Host Ubiquitin System Research Articles

Host-mediated ubiquitination of a mycobacterial protein suppresses immunity.

Mycobacterium tuberculosis is an intracellular pathogen that uses several strategies to interfere with the signalling functions of host immune molecules. Many other bacterial pathogens exploit the host ubiquitination system to promote pathogenesis1,2, but whether this same system modulates the ubiquitination of M. tuberculosis proteins is unknown. Here we report that the host E3 ubiquitin ligase ANAPC2-a core subunit of the anaphase-promoting complex/cyclosome-interacts with the mycobacterial protein Rv0222 and promotes the attachment of lysine-11-linked ubiquitin chains to lysine 76 of Rv0222 in order to suppress the expression of proinflammatory cytokines. Inhibition of ANAPC2 by specific short hairpin RNA abolishes the inhibitory effect of Rv0222 on proinflammatory responses. Moreover, mutation of the ubiquitination site on Rv0222 impairs the inhibition of proinflammatory cytokinesby Rv0222 and reduces virulence during infection in mice. Mechanistically, lysine-11-linked ubiquitination of Rv0222 by ANAPC2 facilitates the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6, preventing the lysine-63-linked ubiquitination and activation of TRAF6. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2-3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection.

Toxoplasma gondii is an intracellular parasite that infects humans and other warm-blooded animals. Exosomes are endocytic-derived vesicles released by cells, representing an important mode of intercellular communication. In exosomes, specific molecules of proteins, lipids, and mRNAs or miRNAs have been detected, some of which are capable of transferring biologically active molecules to recipient cells. Dendritic cells (DCs) are the only antigen-presenting cells (APCs) that activate the initial immune response. In this study, high-throughput sequencing was used to analyze the exosomal miRNA profile of DC2.4 cells infected with Toxoplasma gondii for 28 h, compared with those of uninfected DC2.4 cells. Differential exosomal miRNAs (DEmiRs) from these two cell groups were analyzed. Through high-throughput sequencing, 3434 DEmiRs were obtained, and 12 stably enriched DEmiRNAs were verified by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and selected for further analysis. The target genes of these 12 miRNAs were predicted with online analysis software and subjected to bioinformatics analyses including protein–protein interaction (PPI) network analysis, key driver analysis (KDA), gene ontology (GO) enrichment, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. These DEmiRs were found to be associated with a variety of biological processes and signaling pathways involved in host ubiquitin system, innate immunity, biosynthesis, and transferase activity and could be potential biomarkers for T. gondii infection.

Revisiting Bacterial Ubiquitin Ligase Effectors: Weapons for Host Exploitation.

Protein ubiquitylation plays a central role in eukaryotic cell physiology. It is involved in several regulatory processes, ranging from protein folding or degradation, subcellular localization of proteins, vesicular trafficking and endocytosis to DNA repair, cell cycle, innate immunity, autophagy, and apoptosis. As such, it is reasonable that pathogens have developed a way to exploit such a crucial system to enhance their virulence against the host. Hence, bacteria have evolved a wide range of effectors capable of mimicking the main players of the eukaryotic ubiquitin system, in particular ubiquitin ligases, by interfering with host physiology. Here, we give an overview of this topic and, in particular, we detail and discuss the mechanisms developed by pathogenic bacteria to hijack the host ubiquitination system for their own benefit.

The species-spanning family of LPX-motif harbouring effector proteins

The delivery of effector proteins into infected eukaryotic cells represents a key virulence feature of many microbial pathogens in order to derail essential cellular processes and effectively counter the host defence system. Although bacterial effectors are truly numerous and exhibit a wide range of biochemical activities, commonalities in terms of protein structure and function shared by many bacterial pathogens exist. Recent progress has shed light on a species-spanning family of bacterial effectors containing an LPX repeat motif as a subtype of the leucine-rich repeat superfamily, partially combined with a novel E3 ubiquitin ligase domain. This review highlights the immunomodulatory effects of LPX effector proteins, with particular emphasis on the exploitation of the host ubiquitin system.

The host ubiquitin system in innate immunity and virus replication: proviral and antiviral functions of the host E3-ubiquitin ligase TRIM family

Abstract Type-I Interferons (IFN-I), which are innate immune cytokines with broad-spectrum antiviral effects, are regulated by the host Ubiquitin (Ub) system. Evidence has shown that new forms of poly-Ub chains, which are not covalently attached to any protein (termed unanchored poly-Ub), are important activators of kinase activity in vitro. We recently reported that unanchored K48-linked poly-Ub synthesized by the E3-Ub ligase TRIM6 promote IFN-mediated antiviral innate immunity in vivo by activating the IKKɛ kinase. Intriguingly, we found that two different highly pathogenic viruses, Nipah (NiV; Paramyxoviridae family) and Ebola (EBOV; Filoviridae family), hijack TRIM6 to promote virus replication through mechanisms that involve both covalent and unanchored Ub. This indicates that host antiviral factors can have direct pro-viral functions. We also found that Zika virus (ZIKV; Flaviviridae family), which can cause congenital microcephaly in newborns, requires ubiquitination of viral proteins for efficient replication. Importantly, ZIKV ubiquitination occurs in vivo in a tissue-specific manner. Recombinant infectious ZIKV mutants that lack ubiquitination on specific sites are attenuated in mice and appear to differentially replicate in tissues. As compared to other flaviviruses, ZIKV acquired an additional ubiquitination site, which may be responsible for providing ZIKV an advantage to replicate in brain and reproductive tissue. This would be the first example of a flavivirus utilizing the Ub system for tissue tropism. Taken together, our work suggests that divergent viruses hijack similar innate immune host factors to enhance their replication. This information could help design novel broad-spectrum antiviral strategies

Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins.

Ubiquitination is one of the most important posttranslational modifications in eukaryotic cells where it regulates the activity, cellular localization, and half-life of proteins. Ubiquitination thus affects many essential cellular processes, including vesicle trafficking, cell cycle, DNA repair, immune response, and protein homeostasis. The ubiquitin system is exclusive to eukaryotes; however, pathogenic bacteria have developed effective strategies to influence the host ubiquitin system for their own benefit. Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. This bacterium utilizes a type IV secretion system to translocate more than 300 effector proteins into host cells. These virulence factors modulate a wide spectrum of host processes to support its intracellular survival and replication. Hijacking of host ubiquitin system is an important theme in Legionella virulence, and a number of L. pneumophila effector proteins have been shown to possess ubiquitin ligase or deubiquitinase activity. Among these, members of the SidE family effector proteins (SidEs) catalyze ubiquitination of several ER-associated Rab small GTPases by a mechanism that bypasses the requirement of ATP and the E1, E2 enzymes. Here, we summarize the experimental details of Rab small GTPases ubiquitination catalyzed by SdeA, a member of the SidE family.

Tissue‐specific expression patterns of tau and ubiquitin proteins coordinated with Yersinia pestis pathogenesis in a non‐human primate model

Early identification of molecular markers in conjunction with pathogenic exposure offers a potential means to initiate treatment during a timeframe when it would most be effective. The identification of molecular markers could also allow for novel therapeutic strategies, which become all the more critical as antibiotic resistance continually becomes more widespread. In order to examine pre‐symptomatic illness, African green monkeys were challenged intranasally with aerosolized Yersinia pestis strain CO92. Various tissues and blood samples were collected in short intervals from 6 h till 42 h post‐exposure. Transcriptomic profiles of blood indicated early perturbation as the number of differentially expressed genes increased until 24 h post‐exposure. As suggested by functional analyses, Y. pestis had potentially paralyzed the host defense much earlier than bacteria was detected in the blood. Early activation of the apoptotic networks possibly facilitated the pathogen to overwhelm host defense mechanisms, circumventing the activated pro‐inflammatory response and ubiquitin‐ and microtubule‐mediated defense. Pathogens target the host ubiquitin system in order to manipulate immunity. For instance, Yersinia species have been reported to trigger deubiquitylation by inhibiting NF‐κB activity. Microtubule dynamics are known to be another marked target for early pathogenic intervention. The host‐ubiquitin and host‐microtubule relationships were studied herein for better understanding of Yersinia pathogenicity and how the host succumbs, despite increasing early defense machinery. Lungs and adjacent lymph nodes, such as submandibular and mediastinal lymph nodes, are the port‐of‐entry for the intranasal exposure of Y. pestis. These tissues indicated signs of infection much earlier than the onset of serological infection. The expressions of ubiquitin were probed in these tissues along with the microtubule‐associated tau protein. Longitudinal analysis of tissue‐specific transcriptomic profiles associated with these two protein networks was integrated. Tissue‐specific tau‐ubiquitin protein expression patterns potentially manifested the temporal character of the host defense. For instance, a weak early response to Y. pestis assault was followed by a steady increase of ubiquitin load in the lungs. A completely opposite trend was found in tau expression which showed a gradual reduction with time. An orthogonal pattern was observed in the submandibular lymph node, where ubiquitin decreased and tau increased with time. Integration of these proteomic patterns with the gene‐protein co‐enriched networks indicated potential regulators were mediating the pathogen proliferation and host defense manipulation.The present longitudinal tissue‐specific study can explain the key molecular underpinnings associated with the pathogenesis of pneumonic plague. The significance of this result is underlined by the fact that most of these molecular perturbations occurred at the asymptomatic state, even before blood showed signs of bacterial infection. Hence, potential early markers of Y. pestis pathogenesis could be mined from this analysis.Support or Funding InformationThis work was supported by Defense Threat Reduction Agency, Funding #: TMTI0029_09_WR_T

The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection.

Macrophage-mediated innate immune responses play crucial roles in host defense against pathogens. Recent years have seen an explosion of host proteins that act as restriction factors blocking viral replication in infected cells. However, the essential factors restricting Mycobacterium tuberculosis (Mtb) and their regulatory roles during mycobacterial infection remain largely unknown. We previously reported that Mtb tyrosine phosphatase PtpA, a secreted effector protein required for intracellular survival of Mtb, inhibits innate immunity by co-opting the host ubiquitin system. Here, we identified a new PtpA-interacting host protein TRIM27, which is reported to possess a conserved RING domain and usually acts as an E3 ubiquitin ligase that interferes with various cellular processes. We further demonstrated that TRIM27 restricts survival of mycobacteria in macrophages by promoting innate immune responses and cell apoptosis. Interestingly, Mtb PtpA could antagonize TRIM27-promoted JNK/p38 MAPK pathway activation and cell apoptosis through competitively binding to the RING domain of TRIM27. TRIM27 probably works as a potential restriction factor for Mtb and its function is counteracted by Mtb effector proteins such as PtpA. Our study suggests a potential tuberculosis treatment via targeting of the TRIM27-PtpA interfaces.

HIV-1 Vpr increases HCV replication through VprBP in cell culture

Coinfection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs at a high frequency, in which HIV shows a promotion of HCV-derived liver diseases. However, the mechanism of how this occurs is not well understood. Our previous work has demonstrated that the HIV-1 accessory protein Vpr enhances HCV RNA replication in cell culture. Because Vpr performs most of its functions through host protein VprBP (DCAF1), the role of VprBP in the regulation of HCV by Vpr was investigated in this study. We found that the Vpr mutant Q65R, which is deficient in VprBP binding, could not enhance HCV replication. Furthermore, Vpr-mediated enhancement of HCV replication was severely diminished in VprBP knockdown cells. In addition, an inhibitor of Cullin RING E3 ligases, MLN4924, impaired the function of Vpr during HCV replication. Together, these results suggest that Vpr promotes HCV replication in a VprBP-dependent manner, and that the activity of Cullin RING E3 ligases is essential to this process. In conclusion, our findings demonstrate that HIV-1 Vpr makes the cellular environment more suitable for HCV replication, which might relate with the host ubiquitination system.

Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses.

Anti-infective Activity of 2-Cyano-3-Acrylamide Inhibitors with Improved Drug-Like Properties against Two Intracellular Pathogens.

Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection.

Midori-ishi Cyan/monomeric Kusabira-Orange-based fluorescence resonance energy transfer assay for characterization of various E3 ligases.

Many bacterial pathogens hijack the host ubiquitin system for their own benefit by delivering effectors with ubiquitin ligase (E3) into host cells via the type III secretion system. Therefore, screening for small compounds that selectively inhibit bacterial but not mammalian E3 ligases is a promising strategy for identifying molecules that could substitute for antibiotics. To facilitate high-throughput screening for bacterial E3 ligase inhibitors, we developed a MiCy/mKO (Midori-ishi Cyan/monomeric Kusabira-Orange)-based FRET (fluorescence resonance energy transfer) assay and validated it on Shigella IpaH E3 ligase effectors. We showed the feasibility of using the MiCy/mKO-based FRET assay to identify the most appropriate ubiquitin-conjugating enzymes (E2s) and determine the lysine specificity of a given E3, both hallmarks of E3 activity. Furthermore, we showed the usefulness of the FRET assay in characterizing mammalian E3 ligases, such as TNF receptor-associated factor 6 (TRAF6) and mouse double minute 2 homologue (MDM2). In addition, we confirmed the feasibility of determining the efficiency of inhibition of E3 ligase activity using inhibitors of E1 ubiquitin-activating enzymes, such as UBE1-41, by measuring the IC50 . Based on these results, we concluded that the MiCy/mKO-based FRET assay is useful for characterizing E3 enzyme activity, as well as for high-throughput E3 inhibitor screening.

Modulation of host ubiquitin system genes in human endometrial cell line infected with Mycobacterium tuberculosis.

Endometrium is one of the most commonly affected sites in genital tuberculosis. The understanding of its interaction with the tubercle bacilli is of paramount importance for studying the pathogenesis of this disease. The main objective of this work was to study the interplay between Mycobacterium tuberculosis and host endometrial epithelial cell lines (Ishikawa cell lines), and to identify the differentially expressed genes upon tuberculosis infection. To study this, suppression subtractive hybridization library was constructed using M. tuberculosis H37Rv-infected Ishikawa cell line harvested 24h post-infection. The subtracted cDNA library was screened, and 105 differentially expressed genes were identified and grouped based on their functions. Since ubiquitination process has gained importance in targeting M. tuberculosis to xenophagy, ubiquitin system genes obtained in the library were selected, and time course analysis of their gene expression was performed. We observed an upregulation of mkrn1 and cops5 and downregulation of zfp91, ndfip2, ube2f, rnft1, psmb6, and psmd13 at 24h post-infection. From the results obtained, we surmise that ubiquitination pathway genes may have roles in combating tuberculosis which are yet uncharted.

Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system.

Mycobacterium tuberculosis PtpA, a secreted tyrosine phosphatase essential for tuberculosis pathogenicity, could be an ideal target for a drug against tuberculosis, but its active-site inhibitors lack selectivity over human phosphatases. Here we found that PtpA suppressed innate immunity dependent on pathways of the kinases Jnk and p38 and the transcription factor NF-κB by exploiting host ubiquitin. Binding of PtpA to ubiquitin via a region with no homology to human proteins activated it to dephosphorylate phosphorylated Jnk and p38, leading to suppression of innate immunity. Furthermore, the host adaptor TAB3 mediated NF-κB signaling by sensing ubiquitin chains, and PtpA blocked this process by competitively binding the ubiquitin-interacting domain of TAB3. Our findings reveal how pathogens subvert innate immunity by coopting host ubiquitin and suggest a potential tuberculosis treatment via targeting of ubiquitin-PtpA interfaces.

The bacterial pathogen-ubiquitin interface: lessons learned from Shigella.

Shigella species are the aetiological agents of shigellosis, a severe diarrhoeal disease that is a significant cause of morbidity and mortality worldwide. Shigellosis causes massive colonic destruction, high fever and bloody diarrhoea. Shigella pathogenesis is tightly linked to the ability of the bacterium to invade and replicate intracellularly within the colonic epithelium. Shigella uses a type 3 secretion system to deliver its effector proteins into the cytosol of infected cells. Among the repertoire of Shigella effectors, many are known to target components of the actin cytoskeleton to promote bacterial entry. An emerging alternate theme for effector function is the targeting of the host ubiquitin system. Ubiquitination is a post-translational modification restricted to eukaryotes and is involved in many essential host processes. By virtue of sheer number of ubiquitin-modulating effector proteins, it is clear that Shigella has invested heavily into subversion of the ubiquitin system. Understanding these host-pathogen interactions will inform us about the strategies used by successful pathogens and may also provide avenues for novel antimicrobial strategies.

Bacterial Effectors and Their Functions in the Ubiquitin-Proteasome System: Insight from the Modes of Substrate Recognition

Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

Vaccinia virus virulence factor N1 can be ubiquitylated on multiple lysine residues.

Ubiquitylation is a covalent post-translational modification that regulates protein stability and is involved in many biological functions. Proteins may be modified with mono-ubiquitin or ubiquitin chains. Viruses have evolved multiple mechanisms to perturb the cell ubiquitin system and manipulate it to their own benefit. Here, we report ubiquitylation of vaccinia virus (VACV) protein N1. N1 is an inhibitor of the nuclear factor NF-κB and apoptosis that contributes to virulence, has a Bcl-2-like fold, and is highly conserved amongst orthopoxviruses. The interaction between N1 and ubiquitin occurs at endogenous protein levels during VACV infection and following ectopic expression of N1. Biochemical analysis demonstrated that N1 is covalently ubiquitylated, and heterodimers of ubiquitylated and non-ubiquitylated N1 monomers were identified, suggesting that ubiquitylation does not inhibit N1 dimerization. Studies with other VACV Bcl-2 proteins, such as C6 or B14, revealed that although these proteins also interact with ubiquitin, these interactions are non-covalent. Finally, mutagenesis of N1 showed that ubiquitylation occurs in a conventional lysine-dependent manner at multiple acceptor sites because only an N1 allele devoid of lysine residues remained unmodified. Taken together, we described a previously uncharacterized modification of the VACV protein N1 that provided a new layer of complexity to the biology of this virulence factor, and provided another example of the intricate interplay between poxviruses and the host ubiquitin system.

Exploitation of the host ubiquitin system by human bacterial pathogens.

Ubiquitylation is a crucial post-translational protein modification that regulates several cellular processes in eukaryotes, including inflammatory responses, endocytic trafficking and the cell cycle. Importantly, ubiquitylation also has a central role in modulating eukaryotic defence systems; however, accumulating evidence shows that many bacterial pathogens exploit host ubiquitin systems for their own benefit. In this Review, we highlight the ways in which human bacterial pathogens target ubiquitylation to subvert and manipulate host defence systems, with a focus on the role of molecular mimicry and secreted bacterial effector proteins. These strategies enable bacterial pathogens to maximize effector function and obtain nutrients, thereby promoting bacterial proliferation.

Identification of the Major Ubiquitin-binding Domain of the Pseudomonas aeruginosa ExoU A2 Phospholipase

Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A2 phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex.