Fabienne Guérard

Assessment of the spatial variability of phenolic contents and associated bioactivities in the invasive alga Sargassum muticum sampled along its European range from Norway to Portugal

Sargassum muticum, an invasive brown macroalga presently distributed along European Atlantic coasts from southern Portugal to the south coast of Norway, was studied on a large geographical scale for its production of phenolic compounds with potential industrial applications and their chemical and biological activities. S. muticum can produce high biomass in Europe, which could be exploited to supply such compounds. S. muticum was collected in Portugal, Spain, France, Ireland and Norway (three sites/country) to examine the effect of the latitudinal cline and related environmental factors. Assays focused particularly on polyphenols and their activities. Crude acetone–water extracts were purified using solid phase extraction (SPE) and antioxidant and antimicrobial activities of crude extracts and semi-purified fractions measured. Total phenolic content was assessed by colorimetric Folin–Ciocalteu assay and reactive oxygen species activities by 2,2-diphenyl-1-picrylhydrazyl, reducing power, β-carotene bleaching method and xanthine oxidase assay. Antibacterial activities were tested on terrestrial and marine strains to evaluate potential use in biomedical and aquaculture fields. Purified active phlorotannins, isolated by SPE, were identified using NMR. Phenolic contents differ clearly among countries and among sites within countries. Quality did not change between countries, however, although there were some slight differences in phlorethol type. Additionally, some fractions, especially from the extreme north and south, were very active. We discuss this in relation to environmental conditions and the interest of these compounds. S. muticum represents a potential natural source of bioactive compounds and its collection could offer an interesting opportunity for the future management of this species in Europe.

Phlorotannins in sargassaceae species from brittany (France): Interesting molecules for ecophysiological and valorisation purposes

Phlorotannins are metabolites synthesised by brown algae to protect against environmental stresses. Only a few studies presented structural elucidation of native molecules extracted from macroalgae, contrary to previous published studies, which characterised acetylated phlorotannins. The present work introduces quantitative and qualitative studies to characterise phlorotannins from the eight species of Sargassaceae settled in Brittany, using a colorimetric method used for quantification of total phenolic content (TPC), and one- and two-dimensional nuclear magnetic resonance (NMR) (1H, heteronuclear multiple bond correlation) together with in vivo NMR (high-resolution magic-angle spinning (HR-MAS) NMR) analyses, with the aim to elucidate structural and fingerprint of phlorotannins signals. Halidrys siliquosa, which settles in low tide rock pools, exhibited high TPC (17.77±1.68% dry weight (DW)) while the other species showed lower TPC (ranged from 0.85±0.11% DW for Cystoseira baccata growing in low tide rock pools to 5.53±0.63% DW in Cystoseira humilis growing in high tide rock pools). No relation between TPC and the position of the algae along the shore could be highlighted. Fingerprints using NMR HR-MAS were useful to compare the eight species in terms of phenolic content and in terms of phenolic signals as the species from Brittany produced different phlorotannins. Our study demonstrated that Cystoseira tamariscifolia produced the monomer (phloroglucinol), C. humilis, phloroglucinol and a phlorethol, C. baccata and Cystoseira nodicaulis produced many compounds, i.e. traces of phloroglucinol together with fucols, phlorethols, and fucophlorethols and finally fuhalols were identified in Bifurcaria bifurcata and H. siliquosa. The putative ecophysiological roles of these phlorotannins from Sargassaceae are discussed, together with their potential bioactivities.

Sunscreen, antioxidant, and bactericide capacities of phlorotannins from the brown macroalga Halidrys siliquosa

The present study focused on a brown macroalga (Halidrys siliquosa), with a particular emphasis on polyphenols and their associated biological activities. Two fractions were obtained by liquid/liquid purification from a crude hydroethanolic extract: (i) an ethyl acetate fraction and (ii) an aqueous fraction. Total phenolic contents and antioxidant activities of extract and both fractions were assessed by in vitro tests (Folin–Ciocalteu test, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power assay, superoxide anion scavenging assay, and β-carotene–linoleic acid system). For the most active fraction, i.e., the ethyl acetate fraction, the oxygen radical absorbance capacity (ORAC) value, antibacterial activities, and sunscreen potential (Sun Protection Factor and UV-A-Protection Factor) were tested in vitro. A high correlation found between antioxidant activities and total phenolic content was interpreted as the involvement of polyphenolic compounds in antioxidant mechanisms. Interestingly, the ethyl acetate fraction appeared to be a broad-spectrum UV absorber and showed a strong bactericidal activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. In this fraction, four phenolic compounds (trifuhalols and tetrafuhalols and, for the first time, diphlorethols and triphlorethols) were identified using 1D and 2D nuclear magnetic resonance (NMR) and MS analysis. These findings are promising for the use of H. siliquosa, abundant in Brittany, as a valuable source of photoprotectant molecules for sunscreen and cosmetic applications.

Characterization of the Secretomes of Two Vibrios Pathogenic to Mollusks

Vibrio tapetis causes the brown ring disease in the Japanese clam Ruditapes philippinarum while Vibrio aestuarianus is associated with massive oyster mortalities. As extracellular proteins are often associated with the virulence of pathogenic bacteria, we undertook a proteomic approach to characterize the secretomes of both vibrios. The extracellular proteins (ECPs) of both species were fractionated by SEC-FPLC and in vitro assays were performed to measure the effects of each fraction on hemocyte cellular parameters (phagocytosis and adhesion). Fractions showing a significant effect were subjected to SDS-PAGE, and proteins were identified by nano LC-MS/MS. 45 proteins were identified for V. aestuarianus and 87 for V. tapetis. Most of them belonged to outer membrane or were periplasmic, including porins or adhesins that were already described as virulence factors in other bacterial species. Others were transporter components, flagella proteins, or proteins of unknown function (14 and 15 respectively). Interestingly, for V. aestuarianus, we noted the secretion of 3 extracellular enzymes including the Vam metalloprotease and two other enzymes (one putative lipase and one protease). For V. tapetis, we identified five extracellular enymes, i.e. two different endochitinases, one protease, one lipase and an adhesin. A comparison of both secretomes also showed that only the putative extracellular lipase was common to both secretomes, underscoring the difference in pathogenicity mechanisms between these two species. Overall, these results characterize for the first time the secretomes of these two marine pathogenic vibrios and constitute a useful working basis to further analyze the contribution of specific proteins in the virulence mechanisms of these species.

Laccase-like activity in the hemolymph of Venerupis philippinarum: characterization and kinetic properties.

The phenoloxidases (POs) include tyrosinases (EC 1.14.18.1), catecholases (EC 1.10.3.1) and laccases (EC 1.10.3.2) and are known to play a role in the immune defences of many invertebrates. For the Manila clam, Venerupis philippinarum, the exact role is not known, especially with regard to defences against Brown Ring Disease (BRD), which leads to high mortalities along European coasts. In order to understand the role and functioning of PO in V. philippinarum, the first step, and aim of this study, was to biochemically characterize the PO activity in the circulating hemolymph. Various substrates were tested and the common PO substrates L-DOPA, Catechol and dopamine exhibited good affinity with the enzyme and consequent low K(m) values (3.75, 1.97, 4.91 mM, respectively). A single tyrosinase-specific substrate, PHPPA, was oxidized, but the affinity for it was low (K(m) = 47.33 mM). Three tested laccase-specific substrates were oxidized by V. philippinarum PO (PPD, OPD and hydroquinone) and affinity was higher than for PHPPA. The results obtained with the substrate were confirmed by the use of different inhibitors: CTAB, a laccase-specific inhibitor inhibited PO activity greatly but not completely, whereas 4-Hr, specific to catecholases and tyrosinases, inhibited PO activity to a lesser extent. The results lead us to conclude that V. philippinarum PO activity in the circulating hemolymph, is mainly a laccase-like activity but there is also a smaller-scale tyrosinase-like activity. The inhibition mechanisms were also determined using dose-response substrate concentration for an inhibitor concentration equal or close to the IC50. Optimal conditions for the enzyme activity were also determined using L-DOPA as substrate, showing that its optimal temperature and pH are around 40 °C and 8.4 respectively. The enzyme is denatured for temperatures above 50 °C.

Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis--part II: combined effect of temperature and two V. tapetis strains.

Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally infected with two different bacterial strains and challenged with two different temperatures. Bacterial strains used in this study were Vibrio tapetis strain CECT4600(T), the causative agent of Brown Ring Disease (BRD) and V. tapetis strain LP2, supposed less virulent to V. philippinarum. V. tapetis is considered to proliferate at low temperatures, i.e. under 21 °C. In a global warming context we could hypothesize a decrease of mass mortalities caused by V. tapetis but these thermal changes could also directly impact the immune system of the host V. philippinarum. Thus, the aim of this study was to investigate the effects of the extrapallial injection with V. tapetis combined with temperature challenge on two enzymes activities in V. philippinarum. More precisely, after infection, phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immune response, were studied for 30 days in two compartments: the mantle and the hemolymph. Conchyolin Deposit Stages (CDS) and Shell Repair Stages (SRS) were also determined 30 days post-injection as a proxy of the virulence of the tested strains. In this study, we highlighted that host-pathogen interaction in a varying environment affects the enzymatic response of the host. The coupled effect of V. tapetis injection and temperature challenge was detected 30 days post injection and resulted in virulence differences. These findings were supported by CDS and SRS determination in clams and lead to the conclusion that clams immunity could be enhanced at 22 °C while V. tapetis virulence is lowered at this temperature. Another result of our study was the increase of PO and SOD basal activities as clams are exposed to warmer temperature.