SummaryBacterial 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, selfamplifying 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.

f AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-B. 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-B 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.A IP56 (apoptosis-inducing protein of 56 kDa) is a plasmidencoded toxin of Photobacterium damselae subsp. piscicida (1), a Gram-negative bacterium that infects economically important fish species (2, 3) and is considered one of the most relevant pathogens in mariculture (2-5). In acute infections, a rapid septicemia develops, causing very high mortalities (2, 4, 6). Histopathological analysis of the diseased animals revealed cytotoxic alterations (4, 7-10) that were found to result from pathogeninduced apoptotic death of macrophages and neutrophils (11) and later associated with the activity of AIP56 (1). Indeed, it has been shown that in infected fish, the toxin is systemically distributed and depletes macrophages and neutrophils by postapoptotic secondary necrosis (12), leading to the impairment of the phagocytic defense of the host and consequently favoring P. damselae subsp. piscicida survival and dissemination. Furthermore, the occurrence of a secondary necrotic process in which the phagocytes undergoing apoptosis lyse and release their cytotoxic contents contributes to the genesis of the infection-associated necrotic lesions (12, 13). These observations, together with the fac...

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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.

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