotation platform to search against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and tentatively annotate 650 unique metabolites, which colocalized metabolomic signatures with distinct microscopic features. The MALDI images mapped the presence of phenylpropanoids, benzaldehydes, flavonoids, plant hormones, Krebs cycle compounds, sugars, amino acids, and other molecules that were produced by the complex community in the fungal garden. Differential relative abundance and accumulation of low molecular weight lignin products (coniferyl alcohol, coniferyl aldehyde, sinapoyl aldehyde, cinnamate, ferulate, caffeate, vanillin, etc.) were observed indicating specific spatial patterns. Another observed spatial pattern colocalized with a unique ant wing-like feature that was characterized mainly by primary metabolites, such as soluble sugars, amino acids, and fatty acids. Informed by the metabolomic specific features, the team selected the wing and three additional ROIs characterized as lignocellulose degradation hotspots for subsequent microPOTS metaproteomics analyses. Selected ROIs and their biological replicates were dissected using a laser capture microdissection system and collected in individual wells of the microPOTS chip. Peptides resulting from on-chip sample preparation were analyzed by liquid cryotomography (LC)-MS/MS analyses. For the metaproteomic analyses, a reference database was first curated from 50 million proteins of known members in the consortium that were grouped into >24 million clusters based on sequence similarity to annotate the high-resolution tandem MS spectra with stringent matching criteria. A total of 7,392 non-conservative and taxon-specific peptides that mapped to 2,239 unique protein clusters were detected, unveiling a complex community with relatively high representation of arthropod peptides (5,178) observed only in the wing ROI, while fungal peptides (1,825) and comparatively low abundant plant (552) and bacterial (47) peptides were localized in the other three ROIs mapped as lignocellulose degradation zones. Metaproteomics data revealed the presence of a fungal ligninolytic auxiliary enzyme and several fungal carbohydrate-active enzymes such as hemicellulases, cellulases, pectinases, and amylases in the lignocellulose degradation hotspots ROIs. The metabolic functions detected at the microscale provide more direct evidence that fungi cultivated by leafcutter ants such as Leucoagaricus gongylophorus degrade plant cell walls in the leafcutter ant garden ecosystem.
Leveraging MIPI capability to spatially profile a plethora of metabolites and peptides provided some molecular insights and understanding of species-specific activities in this multimember heterogenous ecosystem. Integration of MIPI data unraveled some of the processes in this complex ecosystem by reconstructing crucial parts of the lignocellulose decomposition pathways in distinct microscopic ROIs. MIPI enzyme-metabolites integration showed a strong correlation comparing abundance and spatial localization between two omics modalities. Mechanistic understanding of this symbiotic system can aid in the biological production of biofuel precursors and bioproducts from plant biomass. This novel MS micron-scale multiomic workflow can be applied to other complex and heterogenous biological systems to enhance the understanding of community member interactions and dynamics.
Khadempour, L., et al. 2021. “From Plants to Ants: Fungal Modification of Leaf Lipids for Nutrition and Communication in the Leaf-Cutter Ant Fungal Garden Ecosystem,” mSystems 6(2). DOI:10.1128/mSystems.01307-20.
Pacific Northwest National Laboratory (PNNL) is a multiprogram national laboratory operated by Battelle for the U.S. Department of Energy (DOE) under Contract DE-AC05-76RLO 1830. This program is supported by the DOE, Biological and Environmental Research (BER) Program under the Early Career Award Program. A part of this work was performed in the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility at PNNL.