Maria M. Costa

Individual sequence variability and functional activities of fibrinogen-related proteins (FREPs) in the Mediterranean mussel (Mytilus galloprovincialis) suggest ancient and complex immune recognition models in invertebrates.

In this paper, we describe sequences of fibrinogen-related proteins (FREPs) in the Mediterranean mussel Mytilus galloprovincialis (MuFREPs) with the fibrinogen domain probably involved in the antigen recognition, but without the additional collagen-like domain of ficolins, molecules responsible for complement activation by the lectin pathway. Although they do not seem to be true or primive ficolins since the phylogenetic analysis are not conclusive enough, their expression is increased after bacterial infection or PAMPs treatment and they present opsonic activities similar to mammalian ficolins. The most remarkable aspect of these sequences was the existence of a very diverse set of FREP sequences among and within individuals (different mussels do not share any identical sequence) which parallels the extraordinary complexity of the immune system, suggesting the existence of a primitive system with a potential capacity to recognize and eliminate different kind of pathogens.

Evidence of high individual diversity on myticin C in mussel (Mytilus galloprovincialis)

Several antimicrobial peptides (AMP) have been described in Mytilus galloprovincialis. However, only in myticin C a high variability on the nucleotide sequence was detected. To determine the individual variability of this AMP, the myticin C present in more than 100 mussels was analyzed by denaturing gradient gel electrophoresis (DGGE). This technique helped us to describe a very high myticin C diversity as compared with a non-immune related gene such as the beta-actin. Moreover, each mussel showed a specific and exclusive myticin C band pattern. Our results showed that the individual sequences of myticin C are unique for each mussel, independently of their geographic origin, age, sex, gonad maturation stage or aggregate where they group together on the wild. Only the animals belonging to the same family shared myticin C sequences. The comparative analysis of genomic DNA and cDNA sequences from the same individual showed that all detected variants shared a very high homology with the more frequent genomic isoforms, suggesting that all the variations were generated from the more common sequences, through a mechanism not yet determined. The fact that myticin C possesses characteristics of an immune gene, its potential antimicrobial effects, molecular diversity, as well as its early and ubiquitous expression, led us to suggest that myticin C might play an important role in innate immune defense in mussels.

Characterization and gene expression analysis of the two main Th17 cytokines (IL-17A/F and IL-22) in turbot, Scophthalmus maximus

This report describes the cloning, characterization and gene expression pattern of two Th17 cytokines, interleukin (IL)-17A/F and -22, in turbot Scophthalmus maximus. The turbot IL-17A/F cDNA contains a 516 bp open reading frame encoding a deduced IL-17A/F protein of 171 amino acid (aa) residues, containing a predicted signal peptide of 31 aa. Turbot IL-22 had a 564 bp ORF coding for a 187 aa protein with a 33 aa signal peptide. The turbot IL-22 protein contained a typical IL-10 family signature. Both cytokines had highest expression levels in the intestine followed by head kidney and gills. Stimulation with the Gram negative bacterium Aeromonas salmonicida was able to modulate IL-17A/F and IL-22 expression in head kidney, spleen and liver but not the intestine. PMA and PHA were also able to induce the expression of both cytokines, suggesting that, as expected, T-cells are likely the main producers of these molecules in turbot as in mammals.

Interferon-Induced Genes of the Expanded IFIT Family Show Conserved Antiviral Activities in Non-Mammalian Species

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in the protective response to viral infection, although the precise mechanism of IFITs for reducing viral proliferation is currently unknown. The interaction with the translation initiation factor eIF-3 or viral proteins and the sequestering of viral RNA have been proposed as potential antiviral functions for these proteins. In humans, four members of this family have been characterized. Nevertheless, information about these proteins in fish is almost non-existent. Exploiting the conservation of synteny between human and zebrafish genomes, we have identified ten members of the IFIT family located on four different chromosomes. The induction of these genes was examined both in vitro and in vivo after interferon (IFN) administration and rhabdovirus challenge. Whereas an induction of IFIT genes was observed after interferon treatments (IFNΦ1, IFNΦ2 and IFNΦ3), the viral infection did not affect these IFN-induced genes in vitro, and even reduced the IFN-induced expression of these genes. The response was largely different in vivo, with a broad up-regulation of IFIT genes after viral challenge. In addition, three selected IFITs were cloned in an expression vector and microinjected into zebrafish larvae to examine the protective effect of IFITs upon viral infection. Reduction in the mortality rate was observed confirming a conserved antiviral function in non-mammalian species.

IL-22 is a key player in the regulation of inflammation in fish and involves innate immune cells and PI3K signaling

IL-22 plays a role in various disorders in mammals, including mucosal-associated infections and inflammatory diseases. No functional IL-22 studies have been conducted on non-mammals to date. In this study, recombinant IL-22 (rIL-22) from turbot was produced to investigate its effects as a bioactive molecule. The expression of several pro-inflammatory cytokines was increased after rIL-22 treatment and reduced by pre-treatment with a JAK/STAT inhibitor. The involvement of the PI3K pathway in IL-22 induction was demonstrated. rIL-22 reduced the mortality in Aeromonas salmonicida-infected turbot, while higher Aeromonas hydrophila- or LPS-induced mortality was observed when IL-22 was blocked in zebrafish embryos. IL-22 knockdown increased pro-inflammatory cytokine expression in bacteria-stimulated fish. In zebrafish, IL-22 expression was detected primarily in the myeloid innate linage. It was found during early developmental stages when the adaptive immune response is not yet functional and in rag1(-)/(-) fish that lack an adaptive immune system. Our results clarify the conserved role of IL-22 in lower vertebrates. We suggest for the first time that IL-22 constitutes a key regulator of inflammatory homeostasis even in distant species such as teleosts, which diverged from mammals more than 350 million years ago.

Occurrence, seasonality and infectivity of Vibrio strains in natural populations of mussels Mytilus galloprovincialis

Widespread and large-scale mortalities of bivalve molluscs significantly affect their production. A number of pathogens have been identified as the primary causes of death in oysters or clams, especially bacteria of the genus Vibrio. We evaluated the occurrence, seasonality and infectivity of Vibrio strains associated with natural mussel (Mytilus galloprovincialis) populations. In particular, different isolates of V. splendidus and V. aestuarianus were analysed because they were associated with major oyster mortalities in areas where mussels are cultured without presenting mortalities. The presence of both Vibrio spp. was analysed bimonthly in mussels, water, sediment, plankton and other associated fauna from 2 sites in Galicia (NW Spain), the region with the highest mussel production in Europe. Environmental factors were also considered. The pathogenicity of different Vibrio isolates was analysed by performing experimental infections in mussels with strains isolated from the field. Results showed that Vibrio populations were mainly influenced by changes in water temperature and salinity. V. splendidus was dominant during the warm months and V. aestuarianus was predominant throughout the cold season. The sediment was the most important natural reservoir for bacteria. Experimental infections showed the extreme resistance of mussels to bacterial pathogens. Isolates of V. splendidus and V. aestuarianus were only moderately pathogenic for mussels in intramuscular infections and bath infections, and mortalities only occurred when animals were infected with a high bacterial concentration in adverse environmental conditions (hypoxia and 25°C). Although the pathogenicity of the Vibrio strains isolated from the wild was low for mussels, their potential risk for other bivalves cannot be ignored.

Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses

ABSTRACT Little is known about the antiviral response in mollusks. As in other invertebrates, the interferon signaling pathways have not been identified, and in fact, there is a debate about whether invertebrates possess antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naive mussels confer resistance in oysters to ostreid herpesvirus 1 (OsHV-1) when oyster hemocytes are incubated with mussel hemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel hemolymph and hemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel hemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible for the antiviral activity of mussel hemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting mollusks. Moreover, myticin C peptides showed antiviral activity against human herpes simplex viruses 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications. IMPORTANCE Several bioactive molecules that have potential pharmaceutical or industrial applications have been identified and isolated from marine invertebrates. Myticin C, an antimicrobial peptide from the Mediterranean mussel (Mytilus galloprovincialis) that was identified by proteomic techniques in both mussel hemolymph and hemocytes, showed potential as an antiviral agent against ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster-farming sector. Both hemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster hemocytes. Additionally, a modified peptide derived from myticin C or the nanoencapsulated normal peptide also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of mollusks.

The Involvement of Cholesterol in Sepsis and Tolerance to Lipopolysaccharide Highlighted by the Transcriptome Analysis of Zebrafish (Danio rerio)

Septic shock is the most common cause of death in intensive care units due to an aggressive inflammatory response that leads to multiple organ failure. However, a lipopolysaccharide (LPS) tolerance phenomenon (a nonreaction to LPS), is also often described. Neither the inflammatory response nor the tolerance is completely understood. In this work, both of these responses were analyzed using microarrays in zebrafish. Fish that were 4 or 6 days postfertilization (dpf) and received a lethal dose (LD) of LPS exhibited 100% mortality in a few days. Their transcriptome profile, even at 4 dpf, resembled the profile in humans with severe sepsis. Moreover, we selected 4-dpf fish to set up a tolerance protocol: fish treated with a nonlethal concentration of Escherichia coli LPS exhibited complete protection against the LD of LPS. Most of the main inflammatory molecules described in mammals were represented in the zebrafish microarray experiments. Additionally and focusing on this tolerance response, the use of cyclodextrins may mobilize cholesterol reservoirs to decrease mortality after a LD dose of LPS. Therefore, it is possible that the use of the whole animal could provide some clues to enhance the understanding of the inflammatory/tolerance response and to guide drug discovery.

β-glucan administration induces metabolic changes and differential survival rates after bacterial or viral infection in turbot (Scophthalmus maximus)

Abstract The innate immune response is able to ward off pathogens and remember previous infections using different mechanisms; this kind of immune reaction has been called “trained immunity”. Changes in cellular metabolism (aerobic glycolysis) have been observed during training with some immunostimulants like &bgr;‐glucans or during viral and bacterial infections. We hypothesize that &bgr;‐glucans can induce metabolic changes used by the host to fight pathogens. Accordingly, we evaluated changes in metabolic parameters in turbot that could affect their survival after a previous intraperitoneal treatment with &bgr;‐glucans and subsequent administration of Viral Hemorrhagic Septicemia Virus (VHSV) or bacteria (Aeromonas salmonicida subsp. salmonicida). The results obtained support that &bgr;‐glucans, VHSV and A. salmonicida induce changes in lactate, glucose and ATP levels in plasma, head kidney and liver and in the mRNA expression of enzymes related to glucose and fatty acid metabolism in head kidney. Additionally, the metabolic changes induced by &bgr;‐glucans are beneficial for VHSV replication, but they are harmful to A. salmonicida, resulting in reduced mortality. &bgr;‐glucans appear to have great therapeutic potential and can induce trained immunity against bacterial disease but not against viral disease, which seems to take advantage of &bgr;‐glucan metabolic alterations. Highlights&bgr;‐glucans, VHSV and A. salmonicida induce metabolic changes in turbot.&bgr;‐glucans induce different changes in viral and bacterial infections.Fish treated with &bgr;‐glucans are not protected against the infection with VHSV.&bgr;‐glucans have great therapeutic potential against bacterial disease.