The Apoptogenic Toxin AIP56 Is a Metalloprotease A-B Toxin that Cleaves NF-κb P65

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 Cys39-Glu40 peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.


Introduction
The NF-kB family of transcription factors is evolutionarily conserved and comprises five members (NF-kB 1 (p50), NF-kB 2 (p52), RelA (p65), RelB and cRel), which form different combinations of homo-or hetero-dimers [1]. Under normal physiological conditions, NF-kB complexes remain inactive in the cytosol through association with the IkB proteins that mask the nuclear localization domains on NF-kB subunits. A variety of stimuli, including bacterial and viral products and cytokines, acting via cellular receptors such as Toll-like receptors (TLRs), Interleukin-1 receptor (IL-1R) and TNF receptors (TNFRs), trigger a signalling cascade that leads to phosphorylation and degradation of the inhibitory IkB proteins with rapid activation and transport of the NF-kB complexes to the nucleus, resulting in the up-regulation of inflammatory and anti-apoptotic genes [2].
NF-kB activation is considered to be the central initiating event of host responses to microbial pathogen invasion [2]. Therefore, it is not surprising that successful microbial pathogens have evolved complex strategies to interfere with NF-kB signalling. A number of pathogenic bacteria were recently found to interfere with this pathway by targeting different intermediates of the NF-kB activation cascade [2][3][4].
Photobacterium damselae piscicida (Phdp) is a Gram-negative bacterium that infects several warm water fish species worldwide and is recognized as one of the most threatening pathogens in mariculture [5][6][7][8]. In acute infections, a rapid septicemia develops and causes very high mortality [5,6,9]. Early descriptions of the histopathology of Phdp infection recognized the occurrence of cytotoxic alterations [5,[10][11][12][13][14][15][16] that we found to result from pathogen-induced macrophage and neutrophil apoptotic death [17,18] by a process that uses mechanisms of the intrinsic and extrinsic apoptotic pathways [19]. The phagocyte destruction observed in Phdp infections occurs systemically and culminates in a secondary necrotic process with lysis of the apoptosing cells [17,18,20]. This leads to the impairment of host immune defences and to the release of the cytotoxic contents of the phagocytes, contributing to the formation of the necrotic lesions observed in the diseased animals.
We have previously shown that phagocyte apoptosis observed in Phdp infections results from the activity of AIP56, a plasmidencoded exotoxin secreted by virulent strains, and that passive immunization with anti-AIP56 rabbit serum protects against Phdp infection [17,21]. These results implicated AIP56 as a key virulence factor of Phdp. However, the molecular target(s) of the toxin remained unidentified and nothing was known about its structure-function relationship.
AIP56 is synthesized as a precursor protein with a cleavable Nterminal signal peptide that is removed during secretion, originating a 497-amino acid mature toxin with the conserved HEIVH zinc-binding motif within its N-terminal region [21], similarly to tetanus neurotoxin [22]. The N-terminal region of AIP56 is homologous to NleC [21,23], a type III secreted effector present in several enteric pathogenic bacteria, while the Cterminal region is highly similar to an uncharacterized hypothetical protein of Acrythosiphon pisum bacteriophage APSE-2 [24] and to the C-terminal portion of a hypothetical protein of the monarch butterfly Danaus plexippus ( Figure S1). This suggested that AIP56 is a two domain protein, belonging to the group of A-B type toxins that includes diphtheria and tetanus toxins [23,25,26].
Here, we show that AIP56 is a zinc-metalloprotease that cleaves NF-kB p65 and that its enzymatic and apoptogenic activities are correlated. In contrast to NleC, which is delivered into the host cell's cytosol through a type III secretion system, AIP56 is an A-Btype exotoxin with an N-terminal domain responsible for the proteolytic activity and a C-terminal domain involved in binding and internalisation into target cells.

Results
The metalloprotease signature of AIP56 is essential for its apoptogenic activity In order to clarify the role played by the zinc metalloprotease activity of AIP56, a mutant (AIP56 AAIVAA ) containing a disrupted putative zinc-binding motif was produced. The oligomerization state and secondary structure content of the toxin were undisturbed by the introduced mutations ( Figure S2) and atomic absorption spectroscopy did not detect zinc in AIP56 AAIVAA , while in AIP56 equimolar amounts of zinc (0.9360.04 mol zinc/mol protein) were present. When tested ex vivo, AIP56 AAIVAA failed to induce apoptosis of sea bass phagocytes, whilst a large number of cells with apoptotic morphology were observed after treatment with AIP56 ( Figure 1A). These results indicate that an intact metalloprotease domain is essential for the apoptogenic activity of AIP56.
It is worth noting that the AIP56 concentrations used in the present work are biologically relevant, since they are similar to those detected in the plasma of infected fish ( Figure S3).
AIP56 is a zinc dependent metalloprotease that cleaves NF-kB p65 at the Cys 39 -Glu 40 peptide bond When incubated with sea bass cell lysates, AIP56 cleaved p65 with the appearance of a lower MW fragment ( Figure 1B). Proteolysis of p65 did not occur in cell lysates incubated with AIP56 AAIVAA or with AIP56 in the presence of the metalloprotease inhibitor 1,10-phenanthroline ( Figure 1B). The p65 fragment was recognised by an antibody specific for a peptide located at the Cterminal region of p65 indicating that the AIP56-mediated p65 cleavage occurred within the N-terminal region, where the Relhomology domain is located. To map the cleavage site, recombinant sea bass p65Rel domain (sbp65Rel) was incubated with the toxin. SDS-PAGE analysis showed that AIP56 cleaved recombinant sbp65Rel in vitro ( Figure 1C), and N-terminal sequencing of the cleaved fragment revealed that the cleavage occurred at the Cys 39 -Glu 40 peptide bond, similar to what was described for NleC [27]. Experiments using in vitro synthesised 35 Slabeled sea bass p65Rel domain (sbp65Rel) and three sbp65Rel mutants (sbp65RelC39A, sbp65RelE40A and sbp65CE39-40AA) showed that mutation of the evolutionarily conserved Cys 39 had no effect on p65 cleavage by either AIP56 or NleC ( Figure S4). However, mutation of the following Glu 40 inhibited cleavage and double mutation of Cys 39 and Glu 40 completely abolished p65 proteolysis by AIP56 and NleC ( Figure S4).
To determine if cellular intoxication by AIP56 involves cleavage of NF-kB p65, sea bass peritoneal leukocytes were incubated with wild type toxin or with AIP56 AAIVAA mutant and p65 proteolysis assessed by Western blotting. Wild type AIP56 caused NF-kB p65 depletion, whilst AIP56 AAIVAA was inactive ( Figure 1D). It has been reported that caspase-3 can cleave p65 [32,33]. To investigate whether caspases are involved in AIP56-dependent cleavage of p65, cells were incubated with the toxin in the presence or absence of the pan-caspase inhibitor ZVAD-FMK ( Figure 1E), previously shown to block AIP56-induced apoptosis [19]. In these experiments, ZVAD-FMK was effective in protecting cells from AIP56-induced apoptosis (data not shown), but did not affect NF-kB p65 cleavage ( Figure 1E), indicating that AIP56-mediated p65 depletion is a caspase-independent event. Taken together, the above results demonstrate that the metalloprotease activity of AIP56 is responsible for the cleavage of NF-kB p65 at the Cys 39 -Glu 40 peptide bond.

AIP56 has two domains
The primary structure of AIP56 suggests that this toxin comprises two functional domains and could be an A-B toxin with its two moieties linked by a single disulphide bond ( Figure S1) [23]. Therefore, in order to define domain boundaries within the toxin, limited proteolysis experiments were performed. SDS-PAGE analysis of AIP56 digested with chymotrypsin, trypsin or proteinase K revealed that the toxin is highly resistant to trypsin digestion, whereas chymotrypsin and proteinase K cleaved AIP56 into two major fragments with approximately 32 and 24 kDa (Figure 2A). These two fragments were only detected upon treatment with the reducing agent DTT, suggesting that they are linked by a disulphide bridge ( Figure 2B). N-terminal Edman

Author Summary
The apoptosis inducing protein of 56 kDa (AIP56) is a key virulence factor secreted by Photobacterium damselae piscicida (Phdp), a Gram-negative bacterium that causes septicaemic infections in economically important marine fish species. It is known that AIP56 induces massive destruction of the phagocytic cells of the infected host, allowing the extracellular multiplication of the bacteria and contributing to the genesis of the pathology. Here we show that AIP56 acts by cleaving NF-kB p65. The NF-kB family of transcription factors is evolutionarily conserved and plays a central role in the host responses to microbial pathogen invasion, regulating the expression of inflammatory and anti-apoptotic genes. Pathogenic bacteria have evolved complex strategies to interfere with NF-kB signalling, usually by injecting protein effectors directly into the cell's cytosol through bacterial secretion machineries that require contact with host cells. In contrast, AIP56 acts at distance and has an intrinsic ability to reach the cytosol due to the presence of a C-terminal domain that functions as ''delivery module.'' Recombinant sea bass p65Rel (7.5 mM) was incubated in the presence or absence of 1 mM of AIP56 for 3 h at 22uC and analysed by SDS-PAGE. Edman degradation of the cleaved sbp65Rel (cl-sbp65Rel) identified the sequence E 40 GRSA 44 showing that cleavage occurred after the conserved C 39 . (D) Incubation of leukocytes with AIP56, but not with AIP56 AAIVAA , AIP56 1-285C262S , AIP56 286-497C298S , nicked AIP56 (AIP56nic) or reconstituted AIP56 (AIP56rct) leads to p65 depletion. Leukocytes were incubated with 10 mg/ml of the indicated proteins for 2 h at 22uC and p65 cleavage was assessed AIP56 Is an A-B Toxin that Cleaves NF-kB p65 PLOS Pathogens | www.plospathogens.org sequencing revealed that chymotrypsin cleavage occurred between Phe 285 and Phe 286 , in the amino-acid stretch flanked by the two unique cysteine residues (Cys 262 and Cys 298 ) of AIP56 ( Figure 2C). Altogether, these results indicate that AIP56 is composed of two domains linked by a disulphide bridge.
The N-terminal domain is responsible for the catalytic activity and the C-terminal domain is implicated in binding to target cells To better understand the function of the two AIP56 domains, constructs corresponding to the N-and C-terminal portions of the toxin (AIP56 1-285 and AIP56 286-497 , respectively) were designed, taking into account the boundary defined by the chymotrypsin cleavage site ( Figure 2C). Purification of these two recombinant proteins using the experimental conditions used for the full-length toxin revealed that they display a major propensity to oxidize leading to the formation of DTT-sensitive dimers ( Figure S5A), a phenomenon that could have a functional impact and complicate subsequent analyses. Therefore, versions of the constructs with the single cysteine replaced by serines (AIP56 1-285C262S and AIP56 286-497C298S , respectively) were produced. The mutants are undistinguishable from the non-mutant proteins, as assessed by CD ( Figure  S5B and S5C) with the N-terminal domain composed mainly of ahelices, whereas b-sheet is the predominant secondary structure of the C-terminal moiety ( Figure 2D). Furthermore, the weighted sum of the CD spectra of the N-and C-terminal domains reproduces the spectrum of the entire protein ( Figure 2D), indicating conservation of the native structure.
To test the catalytic activities of the AIP56 N-and C-terminal domains, AIP56 1-285C262S and AIP56 286-497C298S were incubated with fish leukocyte lysates ( Figure 1B) or with in vitro translated 35 Slabeled sbp65Rel ( Figure S5D). The C-terminal construct did not display catalytic activity, whereas the N-terminal domain cleaved p65, similarly to the full-length toxin. However, neither changes in cellular p65 levels ( Figure 1D) nor apoptosis ( Figure 3A) were observed in sea bass leukocytes incubated with the N-or Cterminal truncate or with a mixture of both. This indicates that the two AIP56 domains are non-toxic and suggests that they need to be part of the same molecule to elicit a biological effect.
The cytosolic location of NF-kB p65 could mean that the lack of toxicity of the N-terminal domain was related to its inability to enter the cells and reach its target. Hence, a strategy to deliver the N-terminal domain into the cell cytosol was designed. Chimeric proteins consisting of the N-terminal portion of Bacillus anthracis LF fused to the AIP56 protease domain (LF 11-263 NAIP56 1-261 ) or to the C-terminal domain (LF 11-263 NAIP56 299-497 ) were produced. Intoxication assays were performed in the presence of PA, the receptor-binding subunit for LF [34]. In cells incubated with LF 11-263 NAIP56 1-261 the p65 levels were significantly reduced, confirming that LF 11-263 NAIP56 1-261 was successfully delivered into the cell cytosol, while no changes in p65 levels were observed in cells incubated with LF 11-263 NAIP56 299-497 ( Figure 3B). Accordingly, LF 11-263 NAIP56 299-497 did not display apoptogenic activity, while incubation with LF 11-263 NAIP56 1-261 resulted in an increased number of cells with apoptotic morphology ( Figure 3B), similar to what was observed in cells incubated with AIP56. Thus, delivery of the AIP56 N-terminal domain into the cytosol reproduces the toxic effect of the full length toxin, confirming that this domain is responsible for the toxin's catalytic and apoptogenic activities. These results also suggest that the C-terminal domain of AIP56 is involved/required for entrance of the toxin into cells. To investigate this possibility, AIP56 AAIVAA , AIP56 1-285C262S or AIP56 286-497C298S were used in competition experiments with AIP56. Both p65 cleavage and apoptosis were monitored in these experiments. AIP56 286-497C298S and AIP56 AAIVAA , but not AIP56 1-285C262S , were able to inhibit the apoptogenic activity of wild type AIP56 in a dose-dependent manner ( Figure 3C, left panel). Furthermore, AIP56 AAIVAA and AIP56 286-497C298S inhibited AIP56-mediated p65 degradation, whereas no effect could be observed when AIP56 1-285C262S was used as competitor ( Figure 3C, right panel). These results indicate that the C-terminal domain mediates binding of the toxin to the cell surface and entry into the cells.
Translocation of AIP56 requires integrity of the Cys 262 -Cys 298 linker but the disulphide bridge is not an absolute requirement for toxicity Results obtained in experiments using N-and C-terminal truncates of AIP56 suggested that the two domains must be part of the same molecule to display toxicity. In order to investigate if the two domains of the toxin bound by a disulphide bridge are able to intoxicate cells, we nicked the toxin with chymotrypsin. Nicking of the toxin and integrity of the disulphide bridge linking the two fragments were confirmed by reducing and non-reducing SDS-PAGE ( Figure S6A). Surprisingly, no changes in p65 cellular levels ( Figure 1D) and no apoptosis ( Figure 4A) were observed upon incubation of sea bass peritoneal cells with nicked toxin. Similar results were obtained using a reconstituted version of the toxin ( Figure 1D and 4A) consisting of disulphide-bound AIP56 1-285 / AIP56 286-497 along with trace amounts of AIP56 1-285 and of AIP56 286-497 homodimers and monomers ( Figure S6A). Although nicking abolished cellular toxicity, it did not induce major structural changes ( Figure S6B) and only a 1uC decrease in Tm (3960.13uC for AIP56 and 3860.25uC for nicked AIP56; mean6SD of 16 measurements in four independent experiments) was measured by DSF. More importantly, nicked AIP56 retained both proteolytic activity against p65 in vitro ( Figure 1B and 4B) and cell binding ability, as indicated by the partial inhibition of the AIP56-mediated p65 cleavage and apoptosis in competition experiments ( Figure 4C). These results suggest that the integrity of the linker region between the two cysteine residues is needed for toxin internalization, in contrast to what is known for the diphtheria, tetanus and botulinum toxins, where nicking of the inter-cysteine loop is required for toxicity [26,35].
In tetanus and botulinum neurotoxin type A, it has been shown that the disulphide bridge is essential for neurotoxicity [36,37]. We found that disruption of the disulphide bridge linking Cys 262 and Cys 298 of AIP56 by alkylation ( Figure S6D) did not affect the catalytic activity of the toxin in vitro ( Figure 4B), but partially compromised its toxicity ( Figure 4D), suggesting that in AIP56 the disulphide bridge plays a role in the intoxication process but is not an absolute requirement for toxicity.

Discussion
In this study, we report the structural and functional characterization of AIP56 as an A-B type bacterial exotoxin that cleaves by Western blotting. (E) AIP56-mediated p65 cleavage is caspase-independent. Leukocytes were incubated with 2 mg/ml AIP56 in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK for 2 h at 22uC, and p65 cleavage was assessed by Western blotting. Numbers to the left of the panels refer to the position and mass of the molecular weight markers, in kDa. doi:10.1371/journal.ppat.1003128.g001 AIP56 Is an A-B Toxin that Cleaves NF-kB p65 PLOS Pathogens | www.plospathogens.org NF-kB p65. Considering the anti-apoptotic functions of NF-kB, and in particular, of its p65 subunit [38][39][40], AIP56-mediated depletion of NF-kB p65 likely explains the disseminated phagocyte apoptosis observed in Phdp infections that contributes to subvert the host immune response and determines the outcome of the infection [17,19,21]. This adds to a general theme of hostpathogen interaction that has recently emerged, consisting in the induction of apoptosis of the host immune cells to the pathogen advantage [41][42][43].
Here we demonstrate that, similar to the anthrax lethal factor and to the clostridial neurotoxins [44,45], AIP56 is a zincendopeptidase but with a catalytic activity towards NF-kB p65. Furthermore, we show that AIP56 is organized into two distinct domains linked by a single disulphide bond. The N-terminal domain of AIP56 harbours the catalytic activity of the toxin and cleaves NF-kB p65 at the Cys 39 -Glu 40 peptide bond, within the p65 N-terminal Rel homology domain, where several key residues of p65 known to be involved in DNA interaction are located [46][47][48]. In the last decade, several reports revealed that Cys 38 of human p65 (Cys 39 in sea bass p65) interacts with the phosphate backbone of NF-kB binding sites [46], that its oxidation and nitrosylation inhibit DNA binding [49] and that it is targeted by several inhibitors of NF-kB with anti-inflammatory and/or anticancer properties [50][51][52][53][54][55][56][57][58][59][60]. More recently, it was shown that hydrogen sulphide-linked sulfhydration of Cys 38 of human p65 plays a key role in regulating the anti-apoptotic actions of NF-kB [40]. Therefore, cleavage of sea bass p65 by AIP56 disrupts a segment crucial for DNA interaction. Considering that the proteolytic activity of AIP56 towards p65 is similar to the one previously described for NleC (both proteases cleave p65 at the same peptide bond), and based on the observation that p65 cleavage by NleC compromises NF-kB dependent transcription  [27,28,30], it is likely that AIP56 also affects NF-kB transcriptional activity.
Although in many cell types down-regulation of NF-kB is not sufficient to trigger apoptosis, it is widely recognised that cells with inactivated NF-kB are more prone to commit suicide in response to different stimuli, including TNF-a and TLRs ligands [38,[61][62][63], and that the anti-apoptotic actions of NF-kB can be largely attributed to its p65 subunit [38][39][40]. In the context of bacterial infections, inhibition of NF-kB function usually leads to impairment of the inflammatory responses [2]. The induction of apoptosis by bacterial effectors through interference with NF-kB activity has also been described, but is a far less common scenario. Examples are Yersinia YopP/J [64][65][66] and Aeromonas salmonicida Aop [67], both inhibiting the degradation of the inhibitory IkB proteins [64,[68][69][70][71], and V. parahaemolyticus protein VP1686 that interacts with and suppresses DNA binding activity of NF-kB [72]. It remains to be determined whether AIP56-mediated depletion of p65 is sufficient to induce apoptosis, in resemblance to what has been suggested for the macrophage apoptosis induced by V. parahaemolyticus type III secreted effector VP1686 [72], or if it requires an additional stimulus.
Almost all bacterial effectors that have been described to target NF-kB signalling are injected directly into the host cell cytosol by type III or type IV secretion systems (see reviews by [2,4]). In contrast, we have found that the AIP56 N-terminal metalloprotease can only act when linked to a C-terminal binding domain that, by analogy with other A-B toxins, may assist the protease domain in its membrane translocation into the cytosol [34,73,74].
Bacterial A-B toxins are often secreted as a single polypeptide chain that is cleaved into the disulphide-bound A and B domains [26,74]. In these toxins, proteolytic nicking and integrity of the disulphide bond linking the A and B domains are essential for toxicity [19,35,36,[75][76][77]. In contrast, AIP56 toxicity is abolished by proteolytic nicking and only mildly compromised by disruption of the disulphide bridge by alkylation. Considering that nicked AIP56 retains the ability to interact with the cell membrane, these observations suggest that the linker region (between Cys 262 and Cys 298 ) is involved in translocating the toxin into the host cell cytosol. The decreased toxicity resulting from alkylation suggests that the integrity of the disulphide bond is important, although not absolutely required, for AIP56 intoxication. The disulphide bond may be involved in stabilizing the spatial relationship between the domains. In addition, that bond is hydrophobic and polarizable and its alkylation can have implications in membrane insertion, as reported for tetanus and botulinum neurotoxins [78].
AIP56 is synthesised as a single polypeptide and, contrary to what has been reported for most A-B toxins, there is no evidence of proteolytically processed toxin in the bacterial culture supernatants or in the serum of infected fish [21]. Furthermore, despite several attempts, we were unable to detect proteolytic processing of AIP56 upon its interaction with host cells. If AIP56 needs to be processed in order to exert its effect, the lack of detection of processed toxin may result from a very small amount of processed toxin (not detectable in our experiments) being sufficient to intoxicate the cells, similarly to what was described for other toxins [79]. Alternatively, after endocytosis, unprocessed AIP56 may be translocated into the cytosol as described for Pseudomonas exotoxin A [80,81] or may localize in an endomembrane (e.g. endosomal membrane) with the catalytic domain facing the cytosolic compartment where it can interact with and cleave p65. Studies aiming at discriminating between these hypotheses will be developed in the future.
It is now recognised that horizontal transfer of entire genes or portion of genes plays a key role in generating diversity in pathogens by allowing them to acquire novel phenotypic characteristics. Indeed, there are several examples of bacterial genes with a mosaic structure, composed of diverse segments with different origins [82][83][84][85][86][87]. The structure of AIP56 suggests that the toxin has a chimeric structure, having an N-terminal catalytic domain highly identical to the type III effector NleC and a Cterminal domain homologous to a hypothetical protein of the bacteriophage APSE-2. The actual transfer events that gave rise to such a chimeric protein toxin remain to be disclosed.
The AIP56 catalytic domain and NleC have the same NF-kB p65 cleavage activity. However, NleC requires a type III secretion machinery for activity, while AIP56 has an intrinsic ability to reach the cytosol, due to the presence of the additional C-terminal domain that functions as a ''delivery module''. This difference may have relevant implications when considering the use of both pathogen-derived molecules as therapeutic agents in situations associated with uncontrolled activation of NF-kB such as inflammatory diseases and cancer.

Materials and Methods
Additional details can be found in Supporting Information.

Ethics statement
This study was carried out in accordance with European and Portuguese legislation for the use of animals for scientific purposes

Circular dichroism spectroscopy (CD)
Far UV CD spectra were acquired on an Olis DSM 20 circular dichroism spectropolarimeter controlled by the Globalworks software. Each spectrum is the average of three scans collected at 20uC with a 0.2 mm path length cuvette and with an integration time of 4 seconds. Proteins were dissolved in 10 mM Tris-HCl, 50 mM NaCl, pH 8.0 and concentrations were determined by absorbance measurements. Analysis of the protein secondary structure was performed using the Globalworks software algorithm.

Cells
Sea bass peritoneal leukocytes were obtained as previously described [19] and used at a density of 2610 6 cells/ml. The peritoneal population of cells consists of approximately 70% macrophages and 20% neutrophils with the presence of small numbers of eosinophilic granular cells, lymphocytes and erythrocytes [19].

Apoptogenic activity assays
Cells were incubated for 4 h at 22uC with AIP56 or AIP56 derived proteins at the indicated doses. Where indicated, the cells were pre-treated for 30 min at 22uC with 25 mM of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK). In experiments using LF chimeric proteins, cells were incubated with the indicated concentrations of LF 11-263 NAIP56 1-261 or LF 11-263 NAIP56 299-497 with or without 10 nM of anthrax protective antigen (PA) obtained as described [88]. Mockand AIP56-treated cells were used as controls. Apoptosis was assessed as described [18], by light microscopy morphological analysis of cytospin preparations stained with Hemacolor (Merck) after labelling neutrophils using Antonow's technique [89,90].

NF-kB p65 cleavage assays
In vitro. p65 cleavage was assessed using sea bass peritoneal cell lysates, recombinant or in vitro translated 35 S-labeled sbp65Rel. To prepare cell lysates, cells were incubated in 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% (v/v) Triton X-100 and 10% (v/v) glycerol for 30 min on ice, briefly sonicated and centrifuged. The supernatant of 2610 6 cells was incubated with 1 mM of the indicated proteins for 2 h at 22uC and p65 cleavage evaluated by Western blotting using an anti-sea bass NF-kB p65 rabbit serum (produced using the peptide SIFNSGNPARFVS located at the Cterminal region of sea bass p65 as antigen). Recombinant sbp65Rel (7.5 mM) was incubated for 3 h at 22uC with 1 mM AIP56 and p65 cleavage evaluated by SDS-PAGE. In vitro translated 35 S-labeled sbp65Rel and 35 S-labeled sbp65Rel mutants (sbp65RelC39A, sbp65RelE40A, and sbp65C39E40AA) were incubated with 0.1, 1, 10 or 100 nM of the different proteins in 10 mM Tris-HCl pH 8.0, 150 mM NaCl and 10% (v/v) glycerol for 2 h at 22uC and p65 cleavage assessed by autoradiography. When specified, 1,10-phenanthroline (Sigma) was used at 5 mM.
Ex vivo. peritoneal leukocytes were treated for 2 or 4 h as described above in the section ''Apoptogenic activity assays'', collected by centrifugation, washed, resuspended in sbPBS and lysed by addition of SDS-PAGE sample buffer. Cleavage of p65 was evaluated by Western blotting, as described above.
Competition assays AIP56 AAIVAA , AIP56 1-285C262S and AIP56 286-497C298S were tested for their ability to inhibit AIP56's apoptogenic activity and AIP56-mediated p65 cleavage. Cells were pre-incubated for 15 min on ice with different concentrations (350 nM to 3.5 mM) of AIP56 AAIVAA , AIP56 1-285C262S , AIP56 286-497C298S or nicked AIP56, followed by incubation for further 15 min on ice with 8.75 nM of AIP56 in the presence of the competitors. Unbound proteins were removed by washing with ice cold supplemented L-15 medium [19] and the cells incubated at 22uC for 4 h.

Statistical analysis
Statistical analysis was performed using a randomized block design, where fish are treated as blocks and the concentration of the treatments/competitors as a factor. The data, percentage of apoptotic cells, have been transformed using the arcsine transformation. Post-hoc comparisons were performed using the Tukey's Honest Significant Difference test. Significance was defined for p,0.05. Figure S1 Schematic diagram of the primary structure of AIP56 and AIP56-related proteins. Grey: signal peptides (experimentally determined for AIP56 [21] and predicted for the remaining proteins using SignalP at http://www.cbs.dtu.dk/services/ SignalP/ [91,92]; Yellow: regions with high identity to NleC and AIP56 N-terminal catalytic domain; Green; regions with high identity to AIP56 linker polypeptide; Orange: regions with high identity to APSE-2 and AIP56 C-terminal domain; Red: zincmetalloprotease signature HEXXH; White: regions with low identity to AIP56 domains, NleC or APSE-2. Conserved zincmetalloprotease signature HEXXH, cysteine residues, and other signalled amino acids are represented at their relative positions. AIP56-related proteins were retrieved by Blast analysis of the AIP56 protein sequence against the non-redundant protein sequences database (updated from [23]). (TIF) Figure S2 Disruption of the zinc-metalloprotease signature does not induce major structural changes in AIP56. Native-PAGE (A) bar corresponds to the median and the lower and upper side of the boxes, the first and third quartiles; circles and diamonds signal extreme observations). When used at the same concentration, AIP56alk resulted in lower percentage of apoptotic cells than AIP56, except for the dose of 0.5 mg/ml, where no statistical differences were observed. doi:10.1371/journal.ppat.1003128.g004 and size exclusion chromatography (B) of AIP56 and AIP56 AAIVAA showing that disruption of the zinc-binding motif did not affect the monodispersity/stokes radius of the protein. In Native-PAGE, BSA electrophoretic mobility is shown for reference purposes. (C) Far-UV CD spectra of wild-type AIP56 (thick line) and AIP56 AAIVAA (thin line) showing that the secondary structure content of the toxin was also unaffected by the introduced mutations. (TIF) Materials and Methods S1 Additional details about the constructs used in this study, protein production and purification, protein quantification, PAGE and Western blotting, analysis of the Zinc content, analytical size exclusion chromatography, circular dichroism spectroscopy (CD), differential scanning fluorimetry (DSF), determination of AIP56 concentration in the plasma of infected fish, are provided as Supporting Materials and Methods. (DOCX)