| Chromobacterium violaceum is an emerging human pathogen that causes severe in-fection with a rapid disease course,high mortality,and multi-drug resistance.However,people know little about the pathogenic mechanism of this pathogen,which increases the difficulty of treating the diseases caused by the bacterium.Revealing the pathogenicity of Chromobacterium violaceum is one of the breakthroughs in preventing and curing infec-tious diseases.The most important virulence of Chromobacterium violaceum comes from its two sets of type III secretion systems,Cpi-1/-1a and Cpi-2,which can deliver the viru-lence factors called effectors into host cells to manipulate host cell signaling pathways,thus facilitating their survival and reproduction.This study screened a Cpi-1/-1a-encoded effector Cop C from Chromobacterium vio-laceum,which can inhibit the activity of caspase-3/-7 in host cells,and proved caspase-3/-7 as the direct targets of Cop C in the host.Cop C expressed in eukaryotes could inhibit the protease activity of caspase-3 while expressed in prokaryotes could not,suggesting that some eukaryotes-specific cofactors contribute to Cop C function.Subsequently,calmodulin(Ca M)was proved to be a host-specific cofactor of Cop C by IP-MS and biochemical veri-fication.Consistently,Cop C co-expressed with Ca M in prokaryotes could also inhibit the protease activity of caspase-3,and prokaryotically purified Ca M,Cop C,and caspase-3 pro-teins could form a stable ternary complex.The small subunit p12 of cleaved caspase-3 co-expressed with Cop C in eukaryotes has an upward shift,suggesting that Cop C may produce post-translational modifications to caspase-3.Both the total mass of caspase-3 co-expressed with Cop C in eukaryotes and co-expressed with Ca M and Cop C in prokaryotes generated a 524 Da mass increase analyzed by electrospray ionization mass spectrometry,which in-dicated that Cop C indeed produced a 524 Da-posttranslational modification to caspase-3.However,the 524 Da mass increase has never been previously reported,suggesting a novel posttranslational modification.In combination with in vitro biochemical experiments and mass spectrometry analysis,the 524 Da modification was confirmed to be attached to the arginine 207 of caspase-3.This modification involves deamination and cyclization from ADP-ribosylation,so it was named ADPR-deacylization,and ADPR-deacylization is re-sistant to the hydrolysis of ADP-ribosylhydrolases in host cells.We further revealed that Cop C can modify the conserved arginine of caspase-3/-/7-/8/-9 in their small subunits with ADPR-deacylization and inactivates their protease activity,thus inhibiting apoptosis and caspase-3-GSDME-mediated pyroptosis,and activating necroptosis.The homologous proteins of Cop C were found to widely exist in various bacteria through multiple sequence alignment analysis and functional verification,and Cop C represents a family of enzymes that catalyze ADPR-deacylization.Finally,the determination of the cryo-EM structures of Ca M-Cop C-caspase-3 ternary complex in pre-reaction,transition,and post-reaction states revealed the mechanisms by which Cop C recognizing ligand NAD~+,substrate caspase-3,and cofactor Ca M,and provided structural evidence for the process of Cop C-catalyzed ADPR-deacylization.This study identified a novel protein post-translational modification and is the first report that pathogen effectors can act on caspase-3/-7/-8/-9 in a post-translational modifi-cation manner.This study also provides new insights into the pathogenesis of Chromobac-terium violaceum and offers a potential target for the development of specific antibacterial drugs targeting the"pathogen-host"interaction process. |
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