Cyanobacteria are photosynthetic bacteria found in almost every terrestrial and aquatic habitat and that are known to be rich sources of potentially valuable secondary metabolites. Such compounds, often referred to as natural products, possess relevant properties for industrial biotechnology, cosmeceuticals and pharmaceuticals. Secondary metabolites are encoded by groups of genes called biosynthetic gene clusters (BGCs). However, an estimated 80-90% of BGCs are not usually expressed under standard laboratory conditions, an artificial environment that presents little hardship or competition. As a consequence, cultivation of individual microorganisms in a laboratory setting may only reveal a small fraction of the metabolic space encountered in nature. Investigating microbial interactions from an ecological perspective is therefore a particularly fruitful approach to unveil both new chemistry and bioactivity.

Cyanobacteria and protozoa have been known for a long time to co-exist in aquatic environments however the interactions between these organisms are poorly understood. The protozoa amoebae are unicellular organisms that are ubiquitously distributed in the environment and are recognized as microbe grazers. Even so, several studies reported that a number of cyanobacterial strains showed different susceptibilities to amoeba grazing: whilst some cyanobacteria can escape predation after being ingested, others are consumed by these eukaryotic organisms. Some studies showed that cyanobacterial morphology or surface proteins can affect amoebal grazing. In other studies, it has been hypothesized that the different susceptibilities of cyanobacteria towards amoebae grazing are more likely be the result of the release of yet unknown cyanobacterial natural products, i.e. supporting a chemically-mediated defense against free-living amoeba grazers. The well-known cyanotoxin microcystin was shown not to be an effective defense mechanism against protozoa predation, since these eukaryotic organisms readily feed on microcystin-producing cyanobacterial strains and pure mycrocystin had no effect on amoeba. Since interactions between cyanobacteria and amoebae are known to occur, but their mediators are unknown, the goal of this study is to discover new chemical interactions between these organisms that are mediated by small molecules, which we aim to characterize. For that purpose, several strains of cyanobacteria will be screened against grazing of different strains of amoebae, using different methodologies such as microscopy and amoebal plaque assays. The strains showing resistance to grazing will be selected for small-scale growth in the presence and absence of amoebae for further organic extraction of the culture medium and cyanobacterial cells. The cyanobacterial crude extracts showing bioactivity against amoebae will be further cultivated in large-scale for isolation and structure elucidation of the compounds responsible for the observed activity, using different chromatographic techniques.

Ultimately, the bioactivity profiling of the pure metabolites that mediate the interaction between cyanobacteria and amoebae will be examined in ecologically-and pharmacologically-relevant assays. It is anticipated that in the time provided to execute this project, one metabolite (or family) conferring resistance towards amoebal grazing will be identified and characterized in this project. This will generate new knowledge regarding the chemical ecology of cyanobacteria and aquatic ecology in general and may promoting a new methodology to find new NPs. In this project the potential activity of these newly-found compounds will be tested against other protozoans, namely parasites responsible for important animal and human diseases such as Leishmania and Plasmodium, a collaboration recently initiated with the head of the parasites disease research group at i3s. Therefore, as there are only a few drugs available for such diseases, the proposed research may also contribute to tackle this important pharmacological issue.

To sum up, CYAmoeba will exploit a unique biological resource (LEGEcc) to focus on the underexplored interactions between cyanobacteria and amoeba. The ecological and biochemical strategies associated with these interactions will be illuminated and will guide discovery of novel NPs and associated biological activities.