Periprosthetic joint infection and immunity: Current understanding of host-microbe interplay.

Periprosthetic joint infection (PJI) is a major complication of total joint arthroplasty. Even with current treatments, failure rates are unacceptably high with a 5-year mortality rate of 26%. Majority of the literature in the field has focused on development of better biomarkers for diagnostics and treatment strategies including innovate antibiotic delivery systems, antibiofilm agents, and bacteriophages. Nevertheless, the role of the immune system, our first line of defense during PJI, is not well understood. Evidence of infection in PJI patients is found within circulation, synovial fluid, and tissue and include numerous cytokines, metabolites, antimicrobial peptides, and soluble receptors that are part of the PJI diagnosis workup. Macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs) are initially recruited into the joint by chemokines and cytokines produced by immune cells and bacteria and are activated by pathogen-associated molecular patterns. While these cells are efficient killers of planktonic bacteria by phagocytosis, opsonization, degranulation, and recruitment of adaptive immune cells, biofilm-associated bacteria are troublesome. Biofilm is not only a physical barrier for the immune system but also elicits effector functions. Additionally, bacteria have developed mechanisms to evade the immune system by inactivating effector molecules, promoting killing or anti-inflammatory effector cell phenotypes, and intracellular persistence and dissemination. Understanding these shortcomings and the mechanisms by which bacteria can subvert the immune system may open new approaches to better prepare our own immune system to combat PJI. Furthermore, preoperative immune system assessment and screening for dysregulation may aid in developing preventative interventions to decrease PJI incidence.

Select symbionts drive high IgA levels in the mouse intestine.

Immunoglobulin A (IgA) is an important factor in maintaining homeostasis at mucosal surfaces, yet luminal IgA levels vary widely. Total IgA levels are thought to be driven by individual immune responses to specific microbes. Here, we found that the prebiotic, pectin oligosaccharide (pec-oligo), induced high IgA levels in the small intestine in a T cell-dependent manner. Surprisingly, this IgA-high phenotype was retained after cessation of pec-oligo treatment, and microbiome transmission either horizontally or vertically was sufficient to retain high IgA levels in the absence of pec-oligo. Interestingly, the bacterial taxa enriched in the overall pec-oligo bacterial community differed from IgA-coated microbes in this same community. Rather, a group of ethanol-resistant microbes, highly enriched for Lachnospiraceae bacterium A2, drove the IgA-high phenotype. These findings support a model of intestinal adaptive immunity in which a limited number of microbes can promote durable changes in IgA directed to many symbionts.

Configuration-Specific Antibody for Bacterial Heptosylation: An Antiadhesion Therapeutic Strategy.

Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.

Mesothelium-Derived Factors Shape GATA6-Positive Large Cavity Macrophages.

The local microenvironment shapes macrophage differentiation in each tissue. We hypothesized that in the peritoneum, local factors in addition to retinoic acid can support GATA6-driven differentiation and function of peritoneal large cavity macrophages (LCMs). We found that soluble proteins produced by mesothelial cells lining the peritoneal cavity maintained GATA6 expression in cultured LCMs. Analysis of global gene expression of isolated mesothelial cells highlighted mesothelin (Msln) and its binding partner mucin 16 (Muc16) as candidate secreted ligands that potentially regulate GATA6 expression in peritoneal LCMs. Mice deficient for either of these molecules showed diminished GATA6 expression in peritoneal and pleural LCMs that was most prominent in aged mice. The more robust phenotype in older mice suggested that monocyte-derived macrophages were the target of Msln and Muc16. Cell transfer and bone marrow chimera experiments supported this hypothesis. We found that lethally irradiated Msln-/- and Muc16-/- mice reconstituted with wild-type bone marrow had lower levels of GATA6 expression in peritoneal and pleural LCMs. Similarly, during the resolution of zymosan-induced inflammation, repopulated peritoneal LCMs lacking expression of Msln or Muc16 expressed diminished GATA6. These data support a role for mesothelial cell-produced Msln and Muc16 in local macrophage differentiation within large cavity spaces such as the peritoneum. The effect appears to be most prominent on monocyte-derived macrophages that enter into this location as the host ages and also in response to infection.

Local barriers configure systemic communications between the host and microbiota.

Associations between the dynamic community of microbes (the microbiota) and the host they colonize appear to be vital for ensuring host health. Microbe-host communication is actively maintained across physiological barriers of various body sites and is mediated by a range of bidirectional secreted proteins and small molecules. So far, a range of "omics" methods have succeeded in revealing the multiplicity of associations between members of a microbiota and a wide range of host processes and diseases. Although these advances point to possibilities for treating disease, there has not been much translational success thus far. We know little about which organisms are key contributors to host health, the importance of strain differences, and the activities of much of the chemical "soup" that is produced by the microbiota. Adding to this complexity are emerging hints of the role of interkingdom interactions between bacteria, phages, protozoa, and/or fungi in regulating the microbiota-host interactions. Functional approaches, although experimentally challenging, could be the next step to unlocking the power of the microbiota.

Biofilm Formation and Virulence of Shigella flexneri Are Modulated by pH of Gastrointestinal Tract.

Shigella infection remains a public health problem in much of the world. Classic models of Shigella pathogenesis suggest that microfold epithelial cells in the small intestine are the preferred initial site of invasion. However, recent evidence supports an alternative model in which Shigella primarily infects a much wider range of epithelial cells that reside primarily in the colon. Here, we investigated whether the luminal pH difference between the small intestine and the colon could provide evidence in support of either model of Shigella flexneri pathogenesis. Because virulence factors culminating in cellular invasion are linked to biofilms in S. flexneri, we examined the effect of pH on the ability of S. flexneri to form and maintain adherent biofilms induced by deoxycholate. We showed that a basic pH (as expected in the small intestine) inhibited formation of biofilms and dispersed preassembled mature biofilms, while an acidic pH (similar to the colonic environment) did not permit either of these effects. To further elucidate this phenomenon at the molecular level, we probed the transcriptomes of biofilms and S. flexneri grown under different pH conditions. We identified specific amino acid (cysteine and arginine) metabolic pathways that were enriched in the bacteria that formed the biofilms but decreased when the pH increased. We then utilized a type III secretion system reporter strain to show that increasing pH reduced deoxycholate-induced virulence of S. flexneri in a dose-dependent manner. Taken together, these experiments support a model in which Shigella infection is favored in the colon because of the local pH differences in these organs.

Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation.

Intestinal Paneth cells modulate innate immunity and infection. In Crohn's disease, genetic mutations together with environmental triggers can disable Paneth cell function. Here, we find that a western diet (WD) similarly leads to Paneth cell dysfunction through mechanisms dependent on the microbiome and farnesoid X receptor (FXR) and type I interferon (IFN) signaling. Analysis of multiple human cohorts suggests that obesity is associated with Paneth cell dysfunction. In mouse models, consumption of a WD for as little as 4 weeks led to Paneth cell dysfunction. WD consumption in conjunction with Clostridium spp. increased the secondary bile acid deoxycholic acid levels in the ileum, which in turn inhibited Paneth cell function. The process required excess signaling of both FXR and IFN within intestinal epithelial cells. Our findings provide a mechanistic link between poor diet and inhibition of gut innate immunity and uncover an effect of FXR activation in gut inflammation.

Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells.

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.

A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.

Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.

Atg14 protects the intestinal epithelium from TNF-triggered villus atrophy.

Regulation of intestinal epithelial turnover is a key component of villus maintenance in the intestine. The balance of cell turnover can be perturbed by various extrinsic factors including the cytokine TNF, a cell signaling protein that mediates both proliferative and cytotoxic outcomes. Under conditions of infection and damage, defects in autophagy are associated with TNF-mediated cell death and tissue damage in the intestinal epithelium. However, a direct role of autophagy within the context of enterocyte cell death during homeostasis is lacking. Here, we generated mice lacking ATG14 (autophagy related 14) within the intestinal epithelium [Atg14f/f Vil1-Cre (VC)+]. These mice developed spontaneous villus loss and intestinal epithelial cell death within the small intestine. Based on marker studies, the increased cell death in these mice was due to apoptosis. Atg14f/f VC+ intestinal epithelial cells demonstrated sensitivity to TNF-triggered apoptosis. Correspondingly, both TNF blocking antibody and genetic deletion of Tnfrsf1a/Tnfr1 rescued villus loss and cell death phenotype in Atg14f/f VC+ mice. Lastly, we identified a similar pattern of spontaneous villus atrophy and cell death when Rb1cc1/Fip200 was conditionally deleted from the intestinal epithelium (Rb1cc1f/f VC+). Overall, these findings are consistent with the hypothesis that factors that control entry into the autophagy pathway are also required during homeostasis to prevent TNF triggered death in the intestine. Abbreviations: ANOVA: analysis of variance; Atg14: autophagy related 14; Atg16l1: autophagy related 16-like 1 (S. cerevisiae); Atg5: autophagy related 5; cCASP3: cleaved CASP3/caspase-3; cCASP8: cleaved CASP8/caspase-8; CHX: cycloheximide; EdU: 5-ethynyl-2´-deoxyuridine thymidine; f/f: flox/flox; H&E: hematoxylin and eosin; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nec-1: necrostatin-1; Rb1cc1/Fip200: RB1-inducible coiled-coil 1; Ripk1: receptor (TNFRSF)-interacting serine-threonine kinase 1; Ripk3: receptor (TNFRSF)-interacting serine-threonine kinase 3; Tnfrsf1a/Tnfr1: tumor necrosis factor receptor superfamily, member 1a; Tnf/ Tnfsf1a: tumor necrosis factor; VC: Vil1/villin 1-Cre.

Temporal Regulation of the Bacterial Metabolite Deoxycholate during Colonic Repair Is Critical for Crypt Regeneration.

Colonic wound repair is an orchestrated process, beginning with barrier re-establishment and followed by wound channel formation and crypt regeneration. Elevated levels of prostaglandin E2 (PGE2) promote barrier re-establishment; however, we found that persistently elevated PGE2 hinders subsequent repair phases. The bacterial metabolite deoxycholate (DCA) promotes transition through repair phases via PGE2 regulation. During barrier re-establishment, DCA levels are locally diminished in the wound, allowing enhanced PGE2 production and barrier re-establishment. However, during transition to the wound channel formation phase, DCA levels increase to inhibit PGE2 production and promote crypt regeneration. Altering DCA levels via antibiotic treatment enhances PGE2 levels but impairs wound repair, which is rescued with DCA treatment. DCA acts via its receptor, farnesoid X receptor, to inhibit the enzyme cPLA2 required for PGE2 synthesis. Thus, colonic wound repair requires temporally regulated signals from microbial metabolites to coordinate host-associated signaling cascades. VIDEO ABSTRACT.

Sweet Talk: Protein Glycosylation in Bacterial Interaction With the Host.

Pathogenic bacteria encode virulent glycosyltransferases that conjugate various glycans onto substrate proteins via the N- or O-linkage. The HMW system in nontypeable Haemophilus influenzae and the Pgl system in Campylobacter jejuni glycosylate bacterial surface or periplasmic proteins at the eukaryotic-like Asn-X-Ser/Thr motif. The NleB effector from enterobacteria mediates arginine GlcNAcylation of host death-domain proteins to block inflammation, representing an atypical N-glycosylation. The large clostridial cytotoxins and related glucosyltransferase toxins from Legionella and Photorhabdus monoglycosylate a serine/threonine or tyrosine in host Rho GTPase or elongation factor 1A (eEF1A). The emerging bacterial autotransporter heptosyltransferase (BAHT) family of heptosyltransferases also catalyses O-glycosylation and modifies autotransporters for adhesion to the host. These glycosylations, diverse in linkages and glycan structures, determine appropriate functioning of bacterial virulence factors or hijack host cellular processes in pathogenesis.

A polar-localized iron-binding protein determines the polar targeting of Burkholderia BimA autotransporter and actin tail formation.

Intracellular bacterial pathogens including Shigella, Listeria, Mycobacteria, Rickettsia and Burkholderia spp. deploy a specialized surface protein onto one pole of the bacteria to induce filamentous actin tail formation for directional movement within host cytosol. The mechanism underlying polar targeting of the actin tail proteins is unknown. Here we perform a transposon screen in Burkholderia thailandensis and identify a conserved bimC that is required for actin tail formation mediated by BimA from B. thailandensis and its closely related pathogenic species B. pseudomallei and B. mallei. bimC is located upstream of bimA in the same operon. Loss of bimC results in even distribution of BimA on the outer membrane surface, where actin polymerization still occurs. BimC is targeted to the same bacterial pole independently of BimA. BimC confers polar targeting of BimA prior to BimA translocation across bacterial inner membrane. BimC is an iron-binding protein, requiring a four-cysteine cluster at the carboxyl terminus. Mutation of the cysteine cluster disrupts BimC polar localization. Truncation analyses identify the transmembrane domain in BimA being responsible for its polar targeting. Consistently, BimC can interact with BimA transmembrane domain in an iron binding-dependent manner. Our study uncovers a new mechanism that determines the polar distribution of bacteria-induced actin tail in infected host cells.

A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family.

A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal ß-barrel, a catalytic domain, a ß-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through ß-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-ß-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC-TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex.

An iron-containing dodecameric heptosyltransferase family modifies bacterial autotransporters in pathogenesis.

Autotransporters deliver virulence factors to the bacterial surface by translocating an effector passenger domain through a membrane-anchored barrel structure. Although passenger domains are diverse, those found in enteric bacteria autotransporters, including AIDA-I in diffusely adhering Escherichia coli (DAEC) and TibA in enterotoxigenic E. coli, are commonly glycosylated. We show that AIDA-I is heptosylated within the bacterial cytoplasm by autotransporter adhesin heptosyltransferase (AAH) and its paralogue AAH2. AIDA-I heptosylation determines DAEC adhesion to host cells. AAH/AAH2 define a bacterial autotransporter heptosyltransferase (BAHT) family that contains ferric ion and adopts a dodecamer assembly. Structural analyses of the heptosylated TibA passenger domain reveal 35 heptose conjugates forming patterned and solenoid-like arrays on the surface of a ß helix. Additionally, CARC, the AIDA-like autotransporter from Citrobacter rodentium, is essential for colonization in mice and requires heptosylation by its cognate BAHT. Our study establishes a bacterial glycosylation system that regulates virulence and is essential for pathogenesis.

SET-domain bacterial effectors target heterochromatin protein 1 to activate host rDNA transcription.

Transcription of rRNA genes (rDNAs) in the nucleolus is regulated by epigenetic chromatin modifications including histone H3 lysine (de)methylation. Here we show that LegAS4, a Legionella pneumophila type IV secretion system (TFSS) effector, is targeted to specific rDNA chromatin regions in the host nucleolus. LegAS4 promotes rDNA transcription, through its SET-domain (named after Drosophila Su(var)3-9, enhancer of zeste [E(z)], and trithorax [trx]) histone lysine methyltransferase (HKMTase) activity. LegAS4's association with rDNA chromatin is mediated by interaction with host HP1α/γ. L. pneumophila infection potently activates rDNA transcription in a TFSS-dependent manner. Other bacteria, including Bordetella bronchiseptica and Burkholderia thailandensis, also harbour nucleolus-localized LegAS4-like HKMTase effectors. The B. thailandensis type III effector BtSET promotes H3K4 methylation of rDNA chromatin, contributing to infection-induced rDNA transcription and bacterial intracellular replication. Thus, activation of host rDNA transcription might be a general bacterial virulence strategy.

Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses.

Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.

Cysteine methylation disrupts ubiquitin-chain sensing in NF-κB activation.

NF-κB is crucial for innate immune defence against microbial infection. Inhibition of NF-κB signalling has been observed with various bacterial infections. The NF-κB pathway critically requires multiple ubiquitin-chain signals of different natures. The question of whether ubiquitin-chain signalling and its specificity in NF-κB activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-κB signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-κB signalling. NleE harboured an unprecedented S-adenosyl-l-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger (NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF (truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-κB activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-κB signalling.

The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus.

Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.

Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family.

A family of bacterial effectors including Cif homolog from Burkholderia pseudomallei (CHBP) and Cif from Enteropathogenic Escherichia coli (EPEC) adopt a functionally important papain-like hydrolytic fold. We show here that CHBP was a potent inhibitor of the eukaryotic ubiquitination pathway. CHBP acted as a deamidase that specifically and efficiently deamidated Gln40 in ubiquitin and ubiquitin-like protein NEDD8 both in vitro and during Burkholderia infection. Deamidated ubiquitin was impaired in supporting ubiquitin-chain synthesis. Cif selectively deamidated NEDD8, which abolished the activity of neddylated Cullin-RING ubiquitin ligases (CRLs). Ubiquitination and ubiquitin-dependent degradation of multiple CRL substrates were impaired by Cif in EPEC-infected cells. Mutations of substrate-contacting residues in Cif abolished or attenuated EPEC-induced cytopathic phenotypes of cell cycle arrest and actin stress fiber formation.