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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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

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 pathways are involved in the AIP56-induced phagocyte apoptosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., (2010 Elsevier Ltd. All rights reserved.)

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.

Recent in vitro studies have demonstrated that copper may induce apoptosis triggering the activation of caspase-3, a central effector of apoptotic cell death. However, the precise mechanism of copper-induced apoptosis is still unclear, even less so in Oreochromis niloticus where no caspase genes have been reported so far. This study aimed to assess the in vivo role of copper in apoptosis induction on O. niloticus gill, simultaneously contributing to elucidate the mechanism of copper-induced apoptosis. Caspase-3 gene was partially sequenced and, after in vivo exposures to 40 and 400 microgL(-1) of copper, its mRNA expression was evaluated by real-time PCR. Apoptosis was also evaluated by TUNEL assay and cell proliferation identified using an antibody against proliferating cell nuclear antigen (PCNA). The copper concentrations used did not induce the upregulation of caspase-3 gene in O. niloticus gill. In addition, in the gills of fish exposed to copper there was no increase in the estimated relative volume of apoptotic cells, indicating that neither the caspase-3-dependent or caspase-independent apoptotic pathways were induced. On the other hand, the increase in the volumetric density of epithelial proliferating cells suggests a concentration-dependent repair response.

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.

Apoptosis is a genetically controlled and evolutionarily conserved form of active cell death, albeit with an increase in complexity with continuing development. A high conservation at the functional and molecular level has been described between the players of the apoptotic machinery in invertebrates (Caenorhabditis elegans and Drosophila) and mammals. However, fish represent an excellent and advantageous model for the study of vertebrate development and disease, bridging the gap between the C. elegans/Drosophila and mouse/human models. Moreover, contrary to C. elegans and Drosophila, fish can be used for studying the development and function of vertebrate-specific organs and have a fully developed immune system similar to that of mammals. Last but not less important, both the environment and human health will obviously gain by using the knowledge generated through the use of fish models, for developing better prophylactic and therapeutic measures with impact on the aquaculture industry. In the present article, structural and functional data on the most important apoptosis related molecules, namely death-receptor, Bcl-2 and caspase families, and mechanisms are reviewed. The data point to the existence in fish of apoptotic pathways equivalent to those of mammals, making fish useful animal models for studying apoptosis, which may have great applicability for the advance of the knowledge on the role of apoptotic cell death in human apoptosis-related disorders as well as in pharmaceutical design.

The relevance of fish research has been rising due to the expansion of aquaculture and to the increasing use of fish as replacements for mammals in the study of human physiological and pathological issues. Fish have much smaller genomes compared to mammals, and zebrafish, fugu, medaka and spotted green puffer fish have the sequence of their genomes completed or near completion. Fish have several of the virtues of Drosophila melanogaster and Caenorhabditis elegans for apoptosis research, but offer additional advantages because they are vertebrates and have a developed immune system and apoptotic pathways similar to those of mammals. Many phenotypes in the zebrafish resemble human diseases and this fish has been increasingly used in pharmaceutical design of apoptosis modulating drugs. The roles of microRNAs, bcl-2, p53, insulin-like growth factor-binding protein-3, and cellular apoptosis susceptibility (CAS) and c-Myc genes (involved in the interaction apoptosis/cancer), and Abeta peptides, presenilin enhancer 2, cyclin-dependent kinase 5 and tau (factors with relevant roles in apoptosis-associated human neurodegenerative disorders), have also been successfully investigated in fish models. Results of research with fish that have advanced the knowledge on the participation of apoptosis in viral infections and of apoptosis and secondary necrosis in bacterial infections are also reviewed. It is expectable that the use of fish for research on apoptosis-related issues relevant for human physiology and pathology and for the design of apoptosis-modulating drugs will continue to increase.

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.