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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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Related in: MedlinePlus

AIP56 N-terminal domain plays the catalytic role and the C-terminal domain is involved in binding and entry into cells.(A) AIP561–285C262S and AIP56286–497C298S lack apoptogenic activity. Leukocytes collected from 5 animals were incubated with AIP56, AIP561–285C262S, AIP56286–497C298S, or a mixture of AIP561–285C262S and AIP56286–497C298S (50 µg/ml each) for 4 h at 22°C. The percentage of apoptotic phagocytes was determined by morphological analysis of cytospin preparations stained with Hemacolor. (B) Delivery of AIP56 N-terminal domain into the cell's cytosol using B. anthracis LF/PA system reproduces the activity of full length AIP56. Leukocytes from 4 animals were incubated for 4 h at 22°C with 20 nM LF11–263•AIP561–261 or LF11–263•AIP56299–497 in the presence of 10 nM PA. Cells incubated with 2 µg/ml (35 nM) AIP56 were used as positive control. Cleavage of NF-κB p65 was detected by Western blotting and the occurrence of apoptosis by morphological analysis of cytospin preparations stained with Hemacolor. Note the presence of several apoptotic cells in the samples incubated with PA+LF11–263•AIP561–261. (C) AIP56 C-terminal domain is involved in toxin binding and entry into the target cells. AIP56AAIVAA and AIP56286–497C298S, but not AIP561–285C262S, inhibit AIP56-associated p65 cleavage and apoptogenic activity. Leukocytes collected from 7 fish were incubated with AIP56AAIVAA, AIP561–285C262S or AIP56286–497C298S at final concentrations of 0.35, 1.75 or 3.5 µM for 15 min on ice, followed by further 15 min incubation on ice with 8.75 nM (0.5 µg/ml) AIP56 in the presence of the competitors. The competitor:AIP56 molar ratios are indicated. Cells incubated with AIP56 in the absence of competitors or with 3.5 µM of each competitor alone were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h prior to determination of the percentage of apoptotic cells 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 signal extreme observations). The inhibitory effect of the highest dose of each competitor upon AIP56-mediated cleavage of p65 was assessed by Western blotting.
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ppat-1003128-g003: AIP56 N-terminal domain plays the catalytic role and the C-terminal domain is involved in binding and entry into cells.(A) AIP561–285C262S and AIP56286–497C298S lack apoptogenic activity. Leukocytes collected from 5 animals were incubated with AIP56, AIP561–285C262S, AIP56286–497C298S, or a mixture of AIP561–285C262S and AIP56286–497C298S (50 µg/ml each) for 4 h at 22°C. The percentage of apoptotic phagocytes was determined by morphological analysis of cytospin preparations stained with Hemacolor. (B) Delivery of AIP56 N-terminal domain into the cell's cytosol using B. anthracis LF/PA system reproduces the activity of full length AIP56. Leukocytes from 4 animals were incubated for 4 h at 22°C with 20 nM LF11–263•AIP561–261 or LF11–263•AIP56299–497 in the presence of 10 nM PA. Cells incubated with 2 µg/ml (35 nM) AIP56 were used as positive control. Cleavage of NF-κB p65 was detected by Western blotting and the occurrence of apoptosis by morphological analysis of cytospin preparations stained with Hemacolor. Note the presence of several apoptotic cells in the samples incubated with PA+LF11–263•AIP561–261. (C) AIP56 C-terminal domain is involved in toxin binding and entry into the target cells. AIP56AAIVAA and AIP56286–497C298S, but not AIP561–285C262S, inhibit AIP56-associated p65 cleavage and apoptogenic activity. Leukocytes collected from 7 fish were incubated with AIP56AAIVAA, AIP561–285C262S or AIP56286–497C298S at final concentrations of 0.35, 1.75 or 3.5 µM for 15 min on ice, followed by further 15 min incubation on ice with 8.75 nM (0.5 µg/ml) AIP56 in the presence of the competitors. The competitor:AIP56 molar ratios are indicated. Cells incubated with AIP56 in the absence of competitors or with 3.5 µM of each competitor alone were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h prior to determination of the percentage of apoptotic cells 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 signal extreme observations). The inhibitory effect of the highest dose of each competitor upon AIP56-mediated cleavage of p65 was assessed by Western blotting.

Mentions: To test the catalytic activities of the AIP56 N- and C-terminal domains, AIP561–285C262S and AIP56286–497C298S were incubated with fish leukocyte lysates (Figure 1B) or with in vitro translated 35S-labeled 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 C-terminal 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 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 N-terminal domain plays the catalytic role and the C-terminal domain is involved in binding and entry into cells.(A) AIP561–285C262S and AIP56286–497C298S lack apoptogenic activity. Leukocytes collected from 5 animals were incubated with AIP56, AIP561–285C262S, AIP56286–497C298S, or a mixture of AIP561–285C262S and AIP56286–497C298S (50 µg/ml each) for 4 h at 22°C. The percentage of apoptotic phagocytes was determined by morphological analysis of cytospin preparations stained with Hemacolor. (B) Delivery of AIP56 N-terminal domain into the cell's cytosol using B. anthracis LF/PA system reproduces the activity of full length AIP56. Leukocytes from 4 animals were incubated for 4 h at 22°C with 20 nM LF11–263•AIP561–261 or LF11–263•AIP56299–497 in the presence of 10 nM PA. Cells incubated with 2 µg/ml (35 nM) AIP56 were used as positive control. Cleavage of NF-κB p65 was detected by Western blotting and the occurrence of apoptosis by morphological analysis of cytospin preparations stained with Hemacolor. Note the presence of several apoptotic cells in the samples incubated with PA+LF11–263•AIP561–261. (C) AIP56 C-terminal domain is involved in toxin binding and entry into the target cells. AIP56AAIVAA and AIP56286–497C298S, but not AIP561–285C262S, inhibit AIP56-associated p65 cleavage and apoptogenic activity. Leukocytes collected from 7 fish were incubated with AIP56AAIVAA, AIP561–285C262S or AIP56286–497C298S at final concentrations of 0.35, 1.75 or 3.5 µM for 15 min on ice, followed by further 15 min incubation on ice with 8.75 nM (0.5 µg/ml) AIP56 in the presence of the competitors. The competitor:AIP56 molar ratios are indicated. Cells incubated with AIP56 in the absence of competitors or with 3.5 µM of each competitor alone were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h prior to determination of the percentage of apoptotic cells 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 signal extreme observations). The inhibitory effect of the highest dose of each competitor upon AIP56-mediated cleavage of p65 was assessed by Western blotting.
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Related In: Results  -  Collection

Show All Figures
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ppat-1003128-g003: AIP56 N-terminal domain plays the catalytic role and the C-terminal domain is involved in binding and entry into cells.(A) AIP561–285C262S and AIP56286–497C298S lack apoptogenic activity. Leukocytes collected from 5 animals were incubated with AIP56, AIP561–285C262S, AIP56286–497C298S, or a mixture of AIP561–285C262S and AIP56286–497C298S (50 µg/ml each) for 4 h at 22°C. The percentage of apoptotic phagocytes was determined by morphological analysis of cytospin preparations stained with Hemacolor. (B) Delivery of AIP56 N-terminal domain into the cell's cytosol using B. anthracis LF/PA system reproduces the activity of full length AIP56. Leukocytes from 4 animals were incubated for 4 h at 22°C with 20 nM LF11–263•AIP561–261 or LF11–263•AIP56299–497 in the presence of 10 nM PA. Cells incubated with 2 µg/ml (35 nM) AIP56 were used as positive control. Cleavage of NF-κB p65 was detected by Western blotting and the occurrence of apoptosis by morphological analysis of cytospin preparations stained with Hemacolor. Note the presence of several apoptotic cells in the samples incubated with PA+LF11–263•AIP561–261. (C) AIP56 C-terminal domain is involved in toxin binding and entry into the target cells. AIP56AAIVAA and AIP56286–497C298S, but not AIP561–285C262S, inhibit AIP56-associated p65 cleavage and apoptogenic activity. Leukocytes collected from 7 fish were incubated with AIP56AAIVAA, AIP561–285C262S or AIP56286–497C298S at final concentrations of 0.35, 1.75 or 3.5 µM for 15 min on ice, followed by further 15 min incubation on ice with 8.75 nM (0.5 µg/ml) AIP56 in the presence of the competitors. The competitor:AIP56 molar ratios are indicated. Cells incubated with AIP56 in the absence of competitors or with 3.5 µM of each competitor alone were used as controls. Cells were washed, transferred to 22°C and incubated for 4 h prior to determination of the percentage of apoptotic cells 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 signal extreme observations). The inhibitory effect of the highest dose of each competitor upon AIP56-mediated cleavage of p65 was assessed by Western blotting.
Mentions: To test the catalytic activities of the AIP56 N- and C-terminal domains, AIP561–285C262S and AIP56286–497C298S were incubated with fish leukocyte lysates (Figure 1B) or with in vitro translated 35S-labeled 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 C-terminal 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.

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