RESUMEN
Infection by intracellular pathogens can trigger activation of the IRE1α branch of the unfolded protein response (UPR), which then modulates innate immunity and infection outcomes during bacterial or viral infection. However, the mechanisms by which infection activates IRE1α have not been fully elucidated. While recognition of microbe-associated molecular patterns can activate IRE1α, it is unclear whether this depends on the canonical role of IRE1α in detecting misfolded proteins. Here, we report that Candida albicans infection of macrophages results in IRE1α activation through C-type lectin receptor signaling, reinforcing a role for IRE1α as a central regulator of host responses to infection by a broad range of pathogens. However, IRE1α activation was not preceded by protein misfolding in response to either C. albicans infection or lipopolysaccharide treatment, implicating a non-canonical mode of IRE1α activation after recognition of microbial patterns. Investigation of the phenotypic consequences of IRE1α activation in macrophage antimicrobial responses revealed that IRE1α activity enhances the fungicidal activity of macrophages. Macrophages lacking IRE1α activity displayed inefficient phagolysosomal fusion, enabling C. albicans to evade fungal killing and escape the phagosome. Together, these data provide mechanistic insight for the non-canonical activation of IRE1α during infection, and reveal central roles for IRE1α in macrophage antifungal responses.
RESUMEN
Myeloid-derived suppressor cells (MDSCs) can promote tumor progression. In this issue of Immunity, Mohamed et al. show that the unfolded protein response sensor, PERK, enhances MDSC-mediated immunosuppression through the NRF2 transcription factor, preventing oxidative damage, mitochondrial DNA release, and DNA sensor-STING-dependent type I interferon production.
Asunto(s)
Células Mieloides , Células Supresoras de Origen Mieloide , Tolerancia Inmunológica , Terapia de Inmunosupresión , Células Mieloides/inmunología , Células Supresoras de Origen Mieloide/inmunología , Respuesta de Proteína DesplegadaRESUMEN
The mitochondrial network plays a critical role in the regulation of innate immune signaling and subsequent production of proinflammatory cytokines such as IFN-ß and IL-1ß. Dynamin-related protein 1 (DRP1) promotes mitochondrial fission and quality control to maintain cellular homeostasis during infection. However, mechanisms by which DRP1 and mitochondrial dynamics control innate immune signaling and the proinflammatory response are incompletely understood. Here we show that macrophage DRP1 is a positive regulator of TNF-α production during sterile inflammation or bacterial infection. Silencing macrophage DRP1 decreased mitochondrial fragmentation and TNF-α production upon stimulation with lipopolysaccharide (LPS) or methicillin-resistant Staphylococcus aureus (MRSA) infection. The defect in TNF-α induction could not be attributed to changes in gene expression. Instead, DRP1 was required for post-transcriptional control of TNF-α. In contrast, silencing DRP1 enhanced IL-6 and IL-1ß production, indicating a distinct mechanism for DRP1-dependent TNF-α regulation. Our results highlight DRP1 as a key player in the macrophage pro-inflammatory response and point to its involvement in post-transcriptional control of TNF-α production.
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Staphylococcus aureus Resistente a Meticilina , Dinámicas Mitocondriales , Dinaminas , Mitocondrias , Proteínas Mitocondriales/genética , Factor de Necrosis Tumoral alfaRESUMEN
Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.
Asunto(s)
Infecciones por Astroviridae/inmunología , Infecciones por Astroviridae/veterinaria , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Mamastrovirus/fisiología , Tropismo Viral/genética , Animales , Células CACO-2 , Línea Celular , Chlorocebus aethiops , Enterocitos/virología , Gastroenteritis/virología , Humanos , Inmunidad Innata/inmunología , Interferones/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/virología , Intestino Delgado/citología , Intestino Delgado/virología , Mamastrovirus/genética , Mamastrovirus/inmunología , Células Vero , Tropismo Viral/inmunologíaRESUMEN
Bacterial metabolism represents the biochemical space that bacteria can manipulate to produce energy, reducing equivalents and building blocks for replication. Gram-positive pathogens, such as Listeria monocytogenes, show remarkable flexibility, which allows for exploitation of diverse biological niches from the soil to the intracytosolic space. Although the human host represents a potentially rich source for nutrient acquisition, competition for nutrients with the host and hostile host defenses can constrain bacterial metabolism by various mechanisms, including nutrient sequestration. Here, we review metabolism in the model Gram-positive bacterium, L. monocytogenes, and highlight pathways that enable the replication, survival, and virulence of this bacterial pathogen.
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Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Listeria monocytogenes/genética , VirulenciaRESUMEN
The innate immune system senses microbial ligands through pattern recognition and triggers downstream signaling cascades to promote inflammation and immune defense mechanisms. Emerging evidence suggests that cells also recognize alterations in host processes induced by infection as triggers. Protein ubiquitination and deubiquitination are post-translational modification processes essential for signaling and maintenance of cellular homeostasis, and infections can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the impact of ubiquitin homeostasis alteration on macrophage function. Perturbation of ubiquitin homeostasis led to a rapid and transient burst of reactive oxygen species (ROS) that promoted macrophage inflammatory and anti-infective capacity. Moreover, we found that ROS production was dependent on the NOX2 phagocyte NADPH oxidase. Global alteration of the ubiquitin proteome also enhanced proinflammatory cytokine production in mice stimulated with a sub-lethal dose of LPS. Collectively, our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.
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Macrófagos/metabolismo , Estrés Oxidativo/inmunología , Ubiquitinación/fisiología , Animales , Femenino , Homeostasis , Inmunidad Innata/fisiología , Inflamación/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Fagocitos/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics and energetic profiling of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus (MNV) infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in macrophages revealed that glycolysis is an important factor for optimal MNV infection, while inhibiting the PPP and OXPHOS showed a relatively minor impact of these pathways on MNV infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased viral protein production and viral RNA. The requirement of glycolysis was specific for MNV (but not astrovirus) infection, independent of the type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMP-activated protein kinase (AMPK), two master regulators of cellular metabolism, implicating Akt signaling in upregulating host metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates glycolysis as a virulence determinant. They further point to cellular metabolism as a novel therapeutic target for norovirus infections and improvements in current human norovirus culture systems.IMPORTANCE Viruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase glycolysis upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high-efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies.
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Glucólisis , Interacciones Huésped-Patógeno , Norovirus/fisiología , Replicación Viral , Animales , Células CACO-2 , Infecciones por Caliciviridae/virología , Humanos , Macrófagos/virología , Metabolómica , Ratones , Fosforilación Oxidativa , Vía de Pentosa Fosfato , Células RAW 264.7RESUMEN
Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.IMPORTANCEListeria monocytogenes is an intracellular bacterial pathogen that is capable of invading numerous host cells during infection. L. monocytogenes can cross the blood-brain barrier, leading to life-threatening meningitis. Here we show that an L. monocytogenes surface protein, InlF, is necessary for optimal colonization of the brain in mice. Furthermore, in the absence of vimentin, a cytosolic intermediate filament protein that is also present on the surface of brain endothelial cells, colonization of the brain was significantly impaired. We further show that InlF binds vimentin to mediate invasion of host cells. This work identifies InlF as a bacterial surface protein with specific relevance for infection of the brain and underscores the significance of host cell surface vimentin interactions in microbial pathogenesis.
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Encéfalo/parasitología , Endocitosis , Interacciones Huésped-Patógeno , Listeria monocytogenes/fisiología , Listeriosis/parasitología , Vimentina/metabolismo , Animales , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Listeriosis/patología , Ratones , RatasRESUMEN
Bacterial pathogens have evolved to exploit humans as a rich source of nutrients to support survival and replication. The pathways of bacterial metabolism that permit successful colonization are surprisingly varied and highlight remarkable metabolic flexibility. The constraints and immune pressures of distinct niches within the human body set the stage for understanding the mechanisms by which bacteria acquire critical nutrients. In this article we discuss how different bacterial pathogens carry out carbon and energy metabolism in the host and how they obtain or use key nutrients for replication and immune evasion.
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Bacterias/metabolismo , Bacterias/patogenicidad , Interacciones Huésped-Patógeno , Redes y Vías Metabólicas , Animales , Bacterias/crecimiento & desarrollo , Carbono/metabolismo , Metabolismo Energético , Humanos , Evasión Inmune , VirulenciaRESUMEN
Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of â¼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection.
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Antivirales/farmacología , Animales , Enzimas Desubicuitinizantes/metabolismo , Humanos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/metabolismo , Macrófagos/virología , Ratones , Norovirus/efectos de los fármacos , Norovirus/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.
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Proteínas Portadoras/inmunología , Caspasa 2/inmunología , Estrés del Retículo Endoplásmico/inmunología , Inflamasomas/inmunología , Mitocondrias/inmunología , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/inmunología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Western Blotting , Brucella abortus/inmunología , Brucella abortus/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 2/genética , Caspasa 2/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/inmunología , Endorribonucleasas/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN/inmunología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
The global spread of anti-microbial resistance requires urgent attention, and diverse alternative strategies have been suggested to address this public health concern. Host-directed immunomodulatory therapies represent one approach that could reduce selection for resistant bacterial strains. Recently, the small molecule deubiquitinase inhibitor WP1130 was reported as a potential anti-infective drug against important human food-borne pathogens, notably Listeria monocytogenes and noroviruses. Utilization of WP1130 itself is limited due to poor solubility, but given the potential of this new compound, we initiated an iterative rational design approach to synthesize new derivatives with increased solubility that retained anti-infective activity. Here, we test a small library of novel synthetic molecules based on the structure of the parent compound, WP1130, for anti-infective activity in vitro. Our studies identify a promising candidate, compound 9, which reduced intracellular growth of L. monocytogenes at concentrations that caused minimal cellular toxicity. Compound 9 itself had no bactericidal activity and only modestly slowed Listeria growth rate in liquid broth culture, suggesting that this drug acts as an anti-infective compound by modulating host-cell function. Moreover, this new compound also showed anti-infective activity against murine norovirus (MNV-1) and human norovirus, using the Norwalk virus replicon system. This small molecule inhibitor may provide a chemical platform for further development of therapeutic deubiquitinase inhibitors with broad-spectrum anti-infective activity.
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Antiinfecciosos/farmacología , Macrófagos/microbiología , Inhibidores de Proteasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Animales , Antivirales/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Cianoacrilatos/química , Cianoacrilatos/farmacología , Humanos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/crecimiento & desarrollo , Macrófagos/efectos de los fármacos , Ratones , Viabilidad Microbiana/efectos de los fármacos , Peso Molecular , Norovirus/efectos de los fármacos , Piridinas/química , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Programmed cell death (PCD) can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase, however, its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated and caspase-1-independent PCD of infected macrophages. We also discovered that rough attenuated B. suis strain VTRS1 induces a caspase-2-mediated and caspase-1-independent proinflammatory cell death in infected macrophages, which was tentatively coined "caspase-2-mediated pyroptosis". However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1ß production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however, unlike its role in S. typhimurium-induced pyroptosis, pore formation did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical non-proinflammatory apoptosis and caspase-1-mediated proinflammatory pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also regulated caspase-3 and -8 activation, as well as cell death in macrophages treated with each of the three reagents. Taken together, our data has demonstrated that caspase-2 can play an important role in mediating a proinflammatory response and a hybrid cell death that demonstrates features of both apoptosis and pyroptosis.
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Apoptosis , Brucella abortus/inmunología , Caspasa 2/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/enzimologíaRESUMEN
The human gut microbiota is inextricably linked to health and disease. One important function of the commensal organisms living in the intestine is to provide colonization resistance against invading enteric pathogens. Because of the complex nature of the interaction between the microbiota and its host, multiple mechanisms likely contribute to resistance. In this review, we dissect the biological role of short-chain fatty acids (SCFA), which are fermentation end products of the intestinal microbiota, in host-pathogen interactions. SCFA exert an extensive influence on host physiology through nutritional, regulatory, and immunomodulatory functions and can also affect bacterial fitness as a form of acid stress. Moreover, SCFA act as a signal for virulence gene regulation in common enteric pathogens. Taken together, these studies highlight the importance of the chemical environment where the biology of the host, the microbiota, and the pathogen intersects, which provides a basis for designing effective infection prevention and control.
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Ácidos Grasos Volátiles , Intestinos , Bacterias , Fermentación , Tracto Gastrointestinal/microbiología , Interacciones Huésped-Patógeno , Humanos , MicrobiotaRESUMEN
Targeted disruption of the plasma membrane is a ubiquitous form of attack used in all three domains of life. Many bacteria secrete pore-forming proteins during infection with broad implications for pathogenesis. The cholesterol-dependent cytolysins (CDC) are a family of pore-forming toxins expressed predominately by Gram-positive bacterial pathogens. The structure and assembly of some of these oligomeric toxins on the host membrane have been described, but how the targeted cell responds to intoxication by the CDCs is not as clearly understood. Many CDCs induce lysis of their target cell and can activate apoptotic cascades to promote cell death. However, the extent to which intoxication causes cell death is both CDC- and host cell-dependent, and at lower concentrations of toxin, survival of intoxicated host cells is well documented. Additionally, the effect of CDCs can be seen beyond the plasma membrane, and it is becoming increasingly clear that these toxins are potent regulators of signaling and immunity, beyond their role in intoxication. In this review, we discuss the cellular response to CDC intoxication with emphasis on the effects of pore formation on the host cell plasma membrane and subcellular organelles and whether subsequent cellular responses contribute to the survival of the affected cell.
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Proteínas Bacterianas/toxicidad , Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , Citotoxinas/toxicidad , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Transducción de Señal/efectos de los fármacos , Inmunidad Adaptativa , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Muerte Celular/efectos de los fármacos , Membrana Celular/química , Membrana Celular/metabolismo , Citotoxinas/química , Citotoxinas/metabolismo , Endocitosis/efectos de los fármacos , Bacterias Grampositivas/inmunología , Bacterias Grampositivas/metabolismo , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Inmunidad Innata/efectos de los fármacos , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Propiedades de SuperficieRESUMEN
We have reported a new innate-like CD4 T cell population that expresses cell surface makers of effector/memory cells and produce Th1 and Th2 cytokines immediately upon activation. Unlike conventional CD4 T cells that are selected by thymic epithelial cells, these CD4 T cells, named T-CD4 T cells, are selected by MHC class II expressing thymocytes. Previously, we showed that the presence of T-CD4 T cells protected mice from airway inflammation suggesting an immune regulatory role of T-CD4 T cells. To further understand the function of T-CD4 T cells, we investigated immune responses mediated by T-CD4 T cells during bacterial infection because the generation of antigen specific CD4 T cells contributes to clearance of infection and for the development of immune memory. The current study shows a suppressive effect of T-CD4 T cells on both CD8 and CD4 T cell-mediated immune responses during Listeria and Helicobacter infections. In the mouse model of Listeria monocytogenes infection, T-CD4 T cells resulted in decreasedfrequency of Listeria-specific CD8 T cells and the killing activity of them. Furthermore, mice with T-CD4 T cells developed poor immune memory, demonstrated by reduced expansion of antigen-specific T cells and high bacterial burden upon re-infection. Similarly, the presence of T-CD4 T cells suppressed the generation of antigen-specific CD4 T cells in Helicobacter pylori infected mice. Thus, our studies reveal a novel function of T-CD4 T cells in suppressing anti-bacterial immunity.
RESUMEN
Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.
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Ácidos Grasos/metabolismo , Listeria monocytogenes/metabolismo , Estrés Fisiológico/fisiología , Factores de Virulencia/metabolismo , Animales , Línea Celular , Ácidos Grasos/química , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Macrófagos/microbiología , Ratones , Estructura Molecular , Mutación , Virulencia , Factores de Virulencia/genéticaRESUMEN
The cysteine protease caspase-7 has an established role in the execution of apoptotic cell death, but recent findings also suggest involvement of caspase-7 during the host response to microbial infection. Caspase-7 can be cleaved by the inflammatory caspase, caspase-1, and has been implicated in processing and activation of microbial virulence factors. Thus, caspase-7 function during microbial infection may be complex, and its role in infection and immunity has yet to be fully elucidated. Here we demonstrate that caspase-7 is cleaved during cytosolic infection with the intracellular bacterial pathogen, Listeria monocytogenes. Cleavage of caspase-7 during L. monocytogenes infection did not require caspase-1 or key adaptors of the primary pathways of innate immune signaling in this infection, ASC, RIP2 and MyD88. Caspase-7 protected infected macrophages against plasma membrane damage attributable to the bacterial pore-forming toxin Listeriolysin O (LLO). LLO-mediated membrane damage could itself trigger caspase-7 cleavage, independently of infection or overt cell death. We also detected caspase-7 cleavage upon treatment with other bacterial pore-forming toxins, but not in response to detergents. Taken together, our results support a model where cleavage of caspase-7 is a consequence of toxin-mediated membrane damage, a common occurrence during infection. We propose that host activation of caspase-7 in response to pore formation represents an adaptive mechanism by which host cells can protect membrane integrity during infection.
Asunto(s)
Caspasa 7/metabolismo , Membrana Celular/microbiología , Listeria monocytogenes/patogenicidad , Listeriosis/enzimología , Listeriosis/patología , Macrófagos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Toxinas Bacterianas/metabolismo , Proteínas Adaptadoras de Señalización CARD , Caspasa 1/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/inmunología , Listeria monocytogenes/metabolismo , Listeriosis/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.
Asunto(s)
Macrófagos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitrilos/farmacología , Fagosomas/enzimología , Inhibidores de Proteasas/farmacología , Piridinas/farmacología , Animales , Línea Celular , Cianoacrilatos , Endopeptidasas/metabolismo , Escherichia coli/fisiología , Listeria monocytogenes/fisiología , Macrófagos/enzimología , Ratones , Óxido Nítrico Sintasa de Tipo II/genética , Transporte de Proteínas/efectos de los fármacos , Ubiquitina/metabolismoRESUMEN
Although a complete pathway of lipoic acid metabolism has been established in Escherichia coli, lipoic acid metabolism in other bacteria is more complex and incompletely understood. Listeria monocytogenes has been shown to utilize two lipoate-protein ligases for lipoic acid scavenging, whereas only one of the ligases can function in utilization of host-derived lipoic acid-modified peptides. We report that lipoic acid scavenging requires not only ligation of lipoic acid but also a lipoyl relay pathway in which an amidotransferase transfers lipoyl groups to the enzyme complexes that require the cofactor for activity. In addition, we provide evidence for a new lipoamidase activity that could allow utilization of lipoyl peptides by lipoate-protein ligase. These data support a model of an expanded, three-enzyme pathway for lipoic acid scavenging that seems widespread in the Firmicutes phylum of bacteria.