The Apoptogenic Toxin AIP56 Is a Metalloprotease A-B Toxin that Cleaves NF-κb P65
Publication Date
February 28, 2013
Journal
PLOS Pathogens
Authors
Daniela S. Silva, Liliana M. G. Pereira, Ana R. Moreira, Frederico Ferreira Da Silva, et al
Volume
9
Issue
2
Pages
e1003128
DOI
https://dx.plos.org/10.1371/journal.ppat.1003128
Publisher URL
http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1003128
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/23468618
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585134
Europe PMC
http://europepmc.org/abstract/MED/23468618
Web of Science
000321863200001
Scopus
84880954175
Mendeley
http://www.mendeley.com/research/apoptogenic-toxin-aip56-metalloprotease-ab-toxin-cleaves-nf%CE%BAb-p65
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Mendeley | Further Information

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Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/973941"], "description"=>"<p>(<b>A</b>) AIP56<sup>1–285C262S</sup> and AIP56<sup>286–497C298S</sup> lack apoptogenic activity. Leukocytes collected from 5 animals were incubated with AIP56, AIP56<sup>1–285C262S</sup>, AIP56<sup>286–497C298S</sup>, or a mixture of AIP56<sup>1–285C262S</sup> and AIP56<sup>286–497C298S</sup> (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>B</b>) Delivery of AIP56 N-terminal domain into the cell's cytosol using <i>B. anthracis</i> 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 LF<sup>11–263</sup>•AIP56<sup>1–261</sup> or LF<sup>11–263</sup>•AIP56<sup>299–497</sup> 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+LF<sup>11–263</sup>•AIP56<sup>1–261</sup>. (<b>C</b>) AIP56 C-terminal domain is involved in toxin binding and entry into the target cells. AIP56<sup>AAIVAA</sup> and AIP56<sup>286–497C298S</sup>, but not AIP56<sup>1–285C262S</sup>, inhibit AIP56-associated p65 cleavage and apoptogenic activity. Leukocytes collected from 7 fish were incubated with AIP56<sup>AAIVAA</sup>, AIP56<sup>1–285C262S</sup> or AIP56<sup>286–497C298S</sup> 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.</p>", "links"=>[], "tags"=>["n-terminal", "catalytic", "c-terminal", "binding"], "article_id"=>642137, "categories"=>["Microbiology"], "users"=>["Daniela S. Silva", "Liliana M. G. Pereira", "Ana R. Moreira", "Frederico Ferreira-da-Silva", "Rui M. Brito", "Tiago Q. Faria", "Irene Zornetta", "Cesare Montecucco", "Pedro Oliveira", "Jorge E. Azevedo", "Pedro J. B. Pereira", "Sandra Macedo-Ribeiro", "Ana do Vale", "Nuno M. S. dos Santos"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003128.g003", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AIP56_N_terminal_domain_plays_the_catalytic_role_and_the_C_terminal_domain_is_involved_in_binding_and_entry_into_cells_/642137", "title"=>"AIP56 N-terminal domain plays the catalytic role and the C-terminal domain is involved in binding and entry into cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 12:43:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/973929"], "description"=>"<p>(<b>A</b>) Disruption of the zinc-metalloprotease signature abolishes AIP56 apoptogenic activity. Sea bass peritoneal leukocytes collected from 5 animals were incubated with AIP56 or AIP56<sup>AAIVAA</sup>, 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 AIP56<sup>AAIVAA</sup> and in mock-treated cells. The percentage of apoptotic phagocytes, determined by morphological analysis, is depicted in the dot plot. (<b>B</b>) Incubation of cell lysates with AIP56, AIP56<sup>1–285C262S</sup> 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. (<b>C</b>) AIP56 cleaves NF-κB p65 at the Cys<sup>39</sup>-Glu<sup>40</sup> 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 E<sup>40</sup>GRSA<sup>44</sup> showing that cleavage occurred after the conserved C<sup>39</sup>. (<b>D</b>) Incubation of leukocytes with AIP56, but not with AIP56<sup>AAIVAA</sup>, AIP56<sup>1–285C262S</sup>, AIP56<sup>286–497C298S</sup>, 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. (<b>E</b>) 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.</p>", "links"=>[], "tags"=>["zinc-metalloprotease", "cleaves", "p65", "peptide"], "article_id"=>642130, "categories"=>["Microbiology"], "users"=>["Daniela S. Silva", "Liliana M. G. Pereira", "Ana R. Moreira", "Frederico Ferreira-da-Silva", "Rui M. Brito", "Tiago Q. Faria", "Irene Zornetta", "Cesare Montecucco", "Pedro Oliveira", "Jorge E. Azevedo", "Pedro J. B. Pereira", "Sandra Macedo-Ribeiro", "Ana do Vale", "Nuno M. S. dos Santos"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003128.g001", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AIP56_is_a_zinc_metalloprotease_that_cleaves_NF_B_p65_at_the_Cys_39_Glu_40_peptide_bond_/642130", "title"=>"AIP56 is a zinc-metalloprotease that cleaves NF-κB p65 at the Cys<sup>39</sup>-Glu<sup>40</sup> peptide bond.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 12:41:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/973970", "https://ndownloader.figshare.com/files/973978", "https://ndownloader.figshare.com/files/973979", "https://ndownloader.figshare.com/files/973981", "https://ndownloader.figshare.com/files/973984", "https://ndownloader.figshare.com/files/973987", "https://ndownloader.figshare.com/files/973993", "https://ndownloader.figshare.com/files/974001"], "description"=>"<div><p>AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of <i>Photobacterium damselae piscicida</i> (<i>Phdp</i>), 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, <i>in vivo</i>, 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<sup>39</sup>-Glu<sup>40</sup> peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.</p> </div>", "links"=>[], "tags"=>["apoptogenic", "toxin", "aip56", "metalloprotease", "a-b", "cleaves", "p65"], "article_id"=>642155, "categories"=>["Microbiology"], "users"=>["Daniela S. Silva", "Liliana M. G. Pereira", "Ana R. Moreira", "Frederico Ferreira-da-Silva", "Rui M. Brito", "Tiago Q. Faria", "Irene Zornetta", "Cesare Montecucco", "Pedro Oliveira", "Jorge E. Azevedo", "Pedro J. B. Pereira", "Sandra Macedo-Ribeiro", "Ana do Vale", "Nuno M. S. dos Santos"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003128.s001", "https://dx.doi.org/10.1371/journal.ppat.1003128.s002", "https://dx.doi.org/10.1371/journal.ppat.1003128.s003", "https://dx.doi.org/10.1371/journal.ppat.1003128.s004", "https://dx.doi.org/10.1371/journal.ppat.1003128.s005", "https://dx.doi.org/10.1371/journal.ppat.1003128.s006", "https://dx.doi.org/10.1371/journal.ppat.1003128.s007", "https://dx.doi.org/10.1371/journal.ppat.1003128.s008"], "stats"=>{"downloads"=>3, "page_views"=>31, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_Apoptogenic_Toxin_AIP56_Is_a_Metalloprotease_A_B_Toxin_that_Cleaves_NF_b_P65__/642155", "title"=>"The Apoptogenic Toxin AIP56 Is a Metalloprotease A-B Toxin that Cleaves NF-κb P65", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-03-01 12:46:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/973933"], "description"=>"<p>(<b>A</b>) Limited proteolysis of AIP56 with chymotrypsin and proteinase K produces two major fragments. AIP56 (0.6 mg/ml) was incubated with 0.25, 1.25, 6.25 or 25 µg/ml of chymotrypsin, trypsin and proteinase K for 30 min on ice and digests analysed by reducing SDS-PAGE. The proteases (marked as *) and undigested AIP56 (marked as **) were loaded as controls. (<b>B</b>) The two AIP56 digestion fragments are linked by a disulphide bridge. AIP56 was incubated with or without 25 µg/ml chymotrypsin (Chym) for 30 min on ice and digests analysed under reducing (+DTT) or non-reducing (−DTT) SDS-PAGE. Numbers to the left and right of the panels refer to the position and mass of the molecular weight markers, in kDa. (<b>C</b>) Schematic representation of AIP56. (<b>D</b>) Far-UV CD spectra of AIP56 (thick solid line), AIP56<sup>1–285</sup> (thin solid line), AIP56<sup>286–497</sup> (thin dashed line) and the weighted sum of AIP56<sup>1–285</sup> and AIP56<sup>286–497</sup> spectra (thick dashed line).</p>", "links"=>[], "tags"=>["composed", "domains", "linked", "disulphide"], "article_id"=>642133, "categories"=>["Microbiology"], "users"=>["Daniela S. Silva", "Liliana M. G. Pereira", "Ana R. Moreira", "Frederico Ferreira-da-Silva", "Rui M. Brito", "Tiago Q. Faria", "Irene Zornetta", "Cesare Montecucco", "Pedro Oliveira", "Jorge E. Azevedo", "Pedro J. B. Pereira", "Sandra Macedo-Ribeiro", "Ana do Vale", "Nuno M. S. dos Santos"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003128.g002", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AIP56_is_composed_of_two_domains_linked_by_a_disulphide_bridge_/642133", "title"=>"AIP56 is composed of two domains linked by a disulphide bridge.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 12:42:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/973949"], "description"=>"<p>(<b>A</b>) 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>B</b>) Nicked and alkylated AIP56 (AIP56alk) display proteolytic activity <i>in vitro</i> in the same dose range as AIP56. <sup>35</sup>S-labeled sbp65Rel was incubated for 2 h at 22°C with wild type, nicked or alkylated AIP56 and cleavage assessed by autoradiography. (<b>C</b>) 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 AIP56<sup>AAIVAA</sup>, AIP56<sup>1–285</sup> or AIP56<sup>286–497</sup> 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. (<b>D</b>) Disruption of the disulphide bridge linking Cys<sup>262</sup> and Cys<sup>298</sup> 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.</p>", "links"=>[], "tags"=>["toxicity", "requires", "linker", "disulfide", "dispensable"], "article_id"=>642139, "categories"=>["Microbiology"], "users"=>["Daniela S. Silva", "Liliana M. G. Pereira", "Ana R. Moreira", "Frederico Ferreira-da-Silva", "Rui M. Brito", "Tiago Q. Faria", "Irene Zornetta", "Cesare Montecucco", "Pedro Oliveira", "Jorge E. Azevedo", "Pedro J. B. Pereira", "Sandra Macedo-Ribeiro", "Ana do Vale", "Nuno M. S. dos Santos"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003128.g004", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_AIP56_toxicity_requires_integrity_of_the_linker_but_the_disulfide_bridge_is_dispensable_for_intoxication_/642139", "title"=>"AIP56 toxicity requires integrity of the linker but the disulfide bridge is dispensable for intoxication.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 12:44:13"}

PMC Usage Stats | Further Information

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Relative Metric

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