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Imaging Mass Spectrometry of Fungal – Bacterial Interplay

Periodic Reporting for period 1 - FUNBIT (Imaging Mass Spectrometry of Fungal – Bacterial Interplay)

Reporting period: 2016-03-01 to 2018-02-28

"Microorganisms interact with each other by releasing small chemical compounds, referred here as ""natural products"", that we can exploit to treat diseases and fight agricultural pests.

The research on natural products have a huge impact for society, as highlighted by the European Commission in the Horizon 2020 program: on one hand, the challenge of antibiotics resistance is at the top of the list of priorities for the Scientific Panel for Health; on the other hand, the safe and sustainable treatment of agricultural pests is crucial to ensure Food Security and Sustainable use of the European natural resources.

The search for new secondary metabolites is a never-ending quest. Traditional methods based on bioassay-guided fractionation still play an important role, but they are very demanding in time and resources, and usually lead to the re-discovery of known compounds. Nowadays, genome mining (the use of information in the genome to understand the metabolism), has revealed that many microorganisms have a huge hidden biosynthetic potential: the number of genes with the ability to encode the production of natural products surpasses by far the number of discovered compounds. Known natural products are only the tip of the iceberg.

One of the reason explaining why most natural products remain undiscovered is that the genetic machinery responsible for their production in microorganisms, specially in fungi, usually remain silent under standard in-lab microbial cultivation protocols. Mimicking the complex microbial communities in the laboratory can stimulate the microbes to reveal the chemicals involved in their communication and competition.

Since co-cultures of fungi and bacteria can provide a promising source of unexplored natural products, it is surprising how they have been overlooked in the scientific literature. The reason may be that several challenges hinder the discovery of natural products from co-cultures: 1) secondary metabolites may be produced in very low quantities in a very localized area; 2) they can be very difficult to extract from the solid medium where the fungi and bacteria are grown; and 3) once a potentially new secondary metabolite is found, if it is outside of its native environment, its biological meaning may be difficult to trace.

In brief, detecting secondary metabolites involved in interactions between organisms often requires the participants to be caught in the act. A great candidate to study microbial interactions is the emerging technique Imaging Mass Spectrometry (Imaging MS), that allows the in situ and real time monitoring of chemical compounds directly on biological samples such as petri dishes and tissues of animals, plants and fungi, enabling the monitoring of chemical compounds while the interacting organisms are in contact. Thus, FUNBIT aims to enhance the rate of secondary natural product discovery guided by Imaging MS.
"FUNBIT has been involved in several multidisciplinary projects to study microbial interactions. Since 2016, FUNBIT has collaborated with four finished projects that have been published in high impact journals:

1. The group of Christine Beemelmans at HKI investigates fungus gardens in termites mounds to discover the chemical weapons used by Pseudoxylaria to fight against other fungi. Pseudoxylaria seemed to exudate more droplets (a process called ""guttation"") in close proximity to its competitor. The droplets contained several compounds, in high abundance, whose molecular mass had not been reported before. The localization of these compounds on the droplets was also visualized by MALDI - Imaging MS.
Guo, H., Kreuzenbeck, N. B., Otani, S., Garcia-Altares, M., Dahse, H. M., Weigel, C., ... & Beemelmanns, C. (2016). Pseudoxylallemycins A–F, Cyclic Tetrapeptides with Rare Allenyl Modifications Isolated from Pseudoxylaria sp. X802: A Competitor of Fungus-Growing Termite Cultivars. Organic Letters.

2. The stereaceous mushroom BY1 is able to produce polyenes yellow pigments to discourage insect larvae from feeding on it. The group of Prof. Dirk Hoffmeister at HKI unravelled the unusual biosynthesis of these compounds and discovered that a new enzyme (a polyketide synthase) catalyzes all steps, including the unprecedented shift of multiple double bonds, to assemble this anti-larval natural product. We now confirmed these findings by MALDI-mass spectrometry imaging and verified polyene accumulation on the wound. Only traces of the compounds were detected on the healthy area, where the mycelium shows only light yellow pigmentation.
Brandt, P., García‐Altares, M., Nett, M., Hertweck, C., & Hoffmeister, D. (2017). Induced Chemical Defense of a Mushroom by a Double‐Bond‐Shifting Polyene Synthase. Angewandte Chemie International Edition, 56(21), 5937-5941.

3. The group of Severin Sasso and Maria Mittag at Friedrich-Schiller-Universität Jena investigates interplay between microalgae, key contributors to carbon fixation on Earth, and other microorganisms in freshwater and marine ecosystems, using the green unicellular alga Chlamydomonas reinhardtii as a model organism. In a recent study, we showed that the bacterium Pseudomonas protegens strongly inhibits the growth and alters the morphology of Chlamydomonas reinhardtii. By Imaging MS and LC-MS analysis, we discovered that the natural products orfamides are produced by Pseudomonas protegens in the algal–bacterial interaction. Orfamide was discovered to alter cytosolic calcium homeostasis and inmobilization of the algae, which represent a novel biological activity for cyclic lipopeptides.
Aiyar, P., Schaeme, D., García-Altares, M., Flores, D.C. Dathe, H., Hertweck, C., Sasso, S. and Mittag, M., 2017. Antagonistic bacteria disrupt calcium homeostasis and immobilize algal cells. Nature communications, 8(1), p.1756.

4. The group of Pierre Stallforth at HKI focus on the interactions between the eukaryotic soil amoeba Dictyostelium discoideum and various soil bacteria to identify bacterial secondary metabolites that kill the amoebal predator. A recent study in which FUNBIT collaborated was accepted in Proc. Natl. Acad. Sci. describing a new cyclic lipopeptide produced by an inedible strain of Pseudomonas with amoebicidal activities.
Arp J, Götze S, Mukherji R, Mattern DJ, Garcia-Alatres M, Klapper M, Brock DA, Brakhage AA, Strassmann JE, Queller DC, Bardl B, Willing K, Peschel G, Stallforth P (2018) Synergistic activity of co-secreted natural products from amoebae-associated bacteria Proc. Natl. Acad. Sci. USA [Accepted]"
The scope of the project has been extended beyond fungal-bacterial interactions due to more collaboration requests and increasing potential of the technique after optimisation of the sample preparation and measuring protocols. The executed projects include other interactions, such as bacteria-microalgae, fungi-fungi, fungi-bacteria, amoeba-bacteria, marine sponges-bacteria, etc. FUNBIT has collaborated in two projects that lead to the discovery of a new family of natural products (pseudoxylallemycins), a previously uncharacterized type of polyketide synthases , a novel cyclic lipopeptide and a new biological mechanism associated to a known cyclic lipopeptide (orfamides) in an inter-kingdom ecological interaction.
MALDI-Imaging MS of Pseudoxylaria exudating droplets containing pseudoxylallemycins
MALDI-Imaging MS showing polyene accumulation on injured fungi