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Bacterial AB toxins are secreted virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, they evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way.In this work, the resolution of the three-dimensional structure of AIP56 showed that instead of a two-domain organization, as previously predicted, AIP56 has a three-domain organization, with a NleC-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only with endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation. Considering the increasing number of putative AIP56 homologous toxins in databases, the data presented here have implications far beyond the AIP56 intoxication process and the virulence ofPhdp.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen.

This text aims to approach late prehistoric walled enclosures, mainly based on research conducted within a Portuguese context and through the study of the architecture of the sites. The periodization of the sites will be presented, and the problems involved in studying temporality at the “site level” will be addressed. The multiple ways of constructing and using space will also be questioned. Finally, the use of the typological designation “walled enclosure” will be discussed bearing in mind the singularity of each site.

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-contain-ing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition., Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior and Fundação para a Ciência e a Tecnologia (FCT), I.P., within the scope of the Norma Transitória - DL57/2016/CP1355/CT0010. This work had also support from the State Agency for Research (AEI) of Spain cofunded by the FEDER Program from the European Union (grants AGL2016-79738-R and BIO2016-77639-P)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apoptosis-inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram-negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single-chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc-metalloprotease moiety that cleaves the NF-kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase-thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BIA-MIC/2007/2014 (POCI-01-0145-FEDER-016608). Ana do Vale was supported by the FCT fellowship SFRH/BPD/95777/2013. We thank Steve Leppla (Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, USA) for providing PA. The authors acknowledge the support of the BioSciences Screening i3S Scientific Platform and the assistance of André Maia for the image acquisition with IN CELL analyzer. The authors also acknowledge the support of the Biochemical and Biophysical Technologies i3S Scientific Platform in protein quantification and circular dichroism.

The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen Photobacterium damselae subsp. damselae, a marine bacterium of the family Vibrionaceae. However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of rstA and rstB mutations in cell fitness and in diverse virulence-related features. Both rstA and rstB mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in rstA and rstB genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. rstA and rstB mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to eparate after cell division, whereas at 1% NaCl the mutants exhibited normal henotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, rstA and rstB mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of P. damselae subsp. damselae also led to the identification of RstAB-independent potential virulence factors as lipoproteins, Frontiers in sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in P. damselae subsp. damselae This work has been supported by grant AGL2016-79738-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant ED431C 2018/18 is also acknowledged. MT thanks the Brazilian Ministry of Education and CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior) for a predoctoral fellowship. XM thanks Xunta de Galicia for a predoctoral fellowship. AdV was supported by the FCT fellowship SFRH/BPD/95777/2013. The mass spectrometry technique was performed at the Proteomics i3S Scientific Platform with the assistance of Hugo Osório. This work had support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125) SI

Here, we report the draft genome sequences of two strains of the fish pathogen Photobacterium damselae subsp. piscicida, isolated from Salmo salar (SNW-8.1) and Seriola quinqueradiata (PP3). The identification of a type III secretion system in the two genomes furthers our understanding of the pathobiology of this subspecies.

AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-¿B p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BIA-MIC/2007/2014 (POCI-01-0145-FEDER-016608). Ana do Vale was supported by the FCT fellowship SFRH/BPD/95777/2013. We thank Steve Leppla (Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, USA) for providing PA and Alexander E. Lang (Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Germany) for providing His-TccC3hvr. We are grateful to Paula Sampaio for assistance in the fluorescence microscopy and acknowledge the support of the ALM i3S Scientific Platform, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122) and to André Maia of the BioSciences Screening i3S Scientific Platform for assistance in image acquisition with IN Cell analyzer. We also acknowledge the support of the Biochemical and Biophysical Technologies i3S Scientific Platform in protein quantification and circular dichroism.

Photobacterium damselae subsp. piscicida (Phdp) is responsible for disease outbreaks in marine aquaculture worldwide. Solea senegalensis, a valuable fish species for aquaculture in the south of Europe, is frequently affected by this pathogen. It is well established that bacteria respond to environmental signals and, in the case of pathogens, this ability may determine the outcome of their interaction with the host. Determination of gene expression under in vivo conditions constitutes a valuable tool in the assessment of microbial pathogenesis. Considering that different hosts may represent different environments for the pathogen, expression of Phdp virulence and in vivo induced antigen (IVIAT) genes during S. senegalensis infection has been determined in the present work. Increased transcription of genes encoding proteins involved in iron acquisition (Irp1, Irp2, HutB and HutD), oxidative stress defence (AhpC and Sod), adhesion (PDP_0080), toxins (AIP56) and metabolism (Impdh, Shmt and AlaRS) were detected in Phdp infecting S. senegalensis head kidney or liver. The highest increases corresponded to genes involved in survival under iron limiting conditions and oxidative stress, indicating their essential role during infection of sole. Results obtained give insight into Phdp virulence strategies and contribute to the identification of promising targets for the control of photobacteriosis.

AIP56 (apoptosis-inducing protein of 56 kDa) is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), the causative agent of a septicaemia affecting warm water marine fish species. Phdp-associated pathology is triggered by AIP56, a short trip AB toxin with a metalloprotease A domain that cleaves the p65 subunit of NF-κB, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes and plays a central role in host responses to infection. During infection by Phdp, AIP56 is systemically disseminated and induces apoptosis of macrophages and neutrophils, compromising the host phagocytic defence and contributing to the genesis of pathology. Although it is well established that the secretion of AIP56 is crucial for Phdp pathogenicity, the protein secretion systems operating in Phdp and the mechanism responsible for the extracellular release of the toxin remain unknown. Here, we report that Phdp encodes a type II secretion system (T2SS) and show that mutation of the EpsL component of this system impairs AIP56 secretion. This work demonstrates that Phdp has a functional T2SS that mediates secretion of its key virulence factor AIP56 This work is a result of the project Norte-01-0145-FEDER-000012-Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Ana do Vale was supported by the FCT fellowship SFRH/BPD/95777/2013. Work in CRO laboratory is supported by grant AGL2016-79738-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union SI

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative pathogen agent of an important fish septicemia. The key virulence factor of Phdp is the plasmid-encoded exotoxin AIP56, which is secreted by exponentially growing pathogenic strains. AIP56 has 520 amino acids including an N-terminal cleavable signal peptide of 23 amino acid residues, two cysteine residues and a zinc-binding region signature HEXXH that is typical of most zinc metallopeptidases. AIP56 induces in vitro and in vivo selective apoptosis of fish macrophages and neutrophils through a caspase-3 dependent mechanism that also involves caspase-8 and -9. In vivo, the AIP56-induced phagocyte apoptosis progresses to secondary necrosis with release of cytotoxic phagocyte molecules including neutrophil elastase. Fish injected with recombinant AIP56 die with a pathology similar to that seen in the natural infection.

In metazoans apoptosis is a major physiological process of cell elimination during development and in tissue homeostasis and can be involved in pathological situations. In vitro, apoptosis proceeds through an execution phase during which cell dismantling is initiated, with or without fragmentation into apoptotic bodies, but with maintenance of a near-to-intact cytoplasmic membrane, followed by a transition to a necrotic cell elimination traditionally called "secondary necrosis". Secondary necrosis involves activation of self-hydrolytic enzymes, and swelling of the cell or of the apoptotic bodies, generalized and irreparable damage to the cytoplasmic membrane, and culminates with cell disruption. In vivo, under normal conditions, the elimination of apoptosing cells or apoptotic bodies is by removal through engulfment by scavengers prompted by the exposure of engulfment signals during the execution phase of apoptosis; if this removal fails progression to secondary necrosis ensues as in the in vitro situation. In vivo secondary necrosis occurs when massive apoptosis overwhelms the available scavenging capacity, or when the scavenger mechanism is directly impaired, and may result in leakage of the cell contents with induction of tissue injury and inflammatory and autoimmune responses. Several disorders where secondary necrosis has been implicated as a pathogenic mechanism will be reviewed.

Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favor microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signaling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed. This work was supported by FEDER funds through Programa Operational Factores de Competitividade - COMPETE and by national funds through FCT - Fundacao para a Ciencia e a Tecnologia (project PTDC/BIA-MIC/3463/2012 FCOMP-01-0124-FEDER-028364; to AV). Research in the groups of Molecular Microbiology and Fish Immunology and Vaccinology is supported by national funds through FCT Fundacao para Ciencia e a Tecnologia/MEC - Ministerio da Educacao e Ciencia and co-funded by FEDER within the partnership agreement: PT2020 related with the research unit number 4293. The Group of Molecular Microbiology also receives support from a Research Grant 2014 by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (to SS) and the PT2020 research project Infect-ERA/0001/2013 PROANTILIS. AdV received the FCT fellowship SFRH/BPD/95777/2013 by national funds through FCT - Fundacao para a Ciencia e a Tecnologia/MEC - Ministerio da Educacao e Ciencia and co-funded by QREN and POPH (Programa Operational Potential Humano). SS is supported by FCT-Investigator program.

Mycobacterium leprae, the causative agent of leprosy, challenges host defense mechanism by impairing the signal transduction of T cells which leads to downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. In this study we sought to identify how soluble forms of M. leprae antigen(s) or particulate (liposome) delivery of the same antigens with two immunomodulators Murabutide and T cell peptide of Trat protein influence the transcription of IL-2 gene in anergic T cells of lepromatous patients. It was demonstrated that MLCwA/ManLAM stimulated cells of BL/LL patients showed defects in both jun-NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities there by resulting in decreased AP-1 activity. Additionally these cells showed reduced calcium levels, PKC activity and calcineurin (CN) activity. This led to impaired nuclear translocation of NFkappaB and NFAT in these patients. In contrast, when same M. leprae antigen(s) were incorporated with the two immunomodulators in liposomal form, increased transcription of IL-2 gene was observed especially in BL/LL patients which appears to be due to, at least in part, to increased expression of AP-1 Fos and Jun family members, NFkappaB and NFAT1 proteins. The increased expression of these transcription factors correlated with increased ERK/JNK, PKC and CN activities in these patients. Since activation of ERK/JNK/PKC kinases and CN phosphatase are required for stimulation of IL-2 transcription, these data provide a molecular explanation for the block in IL-2 production by M. leprae antigens. Thus the above study revealed suppression of all the three distinct biochemical pathways, viz. Ca-CN-NFAT pathway, PKC-NF-kappaB pathway, and MAPK-AP-1 pathway by M. leprae antigen(s) in anergized T cells of lepromatous patients which were activated by liposomal delivery of M. leprae antigens containing the two immunomodulators leading to optimal induction of IL-2 gene expression, which was required for the activation, and proliferation of T cells in lepromatous patients.

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924 bp cDNA of sea bass caspase-9 was obtained, consisting of 1308 bp open reading frame coding for 435 amino acids, 199 bp of the 5′-UTR and 417 bp of the 3′-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp121, Asp325 and Asp343. The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12 h returning to control levels at 24 h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48 h post-infection, when the fish were with advanced septicaemia.

EnglishThe traditional construction of narratives about recent Prehistory is founded on a set of preconceptions taken as natural by the patriarchal order of the western world. Is it possible to translate objects into activities, and associate each activity with a specific sex? After decades of feminist critic and gender studies, can Portuguese archaeology continue to perpetuate androcentric discourses? How can women be rescued from silence and from the margins of past discourses without falling to the temptation of reproducing the stereotypical image of women in the past (as mother and goddess), and how can a past that talks about human beings (instead of males and females) be constructed? portuguesA construcao dos discursos explicativos acerca da Pre-historia Recente pela Arqueologia tradicional assenta num conjunto de preconceitos dados como naturais (naturalizados) pela ordem patriarcal do mundo ocidental. Importa perguntar: sera possivel traduzir objetos em atividades e associar cada atividade a um sexo? Apos decadas de critica feminista e estudos de genero, podera a Arqueologia portuguesa continuar a perpetuar discursos de matriz androcentrica? Como resgatar a mulher ao silencio e as margens do discurso mas sem cair na tentacao de reproduzir a sua imagem estereotipada (como mae e deusa) no passado? Como construir um passado que fale de seres humanos?

In this work, the gene and cDNA of the sea bass CD8alpha have been isolated and characterized. The coding sequence has an ORF of 666 bp. It retains the Ig motif that interacts with MHC and the two cysteines responsible for an intra-chain disulfide bridge. The hinge region contains the two essential cysteines involved in dimerization. The transmembrane region is well conserved in all analysed sequences. Similar to other teleosts, the cytoplasmic region lacks the consensus p56(lck) motif common in higher vertebrates. Analysis of the expression pattern using RT-PCR shows the highest expression in the thymus. Like in the human gene, the sea bass CD8alpha genomic structure is organized into six exons, which roughly correspond to separate functional domains of the protein. Southern blotting shows that CD8alpha exists as a single copy gene. Together, these results support the concept that the basic structure of CD8alpha has been maintained through evolution.

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

Esta dissertação tem como objetivo refletir sobre o conceito de ativação de marca e a sua operacionalização, e perceber a importância dos eventos ao vivo, mais específico dos festivais de música, na comunicação das marcas. A investigação neste estudo contou, com pesquisas bibliográficas presentes no enquadramento teórico que permitiram criar uma linha condutora para se perceber o assunto em questão; entrevistas a agências de comunicação que já tenham ativado marcas, de médias e grades empresas, que se mostraram fundamentais para se observar, e compreender, em que é que consiste a ativação e a importância que esta ferramenta tem vindo a ganhar nas estratégias de comunicação das marcas; e, por fim, a análise de material publicado na imprensa especializada, que veio contribuir para o reforço da informação retirada nas entrevistas. As ferramentas de recolha de dados foram capazes de dar resposta às questões e aos objetivos iniciais, apesar de se ter verificado nos dados recolhidos acerca da ativação de marca, embora que semelhantes, não são bastantes para se formar uma definição exata. As principais características associadas à ativação de marca mostram que ela consiste numa ferramenta que procura trazer valor ao seu target, através da proximidade, da criação de uma relação/interação individual e inovadora, tendo sido possível concluir que os eventos, em concreto os festivais de música, são um momento de excelência para se ativar uma marca. The main objective in this thesis is to reflect what really is brand activation and understand the importance of the live events, focusing especially in festivals. The investigation method adapted to this study is based on a bibliographic research that makes the theoretical framework, and allowed to create a guideline to realize the matter in question. Interviews to communication agencies that had already activated brands in festivals and that were fundamental to observe and comprehend in what consists the brand activation, and although they are very similar, no one was able to give a concrete definition. The main characteristics show that brand activation is a tool that seeks to bring value to the target trough the proximity, the close relation, the individuality, the innovation and through the interaction. It was possible also to conclude that the events, in concrete the music fests, are a moment of excellence to activate

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway. We are grateful to Cesare Montecucco (Dipartimento di Scienze Biomediche Sperimentali dell'Úniversità di Padova and Instituto di Neuroscienze del CNR, Padua, Italy) for providing the PA and to Didier Cabanes (Group of Molecular Microbiology, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal) for providing the Listeria monocytogenes. Liliana M. G. Pereira and Daniela S. Silva were funded by Fundação para a Ciência e a Tecnologia (FCT) fellowships SFRH/BD/43501/2008 and SFRH/BD/35865/2007, respectively, financed by POPH-QREN and cofunded by Fundo Social Europeu and MCTES. Ana do Vale was supported by Programa Ciência, funded by POPH–QREN, Tipologia 4.2–Promoção do Emprego Científico, cofunded by Fundo Social Europeu, and national funding from MCTES. This work was supported by FEDER funds through the Operational Competitiveness Programme (COMPETE) and national funds through FCT under projects FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011) and FCOMP-01-0124-FEDER-009501 (PTDC/CVT/099544/2008).

Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β.

It has been previously shown that the exotoxin of the important fish pathogen Photobacterium damselae ssp. piscicida is a key pathogenicity factor and is responsible for the extensive systemic apoptosis of macrophages and neutrophils seen in acute fish photobacteriosis. The focus of the present study was to further characterize the AIP56-induced apoptosis of sea bass professional phagocytes by assessing the involvement of caspases, mitochondria and oxidative stress. The resulting data indicate that the apoptotic response in peritoneal macrophages and neutrophils treated ex vivo with AIP56 involves activation of caspase-8, -9 and -3, and mitochondria as shown by loss of mitochondrial membrane potential, release of cytochrome c and over-production of ROS. These results together with previous data from this laboratory suggest that both the extrinsic and intrinsic apoptotic athways are involved in the AIP56-induced hagocyte apoptosis., Fundação para a Ciência e a Tecnologia - (FCT)SFRH/BPD/40928/2007, PTDC/CVT/69086/2006

Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this work, the sea bass counterpart of mammalian caspase-8 was sequenced and characterized, and its involvement in the apoptogenic activity of a toxin from a fish pathogen was assessed. A 2472 bp cDNA of sea bass caspase-8 was obtained, consisting of 1455 bp open reading frame coding for 484 amino acids and with a predicted molecular weight of 55.2 kDa. The sea bass caspase-8 gene has 6639 bp and is organized in 11 introns and 12 exons. Several distinctive features of sea bass caspase-8 were identified, which include two death effector domains, the caspase family domains p20 and p10, the caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. The sea bass caspase-8 sequence revealed a significant degree of similarity to corresponding sequences from several vertebrate taxonomic groups. A low expression of sea bass caspase-8 was detected in various tissues of non-stimulated sea bass. Furthermore, it is shown that stimulation of sea bass with mid-exponential phase culture supernatants from Photobacterium damselae ssp. piscicida (Phdp), known to induce selective apoptosis of macrophages and neutrophils, resulted in an increased expression of caspase-8 in the spleen, one of the main affected organs by Phdp infection.

Bacterial modulation of phagocyte cell death is an emerging theme in pathogenesis. Here we describe the systemic destruction of macrophages and neutrophils by the Gram-negative Photobacterium damselae ssp. piscicida (Phdp) in fish pasteurellosis, a deadly systemic infection. Following experimental inoculation, Phdp spreads by bacteraemia and colonizes the organs, producing a septicaemic infection, and secretes the apoptogenic exotoxin AIP56 which is systemically disseminated. In experimental and natural pasteurellosis, destruction of macrophages and neutrophils by secondary necrosis following caspase-3-associated apoptosis was seen predominantly in the spleen, head kidney and gut lamina propria. Identical phagocyte destruction occurred after injection of rAIP56, but not of heat-inactivated rAIP56, or AIP56-negative Phdp strains, indicating that AIP56 is responsible for phagocyte destruction occurring in pasteurellosis. Active caspase-3 and active neutrophil elastase are present in the blood in advanced infection, indicating that phagocyte lysis by secondary necrosis is accompanied by release of tissue-damaging molecules. The AIP56-induced lysis of phagocytes represents a very efficient, self-amplifying etiopathogenic mechanism, because it results in two effects that operate in concert against the host, namely, evasion of the pathogen from a crucial defence mechanism through the destruction of both professional phagocytes, and release of tissue-damaging molecules. The induction by a bacterial exotoxin of in vivo systemic lysis of both professional phagocytes by secondary necrosis, now described for the first time, may represent an overlooked etiopathogenic mechanism operating in other infections of vertebrates.

Detachment-induced apoptosis of enterocytes (anoikis) has not been investigated in vivo. Here we describe anoikis of fish enterocytes following detachment in a septicemia by Photobacterium damselae subsp. piscicida, or following injection of its exotoxin. The in vivo study was complemented with an ex vivo time-lapse analysis using conditions duplicating the in vivo situation. Linings of enterocytes detached from intestine mucosa dissociate into isolated enterocytes which undergo caspase 3-mediated anoikis with cell rounding, loss of polarization, condensation of chromatin and fragmentation of the nuclear envelope, early swelling of mitochondria with rupture of the outer membrane, and brush border disappearance. One mechanism for brush border loss was shedding of apoptotic bodies incorporating the apical part of the enterocyte. Brush border disappearance was also associated with disassembly of the F-actin microvillar core and involved re-absorption into the cell, or expansion and vesiculation followed by shedding of microvillar fragments. The enterocyte anoikis terminates by secondary necrosis and lysis due to lack of elimination by phagocytosis of apoptosing enterocytes. The conditions prevailing in vivo in the gut lumen accelerate enterocyte secondary necrosis. Our results underscore the importance of analyzing anoikis under conditions similar to those occurring in vivo.

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.

The present manuscript reports for the first time the sequencing and characterisation of sea bass (sb) MHCII alpha and beta chains and Ii chain cDNAs as well as their expression analysis under resting state. 3D homology modelling, using crystal structures from mammalian orthologues, has been used to illustrate and support putative structural homologies of the sea bass counterparts. The sbIi cDNA consists of 96 bp of 5'-UTR, a 843 bp open reading frame (ORF) and 899 bp of 3'-UTR including a canonical polyadenylation signal 16 nucleotides before the polyadenylation tail. The ORF was translated into a 280 amino acid sequence, in which all characteristic domains found in the Ii p41 human form could be identified, including the cytoplasmic N-terminus domain, the transmembrane (TM) region, the CLIP domain, the trimerization domain and the thyroglobulin (Tg) type I domain. The trimerization and Tg domains of sbIi were successfully modelled using the human counterparts as templates. Four different sequences of each class II alpha and beta MHCII were obtained from a single fish, apparently not derived from a single locus. All the characteristic features of the MHCII chain structure could be identified in the predicted ORF of sea bass alpha and beta sequences, consisting of leader peptide (LP), alpha1/beta1 and alpha2/beta2 domains, connecting peptide and TM and cytoplasmic regions. Furthermore, independently of the HLA-DR crystal structure used as template in homology modelling, a similar predicted 3D structure and trimeric quaternary architecture was obtained for sbMHC, with major deviations occurring only within the sea bass MHCII alpha1 domain.

Interleukin-10 (IL-10) is a pleiotropic cytokine generally known for its relevance in the resolution of inflammation, but that also has immunostimulatory properties. Here is described the isolation and characterization of the sea bass IL-10 (sbIL-10) cDNA and gene. The sbIL-10 gene encodes a 187 amino acid protein and comprises a five exon-four intron structure as other known IL-10 genes. Important structural residues are maintained in the sbIL-10 protein, including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10. The 3D structure of sbIL-10 was predicted. This first homology model of a fish IL-10 reveals a high degree of compatibility between the dimeric quaternary architectures of sbIL-10 and its mammalian counterparts. The phylogenetic analysis clusters sbIL-10 with other IL-10s, apart from IL-10-related molecules. The involvement of IL-10 in sea bass immune responses was demonstrated by investigating the expression profiles of IL-1beta and IL-10 in the head-kidney and spleen following intraperitoneal injection of UV-killed Photobacterium damselae ssp. piscicida. Furthermore, involvement of IL-10 in the resolution of inflammation is for the first time suggested in fish, due to the delayed maximal mRNA levels of sbIL-10 compared to those of the pro-inflammatory IL-1beta.

The expression of a pre-B cell receptor (pre-BCR) is required for allelic exclusion and pre-BII cell differentiation. V(H)12 microH chains are unusual in that they form pre-BCRs and mediate allelic exclusion, but most cannot drive pre-BII cell differentiation. To explain this paradox, we examined pre-BCR functions and pre-BII cell differentiation in mice expressing microH chain transgenes encoding a B cell-permissible V(H)12 microH chain (designated 10/G4(6-1)), and a non-permissible V(H)12 microH chain (designated 8/G0). Compared with 10/G4 pre-BCRs, 8/G0 pre-BCRs are expressed at low levels on the cell surface. 8/G0 pre-BCRs mediate allelic exclusion, but 8/G0 pre-BII cells are defective in proliferation and expression of survival factors Bcl-2, Bcl-X(L) and hemokinin 1 (HK1). Increasing 8/G0 microH chain production restores HK1 transcription and improves proliferation of pre-BII cells as well as later stage B cell development. These data reveal a hierarchy of pre-BCR function that determines the development and plasticity of early B cells.

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis -acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1β, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida ( Phdp ). Finally, the importance of nuclear factor (NF)-κB on UV-killed Phdp -induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Caspase-3 is one of the major caspases operating in apoptosis, cleaving and inactivating a number of molecules and largely contributing to the apoptotic phenotype and the dismantling of the apoptoting cell. The opening reading frame of sea bass (Dicentrarchus labrax L.) caspase-3 has 281 amino acids. The complete sequence of caspase-3 shows a very close homology to the correspondent sequence from other vertebrates, in particularly with that of Takifugu rubripes and Oryzias latipes, with 87.7 and 87.9% of similarity, respectively. Furthermore, the sea bass caspase-3 sequence retains the motifs that are functionally important, such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. In the sea bass genome, the caspase-3 gene exists as a single copy gene and is organised in six exons and five introns. A very low expression of caspase-3 was detected by RT-PCR in various organs of non-stimulated sea bass, with slightly higher levels in thymus and heart and was increased in head kidneys of Photobacterium damselae ssp. piscicida infected sea bass. This increased expression was accompanied by the occurrence of high numbers of apoptoting cells with activated caspase-3.

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

The infection of sea bass (Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10(7)per peritoneal cavity for each cell type at 24h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel and very powerful microbial pathogenic strategy.