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

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

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

“…Interestingly, AIP56 seems to have originated from a fusion of two components: one related to NleC, which is a type III secreted effector present in several enteric pathogenic bacteria (14) associated with human illness and death worldwide (15), and another related to a protein of unknown function from the bacteriophage APSE2. In line with this, we recently found that AIP56 is an AB-type toxin, possessing a catalytic A domain at its N terminus, homologous to NleC, and a B domain involved in binding/internalization into target cells at its C-terminal region, homologous to APSE2 (16). The catalytic domain of AIP56 is a zinc-dependent metalloprotease that, similarly to NleC, cleaves NF-B, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes, playing a key role in host responses to microbial pathogen invasion (17).…”

“…The catalytic domain of AIP56 is a zinc-dependent metalloprotease that, similarly to NleC, cleaves NF-B, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes, playing a key role in host responses to microbial pathogen invasion (17). We have shown that during intoxication of sea bass peritoneal leukocytes (sbPL), the catalytic activity of AIP56 results in the depletion of NF-B p65 (16), and this likely explains the disseminated apoptosis observed during P. damselae subsp. piscicida infections.…”

“…4051) were from Cell Signaling. The anti-sea bass NF-B p65 rabbit serum was produced using the peptide SIFNSGNPARFVS, located at the C-terminal region of sea bass p65, as the antigen, as described previously (16). Sheep anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (AP311) and sheep anti-mouse HRP-conjugated secondary antibody (AP271) were from The Binding Site; goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (A9919) and goat antimouse alkaline phosphatase-conjugated secondary antibody (A2429) were from Sigma-Aldrich.…”

“…286 -497C298S , and LF 11-263 · AIP56 were produced and purified as previously described (16). Briefly, AIP56, AIP56 286 -497C298S , and LF 11-263 · AIP56 were purified from the soluble fraction of induced E. coli cells by metal affinity chromatography.…”

“…The PCR products were subcloned into pGEM-T Easy vector (Promega) and cloned into the pET28a(ϩ) plasmid (Promega), yielding the plasmids pET28aAIP56MycHϩ and pET28aAIP56V5Hϩ. AIP56Myc and AIP56V5 were expressed in Escherichia coli BL21(DE3) and purified following the protocol used for AIP56 (16). Recombinant proteins were analyzed by SDS-PAGE (see Fig.…”