Mass Spectrometry Imaging for fungal interaction analysis: Classic versus Imprinting Methods

Abstract

Fungi can produce many bioactive metabolites, which are enhanced when challenged in co-culture competition. For a better evaluation of these metabolites, Mass Spectrometry Imaging (MSI) can be used to provide complementary information about the metabolite spatial localization. However, some adaptations are required on available methodologies in MSI for applications in microorganisms, particularly on sample preparation, due to the characteristics of each type of cell that has to be analyzed. The imprinting method has been shown to be a robust method when applied to sample preparation, but to our knowledge it has never been tested for microbial MALDI-MSI. Herein we applied both Classic and Imprinting MALDI-MSI to compare and analyze metabolites produced by Aspergillus terreus (ATCC® 20542TM) and Pleurotus pulmonarius fungi. For the classic method, the fungi were inoculated for 8 days with PDA medium in a MALDI glass slide. For the imprinting method, fungi were also inoculated for 8 days in a MALDI glass slide and then transferred to a filter paper by manual pressure using a homemade apparatus. Samples were then dehydrated and submitted to HCCA matrix application by sublimation. The chemical images were acquired by MALDI-MSI, and the metabolites were identified by UHPLC-ESI-MS/MS. Twelve ions were detected by MALDI-MSI, using classic (m/z 210.54, 276.99, 307.45, 321.04, 329.70, 346.12, 351.12, 462.41 and 484.02) and imprinting (m/z 313.64, 379.66 and 404.36) methods. Some ions presented a higher intensity in the interaction zone between fungi areas, especially the ions m/z 329.70, 351.12 and 484.02. These ions may be related to metabolites involved in communication between microorganisms, because these fungi formed a mutualistic interaction. All ions were investigated by UHPLC-ESI-MS/MS, and two were identified: adenosine monophosphate (C10H14N5O7P, m/z 346.12, [M-H]-) visualized in the Classic Method, and rubrophen (C22H20O6, m/z 379.66, [M-H]-) visualized in the Imprinting Method. The metabolites from microorganisms are rarely reported in MS/MS databases, which explains the difficulty in the identification of these compounds. Our MSI analysis using the classic method provided a higher number of detected ions. However, both classic and imprinting methods resulted in a complementary information, leading to the detection of ions that were not previously observed on the classic approach. Despite of the challenges encountered on the sample preparation and metabolite identification, using both classic and imprinting MALDI-MSI for bioprospection of fungi metabolites is a promising approach on the analytical field of mass spectrometry which can be later used in biotechnological applications.

Enhancing chemical and biological diversity by co-cultivation

“…The advantage of MALDI Imaging for microbiology is the fact that several metabolites can be simultaneously visualized according to their spatial distribution, enabling the comparison of chemical signatures and the correlation with biological phenotypes of interest ( Stasulli and Shank, 2016 ; Müller et al, 2022 ). In co-culture, IMS enables the observation of previously undetected coculture-metabolites and the microbial strain that produced these compounds ( Watrous and Dorrestein, 2011 ; Bleich et al, 2015 ; Chen et al, 2018 ; Teixeira et al, 2022 ). Moreover, the concepts of molecular cluster families integrated into the ideas of MS/MS networking allowed the annotation of unknown m/z peaks, facilitating the dereplication of analogs produced by the same BGCs ( Moree et al, 2012 ; Watrous et al, 2012 ; Moree et al, 2013 ; Stasulli and Shank, 2016 ; Martin et al, 2019 ; Wang et al, 2022 ).…”