Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination

•C. violaceum blocks host ubiquitination via the type III effector CteC•CteC is an ADP-ribosyltransferase that specifically modifies Ub on T66•Threonine ADP-ribosylation of Ub by CteC disrupts host ubiquitin signaling•CteC represents a family of bacterial effector proteins that ADP-ribosylate ubiquitin Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens. Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens. Ubiquitination is a prevalent posttranslational modification in eukaryotic cells (Kerscher et al., 2006Kerscher O. Felberbaum R. Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins.Annu. Rev. Cell Dev. Biol. 2006; 22: 159-180Crossref PubMed Scopus (1140) Google Scholar). Ubiquitin (Ub) is first activated by the Ub-activating enzyme E1 in an ATP-dependent manner. E1 utilizes ATP to adenylylate the last glycine residue of Ub and then transfers Ub onto its catalytic cysteine residue via a thioester bond. E1-mediated activation of Ub is followed by the transfer of Ub to a catalytic cysteine residue of the Ub-conjugating enzyme E2. Under the catalysis of different types of E3 ligases, Ub is linked to be polyUb chains of different linkages on protein substrates. PolyUb chains of different linkages are sensed by specific Ub-binding domains to determine the substrates to be degraded by the ubiquitin-proteasome system (UPS) or to initiate a special signal transduction cascade (Husnjak and Dikic, 2012Husnjak K. Dikic I. Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions.Annu. Rev. Biochem. 2012; 81: 291-322Crossref PubMed Scopus (452) Google Scholar). Ubiquitination regulates almost all eukaryotic signaling pathways and thereby plays an essential role in eukaryotic cellular processes, including immune responses. In the coevolutionary race with their hosts, bacterial pathogens have evolved special protein secretion systems, such as type III secretion systems (T3SSs), to deliver virulent effector proteins into host cells. The injected effectors target key host signaling molecules to modulate host signaling for bacterial infection (Scott and Hartland, 2017Scott N.E. Hartland E.L. Post-translational Mechanisms of Host Subversion by Bacterial Effectors.Trends Mol. Med. 2017; 23: 1088-1102Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). C. violaceum, an intracellular bacterial pathogen, infects humans with a mortality rate greater than 50% and causes severe abscess formation in the liver and spleen as well as the subsequent development of abscesses in various organs (Batista and da Silva Neto, 2017Batista J.H. da Silva Neto J.F. Chromobacterium violaceum Pathogenicity: Updates and Insights from Genome Sequencing of Novel Chromobacterium Species.Front. Microbiol. 2017; 8: 2213Crossref PubMed Scopus (35) Google Scholar). C. violaceum is extensively used as a model pathogen in studies of inflammation and sepsis (Batista and da Silva Neto, 2017Batista J.H. da Silva Neto J.F. Chromobacterium violaceum Pathogenicity: Updates and Insights from Genome Sequencing of Novel Chromobacterium Species.Front. Microbiol. 2017; 8: 2213Crossref PubMed Scopus (35) Google Scholar, Maltez and Miao, 2016Maltez V.I. Miao E.A. Reassessing the Evolutionary Importance of Inflammasomes.J. Immunol. 2016; 196: 956-962Crossref PubMed Scopus (30) Google Scholar). The pathogenicity of C. violaceum is dependent on its two type III secretion systems (T3SSs): the Chromobacterium pathogenicity island-1/-1a (Cpi-1/-1a)-encoded T3SS, which resembles the Spi-1 T3SS system from Salmonella enterica, and the Chromobacterium pathogenicity island-2 (Cpi-2)-encoded T3SS, which resembles the systems encoded by the Spi-2 gene cluster in S. enterica and by the locus of enterocyte effacement (LEE) in enteropathogenic Escherichia coli (Betts et al., 2004Betts H.J. Chaudhuri R.R. Pallen M.J. An analysis of type-III secretion gene clusters in Chromobacterium violaceum.Trends Microbiol. 2004; 12: 476-482Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, Miki et al., 2010Miki T. Iguchi M. Akiba K. Hosono M. Sobue T. Danbara H. Okada N. Chromobacterium pathogenicity island 1 type III secretion system is a major virulence determinant for Chromobacterium violaceum-induced cell death in hepatocytes.Mol. Microbiol. 2010; 77: 855-872PubMed Google Scholar). However, it is unknown how the effector proteins of these two T3SSs modulate host cell signaling during the infection of C. violaceum in humans. Here, we show that the Cpi-1/-1a T3SS effector CteC of C. violaceum functions as a unique ADP-ribosyltransferase to specifically modify Ub on a threonine residue to block host ubiquitin signaling during infection. CteC represents a family of type III effector proteins with Ub-specific ADP-ribosyltransferase activity in bacterial pathogens of diverse hosts. This study reveals a new posttranslational modification of Ub and uncovers a conserved mechanism by which bacterial pathogens of hosts from different kingdoms block eukaryotic ubiquitination. We tested whether C. violaceum interferes with the host nuclear factor-kappa B (NF-κB) immune signaling pathway during infection. Tumor necrosis factor alpha (TNF-α) treatment can activate NF-κB signaling and induce IκBα degradation. C. violaceum significantly inhibited TNF-α-induced IκBα degradation in HeLa cells (Figure S1A). Inactivation of the Cpi-1/-1a T3SS by gene deletion of its ATPase CivC and the translocator CivA (ΔCpi-1/-1a) abolished the ability of C. violaceum to inhibit TNF-α-induced IκBα degradation, whereas dysfunction of the Cpi-2 T3SS by gene deletion of its ATPase CsaN (ΔCpi-2) had no effect (Figure S1A). The upstream signals in TNF-α-activated NF-κB signaling were then investigated. The TNF-α treatment-induced autoubiquitination of TRAF2 with K63-linked polyUb chains was also inhibited by wild-type C. violaceum and the ΔCpi-2 strain, but not by the ΔCpi-1/-1a strain (Figure S1B), suggesting that the Cpi-1/-1a T3SS of C. violaceum inhibited the synthesis of new K63-linked polyUb chains by TRAF2 during infection. We then carefully examined the total levels of polyUb chains in whole cell lysates of the infected cells by western blotting. The amount of mono-Ub and polyUb chains in the cells infected with wild-type C. violaceum or the ΔCpi-2 strain did not decrease (Figure 1A). However, the mono-Ub and polyUb chains in these two samples exhibited significant upward shifts on 15% SDS-PAGE gels compared to those in uninfected and ΔCpi-1/-1a strain-infected cells (Figure 1A), suggesting that all Ub molecules in the host cells undergo an unknown modification by C. violaceum via the Cpi-1/-1a T3SS. Endogenous free mono-Ub in infected cells was further analyzed on SDS-PAGE and native PAGE gels. The upward shift of Ub on SDS-PAGE gels caused by C. violaceum infection was entirely dependent on the functional Cpi-1/-1a T3SS (Figure 1B). On native PAGE gels, free mono-Ub in the infected cells showed faster migration and underwent a dramatic downward shift, which was also dependent on the functional Cpi-1/-1a T3SS, not the Cpi-2 T3SS (Figure 1C). These data suggest that Ub in the host cells was indeed modified by C. violaceum via the Cpi-1/-1a T3SS. We used a glutathione S-transferase (GST) pull-down assay with Ub-binding domains to check polyUb chains in C. violaceum-infected cells. Rare K63-linked polyUb chains were pulled down by the K63-specific UIMs of Rap80 (Sims and Cohen, 2009Sims J.J. Cohen R.E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80.Mol. Cell. 2009; 33: 775-783Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) from the cells infected with wild-type C. violaceum or the ΔCpi-2 strain, in contrast to the abundant K63-linked polyUb chains extracted from the ΔCpi-1/-1a strain-infected cells (Figure 1A), which suggests that C. violaceum infection disrupted the interactions of the host K63-linked polyUb chains with the UIMs via the unknown modification of Ub by the Cpi-1/-1a T3SS. We also used a GST pull-down assay with the K48 linkage-specific TUBE protein from Rad23 (Hjerpe et al., 2009Hjerpe R. Aillet F. Lopitz-Otsoa F. Lang V. England P. Rodriguez M.S. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.EMBO Rep. 2009; 10: 1250-1258Crossref PubMed Scopus (278) Google Scholar) to check the K48-linked polyUb chains in the infected cells. The interactions of K48-linked polyUb chains with the K48-linkage specific TUBE were also severely eliminated by infection with wild-type C. violaceum or the ΔCpi-2 strain, but not the ΔCpi-1/-1a strain (Figure 1A). The polyUb chains modified in the C. violaceum-infected cells could not be bound by the K63 linkage-specific UIMs or the K48-linkage specific TUBE (Figure 1A). Thus, the unknown modification of Ub by C. violaceum blocked the synthesis of new K63-linked polyUb chains upon TNF-α treatment and prevented the recognition of polyUb chains by the Ub-binding domains, which led to the dysfunction of polyUb chains in host cells. We tested the effects of C. violaceum on the UPS. C. violaceum infection caused significant accumulation of labile GFPu, a green fluorescent protein (GFP) reporter of the UPS (Bence et al., 2001Bence N.F. Sampat R.M. Kopito R.R. Impairment of the ubiquitin-proteasome system by protein aggregation.Science. 2001; 292: 1552-1555Crossref PubMed Scopus (1740) Google Scholar), in cells (Figure 1D). Inactivation of the Cpi-1/-1a T3SS, not the Cpi-2 T3SS, completely abolished the ability of C. violaceum to increase GFPu stability (Figure 1D). Thus, the host UPS was also inhibited by C. violaceum via the Cpi-1/-1a T3SS during infection. Taken together, the results indicate that host ubiquitin signaling was impaired by the unknown modification of Ub by C. violaceum. To investigate whether Ub is directly modified by an effector protein of the Cpi-1/-1a T3SS of C. violaceum, we expressed human Ub in C. violaceum. The recombinant Ub protein produced in C. violaceum indeed exhibited a significant upward shift on SDS-PAGE gels compared with that expressed in E. coli (Figure 1E). To identify which effector protein in C. violaceum is responsible for the Ub modification, five function-unknown effectors of the Cpi-1/-1a T3SS (Miki et al., 2011Miki T. Akiba K. Iguchi M. Danbara H. Okada N. The Chromobacterium violaceum type III effector CopE, a guanine nucleotide exchange factor for Rac1 and Cdc42, is involved in bacterial invasion of epithelial cells and pathogenesis.Mol. Microbiol. 2011; 80: 1186-1203Crossref PubMed Scopus (17) Google Scholar) were cloned and individually transfected into 293T cells. Endogenous mono-Ub in the transfected 293T cells was analyzed by SDS-PAGE and native PAGE. Among the five effectors analyzed, only the effector CteC (protein ID: CV_1467) caused a significant upward shift of Ub in the transfected cells on SDS-PAGE gels (Figure 1F). CteC also induced faster migration and a dramatic downward shift of endogenous mono-Ub on native PAGE gels (Figure 1G), as infection of wild-type C. violaceum did. Moreover, recombinant CteC protein directly interacted with Ub in an isothermal titration calorimetry (ITC) assay with a binding affinity of 78.7 μM (Figure S1C). To examine whether CteC is the sole effector in C. violaceum that modifies Ub, we generated a C. violaceum mutant strain with in-frame deletion of the cteC gene. Gene deletion of CteC in the C. violaceum genome abolished the ability of the bacteria to cause the gel shifts of Ub from infected cells on SDS-PAGE and native PAGE gels (Figure 1H). Complementation of the ΔcteC strain with a plasmid containing the wild-type cteC gene completely rescued the Ub modification during bacterial infection (Figure 1H). These data suggest that CteC is the effector in C. violaceum that directly targets Ub via an unknown modification. We analyzed the sequence of CteC and searched its homologs through PSI-BLAST. CteC shares 66% sequence identity with a homolog from Burkholderia ubonensis (protein ID: KVO09808, hereafter named CHBU), a member of the Burkholderia cepacia complex, which is a group of closely related bacterial pathogens that can cause severe infection in cystic fibrosis patients (Chiarini et al., 2006Chiarini L. Bevivino A. Dalmastri C. Tabacchioni S. Visca P. Burkholderia cepacia complex species: health hazards and biotechnological potential.Trends Microbiol. 2006; 14: 277-286Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) (Figure S2A). CteC also shares 24% sequence identity with a homolog (CHCS) from Corallococcus species, a group of gram-negative bacterial pathogens of fungi (Li et al., 2019Li Z. Xia C. Wang Y. Li X. Qiao Y. Li C. Zhou J. Zhang L. Ye X. Huang Y. Cui Z. Identification of an endo-chitinase from Corallococcus sp. EGB and evaluation of its antifungal properties.Int. J. Biol. Macromol. 2019; 132: 1235-1243Crossref PubMed Scopus (7) Google Scholar) (Figure S2A). Thus, CteC represents a family of effector proteins in bacterial pathogens of diverse hosts from different kingdoms. CteC and its family members do not share sequence homology with any protein of known function. To investigate the enzymatic activity of CteC, we manually analyzed the sequences of CteC and its family members and mutated the conserved polar residues of CteC to look for potential catalytic motifs. Mutation of the conserved residues R65 or E220 to alanine completely abolished the ability of CteC to modify Ub in 293T cells (Figure S2B), suggesting that R65 and E220 are potential catalytic residues of CteC. The requirement of R65 and E220 reminded us of the catalytic arginine and glutamate residues in the “R-S-E” catalytic motif of bacterial C3-like ADP-ribosyltransferases (Deng and Barbieri, 2008Deng Q. Barbieri J.T. Molecular mechanisms of the cytotoxicity of ADP-ribosylating toxins.Annu. Rev. Microbiol. 2008; 62: 271-288Crossref PubMed Scopus (116) Google Scholar), including pertussis toxin, the C3 exoenzyme, and Iota toxin (Figure 2A). Indeed, an ITC assay showed that CteC directly interacted with NAD with a binding affinity of 86.2 μM (Figure S1D). The NAD-binding affinity of CteC is comparable to that of the C3 exoenzyme (60 μM) (Ménétrey et al., 2002Ménétrey J. Flatau G. Stura E.A. Charbonnier J.B. Gas F. Teulon J.M. Le Du M.H. Boquet P. Menez A. NAD binding induces conformational changes in Rho ADP-ribosylating clostridium botulinum C3 exoenzyme.J. Biol. Chem. 2002; 277: 30950-30957Crossref PubMed Scopus (59) Google Scholar) and higher than that of the Legionella effector SdeA (381 μM) (Dong et al., 2018Dong Y. Mu Y. Xie Y. Zhang Y. Han Y. Zhou Y. Wang W. Liu Z. Wu M. Wang H. et al.Structural basis of ubiquitin modification by the Legionella effector SdeA.Nature. 2018; 557: 674-678Crossref PubMed Scopus (28) Google Scholar). In contrast, no detectable interaction was observed between CteC and other cofactors, such as acetyl coenzyme A (AcCoA), S-adenosyl methionine (SAM), inositol hexakisphosphate (IP6), and calmodulin (CaM) (Figure S1E). Therefore, CteC is likely an ADP-ribosyltransferase that transfers the ADP-ribosyl (ADPR) group from NAD onto Ub. The ADP-ribosyltransferase activity of CteC toward Ub was then examined in vitro. In the presence of NAD present in reactions, CteC caused a significant upward shift of Ub on SDS-PAGE gels and a dramatic downward shift on native PAGE gels (Figures 2B and 2C). Mutation of the putative catalytic E220 residue to alanine abolished the CteC-induced gel shifts of Ub on SDS-PAGE and native PAGE gels (Figures 2B and 2C). The modification of Ub by CteC was also impaired when carba-NAD, a nonhydrolyzable NAD analog (Szczepankiewicz et al., 2012Szczepankiewicz B.G. Dai H. Koppetsch K.J. Qian D. Jiang F. Mao C. Perni R.B. Synthesis of carba-NAD and the structures of its ternary complexes with SIRT3 and SIRT5.J. Org. Chem. 2012; 77: 7319-7329Crossref PubMed Scopus (40) Google Scholar), was added to the reactions as the ligand (Figures 2B and 2C). When biotin-labeled NAD was used as the ligand, Ub was efficiently labeled with biotin by wild-type CteC, but not by the CteC E220A mutant protein (Figure 2D). Moreover, the CteC-catalyzed modification of Ub could be detected by a specific antibody toward the ADPR group (Figure S2E). Thus, CteC is indeed an ADP-ribosyltransferase that modifies the host Ub. Enzymatic analyses revealed that CteC exhibited robust ADP-ribosyltransferase activity toward Ub (Figures S2C and S2D). Unlike wild-type CteC, the CteC E220A mutant could not cause an upward shift of Ub in the transfected cells on SDS-PAGE gels (Figure 2E). Complementation of the cteC-deleted C. violaceum strain with the CteC E220A mutant failed to rescue the ability of the bacteria to modify Ub during infection (Figures 2F and 2G). The Ub modified by CteC in vitro exhibited the same downward shift on native PAGE gels as the modified endogenous mono-Ub in CteC-transfected 293T cells (Figure 2H), indicating that Ub underwent the same ADP-ribosylation modification by CteC both in vitro and in vivo. We also examined the CteC-catalyzed ADP-ribosylation of Ub using all known human ADPR hydrolases, including ARH1, ARH3, TARG1, MacroD1, MacroD2, NUDT16, PARG, and ENPP1 (Catara et al., 2019Catara G. Corteggio A. Valente C. Grimaldi G. Palazzo L. Targeting ADP-ribosylation as an antimicrobial strategy.Biochem. Pharmacol. 2019; 167: 13-26Crossref PubMed Scopus (5) Google Scholar, Cohen and Chang, 2018Cohen M.S. Chang P. Insights into the biogenesis, function, and regulation of ADP-ribosylation.Nat. Chem. Biol. 2018; 14: 236-243Crossref PubMed Scopus (90) Google Scholar). The CteC-catalyzed ADP-ribosylation of Ub could not be removed by any human ADPR hydrolase (Figure S2F), which suggests that ADP-ribosylation of Ub by CteC is likely irreversible in cells. To identify the modification site on Ub, we determined the crystal structure of CteC-modified human Ub at a resolution of 2.55 Å (Table S1). The high-resolution crystal structure and the omit electron density map clearly revealed that the ADP-ribosylation modification of Ub by CteC occurred on threonine-66 (T66) (Figures 3A and 3B ). This modification is distinct from the common arginine or glutamine ADP-ribosylation catalyzed by bacterial ADP-ribosyltransferases. We also used high-resolution electron transfer dissociation (ETD) mass spectrometry to analyze CteC-modified Ub. The ETD technique preserves the highly labile bonds in the ADP-ribosyl moiety and allows precise mapping of the ADP-ribosylation site (Leidecker et al., 2016Leidecker O. Bonfiglio J.J. Colby T. Zhang Q. Atanassov I. Zaja R. Palazzo L. Stockum A. Ahel I. Matic I. Serine is a new target residue for endogenous ADP-ribosylation on histones.Nat. Chem. Biol. 2016; 12: 998-1000Crossref PubMed Scopus (110) Google Scholar). The high-resolution ETD spectra confirmed the ADP-ribosylation of Ub at T66 by CteC (Figures 3C and S3C). In addition, a mass spectrometric analysis of the change in the mass weight of the CteC-modified Ub revealed that the molecular weight of CteC-modified Ub was increased by 541 daltons compared with that of unmodified Ub (Figure S3D), which further confirmed that only one ADPR group was added specifically onto T66 of Ub. CteC did not modify the Ub T66A mutant with NAD in vitro (Figures 3D and 3E). When biotin-NAD was added as the ligand in the reactions, Ub T66A failed to be labeled with biotin by CteC (Figure 3F). In 293T cells, the Ub T66A mutant was not modified by cotransfected CteC and did not undergo any gel shift on SDS-PAGE (Figure 3G). T66 specifically exists in Ub and is not conserved in Ub-like proteins, including NEDD8 and SUMO (Figure S3E). Consistently, CteC specifically modified Ub, but not NEDD8 or SUMO (Figure S3F). Thus, CteC is a Ub-specific mono-ADP-ribosyltransferase and catalyzes an unusual threonine ADP-ribosylation on T66. The CteC-modified residue T66 is close to I44, a residue crucial for recognition of Ub and polyUb chains by Ub-binding domains, in the hydrophobic patch of Ub (Figures 3H and S3A). The comparison of the CteC-modified Ub structure with the wild-type Ub structures (PDBs: 1UBQ and 3H7P) revealed that the threonine ADP-ribosylation by CteC caused significant conformational changes in the linking loop L1 between β1 and β2 and in the N-terminal region of the L4 loop adjacent to I44. Although the ADPR moiety on T66 in CteC-modified Ub contacts another Ub molecule via hydrogen bonds in crystal packing (Figure S3B), the modification did not induce dimerization of Ub in solution and had no effects on Ub homogeneity (Figure S3G). We examined the effects of the CteC-catalyzed modification on the three-enzyme cascade of ubiquitination. Wild-type Ub and CteC-modified Ub exhibited similar pyrophosphate (PPi) release patterns during Ub adenylation by E1 in the Ub activation reactions (Figure S4A). The CteC-modified Ub was also efficiently transferred onto the catalytic cysteine residue of E1 after adenylation, similar to wild-type Ub (Figure S4B). Therefore, CteC-catalyzed ADP-ribosylation does not affect E1-mediated Ub activation in the ubiquitination cascade. We then examined the effect of threonine ADP-ribosylation by CteC on the transfer of Ub from E1 to E2. After E1 activation, wild-type Ub was linked to the catalytic cysteine residues of the E2 enzymes UbcH5c, UbcH6, UbcH10, Ubc13, UbcH5a, and UbcH7 via thioester bonds. However, the CteC-modified Ub could not be efficiently conjugated to the catalytic cysteine residues of the E2 enzymes (Figures 4A, 4B, S4C, and S4D), which suggests that ADP-ribosylation by CteC prevents the transfer of Ub from E1 to E2. As a result, ADP-ribosylated Ub could not be efficiently linked to be K48- and K63-linked polyUb chains by the E3 ligases IpaH3 and TRAF6, respectively (Figure 4C). Thus, the CteC-catalyzed threonine ADP-ribosylation of Ub prevents the transfer of Ub from E1 to E2 to synthesize new polyUb chains in the host ubiquitination enzymatic cascade. We also examined the effects of the CteC-catalyzed modification on polyUb chains. CteC ADP-ribosylated various types of polyUb chains, including K48-, K63-, and M1-linked polyUb, which caused significant upward shifts of the polyUb chains on SDS-PAGE gels (Figure S5A). Kinetics analysis revealed that CteC exhibited similar enzymatic activities toward mono-Ub and K48-, K63-, and M1-linked polyUb chains in ε-NAD hydrolysis assays (Figure S5B), suggesting that CteC modifies mono-Ub and polyUb chains without significant substrate preference. ADP-ribosylation of K63- and K48-linked polyUb chains by CteC impaired cleavage by the nonspecific deubiquitinase vOTU (Capodagli et al., 2011Capodagli G.C. McKercher M.A. Baker E.A. Masters E.M. Brunzelle J.S. Pegan S.D. Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin.J. Virol. 2011; 85: 3621-3630Crossref PubMed Scopus (56) Google Scholar) (Figure 4D), suggesting that the ADP-ribosylation of Ub by CteC inhibits the deubiquitinase-mediated reversal of polyUb chains. In 293T cells, transfected wild-type CteC modified all mono-Ub and polyUb chains, whereas the inactive CteC E220A mutant had no such effect (Figure 4E). The CteC-modified polyUb chains could not be bound by the K63-specific UIMs and K48-specific TUBE in GST pull-down assays (Figure 4E). CteC modified linear Ub chains synthesized by the LUBAC complex in vivo (Figure S5C), and the CteC-modified linear Ub chains could not be bound by the UBAN domain of NEMO (Rahighi et al., 2009Rahighi S. Ikeda F. Kawasaki M. Akutsu M. Suzuki N. Kato R. Kensche T. Uejima T. Bloor S. Komander D. et al.Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.Cell. 2009; 136: 1098-1109Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) (Figure S5C). A Ub affimer specific for K33-linked polyUb chains (Michel et al., 2017Michel M.A. Swatek K.N. Hospenthal M.K. Komander D. Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.Mol. Cell. 2017; 68: 233-246.e5Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and the inactive mutant C77A of Usp30, a deubiquitinase specific for K6-linked polyUb chains (Gersch et al., 2017Gersch M. Gladkova C. Schubert A.F. Michel M.A. Maslen S. Komander D. Mechanism and regulation of the Lys6-selective deubiquitinase USP30.Nat. Struct. Mol. Biol. 2017; 24: 920-930Crossref PubMed Scopus (72) Google Scholar), did not pull down polyUb chains from the CteC-transfected cells (Figures S5D and S5E). After in vitro modification by CteC, the linear Ub, K33/K11-, and K6/K48-linked polyUb chains could not be bound by the NEMO UBAN domain, the K33 Ub affimer, or the Usp30 C77A mutant, respectively (Figures S5F, S5G, and S5H). Therefore, the CteC-catalyzed threonine ADP-ribosylation prevents recognition of polyUb chains by Ub-binding domains and inhibits deubiquitinase-mediated reversal, which leads to dysfunction of polyUb chains in cells. We investigated the effects of the CteC-catalyzed modification on host ubiquitin signaling. When directly delivered via the N-terminal domain of anthrax lethal factor (LFN) or transfected into cells stably expressing the GFPu reporter of the UPS, wild-type CteC, but not the inactive E220A mutant, delayed the degradation of GFPu by the UPS, resulting in GFPu accumulation (Figures 4F and S6A). CteC also blocked the degradation of the Ub-GFP reporters of the UPS, including UbG76V-GFP and Ub-R-GFP, but not the control Ub-M-GFP reporter (Dantuma et al., 2000Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (440) Google Scholar) (Figures 4G and S6B). Thus, the CteC-catalyzed threonine ADP-ribosylation of Ub caused dysfunction of the host UPS. Consistently, CteC blocked the UPS-mediated degradation of IκBα induced by TNF-α in transfected 293T cells (Figure S6C). During bacterial infection, endogenous mono-Ub and polyUb chains in the infected cells were completely modified by wild-type C. violaceum within 2 h (Figure 5A). Deletion of the cteC gene in C. violaceum abolished the modifications on mono-Ub and polyUb chains and restored the interactions of K48- and K63-linked polyUb chains with K48-TUBE and K63-UIMs, respectively, in infected cells. Complementation of the deletion strain with wild-type CteC, but not the inactive E220A mutant, rescued the modifications and effects on mono-Ub and polyUb chains (Figure 5A). Infection with the cteC-deleted strain did not change the labile property of the GFPu reporter of the UPS (Figure 5B). Complementation of the deletion strain with wild-type CteC, but not the inactive E220A mutant, highly enhanced the stability of GFPu, as wild-type C. violaceum did (Figure 5B). As expected, CteC inhibited the UPS-mediated degradation of IκBα and the synthesis of new polyUb chains by TRAF2 after TNF-α treatment during infection (Figures S6D and S6E). Thus, host ubiquitin signaling is blocked by the CteC-catalyzed threonine ADP-ribosylation of Ub during infection. We then examined the role of CteC in bacterial proliferation in mice during infection. Deletion of the cteC gene in C. violaceum reduced bacterial survival in mouse livers 5-fold at 3 days postinfection and dramatically reduced bacterial survival 100-fold at 6 days postinfection (Figure 5C). Complementation of the cteC-deleted strain with wild-type CteC, but not the inactive E220A mutant, resulted in similar proliferation of the bacteria in mice as the wild-type C. viola