Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

Abstract

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...

Bacterial Toxins as Pathogen Weapons Against Phagocytes

“…Although mammals are not susceptible to Phdp infection, likely due to temperature and osmolality restrictions, AIP56 is able to intoxicate mouse bone marrow derived macrophages (mBMDM; Pereira et al, 2014 ), through a mechanism similar to the one operating during intoxication of fish cells. Upon encountering susceptible cells, AIP56 binds to a still unidentified cell-surface receptor and is internalized through clathrin-mediated endocytosis ( Pereira et al, 2014 ; Figure 3 ).…”

“…Although mammals are not susceptible to Phdp infection, likely due to temperature and osmolality restrictions, AIP56 is able to intoxicate mouse bone marrow derived macrophages (mBMDM; Pereira et al, 2014 ), through a mechanism similar to the one operating during intoxication of fish cells. Upon encountering susceptible cells, AIP56 binds to a still unidentified cell-surface receptor and is internalized through clathrin-mediated endocytosis ( Pereira et al, 2014 ; Figure 3 ). Once in early endosomes, the toxin either follows the recycling pathway back to the extracellular medium or undergoes low pH-induced translocation across the endosomal membrane into the cytosol to display its toxic activity ( Pereira et al, 2014 ).…”

“…Upon encountering susceptible cells, AIP56 binds to a still unidentified cell-surface receptor and is internalized through clathrin-mediated endocytosis ( Pereira et al, 2014 ; Figure 3 ). Once in early endosomes, the toxin either follows the recycling pathway back to the extracellular medium or undergoes low pH-induced translocation across the endosomal membrane into the cytosol to display its toxic activity ( Pereira et al, 2014 ). AIP56 cleaves an evolutionarily conserved peptide bond of the Rel homology domain of NF-κB p65, removing residues crucial for p65-DNA interaction and compromising NF-κB activity ( Silva et al, 2013 ; Figure 3 ).…”

“…AIP56 cleaves an evolutionarily conserved peptide bond of the Rel homology domain of NF-κB p65, removing residues crucial for p65-DNA interaction and compromising NF-κB activity ( Silva et al, 2013 ; Figure 3 ). During intoxication, the proteolytic activity of AIP56 results in a complete depletion of p65 and leads to the apoptotic death of cells ( Silva et al, 2013 ; Pereira et al, 2014 ) through a process involving quick activation of caspases-8, -9 and -3, loss of mitochondrial membrane potential, translocation of cytochrome c to the cytosol and overproduction of ROS ( do Vale et al, 2007 ; Reis et al, 2007a , b , 2010 ; Costa-Ramos et al, 2011 ).…”

A secreted NlpC/P60 endopeptidase fromPhotobacterium damselaesubsp.piscicidacleaves the peptidoglycan of potentially competing bacteria

“…Although it has been suggested that Phdp remains in a cultivable form in salt water for only [4][5] days (38,39), it was also suggested that it has the ability to enter a dormant, non-cultivable but infectious state in salt water and sediment (40). With regard to the mechanisms responsible for the pathogenicity of Phdp, it was shown that extracellular products (ECPs) play a fundamental role (41,42) although among their components, only the toxin AIP56 has been identified and characterized so far (43)(44)(45)(46)(47).…”