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Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , COVID-19/inmunología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Unión Proteica/inmunología , Dominios Proteicos/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Células VeroRESUMEN
The inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines IL-1ß and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Here, we showed that the interferon-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria. IRGB10 directly targeted cytoplasmic bacteria through a mechanism requiring guanylate-binding proteins. Localization of IRGB10 to the bacterial cell membrane compromised bacterial structural integrity and mediated cytosolic release of ligands for recognition by inflammasome sensors. Overall, our results reveal IRGB10 as part of a conserved signaling hub at the interface between cell-autonomous immunity and innate immune sensing pathways.
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Proteínas de Unión al ADN/metabolismo , Francisella/inmunología , GTP Fosfohidrolasas/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Interacciones Huésped-Patógeno/inmunología , Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Linfocitos B/inmunología , Caspasas/metabolismo , Caspasas Iniciadoras , Citosol/inmunología , Citosol/microbiología , GTP Fosfohidrolasas/genética , Infecciones por Bacterias Gramnegativas/microbiología , Inmunidad Celular , Inmunidad Innata , Inflamasomas/metabolismo , Ligandos , Ratones , Ratones Mutantes , Células Mieloides/inmunología , Linfocitos T/inmunologíaRESUMEN
Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.
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Factor 1 de Ribosilacion-ADP/inmunología , Autofagia/inmunología , Proteínas Portadoras/inmunología , Interferón gamma/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Familia de las Proteínas 8 Relacionadas con la Autofagia , Sistemas CRISPR-Cas , Proteínas Portadoras/metabolismo , Simulación por Computador , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , GTP Fosfohidrolasas/inmunología , GTP Fosfohidrolasas/metabolismo , Edición Génica , Immunoblotting , Inmunoprecipitación , Interferón gamma/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.
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Toxoplasma , Toxoplasma/metabolismo , Fosforilación , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Vacuolas/metabolismoRESUMEN
Caspase activation results in pyroptosis, an inflammatory cell death that contributes to several inflammatory diseases by releasing inflammatory cytokines and cellular contents. Fusobacterium nucleatum is a periodontal pathogen frequently detected in human cancer and inflammatory bowel diseases. Studies have reported that F. nucleatum infection leads to NLRP3 activation and pyroptosis, but the precise activation process and disease association remain poorly understood. This study demonstrated that F. nucleatum infection exacerbates acute colitis in mice and activates pyroptosis through caspase-11-mediated gasdermin D cleavage in macrophages. Furthermore, F. nucleatum infection in colitis mice induces the enhancement of IL-1⺠secretion from the colon, affecting weight loss and severe disease activities. Neutralization of IL-1⺠protects F. nucleatum infected mice from severe colitis. Therefore, F. nucleatum infection facilitates inflammation in acute colitis with IL-1⺠from colon tissue by activating noncanonical inflammasome through gasdermin D cleavage.
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Colitis , Inflamasomas , Humanos , Animales , Ratones , Inflamasomas/metabolismo , Fusobacterium nucleatum/metabolismo , Gasderminas , Colitis/inducido químicamente , Caspasas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
INTRODUCTION: The aims were to investigate the clinical characteristics of Toxoplasma gondii (T. gondii) immunoglobulin (Ig) M-positive mothers and to clarify the incidences of serum T. gondii IgM or blood T. gondii DNA positivity in newborns born to the mothers and the actual congenital T. gondii infection. METHODS: Mothers with T. gondii IgM positivity and newborns born to the mothers from 2013 to 2020 were prospectively investigated. Serum T. gondii IgG and IgM were measured by enzyme-linked immunosorbent assay. Blood T. gondii DNA was detected by semi-nested polymerase chain reaction. Congenital T. gondii infection was diagnosed based on clinical characteristic manifestations with serum T. gondii IgG positivity at any age or T. gondii IgG positivity after 12 months of age. RESULTS: Among 71 T. gondii IgM-positive mothers, including one with triplets, 41% had low T. gondii IgG avidity index and 73% received maternal therapy. Among 73 newborns who were examined for serum T. gondii IgG and IgM at birth, none had clinical manifestations, and one (1.4%) had T. gondii IgM positivity. Among 32 newborns who were examined for blood T. gondii DNA at birth, two (6.3%) were positive. All patients with serum T. gondii IgM or blood T. gondii DNA positivity showed T. gondii IgG negativity within 12 months of age. CONCLUSIONS: A few newborns born to T. gondii IgM-positive mothers were suspected of having congenital T. gondii infection based on serum T. gondii IgM or blood T. gondii DNA testing at birth. However, none developed congenital T. gondii infection.
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Toxoplasma , Anticuerpos Antiprotozoarios , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina M , Recién Nacido , Madres , Embarazo , Estudios Prospectivos , Toxoplasma/genéticaRESUMEN
Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic Toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4(+) T cell activation and Th1 effector cell function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores.
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Complejo 3 de Proteína Adaptadora/inmunología , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Fagosomas/inmunología , Receptores Toll-Like/inmunología , Complejo 3 de Proteína Adaptadora/metabolismo , Animales , Antígenos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Ligandos , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Fagocitosis/inmunología , Fagosomas/metabolismo , Transducción de Señal/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.
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Complejo Antígeno-Anticuerpo/inmunología , Autofagia/inmunología , ADN/inmunología , Interferón Tipo I/biosíntesis , Animales , Humanos , Inmunoglobulina G/inmunología , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Fagocitosis/inmunología , Fagosomas/metabolismo , Transporte de Proteínas , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismoRESUMEN
Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates â¼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.
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Proteínas de Unión al GTP/inmunología , Inmunidad Innata/inmunología , Interferón gamma/inmunología , Macrófagos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Cromosomas de los Mamíferos/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Inmunidad Celular/inmunología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Listeria monocytogenes/inmunología , Mediciones Luminiscentes , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Óxido Nítrico/metabolismo , Toxoplasma/metabolismoRESUMEN
Unc93B1, a multitransmembrane ER-resident protein, controls intracellular trafficking of endosomal Toll-like receptors. In this issue of Immunity, Fukui et al. (2011) revealed that Unc93B1 regulates differential transport of TLR7 and TLR9 into signaling endosomes to prevent autoimmunity.
RESUMEN
Signal peptide peptidase (SPP) is an intramembrane aspartic protease involved in the maturation of the core protein of hepatitis C virus (HCV). The processing of HCV core protein by SPP has been reported to be critical for the propagation and pathogenesis of HCV. Here we examined the inhibitory activity of inhibitors for γ-secretase, another intramembrane cleaving protease, against SPP, and our findings revealed that the dibenzoazepine-type structure in the γ-secretase inhibitors is critical for the inhibition of SPP. The spatial distribution showed that the γ-secretase inhibitor compound YO-01027 with the dibenzoazepine structure exhibits potent inhibiting activity against SPP in vitro and in vivo through the interaction of Val223 in SPP. Treatment with this SPP inhibitor suppressed the maturation of core proteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting antivirals. YO-01027 also efficiently inhibited the propagation of protozoa such as Plasmodium falciparum and Toxoplasma gondii These data suggest that SPP is an ideal target for the development of therapeutics not only against chronic hepatitis C but also against protozoiasis.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antiprotozoarios/farmacología , Antivirales/farmacología , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Dibenzazepinas/farmacología , Hepacivirus/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Animales , Antiprotozoarios/química , Antivirales/química , Línea Celular , Dibenzazepinas/química , Células HEK293 , Hepacivirus/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteasas/química , Relación Estructura-Actividad , Toxoplasma/efectos de los fármacos , Proteínas del Núcleo Viral/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Toxoplasma gondii can infect homoeothermic animals including humans and cause lethal toxoplasmosis in immunocompromised individuals. When hosts are infected with T. gondii, the cells induce immune responses against T. gondii. The pathogen infection is recognized by immune sensors that directly detect T. gondii structural components, leading to production of pro-inflammatory cytokines and chemokines. Antigen-presenting cells such as macrophages and dendritic cells strongly activate T cells and induce development of Th1 cells and antigen-specific killer CD8 T cells. These T cells and Group 1 innate lymphoid cells are main producers of IFN-γ, which robustly stimulates cell-autonomous immunity in cells infected with T. gondii. IFN-γ-inducible effectors such as IFN-inducible GTPases, inducible nitric oxide synthase and indoleamine-2,3-dioxygenase differentially play important roles in suppression of T. gondii growth and its direct killing in anti-T. gondii cell-autonomous immune responses. In this review, we will describe our current knowledge of innate, adaptive and IFN-γ-mediated cell-autonomous immunity against T. gondii infection.
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Inmunidad Celular/inmunología , Interferón gamma/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Animales , Interacciones Huésped-Patógeno/inmunología , HumanosRESUMEN
IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIß, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.
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GTP Fosfohidrolasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Interferón gamma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlorocebus aethiops , Cartilla de ADN/genética , Femenino , Fibroblastos/metabolismo , Inflamación/inmunología , Lípidos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Células VeroRESUMEN
Toll-like receptors (TLRs) traffic to distinct membranes for signaling. TLR7 and TLR9 recognize viral nucleic acids in the endosomes and induce robust anti-viral program. Signaling from these TLRs bifurcate at the level of distinct endosomal compartments, namely VAMP3(+) and LAMP(+) endosomes, to mediate the induction of cytokine and type I interferon (IFN) genes, respectively. The formation of the TLR9 endosome competent for IFNs induction requires AP-3. Phosphoinositides (PIs) mark distinct subcellular membranes and control membrane trafficking. However, their role in TLR trafficking and signaling in different dendritic cell (DC) subsets remains unclear. Here, we examined the role of phosphatidylinositol 3P 5-kinase, PIKfyve, in TLR9 trafficking and signaling. We demonstrate that inhibition of PIKfyve activity preferentially blocks TLR9 signaling for type I IFN induction in FLT3L-bone marrow-derived DCs. By confocal microscopy using RAW264.7 cells, we show that trafficking of both TLR9 and CpG to the LAMP1(+) compartment was blocked by PIKfyve inhibitor treatment, whereas their trafficking to the VAMP3(+) endosome remained intact. Further, AP-3 recruitment to TLR9 endosomes was impaired by PIKfyve inhibition. These data indicate that PIKfyve provides critical PIs necessary for the formation of endosome from which TLR9 signals to induce type I IFNs.
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Interferón Tipo I/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 9/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Endosomas/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas/fisiología , Receptor Toll-Like 7/metabolismo , Factores de Transcripción/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismoRESUMEN
IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti-T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-γ-inducible GTPases. IFN-γ-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ-induced recruitment of the immunity-related GTPases to the intracellular pathogen.
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Autofagia/inmunología , Proteínas Portadoras/inmunología , Interferón gamma/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Toxoplasma/inmunología , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Secuencia de Bases , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Embrión de Mamíferos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/parasitología , Técnicas de Inactivación de Genes , Interacciones Huésped-Parásitos/inmunología , Humanos , Inmunidad Celular/genética , Inmunidad Celular/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Toxoplasma/fisiología , Células VeroRESUMEN
The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-ß) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-ß promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIMS, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.
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Regulación de la Expresión Génica/inmunología , Inmunidad Innata/inmunología , Nucleopoliedrovirus/inmunología , Transgenes/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Cartilla de ADN/genética , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Mariposas Nocturnas/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción GenéticaRESUMEN
The research field to identify and characterize genes essential for in vivo virulence in Toxoplasma gondii has been dramatically advanced by a series of in vivo clustered regularly interspaced short palindromic repeats (CRISPR) screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1,000 proteins remained unassigned, and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several genes essential for in vivo virulence that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations.IMPORTANCEToxoplasma gondii is a protozoan parasite that causes severe infection in immunocompromised patients or newborns. Toxoplasma possesses more than 8,000 genes; however, the genes essential for in vivo virulence were not fully identified. The apicomplexan parasites, including Toxoplasma, developed unique organelles that do not exist in other model organisms; thus, determining the subcellular location of parasite proteins is important for understanding their functions. Here, we used in vivo genetic screens that enabled us to investigate hundreds of genes in Toxoplasma during mouse infection. We screened approximately 600 parasite proteins with previously unknown subcellular localizations. We identified many novel genes that confer parasite virulence in mice. Among the top hits, we characterized two genes essential for in vivo virulence, TgGTPase and TgRimM, which are widely conserved in the phylum Apicomplexa. Our findings will contribute to understanding how apicomplexans adapt to the host environment and cause disease.
RESUMEN
Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2.
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia , Streptococcus pneumoniae , Animales , Ratones , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Macroautofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/metabolismo , Infecciones Neumocócicas/inmunología , Streptococcus pneumoniae/metabolismoRESUMEN
Clinical studies have shown that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is associated with aggressive periodontitis and can potentially trigger or exacerbate rheumatoid arthritis (RA). However, the mechanism is poorly understood. Here, we show that systemic infection with A. actinomycetemcomitans triggers the progression of arthritis in mice anti-collagen antibody-induced arthritis (CAIA) model following IL-1ß secretion and cell infiltration in paws in a manner that is dependent on caspase-11-mediated inflammasome activation in macrophages. The administration of polymyxin B (PMB), chloroquine, and anti-CD11b antibody suppressed inflammasome activation in macrophages and arthritis in mice, suggesting that the recognition of lipopolysaccharide (LPS) in the cytosol after bacterial degradation by lysosomes and invasion via CD11b are needed to trigger arthritis following inflammasome activation in macrophages. These data reveal that the inhibition of caspase-11-mediated inflammasome activation potentiates aggravation of RA induced by infection with A. actinomycetemcomitans. This work highlights how RA can be progressed by inflammasome activation as a result of periodontitis-associated bacterial infection and discusses the mechanism of inflammasome activation in response to infection with A. actinomycetemcomitans.
Asunto(s)
Aggregatibacter actinomycetemcomitans , Artritis Experimental , Caspasas Iniciadoras , Inflamasomas , Macrófagos , Animales , Ratones , Macrófagos/inmunología , Artritis Experimental/microbiología , Artritis Experimental/inmunología , Interleucina-1beta/metabolismo , Artritis Reumatoide , Ratones Endogámicos C57BL , Lipopolisacáridos , Infecciones por Pasteurellaceae/microbiologíaRESUMEN
Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.