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The great biotechnological potential of a marine polychaete: An alliance between toxin and natural fluorescence

Ana P. Rodrigo1, 2*, Ana R. Lopes1, Pedro V. Baptista1, Maria H. Costa2, Alexandra R. Fernandes1 and Pedro M. Costa1
  • 1 Unidade de Ciências Biomoleculares Aplicadas, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Portugal
  • 2 Centro de Ciências do Mar e do Ambiente (IPLeiria), Portugal

Marine Biotechnology has been gaining focus, being, in fact, considered one of pillars of the EU’s Blue Growth Agenda for sustainable exploitation of marine resources (Lillebø et al., 2017). Among the bioproducts that can be extracted from marine organisms, toxins, in particular, are especially important due to their ability to bind to specific receptors and interfere with particular pathways, which makes them appealing for a vast range of applications, from eco-friendly pesticides to new antibiotics and painkillers (Hornbeak and Auerbach, 2017; Molinski et al., 2009). Accordingly, there is an increasing number of poisons, venoms and toxins identified from marine invertebrates (von Reumont et al., 2014). However, keeping in mind that the number of novel bioproducts is most likely proportional to marine biodiversity, we may be facing the sheer tip of the iceberg. Class Polychaeta is no exception, albeit the still reduced number of species reported for their bioproducts (Coutinho et al., 2018). Our case study, the marine polychaete Eulalia viridis, an inhabitant of the Portuguese rocky shore, is a good example. The species has been found to provide several potential bioproducts. Among these, it is highlighted a fluorescent proteinaceous toxin found in the skin mucus, whose photochemical and toxicological characterisation is the main goal of the present work. Worms were collected, reared in the laboratory and the mucous secretions harvested by mechanic stimulation in a non-invasive way. Mucus samples were processed as crude or subjected to dialysis by ultrafiltration to purify the +3 kDa fraction and exchange mucins and seawater with biotechnology-relevant assay buffers: sterilised seawater (surrogate of the natural medium), PBS (compatible with bioassays) and Tris-HCl (suitable for biochemical tests). The purification of the extracts is important because venoms and poisons are complex mixtures of toxins, enzymes, small peptides and salts (Casewell et al., 2013). The toxicity of purified extracts was primarily assessed by the Microtox assay. The analysis of the toxin’s inherent fluorescence was done by spectroscopy on crude and purified mucus. Following preliminary investigations, which showed a reduction of fluorescence intensity with time, we hypothesised that oxidation was the cause. The samples, crude and purified into different buffers were thus subjected to treatments with either reducing (DTT, 10%) or oxidative (H2O2, 2%) agents. To assess cytotoxicity purified protein in PBS was tested in cancer cells cultures (MCF7). Viability assays were determined using MTS assay. The Microtox test (which uses the marine bacterium Vibrio fischeri) revealed that purified toxin-bearing extracts were twice more potent in Tris-HCl and PBS buffers, comparatively to seawater (Figure 1). It also showed that DTT greatly enhanced mortality. The Microtox assay has, thus, proven to be a good toxicological screening test, as seen for other polychaete (Iori et al., 2014). The reduced samples, regardless of medium, were far more fluorescent than the oxidised, showing that exposure to oxygen is the likely culprit (Figure 2A). In fact, in denaturing conditions, as in a SDS-PAGE gel, which involves reducing agents, it can be seen that the protein retains its fluorescence under UV light, until oxidation occurs (Figure 2B). Loss of fluorescence intensity via oxidation occurs in both crude and purified samples, with differences caused by the usage of different media (Figure 3). The reducing agent originated a new emission maximum at 510nm, which is far more obvious in purified samples than in crude mucus, where it could only be observed in samples diluted in Tris-HCl. This new peak shifts the emission colour from violet blue to strong blue-greenish. This is an odd coloration for marine organisms, nevertheless reported before in Chaetopterus sp., a tubicolous polychaete with a fluorescent mucus. Interestingly, Chaetopterus mucus’ fluorescence is differentially modulated by distinct oxidising agents, as well as other factors like temperature (Deheyn et al., 2013). Eulalia most likely secrets the substance in a reduced form. It is thus possible that the worm appears fluorescent to other marine organisms. The differences between crude and purified extracts are likely due to the fact that the former are complex mixtures that can even be contaminated with pigments whose function is to absorb UV light, such as the worms conspicuous green pigments (refer to Rodrigo et al., 2015 and references therein). It was also noticed, in both crude and purified samples, that PBS enhanced emission at 400nm. Cell viability assays showed that MCF7 tumour linage’s IC50 was very low ((IC50 0.154 ± 4 µM). In general, cytoxicity demonstrated similarities to what has been seen in neuronal cells exposed to conotoxins (Fang et al., 2011). The cytotoxicity of purified mucus proteins allied with the natural fluorescence under redox conditions reveals the great biotechnological potential. This combination has in fact been searched for using genomic manipulation (Kuzmenkov et al., 2016). Altogether, the substance harvested from Eulalia represents a novel substance that combines several characteristics with great biotechnological potential. It combines the likely ability to bind to a specific class of ligands, as a toxin, and immediately act as a fluorescent reporter that can be shut on/off by modulating its redox status. Also of great importance is its higher toxicity towards a known MCF7 human adenocarcinoma breast cancer cell line, which can bring bioprospecting for anti-tumour agents and reporters to a next-generation.

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Acknowledgements

The Portuguese Foundation for Science and Technology (FCT) is acknowledged for the funding of the research project GreenTech (PTDC/MAR-BIO/0113/2014) and MARE (UID/MAR/04292/2013). Unidade de Ciências Biomoleculares Aplicadas-UCIBIO is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The grants SFRH/BD/109462/2015 to A.P.R. and IF/00265/2015 to P.M.C. (from FCT) are acknowledged as well.

References

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Keywords: Bioprospecting, Cytotoxicity, Fluorospectroscopy, Phyllodocida, protein

Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018.

Presentation Type: Oral Presentation

Topic: Blue Biotech

Citation: Rodrigo AP, Lopes AR, Baptista PV, Costa MH, Fernandes AR and Costa PM (2019). The great biotechnological potential of a marine polychaete: An alliance between toxin and natural fluorescence. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00015

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Received: 29 Apr 2018; Published Online: 07 Jan 2019.

* Correspondence: Miss. Ana P Rodrigo, Unidade de Ciências Biomoleculares Aplicadas, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Caparica, Portugal, a.rodrigo@campus.fct.unl.pt