The Salmonella Type III Effector SspH2 Specifically Exploits the NLR Co-chaperone Activity of SGT1 to Subvert Immunity

To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.     

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.     

Delivery of biologically active anti-inflammatory cytokines IL-10 and IL-1ra in vivo by the Shigella type III secretion apparatus

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-alpha and IL-1beta mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.     

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of ¹²⁵I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼¹²⁵I-ubiquitin thioester co-substrate in the nanomolar range ([S]_½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]_½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼¹²⁵I-ubiquitin thioester (K_i = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (K_i = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the k_cat but not [S]_½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe³⁹⁵. Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349–10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.     

Structure of an SspH1-PKN1 Complex Reveals the Basis for Host Substrate Recognition and Mechanism of Activation for a Bacterial E3 Ubiquitin Ligase

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.     

Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.     

Shigella hijacks the glomulin–cIAPs–inflammasome axis to promote inflammation

Shigella deploys a unique mechanism to manipulate macrophage pyroptosis by delivering the IpaH7.8 E3 ubiquitin ligase via its type III secretion system. IpaH7.8 ubiquitinates glomulin (GLMN) and elicits its degradation, thereby inducing inflammasome activation and pyroptotic cell death of macrophages. Here, we show that GLMN specifically binds cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), members of the inhibitor of apoptosis (IAP) family of RING‐E3 ligases, which results in reduced E3 ligase activity, and consequently inflammasome‐mediated death of macrophages. Importantly, reducing the levels of GLMN in macrophages via IpaH7.8, or siRNA‐mediated knockdown, enhances inflammasome activation in response to infection by Shigella, Salmonella, or Pseudomonas, stimulation with NLRP3 inflammasome activators (including SiO₂, alum, or MSU), or stimulation of the AIM2 inflammasome by poly dA:dT. GLMN binds specifically to the RING domain of both cIAPs, which inhibits their self‐ubiquitination activity. These findings suggest that GLMN is a negative regulator of cIAP‐mediated inflammasome activation, and highlight a unique Shigella stratagem to kill macrophages, promoting severe inflammation.     

Shigella effector IpaH4.5 targets 19S regulatory particle subunit RPN13 in the 26S proteasome to dampen cytotoxic T lymphocyte activation

Subversion of antigen‐specific immune responses by intracellular pathogens is pivotal for successful colonisation. Bacterial pathogens, including Shigella, deliver effectors into host cells via the type III secretion system (T3SS) in order to manipulate host innate and adaptive immune responses, thereby promoting infection. However, the strategy for subverting antigen‐specific immunity is not well understood. Here, we show that Shigella flexneri invasion plasmid antigen H (IpaH) 4.5, a member of the E3 ubiquitin ligase effector family, targets the proteasome regulatory particle non‐ATPase 13 (RPN13) and induces its degradation via the ubiquitin–proteasome system (UPS). IpaH4.5‐mediated RPN13 degradation causes dysfunction of the 19S regulatory particle (RP) in the 26S proteasome, inhibiting guidance of ubiquitinated proteins to the proteolytically active 20S core particle (CP) of 26S proteasome and thereby suppressing proteasome‐catalysed peptide splicing. This, in turn, reduces antigen cross‐presentation to CD8+ T cells via major histocompatibility complex (MHC) class I in vitro. In RPN13 knockout mouse embryonic fibroblasts (MEFs), loss of RPN13 suppressed CD8+ T cell priming during Shigella infection. Our results uncover the unique tactics employed by Shigella to dampen the antigen‐specific cytotoxic T lymphocyte (CTL) response.     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.     

The CUL7/F-box and WD Repeat Domain Containing 8 (CUL7/Fbxw8) Ubiquitin Ligase Promotes Degradation of Hematopoietic Progenitor Kinase 1

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasome-mediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7/Fbxw8 ubiquitin ligase targeted HPK1 for degradation via the 26 S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1. In addition, we showed that Thr-355 of HPK1 is a key PP4 dephosphorylation site, through which CUL7/Fbxw8 ubiquitin ligase and PP4 regulates HPK1 stability. Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation of pancreatic cancer cells. Our study demonstrated that targeted degradation of HPK1 by the CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain the activity of HPK1 and that CUL7/Fbxw8 ubiquitin ligase promotes pancreatic cancer cell proliferation. CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation revealed a direct link and novel role of CUL7/Fbxw8 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.     

A disulfide driven domain swap switches off the activity of Shigella IpaH9.8 E3 ligase

We show that the monomeric form of Shigella IpaH9.8 E3 ligase catalyses the ubiquitination of human U2AF35 in vitro, providing a molecular mechanism for the observed in vivo effect. We further discover that under non-reducing conditions IpaH9.8 undergoes a domain swap driven by the formation of a disulfide bridge involving the catalytic cysteine and that this dimer is unable to catalyse the ubiquitination of U2AF35. The crystal structure of the domain-swapped dimer is presented. The redox inactivation of IpaH9.8 could be a mechanism of regulating the activity of the IpaH9.8 E3 ligase in response to cell damage so that the host cell in which the bacteria resides is maintained in a benign state suitable for bacterial survival.

Structured summary

MINT-7993779: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0408) by X-ray crystallography (MI:0114) MINT-7993812: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by affinity chromatography technology (MI:0004) MINT-7993790: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by blue native page (MI:0276)     

Sequencing and analysis of the Edwardsiella ictaluri plasmids

To determine possible functions of the Edwardsiella ictaluri plasmids, pEI1 and pEI2, we analyzed the sequence of both plasmids. Plasmid pEI1 is 4807 bp, with 51% G + C, and 23 possible open reading frames of 40 amino acids or greater. Plasmid pEI2 is 5643 bp, with 51% G + C, and 24 possible reading frames. Database searches indicated that pEI1 contains an insertion element and a ROM analog. In addition, pEI1 possesses an open reading frame with strong homology to SlrP, SspH1, and SspH2 of Salmonella typhimurium and IpaH of Shigella flexnari, which have leucine-rich repeat regions and are components of type III secretory systems. pEI2 has a frame with weak homology to Spa15 of S. flexnari 5 and InvB of S. sonnei and S. typhimurium, which are also type III secretory system components, three origins of replication, a Rep analog, and a multimer resolution site.     

Functional Analysis of NopM, a Novel E3 Ubiquitin Ligase (NEL) Domain Effector of Rhizobium sp. Strain NGR234

Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant''s defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.     

病原菌调节宿主细胞泛素化途径的研究进展

泛素化是真核生物特有的蛋白质翻译后修饰,广泛地参与宿主细胞各种信号通路和生理过程.病原菌常通过分泌毒性效应蛋白,对泛素和泛素结合酶进行独特的共价修饰,或者利用泛素连接酶和去泛素化酶的酶学活性,调节宿主泛素化过程,从而干扰宿主细胞的信号转导,促进细菌的感染和生存.本文概述了病原菌效应蛋白调节宿主泛素化途径的主要研究进展和最新发现.     

Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella''s type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.     

Conserved structural mechanisms for autoinhibition in IpaH ubiquitin ligases

The IpaH family of novel E3 ligase (NEL) enzymes occur in a variety of pathogenic and commensal bacteria that interact with eukaryotic hosts. We demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes autoinhibit the enzymatic activity of the adjacent catalytic novel E3 ligase domain by two distinct but conserved structural mechanisms. Autoinhibition is required for the in vivo biological activity of two IpaH enzymes in a eukaryotic model system. Autoinhibition was retro-engineered into a constitutively active IpaH enzyme from Yersinia pestis by introduction of single site substitutions, thereby demonstrating the conservation of autoregulatory infrastructure across the IpaH enzyme family.     

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.