54 results found
Fisch D, Pfleiderer MM, Anastasakou E, et al., 2023, PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection., Science, Vol: 382
Disruption of cellular activities by pathogen virulence factors can trigger innate immune responses. Interferon-γ (IFN-γ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense by attacking intracellular pathogens and by inducing programmed cell death. Working in human macrophages, we discovered that GBP1 expression in the absence of IFN-γ killed the cells and induced Golgi fragmentation. IFN-γ exposure improved macrophage survival through the activity of the kinase PIM1. PIM1 phosphorylated GBP1, leading to its sequestration by 14-3-3σ, which thereby prevented GBP1 membrane association. During Toxoplasma gondii infection, the virulence protein TgIST interfered with IFN-γ signaling and depleted PIM1, thereby increasing GBP1 activity. Although infected cells can restrain pathogens in a GBP1-dependent manner, this mechanism can protect uninfected bystander cells. Thus, PIM1 can provide a bait for pathogen virulence factors, guarding the integrity of IFN-γ signaling.
Mishra V, Crespo Puig A, McCarthy C, et al., 2023, IL-1β turnover by the UBE2L3 ubiquitin conjugating enzyme and HECT E3 ligases limits inflammation, Nature Communications, Vol: 14, Pages: 1-16, ISSN: 2041-1723
The cytokine interleukin-1β (IL-1β) has pivotal roles in antimicrobial immunity, but also incites inflammatory disease. Bioactive IL-1β is released following proteolytic maturation of the pro-IL-1β precursor by caspase-1. UBE2L3, a ubiquitin conjugating enzyme, promotes pro-IL-1β ubiquitylation and proteasomal disposal. However, actions of UBE2L3 in vivo and its ubiquitin ligase partners in this process are unknown. Here we report that deletion ofUbe2l3 in mice reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production, neutrophilic inflammation and disease following inflammasome activation. An unbiased RNAi screen identified TRIP12 and AREL1 E3 ligases of the Homologous to E6 C-terminus (HECT) family in adding destabilising K27-, K29- and K33- poly-ubiquitin chains on pro-IL-1β. We show that precursor abundance determines mature IL-1β production, andUBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1β. Our study uncovers fundamental processes governing IL-1β homeostasis and provides molecular insights that could be exploited to mitigate its adverse actions in disease.
Mishra V, Crespo-Puig A, McCarthy C, et al., 2022, IL-1β turnover by TRIP12 and AREL1 ubiquitin ligases and UBE2L3 limits inflammation
<jats:title>ABSTRACT</jats:title><jats:p>The cytokine interleukin-1β (IL-1β) has pivotal roles in antimicrobial immunity, but also incites inflammatory pathology. Bioactive IL-1β is released following proteolytic maturation of the pro-IL-1β precursor by caspase-1 inflammasomes. UBE2L3/UBCH7, a conserved ubiquitin conjugating enzyme, promotes pro-IL-1β ubiquitylation and proteasomal disposal. However, UBE2L3 actions<jats:italic>in vivo</jats:italic>and ubiquitin ligases involved in this process are unknown. Here we report that deletion of<jats:italic>Ube2l3</jats:italic>in mice markedly reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production, neutrophilic inflammation and disease symptoms following inflammasome activation. A family-wide siRNA screen identified two ubiquitin ligases, TRIP12 and AREL1, which we show add K27-, K29- and K33- poly-ubiquitin chains on lysine residues in the ‘pro’ domain and destabilise pro-IL-1β. Mutation of ubiquitylation sites increased pro-IL-1β stability, but did not affect proteolysis by caspase-1. The extent of mature IL-1β production is therefore determined by precursor abundance, and UBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1β. Our study has uncovered fundamental processes governing IL-1β homeostasis and provided molecular insights that could be exploited to mitigate its adverse actions in disease.</jats:p>
Kennedy C, Goya Grocin A, Kovacic T, et al., 2021, A probe for NLRP3 inflammasome inhibitor MCC950 identifies carbonic anhydrase 2 as a novel target, ACS Chemical Biology, Vol: 16, Pages: 982-990, ISSN: 1554-8929
Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, until now, no off-targets have been identified. Herein, we report the design, synthesis, and application of a novel photoaffinity alkyne-tagged probe for MCC950 (IMP2070) which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, including carbonic anhydrase 2 (CA2) as a specific target of IMP2070, and independent cellular thermal proteomic profiling revealed stabilization of CA2 by MCC950. MCC950 displayed noncompetitive inhibition of CA2 activity, confirming carbonic anhydrase as an off-target class for this compound. These data highlight potential biological mechanisms through which MCC950 and derivatives may exhibit off-target effects in preclinical or clinical studies.
Mylona E, Sanchez Garrido J, Nguyen Hoang Thu T, et al., 2021, Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death, Cellular Microbiology, Vol: 23, Pages: 1-14, ISSN: 1462-5814
Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD‐mediated pyroptosis via activation of caspase‐1, caspase‐4 and caspase‐8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island‐1 (SPI‐1) injectisome, HilA‐mediated overexpression of the SPI‐1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O‐antigen length regulator, which induces the production of very long O‐antigen chains. Using a ΔfepE mutant we established that the very long O‐antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome‐mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar‐specific inflammasome modulation, which mirrors the pro‐ and anti‐inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome‐mediated detection through the elaboration of very long LPS O‐polysaccharides.
Zaveri A, Bose A, Sharma S, et al., 2021, Mycobacterial STAND adenylyl cyclases: the HTH domain binds DNA to form biocrystallized nucleoids, Biophysical Journal, Vol: 120, Pages: 1231-1246, ISSN: 0006-3495
Mycobacteria harbor a unique class of adenylyl cyclases with a complex domain organization consisting of an N-terminal putative adenylyl cyclase domain fused to a nucleotide-binding adaptor shared by apoptotic protease-activating factor-1, plant resistance proteins, and CED-4 (NB-ARC) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal helix-turn-helix (HTH) domain. The products of the rv0891c-rv0890c genes represent a split gene pair, where Rv0891c has sequence similarity to adenylyl cyclases, and Rv0890c harbors the NB-ARC-TPR-HTH domains. Rv0891c had very low adenylyl cyclase activity so it could represent a pseudoenzyme. By analyzing the genomic locus, we could express and purify Rv0890c and find that the NB-ARC domain binds ATP and ADP, but does not hydrolyze these nucleotides. Using systematic evolution of ligands by exponential enrichment (SELEX), we identified DNA sequences that bound to the HTH domain of Rv0890c. Uniquely, the HTH domain could also bind RNA. Atomic force microscopy revealed that binding of Rv0890c to DNA was sequence independent, and binding of adenine nucleotides to the protein induced the formation of higher order structures that may represent biocrystalline nucleoids. This represents the first characterization of this group of proteins and their unusual biochemical properties warrant further studies into their physiological roles in future.
Zhong Q, Roumeliotis T, Kozik Z, et al., 2020, Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells, PLoS Biology, Vol: 18, ISSN: 1544-9173
Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.
Fisch D, Clough B, Domart M-C, et al., 2020, Human GBP1 differentially targets salmonella and toxoplasma to license recognition of microbial ligands and caspase-mediated death, Cell Reports, Vol: 32, Pages: 1-22, ISSN: 2211-1247
Interferon-inducible guanylate-binding proteins (GBPs) promote cell-intrinsic defense through host cell death. GBPs target pathogens and pathogen-containing vacuoles and promote membrane disruption for release of microbial molecules that activate inflammasomes. GBP1 mediates pyroptosis or atypical apoptosis of Salmonella Typhimurium (STm)- or Toxoplasma gondii (Tg)- infected human macrophages, respectively. The pathogen-proximal detection-mechanisms of GBP1 remain poorly understood, as humans lack functional immunity-related GTPases (IRGs) that assist murine Gbps. Here, we establish that GBP1 promotes the lysis of Tg-containing vacuoles and parasite plasma membranes, releasing Tg-DNA. In contrast, we show GBP1 targets cytosolic STm and recruits caspase-4 to the bacterial surface for its activation by lipopolysaccharide (LPS), but does not contribute to bacterial vacuole escape. Caspase-1 cleaves and inactivates GBP1, and a cleavage-deficient GBP1D192E mutant increases caspase-4-driven pyroptosis due to the absence of feedback inhibition. Our studies elucidate microbe-specific roles of GBP1 in infection detection and its triggering of the assembly of divergent caspase signaling platforms.
Sanchez-Garrido J, Shenoy A, 2020, Regulation and repurposing of nutrient sensing and autophagy in innate immunity, Autophagy, Vol: 17, Pages: 1571-1591, ISSN: 1554-8627
Nutrients not only act as building blocks but also as signaling molecules. Nutrient-availability promotes cell growth and proliferation and suppresses catabolic processes, such as macroautophagy/autophagy. These effects are mediated by checkpoint kinases such as MTOR (mechanistic target of rapamycin kinase), which is activated by amino acids and growth factors, and AMP-activated protein kinase (AMPK), which is activated by low levels of glucose or ATP. These kinases have wide-ranging activities that can be co-opted by immune cells upon exposure to danger signals, cytokines or pathogens. Here, we discuss recent insight into the regulation and repurposing of nutrient-sensing responses by the innate immune system during infection. Moreover, we examine how natural mutations and pathogen-mediated interventions can alter the balance between anabolic and autophagic pathways leading to a breakdown in tissue homeostasis and/or host defense.
Sanchez Garrido J, Slater SL, Clements A, et al., 2020, Vying for the control of inflammasomes: the cytosolic frontier of enteric bacterial pathogen - host interactions, Cellular Microbiology, Vol: 22, Pages: 1-19, ISSN: 1462-5814
Enteric pathogen-host interactions occur at multiple interfaces,includingthe intestinal epitheliumand deeper organsof the immune system. Microbial ligands and activities are detected by host sensorsthat elicit a range of immune responses. Membrane-bound Toll-Like Receptors (TLRs) and cytosolic inflammasomepathways are key signal transducers that trigger production of pro-inflammatory molecules such as cytokines and chemokinesand regulate cell deathin response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tusslebetween bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1and are regulated by related caspases, such as caspase-11, -4, -5 and -8.Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasomeactivation(e.g. Listeria monocytogenes, Helicobacter pylori), others(e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system (T3SS) effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasomepathwaysand their immune functions and discuss how enteric bacterial pathogens interact with them.These studies have not only shed light on the inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune
Subbarao S, Sanchez-Garrido J, Krishnan N, et al., 2020, Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages, Scientific Reports, Vol: 10, ISSN: 2045-2322
Mycobacterium tuberculosis infection causes high rates of morbidity and mortality. Host-directed therapy may enhance the immune response, reduce tissue damage and shorten treatment duration. The inflammasome is integral to innate immune responses but over-activation has been described in tuberculosis (TB) pathology and TB-immune reconstitution syndrome. Here we explore how clinical isolates differentially activate the inflammasome and how inflammasome inhibition can lead to enhanced bacterial clearance. Wild-type, Nlrp3−/−/Aim2−/−, Casp1/11−/− and Asc−/− murine bone-marrow derived macrophages (BMDMs) were infected with laboratory strain M. tuberculosis H37Rv or clinical isolates from various lineages. Inflammasome activation and bacterial numbers were measured, and pharmacological inhibition of NLRP3 was achieved using MCC950. Clinical isolates of M. tuberculosis differed in their ability to activate inflammasomes. Beijing isolates had contrasting effects on IL-1β and caspase-1 activation, but all clinical isolates induced lower IL-1β release than H37Rv. Our studies suggest the involvement of NLRP3, AIM2 and an additional unknown sensor in IL-1β maturation. Pharmacological blockade of NLRP3 with MCC950 reduced bacterial survival, and combined treatment with the antimycobacterial drug rifampicin enhanced the effect. Modulating the inflammasome is an attractive adjunct to current anti-mycobacterial therapy that warrants further investigation.
Prasad H, Shenoy AR, Visweswariah SS, 2020, Cyclic nucleotides, gut physiology and inflammation, The Federation of European Biochemical Societies (FEBS) Journal, Vol: 287, Pages: 1970-1981, ISSN: 1742-464X
Misregulation of gut function and homeostasis impinges on the overall well‐being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria‐mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'‐monophosphate and cyclic guanosine 3′,5′‐monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function.
Watson J, Sanchez-garrido J, Goddard P, et al., 2019, Shigella sonnei O-Antigen Inhibits Internalization, Vacuole Escape, and Inflammasome Activation, mBio, Vol: 10, Pages: 1-14, ISSN: 2150-7511
Two Shigella species, flexneri and sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle and high-income countries. S. flexneri is a prototypiccytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-antigen onto its surface compared to other Shigella species.
Mullineaux-Sanders C, Sanchez-Garrido J, Hopkins EGD, et al., 2019, <i>Citrobacter rodentium</i>-host-microbiota interactions: immunity, bioenergetics and metabolism, NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 701-715, ISSN: 1740-1526
Watson JL, Sanchez-Garrido J, Goddard PJ, et al., 2019, <i>Shigella sonnei</i>O-antigen inhibits internalisation, vacuole escape and inflammasome activation
<jats:title>Abstract</jats:title><jats:p>Two<jats:italic>Shigella</jats:italic>species,<jats:italic>flexneri</jats:italic>and<jats:italic>sonnei</jats:italic>, cause approximately 90% of bacterial dysentery worldwide. While<jats:italic>S. flexneri</jats:italic>is the dominant species in low-income countries,<jats:italic>S. sonnei</jats:italic>causes the majority of infections in middle and high-income countries.<jats:italic>S. flexneri</jats:italic>is a prototypic cytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By contrast little is known about the invasion, vacuole escape and induction of pyroptosis during<jats:italic>S. sonnei</jats:italic>infection of macrophages. We demonstrate that<jats:italic>S. sonnei</jats:italic>causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic<jats:italic>S. sonnei</jats:italic>and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen, which in<jats:italic>S. sonnei</jats:italic>is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings suggest that<jats:italic>S. sonnei</jats:italic>has adapted to an extracellular lifestyle by incorporating additional O-antigen into its surface structures compared to other<jats:italic>Shigella</jats:italic>species.</jats:p>
Fisch D, Clough B, Domart M-C, et al., 2019, Differential spatiotemporal targeting of Toxoplasma and Salmonella by GBP1 assembles caspase signalling platforms, Publisher: bioRxiv
Human guanylate binding proteins (GBPs), a family of IFNγ-inducible GTPases, promote cell-intrinsic defence against pathogens and host cell death. We previously identified GBP1 as a mediator of cell death of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella Typhimurium (STm). How GBP1 targets microbes for AIM2 activation during Tg infection and caspase-4 during STm infection remains unclear. Here, using correlative light and electron microscopy and EdU labelling of Tg-DNA, we reveal that GBP1-decorated parasitophorous vacuoles (PVs) lose membrane integrity and release Tg-DNA for detection by AIM2-ASC-CASP8. In contrast, differential staining of cytosolic and vacuolar STm revealed that GBP1 does not contribute to STm escape into the cytosol but decorates almost all cytosolic STm leading to the recruitment of caspase-4. Caspase-5, which can bind LPS and whose expression is upregulated by IFNγ, does not target STm pointing to a key role for caspase-4 in pyroptosis. We also uncover a regulatory mechanism involving the inactivation of GBP1 by its cleavage at Asp192 by caspase-1. Cells expressing non-cleavable GBP1D192E therefore undergo higher caspase-4-driven pyroptosis during STm infection. Taken together, our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in inducing cell death by leading to assembly and regulation of divergent caspase signalling platforms.
Fisch D, Bando H, Clough B, et al., 2019, Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis, EMBO Journal, Vol: 38, ISSN: 0261-4189
The guanylate binding protein (GBP) family of interferon-inducible GTPases promotes antimicrobial immunity and cell death. During bacterial infection, multiple mouse Gbps, human GBP2, and GBP5 support the activation of caspase-1-containing inflammasome complexes or caspase-4 which trigger pyroptosis. Whether GBPs regulate other forms of cell death is not known. The apicomplexan parasite Toxoplasma gondii causes macrophage death through unidentified mechanisms. Here we report that Toxoplasma-induced death of human macrophages requires GBP1 and its ability to target Toxoplasma parasitophorous vacuoles through its GTPase activity and prenylation. Mechanistically, GBP1 promoted Toxoplasma detection by AIM2, which induced GSDMD-independent, ASC-, and caspase-8-dependent apoptosis. Identical molecular determinants targeted GBP1 to Salmonella-containing vacuoles. GBP1 facilitated caspase-4 recruitment to Salmonella leading to its enhanced activation and pyroptosis. Notably, GBP1 could be bypassed by the delivery of Toxoplasma DNA or bacterial LPS into the cytosol, pointing to its role in liberating microbial molecules. GBP1 thus acts as a gatekeeper of cell death pathways, which respond specifically to infecting microbes. Our findings expand the immune roles of human GBPs in regulating not only pyroptosis, but also apoptosis.
Goddard P, Sanchez Garrido J, Slater S, et al., 2019, Enteropathogenic E. coli stimulates effector-driven rapid caspase-4 activation in human macrophages, Cell Reports, Vol: 27, Pages: 1008-1017.e6, ISSN: 2211-1247
Microbial infections can stimulate the assembly of inflammasomes, which activate caspase-1. The gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) causes localized actin polymerization in host cells. Actin polymerization requires the binding of the bacterial adhesin intimin to Tir, which is delivered to host cells via a type 3 secretion system (T3SS). We show that EPEC induces T3SS-dependent rapid non-canonical NLRP3 inflammasome activation in human macrophages. Notably, caspase-4 activation by EPEC triggers pyroptosis and cytokine processing through the NLRP3-caspase-1 inflammasome. Mechanistically, caspase-4 activation requires the detection of LPS and EPEC-induced actin polymerization, either via Tir tyrosine phosphorylation and the phosphotyrosine-binding adaptor NCK or Tir and the NCK-mimicking effector TccP. An engineered E. coli K12 could reconstitute Tir-intimin signaling, which is necessary and sufficient for inflammasome activation, ruling out the involvement of other virulence factors. Our studies reveal a crosstalk between caspase-4 and caspase-1 that is cooperatively stimulated by LPS and effector-driven actin polymerization.
Ahmad L, Mashbat B, Leung C, et al., 2019, Human TANK-binding kinase 1 is required for early autophagy induction upon herpes simplex virus 1 infection, Journal of Allergy and Clinical Immunology, Vol: 143, Pages: 765-769.e7, ISSN: 0091-6749
Sanchez-Garrdio J, Sancho-Shimizu V, Shenoy A, 2018, Regulated proteolysis of p62/SQSTM1 enables differential control of autophagy and nutrient sensing, Science Signaling, Vol: 11, ISSN: 1937-9145
The multidomain scaffold protein p62 (also called sequestosome-1) is involved in autophagy, antimicrobial immunity, and oncogenesis. Mutations in SQSTM1, which encodes p62, are linked to hereditary inflammatory conditions such as Paget’s disease of the bone, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, and distal myopathy with rimmed vacuoles. Here, we report that p62 was proteolytically trimmed by the protease caspase-8 into a stable protein, which we called p62TRM. We found that p62TRM, but not full-length p62, was involved in nutrient sensing and homeostasis through the mechanistic target of rapamycin complex 1 (mTORC1). The kinase RIPK1 and caspase-8 controlled p62TRM production and thus promoted mTORC1 signaling. An FTD-linked p62 D329G polymorphism and a rare D329H variant could not be proteolyzed by caspase-8, and these noncleavable variants failed to activate mTORC1, thereby revealing the detrimental effect of these mutations. These findings on the role of p62TRM provide new insights into SQSTM1-linked diseases and mTORC1 signaling.
Shenoy AR, Furniss RCD, Goddard PJ, et al., 2018, Modulation of host cell processes by T3SS effectors, Escherichia coli, a Versatile Pathogen, Editors: Frankel, Ron, Publisher: Springer Verlag
Two of the enteric Escherichia coli pathotypes-enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC)-have a conserved type 3 secretion system which is essential for virulence. The T3SS is used to translocate between 25 and 50 bacterial proteins directly into the host cytosol where they manipulate a variety of host cell processes to establish a successful infection. In this chapter, we discuss effectors from EPEC/EHEC in the context of the host proteins and processes that they target-the actin cytoskeleton, small guanosine triphosphatases and innate immune signalling pathways that regulate inflammation and cell death. Many of these translocated proteins have been extensively characterised, which has helped obtain insights into the mechanisms of pathogenesis of these bacteria and also understand the host pathways they target in more detail. With increasing knowledge of the positive and negative regulation of host signalling pathways by different effectors, a future challenge is to investigate how the specific effector repertoire of each strain cooperates over the course of an infection.
Bist P, Cheong WS, Ng A, et al., 2017, E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP, Nature Communications, Vol: 8, ISSN: 2041-1723
Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.
Mazon-Moya MJ, Willis AR, Torraca V, et al., 2017, Septins restrict inflammation and protect zebrafish larvae from Shigella infection, PLoS Pathogens, Vol: 13, Pages: 1-23, ISSN: 1553-7366
Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation.
Pallett MA, Crepin VF, Serafini N, et al., 2017, Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells, Mucosal Immunology, Vol: 10, Pages: 602-612, ISSN: 1935-3456
The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.
Eldridge MJG, Sanchez Garrido J, Hoben GF, et al., 2017, The atypical ubiquitin E2 conjugase UBE2L3 is an indirect caspase-1 target and controls IL-1beta secretion by inflammasomes, Cell Reports, Vol: 18, Pages: 1285-1297, ISSN: 2211-1247
Caspase-1 activation by inflammasome signalling scaffoldsinitiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-IL-1α into its mature form and directs its secretion, triggers pyroptosis and the release of non-substrate alarmins such as IL-1α and HMGB1. While somecaspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here we report using unbiased proteomics that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, controlled pyroptosis-and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acted post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increased pro-IL-1β levels and mature-IL-1βsecretion by inflammasomes. These findings on UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms.
Thurston T, Matthews S, Jennings E, et al., 2016, Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death, Nature Communications, Vol: 7, ISSN: 2041-1723
Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.
Surana S, Shenoy AR, Krishnan Y, 2015, Designing DNA nanodevices for compatibility with the immune system of higher organisms, Nature Nanotechnology, Vol: 10, Pages: 741-747, ISSN: 1748-3395
DNA is proving to be a powerful scaffold to construct molecularly precise designer DNA devices. Recent trends reveal their ever-increasing deployment within living systems as delivery devices that not only probe but also program and re-program a cell, or even whole organisms. Given that DNA is highly immunogenic, we outline the molecular, cellular and organismal response pathways that designer nucleic acid nanodevices are likely to elicit in living systems. We address safety issues applicable when such designer DNA nanodevices interact with the immune system. In light of this, we discuss possible molecular programming strategies that could be integrated with such designer nucleic acid scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms.
Mostowy S, Shenoy AR, 2015, The cytoskeleton in cell-autonomous immunity: structural determinants of host defence, Nature Reviews Immunology, Vol: 15, Pages: 559-573, ISSN: 1474-1741
Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence.
Eldridge MJG, Shenoy AR, 2015, Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens, CURRENT OPINION IN MICROBIOLOGY, Vol: 23, Pages: 32-41, ISSN: 1369-5274
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