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1.
EMBO J ; 38(13): e100926, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31268602

RESUMEN

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.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Macrófagos/parasitología , Toxoplasma/patogenicidad , Toxoplasmosis/metabolismo , Caspasas Iniciadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Macrófagos/metabolismo , Prenilación de Proteína , Piroptosis , Células THP-1 , Toxoplasmosis/parasitología
2.
PLoS Biol ; 18(12): e3000986, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33378358

RESUMEN

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.


Asunto(s)
Calcio/metabolismo , Proteínas de Escherichia coli/metabolismo , Piroptosis/fisiología , Receptores de Superficie Celular/metabolismo , Actinas/metabolismo , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/fisiología , Análisis por Conglomerados , Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Intestinos/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Sistemas de Secreción Tipo III/metabolismo
3.
Cell Microbiol ; 23(5): e13306, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33355403

RESUMEN

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.


Asunto(s)
Inflamasomas/metabolismo , Macrófagos/microbiología , Macrófagos/fisiología , Antígenos O/fisiología , Fiebre Paratifoidea/microbiología , Piroptosis , Salmonella paratyphi A/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Caspasas/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/inmunología , Antígenos O/química , Proteínas de Unión a Fosfato/metabolismo , Salmonella paratyphi A/inmunología , Células THP-1 , Sistemas de Secreción Tipo III/metabolismo , Virulencia , Factores de Virulencia/metabolismo
4.
Biophys J ; 120(7): 1231-1246, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33217386

RESUMEN

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.


Asunto(s)
Adenilil Ciclasas , Proteínas Bacterianas , Mycobacterium/enzimología , Adenilil Ciclasas/genética , ADN/genética
5.
Cell Microbiol ; 22(4): e13184, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32185892

RESUMEN

Enteric pathogen-host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane-bound toll-like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro-inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1 and 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 inflammasome activation (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 effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.


Asunto(s)
Citosol/inmunología , Enterobacteriaceae/patogenicidad , Interacciones Huésped-Patógeno/inmunología , Inflamasomas/genética , Transducción de Señal/inmunología , Animales , Muerte Celular , Citosol/microbiología , Enterobacteriaceae/inmunología , Interacciones Huésped-Patógeno/genética , Humanos , Inflamasomas/inmunología , Macrófagos/microbiología , Ratones , Sistemas de Secreción Tipo III/metabolismo
6.
PLoS Pathog ; 13(6): e1006467, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28650995

RESUMEN

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.


Asunto(s)
Disentería Bacilar/inmunología , Inmunidad Innata/inmunología , Septinas/metabolismo , Animales , Modelos Animales de Enfermedad , Disentería Bacilar/microbiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamación/inmunología , Inflamación/microbiología , Mucosa Intestinal/microbiología , Larva/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiología , Shigella flexneri , Pez Cebra
7.
Curr Top Microbiol Immunol ; 416: 73-115, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30178263

RESUMEN

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.


Asunto(s)
Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Sistemas de Secreción Tipo III/metabolismo , Citoesqueleto de Actina/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , GTP Fosfohidrolasas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata
9.
J Immunol ; 189(2): 813-8, 2012 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-22675202

RESUMEN

Autophagy is a major innate immune defense pathway in both plants and animals. In mammals, this cascade can be elicited by cytokines (IFN-γ) or pattern recognition receptors (TLRs and nucleotide-binding oligomerization domain-like receptors). Many signaling components in TLR- and nucleotide-binding oligomerization domain-like receptor-induced autophagy are now known; however, those involved in activating autophagy via IFN-γ remain to be elucidated. In this study, we engineered macrophages encoding a tandem fluorescently tagged LC3b (tfLC3) autophagosome reporter along with stably integrated short hairpin RNAs to demonstrate IFN-γ-induced autophagy required JAK 1/2, PI3K, and p38 MAPK but not STAT1. Moreover, the autophagy-related guanosine triphosphatase Irgm1 proved dispensable in both stable tfLC3-expressing RAW 264.7 and tfLC3-transduced Irgm1(-/-) primary macrophages, revealing a novel p38 MAPK-dependent, STAT1-independent autophagy pathway that bypasses Irgm1. These unexpected findings have implications for understanding how IFN-γ-induced autophagy is mobilized within macrophages for inflammation and host defense.


Asunto(s)
Autofagia/inmunología , Interferón gamma/fisiología , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/citología , Macrófagos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Autofagia/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Línea Celular , Proteínas de Unión al GTP/deficiencia , Proteínas de Unión al GTP/fisiología , Genes Reporteros/inmunología , Macrófagos/enzimología , Ratones , Ratones Noqueados , Fagosomas/enzimología , Fagosomas/inmunología , Fagosomas/metabolismo , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/fisiología
10.
Nat Commun ; 14(1): 4385, 2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37474493

RESUMEN

The cytokine interleukin-1ß (IL-1ß) has pivotal roles in antimicrobial immunity, but also incites inflammatory disease. Bioactive IL-1ß is released following proteolytic maturation of the pro-IL-1ß precursor by caspase-1. UBE2L3, a ubiquitin conjugating enzyme, promotes pro-IL-1ß 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 of Ube2l3 in mice reduces pro-IL-1ß turnover in macrophages, leading to excessive mature IL-1ß 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-1ß. We show that precursor abundance determines mature IL-1ß production, and UBE2L3, TRIP12 and AREL1 limit inflammation by shrinking the cellular pool of pro-IL-1ß. Our study uncovers fundamental processes governing IL-1ß homeostasis and provides molecular insights that could be exploited to mitigate its adverse actions in disease.


Asunto(s)
Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Animales , Ratones , Inflamación , Interleucina-1beta , Ubiquitina , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética
11.
Science ; 382(6666): eadg2253, 2023 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-37797010

RESUMEN

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.


Asunto(s)
Proteínas de Unión al GTP , Interacciones Huésped-Patógeno , Inmunidad Innata , Interferón gamma , Proteínas Proto-Oncogénicas c-pim-1 , Toxoplasma , Toxoplasmosis , Humanos , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Interferón gamma/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Toxoplasmosis/inmunología , Factores de Virulencia/metabolismo , Macrófagos/inmunología , Proteínas 14-3-3/metabolismo , Interacciones Huésped-Patógeno/inmunología
12.
Autophagy ; 17(7): 1571-1591, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-32627660

RESUMEN

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.Abbreviations: AKT1/PKB: AKT serine/threonine kinase 1; ATG: autophagy related; BECN1: beclin 1; CGAS: cyclic GMP-AMP synthase; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; FFAR: free fatty acid receptor; GABARAP: GABA type A receptor-associated protein; IFN: interferon; IL: interleukin; LAP: LC3-associated phagocytosis; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NLR: NOD (nucleotide-binding oligomerization domain) and leucine-rich repeat containing proteins; PI3K, phosphoinositide 3-kinase; PRR: pattern-recognition receptor; PtdIns3K: phosphatidylinositol 3-kinase; RALB: RAS like proto-oncogene B; RHEB: Ras homolog, MTORC1 binding; RIPK1: receptor interacting serine/threonine kinase 1; RRAG: Ras related GTP binding; SQSTM1/p62: sequestosome 1; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; STK11/LKB1: serine/threonine kinase 11; TBK1: TANK binding kinase 1; TLR: toll like receptor; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; TRIM: tripartite motif protein; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H+-proton-translocating ATPase.


Asunto(s)
Autofagia , Inmunidad Innata , Nutrientes/metabolismo , Animales , Autofagia/fisiología , Humanos , Inmunidad Innata/fisiología , Infecciones/inmunología , Infecciones/metabolismo , Nutrientes/fisiología , Transducción de Señal/inmunología , Transducción de Señal/fisiología
13.
ACS Chem Biol ; 16(6): 982-990, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-34003636

RESUMEN

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.


Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Furanos/farmacología , Indenos/farmacología , Inflamasomas/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Sulfonamidas/farmacología , Anhidrasa Carbónica II/metabolismo , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Furanos/química , Humanos , Indenos/química , Inflamasomas/metabolismo , Macrófagos/metabolismo , Modelos Moleculares , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteómica , Sulfonamidas/química
14.
Sci Rep ; 10(1): 3709, 2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-32111888

RESUMEN

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-1ß and caspase-1 activation, but all clinical isolates induced lower IL-1ß release than H37Rv. Our studies suggest the involvement of NLRP3, AIM2 and an additional unknown sensor in IL-1ß 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.


Asunto(s)
Inflamasomas/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/inmunología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Furanos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Indenos , Inflamasomas/efectos de los fármacos , Inflamasomas/genética , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Sulfonamidas , Sulfonas/farmacología , Tuberculosis/genética , Tuberculosis/microbiología
15.
Cell Rep ; 32(6): 108008, 2020 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-32783936

RESUMEN

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.


Asunto(s)
Caspasas/inmunología , Proteínas de Unión al GTP/inmunología , Salmonella typhimurium/inmunología , Toxoplasma/inmunología , Muerte Celular/inmunología , Células HEK293 , Humanos , Inflamasomas/inmunología , Interferón gamma/farmacología , Ligandos , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Células THP-1 , Toxoplasma/genética , Toxoplasmosis/inmunología , Toxoplasmosis/microbiología , Vacuolas/inmunología
16.
J Mol Evol ; 68(6): 587-602, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19495554

RESUMEN

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity. Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signal transduction.


Asunto(s)
Biología Computacional/métodos , Evolución Molecular , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Fosfotransferasas/genética , Estructura Terciaria de Proteína/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , GMP Cíclico/metabolismo , Bases de Datos Genéticas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido
17.
Nat Rev Microbiol ; 17(11): 701-715, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31541196

RESUMEN

Citrobacter rodentium is an extracellular enteric mouse-specific pathogen used to model infections with human pathogenic Escherichia coli and inflammatory bowel disease. C. rodentium injects type III secretion system effectors into intestinal epithelial cells (IECs) to target inflammatory, metabolic and cell survival pathways and establish infection. While the host responds to infection by activating innate and adaptive immune signalling, required for clearance, the IECs respond by rapidly shifting bioenergetics to aerobic glycolysis, which leads to oxygenation of the epithelium, an instant expansion of mucosal-associated commensal Enterobacteriaceae and a decline of obligate anaerobes. Moreover, infected IECs reprogramme intracellular metabolic pathways, characterized by simultaneous activation of cholesterol biogenesis, import and efflux, leading to increased serum and faecal cholesterol levels. In this Review we summarize recent advances highlighting the intimate relationship between C. rodentium pathogenesis, metabolism and the gut microbiota.


Asunto(s)
Citrobacter rodentium/crecimiento & desarrollo , Citrobacter rodentium/inmunología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/microbiología , Interacciones Microbiota-Huesped , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Inmunidad Adaptativa , Aerobiosis , Animales , Citrobacter rodentium/metabolismo , Metabolismo Energético , Células Epiteliales/inmunología , Células Epiteliales/patología , Glucólisis , Inmunidad Innata , Ratones , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/metabolismo
18.
mBio ; 10(6)2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31848280

RESUMEN

Two Shigella species, Shigella flexneri and Shigella 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 prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here 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 (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.


Asunto(s)
Disentería Bacilar/metabolismo , Disentería Bacilar/microbiología , Interacciones Huésped-Patógeno/inmunología , Inflamasomas/metabolismo , Antígenos O/inmunología , Shigella sonnei/fisiología , Vacuolas/metabolismo , Endocitosis/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Modelos Biológicos , Piroptosis/inmunología , Sistemas de Secreción Tipo III
19.
Cell Rep ; 27(4): 1008-1017.e6, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31018119

RESUMEN

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.


Asunto(s)
Caspasas Iniciadoras/fisiología , Escherichia coli Enteropatógena/patogenicidad , Macrófagos/microbiología , Actinas/metabolismo , Caspasa 1/genética , Caspasa 1/metabolismo , Caspasa 1/fisiología , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inflamasomas/fisiología , Modelos Biológicos , Polimerizacion
20.
J Bacteriol ; 190(11): 3824-34, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18390660

RESUMEN

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3',5'-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the DeltaMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the DeltaMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the DeltaMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.


Asunto(s)
Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/metabolismo , AMP Cíclico/metabolismo , Mycobacterium smegmatis/metabolismo , Adenilil Ciclasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Transducción de Señal/fisiología
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