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The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

Silva DS, Pereira LM, Moreira AR, Ferreira-da-Silva F, Brito RM, Faria TQ, Zornetta I, Montecucco C, Oliveira P, Azevedo JE, Pereira PJ, Macedo-Ribeiro S, do Vale A, dos Santos NM - PLoS Pathog. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Fish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

ABSTRACT
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.

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AIP56 is a zinc-metalloprotease that cleaves NF-κB p65 at the Cys39-Glu40 peptide bond.(A) Disruption of the zinc-metalloprotease signature abolishes AIP56 apoptogenic activity. Sea bass peritoneal leukocytes collected from 5 animals were incubated with AIP56 or AIP56AAIVAA, for 4 h at 22°C. Mock-treated cells were used as controls. Images shown are representative cytospin preparations stained with Antonow's for labelling neutrophils (brown) followed by Hemacolor. Note the presence of several apoptotic cells (arrowheads) in the sample incubated with AIP56 and their absence in cells incubated with AIP56AAIVAA and in mock-treated cells. The percentage of apoptotic phagocytes, determined by morphological analysis, is depicted in the dot plot. (B) Incubation of cell lysates with AIP56, AIP561–285C262S and nicked AIP56 resulted in p65 cleavage. Lysates were incubated for 2 h at 22°C with 1 µM of the indicated proteins in the presence or absence of the metalloprotease inhibitor 1,10-phenanthroline (O-phe) and p65 cleavage assessed by Western blotting. (C) AIP56 cleaves NF-κB p65 at the Cys39-Glu40 peptide bond. Recombinant sea bass p65Rel (7.5 µM) was incubated in the presence or absence of 1 µM of AIP56 for 3 h at 22°C and analysed by SDS-PAGE. Edman degradation of the cleaved sbp65Rel (cl-sbp65Rel) identified the sequence E40GRSA44 showing that cleavage occurred after the conserved C39. (D) Incubation of leukocytes with AIP56, but not with AIP56AAIVAA, AIP561–285C262S, AIP56286–497C298S, nicked AIP56 (AIP56nic) or reconstituted AIP56 (AIP56rct) leads to p65 depletion. Leukocytes were incubated with 10 µg/ml of the indicated proteins for 2 h at 22°C and p65 cleavage was assessed by Western blotting. (E) AIP56-mediated p65 cleavage is caspase-independent. Leukocytes were incubated with 2 µg/ml AIP56 in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK for 2 h at 22°C, 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.
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ppat-1003128-g001: AIP56 is a zinc-metalloprotease that cleaves NF-κB p65 at the Cys39-Glu40 peptide bond.(A) Disruption of the zinc-metalloprotease signature abolishes AIP56 apoptogenic activity. Sea bass peritoneal leukocytes collected from 5 animals were incubated with AIP56 or AIP56AAIVAA, for 4 h at 22°C. Mock-treated cells were used as controls. Images shown are representative cytospin preparations stained with Antonow's for labelling neutrophils (brown) followed by Hemacolor. Note the presence of several apoptotic cells (arrowheads) in the sample incubated with AIP56 and their absence in cells incubated with AIP56AAIVAA and in mock-treated cells. The percentage of apoptotic phagocytes, determined by morphological analysis, is depicted in the dot plot. (B) Incubation of cell lysates with AIP56, AIP561–285C262S and nicked AIP56 resulted in p65 cleavage. Lysates were incubated for 2 h at 22°C with 1 µM of the indicated proteins in the presence or absence of the metalloprotease inhibitor 1,10-phenanthroline (O-phe) and p65 cleavage assessed by Western blotting. (C) AIP56 cleaves NF-κB p65 at the Cys39-Glu40 peptide bond. Recombinant sea bass p65Rel (7.5 µM) was incubated in the presence or absence of 1 µM of AIP56 for 3 h at 22°C and analysed by SDS-PAGE. Edman degradation of the cleaved sbp65Rel (cl-sbp65Rel) identified the sequence E40GRSA44 showing that cleavage occurred after the conserved C39. (D) Incubation of leukocytes with AIP56, but not with AIP56AAIVAA, AIP561–285C262S, AIP56286–497C298S, nicked AIP56 (AIP56nic) or reconstituted AIP56 (AIP56rct) leads to p65 depletion. Leukocytes were incubated with 10 µg/ml of the indicated proteins for 2 h at 22°C and p65 cleavage was assessed by Western blotting. (E) AIP56-mediated p65 cleavage is caspase-independent. Leukocytes were incubated with 2 µg/ml AIP56 in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK for 2 h at 22°C, 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.

Mentions: In order to clarify the role played by the zinc metalloprotease activity of AIP56, a mutant (AIP56AAIVAA) 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 AIP56AAIVAA, while in AIP56 equimolar amounts of zinc (0.93±0.04 mol zinc/mol protein) were present. When tested ex vivo, AIP56AAIVAA 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.


The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

Silva DS, Pereira LM, Moreira AR, Ferreira-da-Silva F, Brito RM, Faria TQ, Zornetta I, Montecucco C, Oliveira P, Azevedo JE, Pereira PJ, Macedo-Ribeiro S, do Vale A, dos Santos NM - PLoS Pathog. (2013)

AIP56 is a zinc-metalloprotease that cleaves NF-κB p65 at the Cys39-Glu40 peptide bond.(A) Disruption of the zinc-metalloprotease signature abolishes AIP56 apoptogenic activity. Sea bass peritoneal leukocytes collected from 5 animals were incubated with AIP56 or AIP56AAIVAA, for 4 h at 22°C. Mock-treated cells were used as controls. Images shown are representative cytospin preparations stained with Antonow's for labelling neutrophils (brown) followed by Hemacolor. Note the presence of several apoptotic cells (arrowheads) in the sample incubated with AIP56 and their absence in cells incubated with AIP56AAIVAA and in mock-treated cells. The percentage of apoptotic phagocytes, determined by morphological analysis, is depicted in the dot plot. (B) Incubation of cell lysates with AIP56, AIP561–285C262S and nicked AIP56 resulted in p65 cleavage. Lysates were incubated for 2 h at 22°C with 1 µM of the indicated proteins in the presence or absence of the metalloprotease inhibitor 1,10-phenanthroline (O-phe) and p65 cleavage assessed by Western blotting. (C) AIP56 cleaves NF-κB p65 at the Cys39-Glu40 peptide bond. Recombinant sea bass p65Rel (7.5 µM) was incubated in the presence or absence of 1 µM of AIP56 for 3 h at 22°C and analysed by SDS-PAGE. Edman degradation of the cleaved sbp65Rel (cl-sbp65Rel) identified the sequence E40GRSA44 showing that cleavage occurred after the conserved C39. (D) Incubation of leukocytes with AIP56, but not with AIP56AAIVAA, AIP561–285C262S, AIP56286–497C298S, nicked AIP56 (AIP56nic) or reconstituted AIP56 (AIP56rct) leads to p65 depletion. Leukocytes were incubated with 10 µg/ml of the indicated proteins for 2 h at 22°C and p65 cleavage was assessed by Western blotting. (E) AIP56-mediated p65 cleavage is caspase-independent. Leukocytes were incubated with 2 µg/ml AIP56 in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK for 2 h at 22°C, 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585134&req=5

ppat-1003128-g001: AIP56 is a zinc-metalloprotease that cleaves NF-κB p65 at the Cys39-Glu40 peptide bond.(A) Disruption of the zinc-metalloprotease signature abolishes AIP56 apoptogenic activity. Sea bass peritoneal leukocytes collected from 5 animals were incubated with AIP56 or AIP56AAIVAA, for 4 h at 22°C. Mock-treated cells were used as controls. Images shown are representative cytospin preparations stained with Antonow's for labelling neutrophils (brown) followed by Hemacolor. Note the presence of several apoptotic cells (arrowheads) in the sample incubated with AIP56 and their absence in cells incubated with AIP56AAIVAA and in mock-treated cells. The percentage of apoptotic phagocytes, determined by morphological analysis, is depicted in the dot plot. (B) Incubation of cell lysates with AIP56, AIP561–285C262S and nicked AIP56 resulted in p65 cleavage. Lysates were incubated for 2 h at 22°C with 1 µM of the indicated proteins in the presence or absence of the metalloprotease inhibitor 1,10-phenanthroline (O-phe) and p65 cleavage assessed by Western blotting. (C) AIP56 cleaves NF-κB p65 at the Cys39-Glu40 peptide bond. Recombinant sea bass p65Rel (7.5 µM) was incubated in the presence or absence of 1 µM of AIP56 for 3 h at 22°C and analysed by SDS-PAGE. Edman degradation of the cleaved sbp65Rel (cl-sbp65Rel) identified the sequence E40GRSA44 showing that cleavage occurred after the conserved C39. (D) Incubation of leukocytes with AIP56, but not with AIP56AAIVAA, AIP561–285C262S, AIP56286–497C298S, nicked AIP56 (AIP56nic) or reconstituted AIP56 (AIP56rct) leads to p65 depletion. Leukocytes were incubated with 10 µg/ml of the indicated proteins for 2 h at 22°C and p65 cleavage was assessed by Western blotting. (E) AIP56-mediated p65 cleavage is caspase-independent. Leukocytes were incubated with 2 µg/ml AIP56 in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK for 2 h at 22°C, 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.
Mentions: In order to clarify the role played by the zinc metalloprotease activity of AIP56, a mutant (AIP56AAIVAA) 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 AIP56AAIVAA, while in AIP56 equimolar amounts of zinc (0.93±0.04 mol zinc/mol protein) were present. When tested ex vivo, AIP56AAIVAA 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Fish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus