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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 toxicity requires integrity of the linker but the disulfide bridge is dispensable for intoxication.(A) Nicked and reconstituted AIP56 are not apoptogenic. Leukocytes collected from 3 animals were incubated with nicked or reconstituted AIP56 (AIP56nic and AIP56rct, respectively) for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Cells treated with AIP56 and mock-treated cells were used as positive and negative controls, respectively. (B) Nicked and alkylated AIP56 (AIP56alk) display proteolytic activity in vitro in the same dose range as AIP56. 35S-labeled sbp65Rel was incubated for 2 h at 22°C with wild type, nicked or alkylated AIP56 and cleavage assessed by autoradiography. (C) Nicked AIP56 competes with intact AIP56 and inhibits its toxicity. Leukocytes collected from 3 animals were incubated with 3.5 µM nicked AIP56 (AIP56nic) for 15 minutes on ice followed by further 15 min incubation on ice with 8.75 nM of AIP56. Mock-treated cells, cells incubated with 8.75 nM AIP56, or cells incubated with 3.5 µM of AIP56AAIVAA, AIP561–285 or AIP56286–497 before incubation with 8.75 nM of AIP56 were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h. The percentage of apoptotic cells was determined by morphological analysis of cytospin preparations stained with Hemacolor and the p65 cleavage was assessed by Western blotting. (D) Disruption of the disulphide bridge linking Cys262 and Cys298 partially compromises AIP56 toxicity. Leukocytes collected from 5 animals were incubated with AIP56 or AIP56alk for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Left panel presents the box plot of percentage of apopotic cells (the middle 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 µg/ml, where no statistical differences were observed.
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ppat-1003128-g004: AIP56 toxicity requires integrity of the linker but the disulfide bridge is dispensable for intoxication.(A) Nicked and reconstituted AIP56 are not apoptogenic. Leukocytes collected from 3 animals were incubated with nicked or reconstituted AIP56 (AIP56nic and AIP56rct, respectively) for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Cells treated with AIP56 and mock-treated cells were used as positive and negative controls, respectively. (B) Nicked and alkylated AIP56 (AIP56alk) display proteolytic activity in vitro in the same dose range as AIP56. 35S-labeled sbp65Rel was incubated for 2 h at 22°C with wild type, nicked or alkylated AIP56 and cleavage assessed by autoradiography. (C) Nicked AIP56 competes with intact AIP56 and inhibits its toxicity. Leukocytes collected from 3 animals were incubated with 3.5 µM nicked AIP56 (AIP56nic) for 15 minutes on ice followed by further 15 min incubation on ice with 8.75 nM of AIP56. Mock-treated cells, cells incubated with 8.75 nM AIP56, or cells incubated with 3.5 µM of AIP56AAIVAA, AIP561–285 or AIP56286–497 before incubation with 8.75 nM of AIP56 were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h. The percentage of apoptotic cells was determined by morphological analysis of cytospin preparations stained with Hemacolor and the p65 cleavage was assessed by Western blotting. (D) Disruption of the disulphide bridge linking Cys262 and Cys298 partially compromises AIP56 toxicity. Leukocytes collected from 5 animals were incubated with AIP56 or AIP56alk for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Left panel presents the box plot of percentage of apopotic cells (the middle 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 µg/ml, where no statistical differences were observed.

Mentions: 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 AIP561–285/AIP56286–497 along with trace amounts of AIP561–285 and of AIP56286–497 homodimers and monomers (Figure S6A). Although nicking abolished cellular toxicity, it did not induce major structural changes (Figure S6B) and only a 1°C decrease in Tm (39±0.13°C for AIP56 and 38±0.25°C for nicked AIP56; mean±SD 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].


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 toxicity requires integrity of the linker but the disulfide bridge is dispensable for intoxication.(A) Nicked and reconstituted AIP56 are not apoptogenic. Leukocytes collected from 3 animals were incubated with nicked or reconstituted AIP56 (AIP56nic and AIP56rct, respectively) for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Cells treated with AIP56 and mock-treated cells were used as positive and negative controls, respectively. (B) Nicked and alkylated AIP56 (AIP56alk) display proteolytic activity in vitro in the same dose range as AIP56. 35S-labeled sbp65Rel was incubated for 2 h at 22°C with wild type, nicked or alkylated AIP56 and cleavage assessed by autoradiography. (C) Nicked AIP56 competes with intact AIP56 and inhibits its toxicity. Leukocytes collected from 3 animals were incubated with 3.5 µM nicked AIP56 (AIP56nic) for 15 minutes on ice followed by further 15 min incubation on ice with 8.75 nM of AIP56. Mock-treated cells, cells incubated with 8.75 nM AIP56, or cells incubated with 3.5 µM of AIP56AAIVAA, AIP561–285 or AIP56286–497 before incubation with 8.75 nM of AIP56 were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h. The percentage of apoptotic cells was determined by morphological analysis of cytospin preparations stained with Hemacolor and the p65 cleavage was assessed by Western blotting. (D) Disruption of the disulphide bridge linking Cys262 and Cys298 partially compromises AIP56 toxicity. Leukocytes collected from 5 animals were incubated with AIP56 or AIP56alk for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Left panel presents the box plot of percentage of apopotic cells (the middle 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 µg/ml, where no statistical differences were observed.
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Related In: Results  -  Collection

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ppat-1003128-g004: AIP56 toxicity requires integrity of the linker but the disulfide bridge is dispensable for intoxication.(A) Nicked and reconstituted AIP56 are not apoptogenic. Leukocytes collected from 3 animals were incubated with nicked or reconstituted AIP56 (AIP56nic and AIP56rct, respectively) for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Cells treated with AIP56 and mock-treated cells were used as positive and negative controls, respectively. (B) Nicked and alkylated AIP56 (AIP56alk) display proteolytic activity in vitro in the same dose range as AIP56. 35S-labeled sbp65Rel was incubated for 2 h at 22°C with wild type, nicked or alkylated AIP56 and cleavage assessed by autoradiography. (C) Nicked AIP56 competes with intact AIP56 and inhibits its toxicity. Leukocytes collected from 3 animals were incubated with 3.5 µM nicked AIP56 (AIP56nic) for 15 minutes on ice followed by further 15 min incubation on ice with 8.75 nM of AIP56. Mock-treated cells, cells incubated with 8.75 nM AIP56, or cells incubated with 3.5 µM of AIP56AAIVAA, AIP561–285 or AIP56286–497 before incubation with 8.75 nM of AIP56 were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h. The percentage of apoptotic cells was determined by morphological analysis of cytospin preparations stained with Hemacolor and the p65 cleavage was assessed by Western blotting. (D) Disruption of the disulphide bridge linking Cys262 and Cys298 partially compromises AIP56 toxicity. Leukocytes collected from 5 animals were incubated with AIP56 or AIP56alk for 4 h at 22°C and the percentage of apoptotic cells determined by morphological analysis of cytospin preparations stained with Hemacolor. Left panel presents the box plot of percentage of apopotic cells (the middle 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 µg/ml, where no statistical differences were observed.
Mentions: 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 AIP561–285/AIP56286–497 along with trace amounts of AIP561–285 and of AIP56286–497 homodimers and monomers (Figure S6A). Although nicking abolished cellular toxicity, it did not induce major structural changes (Figure S6B) and only a 1°C decrease in Tm (39±0.13°C for AIP56 and 38±0.25°C for nicked AIP56; mean±SD 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].

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