The bacterial exotoxin AIP56 induces fish macrophage and neutrophil apoptosis using mechanisms of the extrinsic and intrinsic pathways.

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.

Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis.

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.

Characterization of an equine macrophage cell line: application to studies of EIAV infection.

EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activity, are able to phagocytose fluorescent bioparticles, and produce nitrites in response to LPS. The EML-3C cell line expresses the EIAV receptor for cellular entry (ELR1) and supports replication of the virulent EIAV(PV) biological clone. Thus, EML-3C cells provide a useful cell line possessing equine macrophage related properties for the growth and study of EIAV infection as well as of other equine macrophage tropic viruses.

Systemic macrophage and neutrophil destruction by secondary necrosis induced by a bacterial exotoxin in a Gram-negative septicaemia.

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.

Cytochemical and ultrastructural study of anoikis and secondary necrosis in enterocytes detached in vivo.

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.

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia.

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 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp 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 Asp(121), Asp(325) and Asp(343). 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 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils.

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.

Effect of extracellular products of Pseudoalteromonas atlantica on the edible crab Cancer pagurus.

Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100 degrees C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 microg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 microg g of body weight(-1)) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.

Shell disease syndrome in the edible crab, Cancer pagurus--isolation, characterization and pathogenicity of chitinolytic bacteria.

Chitinolytic bacteria are believed to be the primary aetiological agents of shell disease syndrome in marine crustaceans. The disease principally results from the breakdown of their chitinous exoskeletons by the shell disease pathogens, but pathogenicity may also manifest internally should a breach of the carapace occur. The current study looks at the pathogenicity of a number of bacteria (predominantly from the genus Vibrio) isolated from the edible crab, Cancer pagurus. All chitinase-producing bacteria investigated were capable of growth in a minimal medium consisting of chitin powder from crab shells, but differed in their speed of growth and nature of chitinolytic activity, suggesting that they have different roles within the lesion community. Two isolates (designated I4 and I7) were chosen for studies on internal pathogenicity, which included the effect of the pathogen on crab tissues, the ability of the host to remove the bacteria from circulation and the antibacterial activity of crab blood. Initially, I4 was rapidly removed from circulation, but began to reappear in the blood after 24 h. By 100 h, 100% of crabs were moribund. The septicaemic effects of the isolate were reflected in the low levels of its killing by blood-cell lysate and serum. By contrast, I7 was only slowly removed from circulation and caused the rapid mortality of all crabs in <3 h. A large decline in the number of circulating blood cells following injection of I7 was mirrored by an accumulation of these cells in the gills. Initial experiments suggest that the death of the crabs following injection with I7 may be caused by toxic extracellular bacterial products that exert their effects on the blood cells and nervous system of the crabs.