RESUMO
NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Imunidade Inata/imunologia , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 3 de Interferon/imunologia , Proteínas Mitocondriais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Cultivadas , Ativação Enzimática/imunologia , Células HEK293 , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Fator Regulador 1 de Interferon/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , NF-kappa B/metabolismo , RNA Viral/genética , Vírus Sendai/imunologia , eIF-2 Quinase/metabolismoRESUMO
Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.
Assuntos
DNA Viral/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Nucleicos/metabolismo , Ligação Proteica/imunologiaRESUMO
PURPOSE OF REVIEW: While effective vaccines to prevent invasive infections by Neisseria meningitidis have been deployed around the world, development of a vaccine to prevent Neisseria gonorrhoeae has lagged. After multiple failed vaccine candidates, vaccine development for N. gonorrhoeae is showing promise for the first time in several decades. This review highlights recent progress in the field. RECENT FINDINGS: Vaccines containing outer-membrane vesicles (OMV) have been used to manage outbreaks of the serogroup B N. meningitidis in a number of countries. Epidemiologic studies indicate these vaccination campaigns were associated with reductions in reported N. gonorrhoeae infections. Recently, a serogroup B N. meningitidis vaccine containing both recombinant antigens and OMV has been licensed through much of the world. Epidemiologic studies also demonstrate associations between 4CMenB immunization and reduced N. gonorrhoeae infections. Additionally, mathematical modeling studies have begun to identify potential strategies for vaccine deployment to maximize reduction of infections. SUMMARY: After several decades with little progress towards an effective gonococcal vaccine, large observational studies have provided evidence that a new generation of group B N. meningitidis vaccines containing OMV have serendipitously restarted the field. Ongoing clinical trials will soon provide definitive evidence regarding the efficacy of these vaccines in preventing N. gonorrhoeae infection.
Assuntos
Gonorreia , Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis , Humanos , Infecções Meningocócicas/prevenção & controle , Vacinas Bacterianas , Neisseria gonorrhoeae , Gonorreia/prevenção & controleRESUMO
Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Vírus da Hepatite A , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Vírus da Hepatite A/genética , Vírus da Hepatite A/metabolismo , Mamíferos , Proteínas Virais/metabolismoRESUMO
PURPOSE OF REVIEW: There are an estimated 374 million new sexually transmitted infections (STIs) worldwide every year. Our review article examines the current evidence of how STI acquisition, transmission, and pathogenesis is impacted upon by the genital microbiota, with a focus on epidemiological, biochemical, and immunological features. RECENT FINDINGS: At least in women, a genital microbiota dominated by lactobacilli has long been considered optimal for reproductive health, while depletion of lactobacilli may lead to a genital microenvironment dominated by anaerobic pathogens, which can manifest clinically as bacterial vaginosis. Recent research efforts have characterized genital microbiota composition in greater resolution, sometimes at species-level, using proteomics, metabolomics, and deep sequencing. This has enhanced our understanding of how specific microbiota members influence acquisition or clinical manifestation of STI pathogen infection. Other advances include a steady, though still slow, increase in the number of studies that sought to determine the genital (penile or urethral) microbiota of males and how it may impact that of their female partners' genital microbiota and risk of STI acquisition. Altogether, these data enabled us to explore the concept that genital microbiota may be sexually transmitted and influence pathogenesis and clinical presentation of other STI. SUMMARY: With STI infection rates increasing worldwide, it is important now more than ever to find novel STI prevention strategies. Understanding if and how the genital microbiota is a modifiable risk factor for STI transmission, acquisition, and clinical manifestation may prove to be an important strategy in our efforts to curb morbidity in at risk populations.
Assuntos
Infecções por HIV , Microbiota , Infecções Sexualmente Transmissíveis , Vaginose Bacteriana , Masculino , Feminino , Humanos , Infecções por HIV/prevenção & controle , Infecções Sexualmente Transmissíveis/epidemiologia , Infecções Sexualmente Transmissíveis/prevenção & controle , Vaginose Bacteriana/epidemiologia , GenitáliaRESUMO
Gonorrhea rates and antibiotic resistance are both increasing. Neisseria gonorrhoeae (Ng) is an exclusively human pathogen and is exquisitely adapted to its natural host. Ng can subvert immune responses and undergoes frequent antigenic variation, resulting in limited immunity and protection from reinfection. Previous gonococcal vaccine efforts have been largely unsuccessful, and the last vaccine to be tested in humans was more than 35 years ago. Advancing technologies and the threat of untreatable gonorrhea have fueled renewed pursuit of a vaccine as a long-term sustainable solution for gonorrhea control. Despite the development of a female mouse model of genital gonococcal infection two decades ago, correlates of immunity or protection remain largely unknown, making the gonococcus a challenging vaccine target. The controlled human urethral infection model of gonorrhea (Ng CHIM) has been used to study gonococcal pathogenesis and the basis of anti-gonococcal immunity. Over 200 participants have been inoculated without serious adverse events. The Ng CHIM replicates the early natural course of urethral infection. We are now at an inflexion point to pivot the use of the model for vaccine testing to address the urgency of improved gonorrhea control. Herein we discuss the need for gonorrhea vaccines, and the advantages and limitations of the Ng CHIM in accelerating the development of gonorrhea vaccines.
RESUMO
Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP.
Assuntos
DNA/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Citocinas/biossíntese , Herpes Simples/imunologia , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Interferon Tipo I/biossíntese , Camundongos , Camundongos Knockout , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transporte ProteicoRESUMO
There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development.
Assuntos
Proteção Cruzada/imunologia , Vacinas Meningocócicas/farmacologia , Neisseria gonorrhoeae/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Estudos de Casos e Controles , Reações Cruzadas/imunologia , Feminino , Gonorreia/imunologia , Humanos , Soros Imunes/imunologia , Imunização/métodos , Masculino , Infecções Meningocócicas/microbiologia , Vacinas Meningocócicas/imunologia , Vacinas Meningocócicas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Estudos Retrospectivos , Sorogrupo , Vacinação/métodosRESUMO
CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not by 3M patient-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in defects and sensitizes cells to microtubule damage similarly to loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Instabilidade Genômica , Microtúbulos/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Proteínas Culina/genética , Nanismo/genética , Proteínas F-Box/metabolismo , Genoma Humano , Células HEK293 , Humanos , Hipotonia Muscular/genética , Mutação de Sentido Incorreto , Transporte Proteico , Fuso Acromático/metabolismo , Coluna Vertebral/anormalidadesRESUMO
15 years ago, the fundamental biology of an inflammatory signaling complex eventually dubbed "the inflammasome" began to unravel in chronologic parallel with the discovery that many inflammatory diseases were associated with its hyperactivity. Though the genetic origins of Familial Mediterranean Fever (FMF, caused my mutations in MEFV) were discovered first, it would take nearly two decades before the mechanistic connections to a PYRIN inflammasome were made. In the interim, the intensive study of the NLRP3 inflammasome, and the diseases associated with its hyperactivation, have largely dictated the paradigm of inflammasome composition and function. Despite impressive gains, focusing on NLRP3 left gaps in our understanding of inflammasome biology. Foremost among these gaps were how inflammasomes become activated and the connections between inflammasome structure and function. Fortunately, work in another inflammasome inducer, NLRC4, grew to fill those gaps. The current understanding of the NLRC4 inflammasome is perhaps the most comprehensive illustration of the inflammasome paradigm: trigger (e.g. cytosolic flagellin), sensor (NAIP), nucleator (NLRC4), adaptor (ASC), and effector (CASP1). Detailed work has also identified observations that challenge this paradigm. Simultaneously, the features unique to each inflammasome offer a lesson in contrast, providing perspectives on inflammasome activation, regulation, and function. In this review, we endeavor to highlight recent breakthroughs related to NLRC4 inflammasome structure and activation, important in vivo work in infection and systemic inflammation, and the characterization of a spectrum of human NLRC4-associated autoinflammatory diseases.
Assuntos
Doenças Autoimunes/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Febre Familiar do Mediterrâneo/genética , Infecções/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Pirina/metabolismo , Animais , Doenças Autoimunes/genética , Humanos , Pirina/genética , Transdução de SinaisRESUMO
PURPOSE OF REVIEW: This review provides an update of nonviral, curable sexually transmitted infections (STIs) in pregnancy and summarizes our understanding of the current issues and controversies surrounding risk factors, screening, and treatment of STIs in pregnancy primarily in high-income countries (using the United States and the United Kingdom as examples). The infections covered in this review are syphilis, gonorrhea, chlamydia, trichomoniasis, and Mycoplasma genitalium infections. RECENT FINDINGS: Overall, limited modern data is available to update researchers and clinicians on the epidemiology and care of STIs in pregnancy. Though common risk factors can be identified among these STIs, like socioeconomic status and inadequate antenatal care, specific screening and treatment challenges vary by geography and pathogen. Wherever available, surveillance data and research evidence are often limited to nonpregnant patients, leading to imperfect pregnancy-specific risk estimates and obstetric lags in the development and adoption of new guidelines. We have identified three areas of opportunity that may enhance the effectiveness of current approaches and inform new ones: improved data collection and evidence-based screening practices; prompt and comprehensive therapy, including partner services, and evaluations of new treatment modalities; and equitable antenatal and sexual healthcare for all pregnant persons and their partners. SUMMARY: These findings highlight the need to revisit standards of screening and management of STIs in pregnancy in high-income countries.
Assuntos
Complicações Infecciosas na Gravidez/epidemiologia , Infecções Sexualmente Transmissíveis/epidemiologia , Feminino , Humanos , Gravidez , Complicações Infecciosas na Gravidez/microbiologia , Infecções Sexualmente Transmissíveis/microbiologia , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
Neisseria gonorrhoeae is an exclusive human pathogen that evades the host immune system through multiple mechanisms. We have shown that N. gonorrhoeae suppresses the capacity of antigen-presenting cells to induce CD4+ T cell proliferation. In this study, we sought to determine the gonococcal factors involved in this adaptive immune suppression. We show that suppression of the capacity of antigen-pulsed dendritic cells to induce T cell proliferation is recapitulated by administration of a high-molecular-weight fraction of conditioned medium from N. gonorrhoeae cultures, which includes outer membrane vesicles that are shed during growth of the bacteria. N. gonorrhoeae PorB is the most abundant protein in N. gonorrhoeae-derived vesicles, and treatment of dendritic cells with purified recombinant PorB inhibited the capacity of the cells to stimulate T cell proliferation. This immunosuppressive feature of purified PorB depended on proper folding of the protein. PorB from N. gonorrhoeae, as well as other Neisseria species and other Gram-negative bacterial species, are known to activate host Toll-like receptor 2 (TLR2) signaling. Published studies have demonstrated that purified Neisseria PorB forms proteinacious nanoparticles, termed proteosomes, when detergent micelles are removed. Unlike folded, detergent-solubilized PorB, PorB proteosomes stimulate immune responses. We now demonstrate that the formation of PorB proteosomes from structurally intact PorB eliminates the immunosuppressive property of the protein while enhancing TLR2 stimulation. These findings suggest that gonococcal PorB present in shed outer membrane vesicles plays a role in suppression of adaptive immune responses to this immune-evasive pathogen.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Gonorreia/imunologia , Neisseria gonorrhoeae/imunologia , Porinas/química , Dobramento de Proteína , Linfócitos T CD4-Positivos/microbiologia , Células Cultivadas , Células Dendríticas/microbiologia , Gonorreia/microbiologia , Humanos , Ativação Linfocitária , Porinas/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismoRESUMO
Inflammasomes are multiprotein structures that activate caspase-1, support secretion of pro-inflammatory cytokines, IL-1ß and IL-18, and also induce inflammatory programmed cell death, termed pyoptosis. Inflammasomes are activated in response to the detection of endogenous and microbially derived danger signals and are mediated by several classes of inflammasome-forming sensors. These include several nucleotide-binding proteins of the NOD-like receptor (NLR) family, including NLRP1, NLRP3 and NLRC4, as well as the proteins Absent in Melanoma 2 (AIM2) and Pyrin. Mutations in genes encoding some of these sensors have been found to be associated with gain-of-function monogenetic inflammatory disorders in humans. Genetic, biochemical and structural studies have begun to demonstrate how these proteins sense danger signals and to shed light on the step-by-step processes that are necessary for the assembly of inflammasomes, in both physiologic responses to pathogens and potentially in autoinflammatory conditions. Recent biochemical studies of pro-caspase-1 and an adapter protein known as ASC suggest that inflammasomes act to initiate self-generating effector filaments responsible for activating caspase-1 and initiating downstream signaling. These studies have suggested a model of molecular events from sensor activation to inflammasome formation that may describe processes that are universal to inflammasome formation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inflamassomos/metabolismo , Multimerização Proteica , Animais , Caspase 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Modelos ImunológicosRESUMO
Staphylococcus aureus infections are a growing health burden worldwide, and paramount to this bacterium's pathogenesis is the production of virulence factors, including pore-forming leukotoxins. Leukocidin A/B (LukAB) is a recently discovered toxin that kills primary human phagocytes, though the underlying mechanism of cell death is not understood. We demonstrate here that LukAB is a major contributor to the death of human monocytes. Using a variety of in vitro and ex vivo intoxication and infection models, we found that LukAB activates Caspase 1, promotes IL-1ß secretion and induces necrosis in human monocytes. Using THP1 cells as a model for human monocytes, we found that the inflammasome components NLRP3 and ASC are required for LukAB-mediated IL-1ß secretion and necrotic cell death. S. aureus was shown to kill human monocytes in a LukAB dependent manner under both extracellular and intracellular ex vivo infection models. Although LukAB-mediated killing of THP1 monocytes from extracellular S. aureus requires ASC, NLRP3 and the LukAB-receptor CD11b, LukAB-mediated killing from phagocytosed S. aureus is independent of ASC or NLRP3, but dependent on CD11b. Altogether, this study provides insight into the nature of LukAB-mediated killing of human monocytes. The discovery that S. aureus LukAB provokes differential host responses in a manner dependent on the cellular contact site is critical for the development of anti-infective/anti-inflammatory therapies that target the NLRP3 inflammasome.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Espaço Extracelular/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Leucocidinas/metabolismo , Infecções Estafilocócicas/metabolismo , Fatores de Virulência/fisiologia , Proteínas Adaptadoras de Sinalização CARD , Citometria de Fluxo , Humanos , Imunoensaio , Immunoblotting , Espaço Intracelular/metabolismo , Microscopia Eletrônica de Transmissão , Proteína 3 que Contém Domínio de Pirina da Família NLR , Staphylococcus aureus/metabolismoRESUMO
Staphylococcus aureus is a Gram-positive coccus that interacts with human hosts on a spectrum from quiet commensal to deadly pathogen. S. aureus is capable of infecting nearly every tissue in the body resulting in cellulitis, pneumonia, osteomyelitis, endocarditis, brain abscesses, bacteremia, and more. S. aureus has a wide range of factors that promote infection, and each site of infection triggers a different response in the human host. In particular, the different patterns of inflammasome activation mediate tissue-specific pathogenesis or protection in S. aureus infection. Although still a nascent field, understanding the unique host-pathogen interactions in each infection and the role of inflammasomes in mediating pathogenesis may lead to novel strategies for treating S. aureus infections. Reviews addressing S. aureus virulence and pathogenesis (Thammavongsa et al. 2015), as well as epidemiology and pathophysiology (Tong et al. 2015), have recently been published. This review will focus on S. aureus factors that activate inflammasomes and their impact on innate immune signaling and bacterial survival.
Assuntos
Inflamassomos/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/fisiologia , VirulênciaRESUMO
Inflammasomes are multi-protein complexes that regulate maturation of the interleukin 1ß-related cytokines IL-1ß and IL-18 through activation of the cysteine proteinase caspase-1. NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is a key component of inflammasomes that assemble in response to a wide variety of endogenous and pathogen-derived danger signals. Activation of the NLRP3-inflammasome and subsequent secretion of IL-1ß is highly regulated by at least three processes: transcriptional activation of both NLRP3 and pro-IL-1ß genes, non-transcriptional priming of NLRP3, and final activation of NLRP3. NLRP3 is predominantly expressed in cells of the hematopoietic lineage. Using a yeast two-hybrid screen, we identified the hematopoietic-restricted protein, G protein signaling modulator-3 (GPSM3), as a NLRP3-interacting protein and a negative regulator of IL-1ß production triggered by NLRP3-dependent inflammasome activators. In monocytes, GPSM3 associates with the C-terminal leucine-rich repeat domain of NLRP3. Bone marrow-derived macrophages lacking GPSM3 expression exhibit an increase in NLRP3-dependent IL-1ß, but not TNF-α, secretion. Furthermore, GPSM3-null mice have enhanced serum and peritoneal IL-1ß production following Alum-induced peritonitis. Our findings suggest that GPSM3 acts as a direct negative regulator of NLRP3 function.
Assuntos
Proteínas de Transporte/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Inflamassomos/metabolismo , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/efeitos adversos , Compostos de Alúmen/farmacologia , Animais , Proteínas de Transporte/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite/induzido quimicamente , Peritonite/metabolismo , Peritonite/patologia , Estrutura Terciária de ProteínaRESUMO
Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1ß (IL-1ß) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.
Assuntos
Aciltransferases/fisiologia , Gonorreia/imunologia , Inflamação/imunologia , Lipídeo A/fisiologia , Neisseria gonorrhoeae/imunologia , Acilação/fisiologia , Aciltransferases/genética , Análise de Variância , Animais , Caspase 1/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/imunologia , Neisseria gonorrhoeae/genética , Transdução de Sinais/imunologiaRESUMO
The RIG-like helicase (RLH) family of intracellular receptors detect viral nucleic acid and signal through the mitochondrial antiviral signalling adaptor MAVS (also known as Cardif, VISA and IPS-1) during a viral infection. MAVS activation leads to the rapid production of antiviral cytokines, including type 1 interferons. Although MAVS is vital to antiviral immunity, its regulation from within the mitochondria remains unknown. Here we describe human NLRX1, a highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family member (known as NLR) that localizes to the mitochondrial outer membrane and interacts with MAVS. Expression of NLRX1 results in the potent inhibition of RLH- and MAVS-mediated interferon-beta promoter activity and in the disruption of virus-induced RLH-MAVS interactions. Depletion of NLRX1 with small interference RNA promotes virus-induced type I interferon production and decreases viral replication. This work identifies NLRX1 as a check against mitochondrial antiviral responses and represents an intersection of three ancient cellular processes: NLR signalling, intracellular virus detection and the use of mitochondria as a platform for anti-pathogen signalling. This represents a conceptual advance, in that NLRX1 is a modulator of pathogen-associated molecular pattern receptors rather than a receptor, and identifies a key therapeutic target for enhancing antiviral responses.
Assuntos
Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Vírus/imunologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Interferon beta/metabolismo , Camundongos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , NF-kappa B/metabolismo , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Trifosfato de Adenosina/metabolismo , Imunidade Inata/fisiologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/imunologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Animais , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Células Cultivadas , Cromatografia de Afinidade , Células HEK293 , Humanos , Insetos/citologia , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/imunologia , Ligação Proteica/fisiologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gα(i/o)-Loco (GoLoco) motif containing proteins. GPSM3 acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gα(i) subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gß subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent on GPSM3 phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.