- Open Access
Ultraviolet (IUV) and mass spectrometry (IMS) imaging for the deconvolution of microbial interactions
© The Author(s). 2018
- Published: 20 November 2018
Spatial localization of natural products or proteins during microbial interactions can help to identify new antimicrobials both as offensive or defensive agents. Visible spatial interactions have been used for decades to enhance Drug Discovery processes both in industry and academia.
Herein we describe an automated micro-extraction methodology, that coupled with the previously described HPLC-Studio 2.0 software and the new development, the MASS-Studio 1.0 software, can combine multiple chemical analyses to generate ultraviolet (UV) and mass spectrometry (MS) images from traditional affordable analytical equipment. As a proof of concept, we applied this methodology on two microbial antagonisms observed among co-habitant endophytes isolated from endemic plants of arid areas of the south of Europe.
The use of UV and MS images highlighted interacting naturals products and allowed clear identification of induced molecules of interest not produced by the strains when cultured individually.
- Microbial interactions
Drug Discovery for new chemical entities and innovative compound design relies on natural products for more than half of the drugs in development . So far more than 42% of known bioactive compounds have been described as produced by filamenting fungi and many of these molecules with pharmacological applications were developed for clinical uses, especially as antibiotics and antifungals among other applications [2, 3]. However, a general perception is that the emerging rates of discovery of new molecules, especially new antibiotics, are decreasing after half a century of continued research on fungal diversity and axenic fermentation-based processes .
Fungal genome mining has revealed the high number of gene clusters involved in the biosynthesis of fungal secondary metabolites (SMs) that are not produced in strains cultivated axenically in laboratory conditions [5, 6]. Recently, several approaches have been applied to foster the expression of these unexpressed pathways and to promote their biosynthesis. These strategies have included: the manipulation of medium and growth conditions in miniaturized nutritional arrays , the application of transformation techniques for the generation of gene knockouts, the exchange of native gene promoters with constitutive or inducible promoters or the overexpression of transcription factors , or the co-cultivation of more than one microorganism in constant interaction .
Traditional screening processes to discover new bioactive molecules, involve culturing a single microbial strain, however the use of co-cultures presents new opportunities for the activation of cryptic biosynthetic pathways. Microorganisms can present an antagonistic reaction in presence of other microorganisms that promotes changes in their morphology and the production of SMs, enzymes and other compounds in the interaction zone .
Co-culturing has proved to be an effective tool to simulate the physiological conditions that occur during microbial interaction in their natural environment and may have an enormous potential for the discovery of new molecules with therapeutic approaches [11–13].
Recent detection techniques try to evaluate natural products by Image Mass Spectrometry (IMS) [14, 15] by remarking its high level of suitability for analyzing microbial interactions and detecting the activation of cryptic pathways [16, 17]. Unfortunately, most of these techniques rely on the use of very expensive or innovative ionization heads in the Mass Spectrometer (MS) (ie MALDI, or nano-DESI respectively). Most of the common MS for natural products are electrospray units that cover mass ranges from 150 to 1500 Da, more suitable for natural products extracts. Recently, we have developed in our lab the MASS-Studio 1.0 software tool for high-throughput analysis of batches of samples analyzed by LC-ESI-MS equipment. Herein we decided to combine miniaturized chemical extractions with HPLC-Studio 2.0 [18, 19] and MASS-Studio 1.0  utilities to generate ultraviolet and mass images from LC-ESI-MS analyses.
Endophytic strains were isolated as described previously by Gonzalez-Menendez et al. (2016) . Strains interaction were performed by co-culturing on malt agar (malt extract Difco™ 20 g, agar 20 g and 1000 mL deionized H2O) for 14 days at 22 °C and 70% of relative humidity, and said agar of the positive antagonist was separated into 80 portions corresponding to 80 microplate wells. All co-culture portions were extracted with acetone, shaking at 220 rpm for 1 h and the samples were dried in a Genevac HT-8. Finally, the dried samples were suspended in 500 μl of 20% DMSO. The samples were analyzed by UPLC-UV and by low resolution mass spectrometry (LR-MS) in the range of positive m/z for each extract. Mass ion detection was performed in a ramp from 150 m/z to 1500 m/z in positive and negative modes. MASS-Studio 1.0 software was used for generating the mass spectrometry imaging for each co-culture. Comparison with proprietary database of more than 950 known microbial standards was performed by low resolution (LC-LRMS) using the same raw data that generated the images.
Once chemical evaluation had been performed raw data corresponding to each individual analysis was recorded in ‘cvs’ files by the equipment software and HPLC-Studio 2.0 [18, 19] and MASS-Studio 1.0  were used to combine all individual analyses from ultraviolet or mass spectrometry detection respectively. Typical runs in these studies compare components detected in the samples and identify if they correspond to the same metabolite or not, by bucketing in the time dimension in the case of the HPLC-Studio 2.0 or in the mass direction in the MASS-Studio 1.0 software.
Traditional analytical methods have allowed the detection of changes in the metabolite profiles that vary depending on the interacting fungi [21, 22]. Different co-culturing techniques have been developed for this purpose including liquid and solid media, but all approaches consist on culturing two or more microorganisms in a single confined environment to facilitate interactions and induce further chemical diversity [23, 24].
An automated method based on image mass spectrometry (IMS) has been used for evaluating the presence of different secondary metabolites when a clear antagonistic effect was observed in a fungal co-culture. This approach aims to analyze the microbial interactions in terms of the natural products generated (UV and MS) to evaluate the effect of antagonism and detect molecules that could be produced as a consequence, among others, of communication, attack or defense between both microorganisms. As, some of these molecules may not be produced when these microorganisms are grown axenically, there is a high probability that some of them may show biological activities with possible therapeutic purposes.
In the case of co-culturing Hypoxylon mediterraneum with Bacillus sp. (Fig. 5d) twelve known compounds with antimicrobial activities were dereplicated as possible responsible of part of the interactions observed. These compounds belonged to three main families produced by Bacillus sp.: surfactins (Fig. 5D, o and D, q), iturins (Fig. 5D, p and D, r) and mycosubtilins (Fig. 5D, s and D, t) [21, 22]. Other accumulated ions were found to be produced by H. mediterraneum in response to this antimicrobial attack, as (m/z) 263, 337, 429, 443, 520 or 709, but were not observed when the strain was grown axenically. This agreed with the selective production of several secondary metabolites as a signaling response or a defense mechanism.
A combination of compound management techniques, automated micro-extractions and the HPLC-Studio 2.0 and MASS-Studio 1.0 software tools was used for the development of Ultra Violet and Mass Spectrometry Imaging from microbial interactions. Spatial localization of secondary metabolites resulted in an advanced tool for the evaluation of the antagonistic effects among strains in ecological communities. Mass Spectrometry Imaging (IMS) resulted in a more informative analytical technique than Ultraviolet Imaging (IUV) for the evaluation of these microbial interactions. Moreover, this methodology, when combined with evaluation of antimicrobial properties, can speed up the discovery of bioactive natural products and signaling molecules.
The School of Master Degrees of the University of Granada was involved with this work as part of their PhD. Program: ‘New Therapeutic Targets: Discovery and Development of New Antibiotics’.
This work was supported by Fundación MEDINA. Publication cost of this article was funded by Fundación MEDINA.
About this supplement
This article has been published as part of BMC Systems Biology Volume 12 Supplement 5, 2018: Selected articles from the 5th International Work-Conference on Bioinformatics and Biomedical Engineering: systems biology. The full contents of the supplement are available online at https://bmcsystbiol.biomedcentral.com/articles/supplements/volume-12-supplement-5.
JRT and VGM designed the study. VGM and RS performed the microbiology including images. VGM and FM performed the sample preparation including images. JRT and GM developed the software algorithm, software application and schemas. JM performed the low-resolution MS analyses and high-resolution MS dereplications. JRM processed the data and generated the MS images. JRT wrote the manuscript. VGM and OG reviewed critically the manuscript. All authors contributed and approved the final manuscript.
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