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Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires’ disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires’ disease worldwide.

The COVID-19 outbreak caused by the SARS-CoV-2 virus has developed into a global health emergency. In addition to causing respiratory symptoms following SARS-CoV-2 infection, COVID-19-associated coagulopathy (CAC) is the main cause of death in patients with severe COVID-19. In this study, we performed single-cell sequencing analysis of the right ventricular free wall tissue from healthy donors, patients who died in the hypercoagulable phase of CAC, and patients in the fibrinolytic phase of CAC. Among these, we collected 61,187 cells, which were enriched in 24 immune cell subsets and 13 cardiac-resident cell subsets. We found that in response to SARS-CoV-2 infection, CD9highCCR2highmonocyte-derived mø promoted hyperactivation of the immune system and initiated the extrinsic coagulation pathway by activating CXCR-GNB/G-PI3K-AKT. This sequence of events is the main process contributing the development of coagulation disorders subsequent to SARS-CoV-2 infection. In the characteristic coagulation disorder caused by SARS-CoV-2, excessive immune activation is accompanied by an increase in cellular iron content, which in turn promotes oxidative stress and intensifies intercellular competition. This induces cells to alter their metabolic environment, resulting in an increase in sugar uptake, such as that via the glycosaminoglycan synthesis pathway, in CAC coagulation disorders. In addition, high levels of reactive oxygen species generated in response elevated iron levels promote the activation of unsaturated fatty acid metabolic pathways in endothelial cell subgroups, including vascular endothelial cells. This in turn promotes the excessive production of the toxic peroxidation by-product malondialdehyde, which exacerbates both the damage caused to endothelial cells and coagulation disorders.

Objective A colistin-resistant Escherichia coli strain isolated from dog feces was characterized in this study. Methods and results A multiplex PCR assay was used to detect the presence of colistin-resistant mcr genes; it was found that E. coli QDFD216 co-harbored the mcr-1 and mcr-3 genes. Whole-genome sequencing and further bioinformatics analysis revealed that E. coli QDFD216 belonged to serotype O176:H11, fimH1311 type and ST132. The resistance genes bla CTX-M-14, mdfA, dfrA3, acrA, acrB, tolc, and sul3 were present in the chromosome. The mcr-1.1 and mcr-3.7 genes were located in two plasmids of different incompatibility groups. mcr-1.1 was carried by a IncX4-type plasmid within an typical IS26-parA-mcr-1.1-pap2 cassette, while mcr-3.7 was encoded by an IncP1-type plasmid with a genetic structure of TnAs2-mcr-3.7-dgkA-IS26. No additional antibiotic resistance genes were carried by either plasmid. Conclusion This is the first report of an E. coli isolate co-harboring a mcr-1.1-carrying IncX4 plasmid and a mcr-3.7-carrying IncP1 plasmid. The evolution and mechanism of mcr gene co-existence need further study to assess its impact on public health.

Background The emergence and prevalence of plasmid-mediated colistin-resistant bacterial strains in recent years have raised great concerns in clinical medicine. It is urgently needed to develop a cheaper, faster, simpler, sensitive, and specific molecular detection method to identify and monitor the dissemination of the transferable resistant determinants. Methods and results Herein, eight pairs of primers were designed to set up a multiplex PCR method for the rapid and efficient determination of reported mcr genes. This assay can give results within 85 min (35 min for amplification and 50 min for electrophoresis). We validated the feasibility of this assay by testing the presence of mcr genes in 60 colistin-resistant isolates. Conclusion Our multiplex PCR technique exhibits remarkable advantages in the light of clear identification, efficiency of amplification, as well as the time consuming for detection, and thus could be useful for the surveillance and epidemiological research of plasmid-mediated colistin resistance, particularly for the under-resourced laboratories.

Chlamydia trachomatis is the cause of several diseases such as sexually transmitted urogenital disease and ocular trachoma. The pathogen contains a small genome yet, upon infection, expresses two enzymes with deubiquitinating activity, termed ChlaDUB1 and ChlaDUB2, presumed to have redundant deubiquitinase (DUB) function because of the similarity of the primary structure of their catalytic domain. Previous studies have led to structural characterization of the enzymatic properties of ChlaDUB1; however, ChlaDUB2 has yet to be investigated thoroughly. In this study, we investigated the deubiquitinase properties of ChlaDUB2 and compared them to those of ChlaDUB1. This revealed a distinct difference in hydrolytic activity with regard to di- and polyubiquitin chains while showing similar ability to cleave a monoubiquitin-based substrate, ubiquitin aminomethylcoumarin (Ub-AMC). ChlaDUB2 was unable to cleave a diubiquitin substrate efficiently, whereas ChlaDUB1 could rapidly hydrolyze this substrate like a prototypical prokaryotic DUB, SdeA. With polyubiquitinated green fluorescent protein substrate (GFP-Ubn), whereas ChlaDUB1 efficiently disassembled the polyubiquitin chains into the monoubiquitin product, the deubiquitination activity of ChlaDUB2, while showing depletion of the substrate, did not produce appreciable levels of the monoubiquitin product. We report the structures of a catalytic construct of ChlaDUB2 and its complex with ubiquitin propargyl amide. These structures revealed differences in residues involved in substrate recognition between the two Chlamydia DUBs. On the basis of the structures, we conclude that the distal ubiquitin binding is equivalent between the two DUBs, consistent with the Ub-AMC activity result. Therefore, the difference in activity with longer ubiquitinated substrates may be due to the differential recognition of these substrates involving additional ubiquitin binding sites.

The production of metallo-β-lactamases (MBLs) is one of the major mechanisms adopted by bacterial pathogens to resist carbapenems. One promising strategy to overcome MBLs-mediated carbapenems resistance is to develop effective inhibitors. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost-effective approach to fight infections caused by carbapenem resistant pathogens. Here, twelve FDA-approved compounds were screened to neutralize the ability of NDM-1. Among these compounds, dexrazoxane, embelin, candesartan cilexetil (CAN) and nordihydroguaiaretic acid (NDGA) were further demonstrated to inhibit all tested MBLs, and showed an in vitro synergistic bactericidal effect with meropenem against MBLs-producing bacteria. Mechanistic studies revealed that dexrazoxane, embelin and CAN are metal ion chelating agents, while the inhibition of NDM-1 by NDGA involves its direct binding with the active region of NDM-1. Furthermore, dexrazoxane, embelin and CAN and NDGA dramatically rescued the treatment efficacy of meropenem in three infection models. Our observations indicated that dexrazoxane, embelin, CAN and NDGA are promising carbapenem adjuvants against MBLs-positive carbapenem resistant bacterial pathogens.

Significance Many effectors of the Coxiella burnetii Dot/Icm transporter are important for its virulence, but the lack of understanding of their biochemical activity prevents the use of them as potential therapeutic targets. Here, we found that the effector Cbu0513 (CinF) is a protein phosphatase that attacks IκBα, a key regulatory protein in NF-κB signaling. Unlike its homologs such as ST0318 from the archaeon Sulfolobus tokodaii, CinF has lost the enzymatic activity as a fructose-1,6-bisphosphate aldolase/phosphatase. Instead, it specifically targets IκBα to make it resistant to proteasome-mediated degradation in cells stimulated with NF-κB agonists. Our finding has expanded the strategy used by bacterial pathogens to inhibit host immunity, which may provide leads for the development of immune modulators for disease treatment., Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella-containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii, CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity.

Polymyxin B has been re‐applied to the clinic as the final choice for the treatment of multidrug‐resistant gram‐negative pathogenic infections, but the use of polymyxin B has been re‐assessed because of the emergence and spread of the plasmid‐mediated mcr‐1 gene. The purpose of this study was to search for an MCR inhibitor synergistically acting with polymyxin to treat the infection caused by this pathogen. In this study, we used the broth microdilution checkerboard method to evaluate the synergistic effect of isoalantolactone (IAL) and polymyxin B on mcr‐1‐positive Enterobacteriaceae. Growth curve analysis, time‐killing assays and a combined disc test were used to further verify the efficacy of the combined drug. Colonization of the thigh muscle in mice, survival experiments and lung tissue section observations was used to determine the effect of synergy in vivo after Klebsiella pneumoniae and Escherichia coli infection. We screened a natural compound, IAL, which can enhance the sensitivity of polymyxin B to mcr‐1‐positive Enterobacteriaceae. The results showed that the combined use of polymyxin B and IAL has a synergistic effect on mcr‐1‐positive Enterobacteriaceae, such as K pneumoniae and E coli, not only in vitro but also in vivo. Our results indicate that IAL is a natural compound with broad application prospects that can prolong the service life of polymyxin B and make outstanding contributions to the treatment of gram‐negative Enterobacteriaceae infections resistant to polymyxin B.

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2-4. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ5; however, the mechanism of this regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA-a member of the SidE family-at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the α-phosphate position by SidJ, which-in the absence of glutamate or modifiable SdeA-undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.

The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4-phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella-containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3-kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3-phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P-binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.

Streptococcus pneumoniae is an important pathogen that causes otitis media, pneumonia, meningitis and bacteremia. As an important virulence factors of S. pneumoniae, pneumolysin (PLY) can penetrate cell membranes and lead to cell lysis and inflammation, which is one of the main causes of infection and damage of S. pneumoniae. Therefore, using pneumolysin as a target to study its inhibitors can provide a new treatment strategy for pneumococcal disease. This study analyzed the inhibitory effect of the natural compound hederagenin on PLY in vitro. The results show that hederagenin has great potential as a new strategy for the treatment of pneumococcal diseases.

Legionella pneumophilaextensively modulates the host ubiquitin network to create theLegionella-containing vacuole (LCV) for its replication. Many of its virulence factors function as ubiquitin ligases or deubiquitinases (DUBs). Here we identified Lem27 as a DUB that displays a preference for diubiquitin formed by K6, K11 or K48. Lem27 is associated with the LCV where it regulates Rab10 ubiquitination in concert with SidC and SdcA, two bacterial E3 ubiquitin ligases. Structural analysis of the complex formed by an active fragment of Lem27 and the substrate-based suicide inhibitor ubiquitin-propargylamide (PA) reveals that it harbors a fold resembling those in the OTU1 DUB subfamily with a Cys-His catalytic dyad and that it recognizes ubiquitin via extensive hydrogen bonding at six contact sites. Our results establish Lem27 as a deubiquitinase that functions to regulate protein ubiquitination on L. pneumophila phagosomes by counteracting the activity of bacterial ubiquitin E3 ligases.

Chlamydia trachomatis is the cause of several diseases worldwide in the form of a sexually transmitted urogenital disease or ocular trachoma. The pathogen contains a small genome yet, upon infection, expresses two enzymes with deubiquitinating activity, termed ChlaDUB1 and ChlaDUB2, presumed to have redundant deubiquitinase (DUB) function on account of similarity of primary structure of their catalytic domain. Previous studies have led to structural characterization of enzymatic properties of ChlaDUB1 however, ChlaDUB2 has yet to be investigated thoroughly. In this study, we investigated the deubiquitinase properties of ChlaDUB2 and compared them to that of ChlaDUB1. This revealed a distinct difference in hydrolytic activity with regards to di- and polyubiquitin chains, while showing similar ability to cleave a monoubiquitin-based substrate, ubiquitin aminomethylcoumarin (Ub-AMC). ChlaDUB2 was unable to cleave a diubiquitin substrate efficiently whereas ChlaDUB1 could rapidly hydrolyze this substrate comparable to a prototypical prokaryotic DUB, SdeA. With polyubiquitinated green fluorescent protein substrate (GFP-Ub(n)), whereas ChlaDUB1 efficiently disassembled the polyubiquitin chains into monoubiquitin product, the deubiquitination activity of ChlaDUB2, while showing a depletion of the substrate, did not produce appreciable levels of the monoubiquitin product. We report the structures of a catalytic construct of ChlaDUB2 and its complex with ubiquitin propargyl amide. These structures revealed differences in residues involved in substrate recognition between the two Chlamydia DUBs. Based on the structures we conclude that the distal ubiquitin binding is equivalent between the two DUBs, consistent with the Ub-AMC activity result. Therefore, the difference in activity with longer ubiquitinated substrates may be due to differential recognition of these substrates involving additional ubiquitin binding sites.

Background The emergence and prevalence of plasmid-mediated colistin-resistant bacterial strains in recent years have raised great concerns in clinical medicine. It is urgently needed to develop a cheaper, faster, simpler, sensitive, and specific molecular detection method to identify and monitor the dissemination of the transferable resistant determinants. Methods and Results Herein, eight pairs of primers were designed to set up a multiplex PCR method for the rapid and efficient determination of reported mcr genes. This assay can give results within 85 min (35 min for amplification and 50 min for electrophoresis). We validated the feasibility of this assay by testing the presence of mcr genes in 60 colistin-resistant isolates. Conclusion Our multiplex PCR technique exhibits remarkable advantages in the light of clear identification, efficiency of amplification, as well as the time consuming for detection, and thus could be useful for the surveillance and epidemiological research of plasmid-mediated colistin resistance, particularly for the under-resourced laboratories.

Colistin (polymyxin E) is an effective antibiotic for the treatment of most multidrug-resistant Gram-negative bacteria. However, some bacteria, including bacterial spp. belonging to the Enterobacteriaceae family, have an acquired resistance against polymyxins, which is attributed to they possess plasmid-carried resistance genes (mcr-1 and its variants). So, there is an urgent need to develop new therapeutic strategies to target broad spectrum resistant spp. from Enterobacteriaceae family in response to the loss of the protective barrier of last-line antibiotics. Here, we report the adjuvant capacity of nordihydroguaiaretic acid (NDGA) for restoring the antibacterial activity of colistin against MCR-1-positive E. coli ZJ487 in vivo/in vitro.A checkerboard assay, time-killing analysis, isobolograms, growth curves and inducible resistance test showed the effect of NDGA combined with colistin in vitro. TLC was used to detect the inhibitory effect of NDGA on MCR-1. Colony determination and hematoxylin and eosin (HE) staining were used to assess the synergistic effect of NDGA and colistin in mice.Our results showed that NDGA in combination with colistin showed a synergistic bactericidal action without inducing resistance. NDGA directly inhibited MCR-1 activity and resulted in measurable injury to the bacterial cell membrane to recover the antibacterial effect of colistin. Most importantly, NDGA in combination with colistin exhibited an in vivo synergistic effect in murine peritonitis infection models, as evidenced by the survival rate of MCR-1-positive E. coli ZJ487-infected mice which increased from 6.67 to 50.0%.Our study demonstrated that NDGA effectively rescues the efficiency of colistin against MCR-positive E. coli ZJ487 by simultaneously inhibiting both, the MCR activity and the injury to the cell membrane of bacteria.

Summary Ubiquitination is a post-translational modification that regulates a myriad of cellular processes in eukaryotes1–4. The conventional ubiquitination cascade culminates in a covalent linkage between the C-terminus of ubiquitin (Ub) and a target protein, most often on a lysine sidechain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a novel mode of ubiquitination in which a phosphoribosylated ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6–8. To uncover the molecular mechanism of this unique post-translational modification, we determined the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain (PDE) and a mono-ADP-ribosyltransferase (mART) domain. Biochemical analysis shows that the mART domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the PDE domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous PDE domain from the SidE family member SdeD9 in complex with Ub or ADPR-Ub. The structures suggest an intriguing mechanism for how SdeA processes ADPR-Ub to PR-Ub plus AMP and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-ubiquitination (PR-ubiquitination) and paves the way for future studies of this unusual type of ubiquitination in eukaryotes.

The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD+-dependent ubiquitination of certain host proteins independent of the canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA’s modulatory functions during Legionella infection.

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies have revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attack several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. In this case, ubiquitin is first activated by ADP-ribosylation at Arg42 by a mono-ADP-ribosyltransferase activity; the intermediate is then cleaved by a phosphodiesterase activity also residing within SdeA, concomitant with the attachment of ubiquitin to serine residues of substrate proteins via a phosphoribosyl linker. Here we demonstrate that the effect of SidEs is antagonized by SidJ, an effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ reverses ubiquitination of SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. SidJ also displays classical deubiquitinase activity but does not require catalytic cysteine residues. Further, these deubiquitinase activities of SidJ are essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a ubiquitin-deconjugating enzyme that functions to impose temporal regulation on the activity of SidE effectors. SidJ may be important in future studies of signaling cascades mediated by this unique ubiquitination, one that also potentially regulates cellular processes in eukaryotic cells.

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 A resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.

Ubiquitination is a prevalent protein modification catalyzed by E1, E2, and E3 enzymes that activate, conjugate, and ligate, respectively, the ubiquitin protein to substrate protein. In order to establish a mutualistic or parasitic relationship with their eukaryotic hosts, many microorganisms hijack different aspects of the ubiquitination machinery using bacterial proteins that function as E3 ligases or as enzymes that modify E2s or ubiquitin. Recently, the SidE family of effector proteins (SidEs) from the intracellular bacterial pathogen Legionella pneumophila was found to catalyze ubiquitination by a mechanism unrelated to the classical three-enzyme cascade. Instead of utilizing ATP, SidEs-catalyzed ubiquitination reactions are energized by nicotinamide adenine dinucleotide (NAD). Ubiquitin is first activated by ADP-ribosylation at residue Arg42 to form ADP-ribosylated ubiquitin (ADPR-Ub). ADPR-Ub is then cleaved by an activity conferred by a phosphodiesterase (PDE)-related domain also embedded in the SidE family proteins. ADPR-Ub cleavage is coupled to covalent attachment of phosphoribosylated ubiquitin to serine residues of target proteins and the release of AMP. Furthermore, SidE-induced ubiquitination can be reversed by SidJ, another virulence factor from L. pneumophila. Here, we describe the experimental details for SdeA-induced ubiquitination of the small GTPase Rab33b and its reversal by SidJ.

Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the e-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

Protein ubiquitination is one of the most prevalent posttranslational modifications; it regulates a wide range of critical cellular processes in eukaryotes. This modification occurs by covalent attachment of the ubiquitin molecule to other proteins via an isopeptide bond in reactions typically catalyzed by sequential actions of three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Ubiquitination is a reversible process catalyzed by a group of proteins known as deubiquitinase (DUB), which specifically cleaves the isopeptide bond between ubiquitin and modified proteins. Recently, a novel form of ubiquitination catalyzed by the SidE family of effectors from the bacterial pathogen Legionella pneumophila was reported. These proteins ubiquitinate structurally diverse host proteins such as reticulons and ER-associated Rab small GTPases by a two-step mechanism that uses NAD as the energy source for ubiquitin activation prior to being transferred to serine residues in target proteins. This process bypasses the need for E1 and E2 enzymes. Intriguingly, ubiquitination induced by SidEs is regulated by SidJ, another L. pneumophila effector protein which reverses the modification by functioning as an unconventional DUB. Here, we summarize the experimental details of Rab small GTPases (use Rab33b as an example) ubiquitination catalyzed by SidEs (use SdeA as an example) as well as deubiquitination catalyzed by SidJ.

Ubiquitination is a prevalent protein modification catalyzed by E1, E2, and E3 enzymes that activate, conjugate, and ligate, respectively, the ubiquitin protein to substrate protein. In order to establish a mutualistic or parasitic relationship with their eukaryotic hosts, many microorganisms hijack different aspects of the ubiquitination machinery using bacterial proteins that function as E3 ligases or as enzymes that modify E2s or ubiquitin. Recently, the SidE family of effector proteins (SidEs) from the intracellular bacterial pathogen Legionella pneumophila was found to catalyze ubiquitination by a mechanism unrelated to the classical three-enzyme cascade. Instead of utilizing ATP, SidEs-catalyzed ubiquitination reactions are energized by nicotinamide adenine dinucleotide (NAD). Ubiquitin is first activated by ADP-ribosylation at residue Arg42 to form ADP-ribosylated ubiquitin (ADPR-Ub). ADPR-Ub is then cleaved by an activity conferred by a phosphodiesterase (PDE)-related domain also embedded in the SidE family proteins. ADPR-Ub cleavage is coupled to covalent attachment of phosphoribosylated ubiquitin to serine residues of target proteins and the release of AMP. Furthermore, SidE-induced ubiquitination can be reversed by SidJ, another virulence factor from L. pneumophila. Here, we describe the experimental details for SdeA-induced ubiquitination of the small GTPase Rab33b and its reversal by SidJ.

Protein ubiquitination is one of the most prevalent posttranslational modifications; it regulates a wide range of critical cellular processes in eukaryotes. This modification occurs by covalent attachment of the ubiquitin molecule to other proteins via an isopeptide bond in reactions typically catalyzed by sequential actions of three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Ubiquitination is a reversible process catalyzed by a group of proteins known as deubiquitinase (DUB), which specifically cleaves the isopeptide bond between ubiquitin and modified proteins. Recently, a novel form of ubiquitination catalyzed by the SidE family of effectors from the bacterial pathogen Legionella pneumophila was reported. These proteins ubiquitinate structurally diverse host proteins such as reticulons and ER-associated Rab small GTPases by a two-step mechanism that uses NAD as the energy source for ubiquitin activation prior to being transferred to serine residues in target proteins. This process bypasses the need for E1 and E2 enzymes. Intriguingly, ubiquitination induced by SidEs is regulated by SidJ, another L. pneumophila effector protein which reverses the modification by functioning as an unconventional DUB. Here, we summarize the experimental details of Rab small GTPases (use Rab33b as an example) ubiquitination catalyzed by SidEs (use SdeA as an example) as well as deubiquitination catalyzed by SidJ.