RESUMO
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
Assuntos
Proteínas de Transporte/imunologia , Caspase 1/imunologia , Inflamassomos/imunologia , Glicoproteínas de Membrana/imunologia , NADPH Oxidases/imunologia , Fagossomos/imunologia , Animais , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Células Cultivadas , Ativação Enzimática/imunologia , Citometria de Fluxo , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Concentração de Íons de Hidrogênio , Immunoblotting , Inflamassomos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagocitose/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Fagossomos/ultraestrutura , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologiaRESUMO
Although infections with virulent pathogens often induce a strong inflammatory reaction, what drives the increased immune response to pathogens compared to nonpathogenic microbes is poorly understood. One possibility is that the immune system senses the level of threat from a microorganism and augments the response accordingly. Here, focusing on cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli-derived effector molecule, we showed the host indirectly sensed the pathogen by monitoring for the effector that modified RhoGTPases. CNF1 modified Rac2, which then interacted with the innate immune adaptors IMD and Rip1-Rip2 in flies and mammalian cells, respectively, to drive an immune response. This response was protective and increased the ability of the host to restrict pathogen growth, thus defining a mechanism of effector-triggered immunity that contributes to how metazoans defend against microbes with pathogenic potential.
Assuntos
Transdução de Sinais , Proteínas rac de Ligação ao GTP/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila the Imd pathway detects Gram-negative bacterial infections through recognition of diaminopimelic acid (DAP)-type peptidoglycan and activation of the NF-κB precursor Relish, which drives robust antimicrobial peptide gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and Lys-63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. Here, we demonstrate that Imd is rapidly Lys-63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 and Ubc13-Uev1a, in conjunction with the E3 ligase Diap2. Lys-63-ubiquitination activates the TGFß-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of Lys-63 chains and the addition of Lys-48 polyubiquitin. This ubiquitin-editing process results in the proteasomal degradation of Imd, which we propose functions to restore homeostasis to the Drosophila immune response.
Assuntos
Proteínas de Drosophila/imunologia , Imunidade Inata , MAP Quinase Quinase Quinases/imunologia , Transdução de Sinais/imunologia , Ubiquitinação/imunologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , MAP Quinase Quinase Quinases/genética , Poliubiquitina/genética , Poliubiquitina/imunologia , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitinação/genéticaRESUMO
Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappaB signaling pathways-IMD and Toll. The IMD pathway is triggered by DAP-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. We now show that PGN stimulation causes caspase-mediated cleavage of the imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMD with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappaB signaling.
Assuntos
Caspases/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , NF-kappa B/metabolismo , Transdução de Sinais , Alelos , Motivos de Aminoácidos , Animais , Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Alinhamento de Sequência , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-κB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-κB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition.
Assuntos
Proteínas de Bactérias/metabolismo , Imunidade Inata , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Serina O-Acetiltransferase/metabolismo , Yersinia pestis/enzimologia , Acetilação , Animais , Proteínas de Bactérias/imunologia , Drosophila melanogaster , Células HEK293 , Humanos , MAP Quinase Quinase Quinases/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Peste/imunologia , Peste/metabolismo , Serina O-Acetiltransferase/imunologia , Yersinia pestis/imunologia , Yersinia pestis/patogenicidadeRESUMO
Immunosuppression via cell-cell contact with apoptotic cells is a well studied immunological phenomenon. Although the original studies of immune repression used primary cells, which undergo spontaneous cell death or apoptosis in response to irradiation, more recent studies have relied on chemotherapeutic agents to induce apoptosis in cell lines. In this work, we demonstrate that Jurkat cells induced to die with actinomycin D suppressed inflammatory cytokine production by macrophages, whereas cells treated with etoposide did not. This immune repression mediated by actinomycin D-treated cells did not require phagocytosis or cell-cell contact and thus occurs through a different mechanism from that seen with primary apoptotic neutrophils. Moreover, cells induced to die with etoposide and then treated for a short time with actinomycin D also suppressed macrophage responses, indicating that suppression was mediated by actinomycin D independent of the mechanism of cell death. Finally, phagocytosis of actinomycin D-treated cells caused apoptosis in macrophages, and suppression could be blocked by inhibition of caspase activity in the target macrophage. Together, these data indicate that apoptotic cells act as "Trojan horses," delivering actinomycin D to engulfing macrophages. Suppression of cytokine production by macrophages is therefore due to exposure to actinomycin D from apoptotic cells and is not the result of cell-receptor interactions. These data suggest that drug-induced death may not be an appropriate surrogate for the immunosuppressive activity of apoptotic cells. Furthermore, these effects of cytotoxic drugs on infiltrating immune phagocytes may have clinical ramifications for their use as antitumor therapies.
Assuntos
Apoptose/imunologia , Citocinas/imunologia , Mediadores da Inflamação/imunologia , Macrófagos/imunologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Comunicação Celular/imunologia , Linhagem Celular , Técnicas de Cocultura , Citocinas/metabolismo , Dactinomicina/farmacologia , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Mediadores da Inflamação/metabolismo , Células Jurkat , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fagocitose/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nuclear Factor-κB (NF-κB)/Rel transcription factors form an integral part of innate immune defenses and are conserved throughout the animal kingdom. Studying the function, mechanism of activation and regulation of these factors is crucial for understanding host responses to microbial infections. The fruit fly Drosophila melanogaster has proved to be a valuable model system to study these evolutionarily conserved NF-κB mediated immune responses. Drosophila combats pathogens through humoral and cellular immune responses. These humoral responses are well characterized and are marked by the robust production of a battery of anti-microbial peptides. Two NF-κB signaling pathways, the Toll and the IMD pathways, are responsible for the induction of these antimicrobial peptides. Signal transduction in these pathways is strikingly similar to that in mammalian TLR pathways. In this chapter, we discuss in detail the molecular mechanisms of microbial recognition, signal transduction and NF-κB regulation, in both the Toll and the IMD pathways. Similarities and differences relative to their mammalian counterparts are discussed, and recent advances in our understanding of the intricate regulatory networks in these NF-κB signaling pathways are also highlighted.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/imunologia , Imunidade Humoral , NF-kappa B/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Imunidade Adaptativa , Animais , Receptores Toll-Like/fisiologiaRESUMO
A common defining characteristic of pathogenic bacteria is the expression of a repertoire of effector molecules that have been named virulence factors. These bacterial factors include a -variety of proteins, such as toxins that are internalized by receptors and translocate across endosomal membranes to reach the cytosol, as well as others that are introduced directly into the cell by means of bacterial secretory apparatuses. Given the importance of these effectors for understanding bacterial pathogenicity, significant effort has been made to dissect their molecular mechanisms of action and their respective roles during infection. Herein we will discuss how Drosophila have been used as a model system to study these important microbial effectors, and to understand their contribution to pathogenicity.
Assuntos
Bactérias/patogenicidade , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Proteínas Ativadoras de GTPase/metabolismo , Imunidade Inata/imunologia , Inflamação , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase (IKK) and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. In this report, we show that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish.
Assuntos
Anti-Infecciosos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Quinase I-kappa B/fisiologia , Peptídeos/genética , Fatores de Transcrição/metabolismo , Animais , Drosophila , Proteínas de Drosophila/química , Epistasia Genética , Quinase I-kappa B/química , Fosforilação , Regiões Promotoras Genéticas , Serina/metabolismoRESUMO
Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Imunidade Inata/fisiologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Drosophila melanogaster , Pectobacterium carotovorum/imunologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologiaRESUMO
Peptidoglycan recognition proteins (PGRPs) are mediators of innate immunity and recently have been implicated in developmental regulation. To explore the interplay between these two roles, we characterized a PGRP in the host squid Euprymna scolopes (EsPGRP1) during colonization by the mutualistic bacterium Vibrio fischeri. Previous research on the squid-vibrio symbiosis had shown that, upon colonization of deep epithelium-lined crypts of the host light organ, symbiont-derived peptidoglycan monomers induce apoptosis-mediated regression of remote epithelial fields involved in the inoculation process. In this study, immunofluorescence microscopy revealed that EsPGRP1 localizes to the nuclei of epithelial cells, and symbiont colonization induces the loss of EsPGRP1 from apoptotic nuclei. The loss of nuclear EsPGRP1 occurred prior to DNA cleavage and breakdown of the nuclear membrane, but followed chromatin condensation, suggesting that it occurs during late-stage apoptosis. Experiments with purified peptidoglycan monomers and with V. fischeri mutants defective in peptidoglycan-monomer release provided evidence that these molecules trigger nuclear loss of EsPGRP1 and apoptosis. The demonstration of a nuclear PGRP is unprecedented, and the dynamics of EsPGRP1 during apoptosis provide a striking example of a connection between microbial recognition and developmental responses in the establishment of symbiosis.
Assuntos
Aliivibrio fischeri/imunologia , Aliivibrio fischeri/fisiologia , Proteínas de Transporte/imunologia , Decapodiformes/imunologia , Decapodiformes/microbiologia , Peptidoglicano/imunologia , Simbiose , Aliivibrio fischeri/genética , Sequência de Aminoácidos , Animais , Apoptose , Proteínas de Transporte/metabolismo , Núcleo Celular/química , Células Epiteliais/química , Células Epiteliais/microbiologia , Deleção de Genes , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptidoglicano/genética , Peptidoglicano/metabolismoRESUMO
BACKGROUND: Wound healing is a goal for advanced technology in the surgical space to benefit clinical outcomes. Surgical staplers are commonly used in a variety of open and minimally invasive abdominal and thoracic procedures. Assessment of wound healing traits, such as perfusion, has been challenging due to technical limitations. A novel technique that utilizes micro-computed tomography methodology to measure perfusion was designed to compare the micro-perfusion of staple lines between commercial stapler reloads that employ different staple height strategies. MATERIALS AND METHODS: Following an Institutional Animal Care and Use Committee-approved protocol, rats were euthanized and immediately heparinized prior to a subtotal gastrectomy with either graduated-height or single-height staples. Rats were then perfused with barium, following which stomachs were removed and immediately fixed in formalin to prevent degradation. Stomachs were then imaged using micro-computed tomography and subsequent analysis was utilized to quantify fluid volume and patent vasculature proximity to staples within the staple line region for each group. RESULTS: Average perfusion volume was significantly higher with graduated-height staples (0.33% ± 0.18%) compared to single-height staples (0.16% ± 0.09%, P=0.011). Average vessel-to-staple line distance was not significant but trended lower with graduated-height staples (0.35±0.02 mm) compared to single-height staples (0.36±0.03 mm, P=0.18). DISCUSSION: Graduated-height staples had significantly higher perfusion volume than single-height staples, which likely has a downstream benefit on wound healing and clinical outcomes. CONCLUSION: This study shows a higher perfusion volume around the staple lines using graduated-height staples as compared to single-height staples and this may contribute to better wound healing in patients.
RESUMO
Pertussis may go unrecognized during respiratory syncytial virus (RSV) epidemics. Nosocomially transmitted pertussis can be severe in infants. Polymerase chain reaction (PCR) screening may identify infants with pertussis on admission, allowing for preemptive isolation. In a random sample, 1 (0.6%) of 166 children admitted to the hospital during RSV season were Bordetella pertussis PCR positive during a nonepidemic period. These data show that screening may not be useful when pertussis prevalence is low.
Assuntos
Bordetella pertussis/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/epidemiologia , Coqueluche/epidemiologia , Baltimore/epidemiologia , Estudos de Coortes , Hospitalização , Humanos , Lactente , Programas de Rastreamento , Reação em Cadeia da Polimerase , Prevalência , Infecções por Vírus Respiratório Sincicial/complicações , Estações do Ano , Coqueluche/complicações , Coqueluche/diagnósticoRESUMO
OBJECTIVE: To assess the safety and efficacy of a chlorine dioxide water treatment system in controlling Legionella in a hospital water supply. DESIGN: For 17 months following installation of the system, we performed regular water cultures throughout the building, assessed chlorine dioxide and chlorite levels, and monitored metal corrosion. RESULTS: Sites that grew Legionella species decreased from 41% at baseline to 4% (P = .001). L. anisa was the only species recovered and it was found in samples of both hot and cold water. Levels of chlorine dioxide and chlorite were below Environmental Protection Agency (EPA) limits for these chemicals in potable water. Further, enhanced carbon filtration effectively removed the chemicals, even at chlorine dioxide levels of more than twice what was used to treat the water. After 9 months, corrosion of copper test strips exposed to the chlorine dioxide was not higher than that of control strips. During the evaluation period, there were no cases of nosocomial Legionella in the building with the system, whereas there was one case in another building. CONCLUSIONS: Our results indicate that operation of a chlorine dioxide system effectively removed Legionella species from a hospital water supply. Furthermore, we found that the system was safe, as levels of chlorine dioxide and chlorite were below EPA limits. The system did not appear to cause increased corrosion of copper pipes. Our results indicate that chlorine dioxide may hold promise as a solution to the problem of Legionella contamination of hospital water supplies.
Assuntos
Compostos Clorados , Infecção Hospitalar/prevenção & controle , Desinfecção/métodos , Doença dos Legionários/prevenção & controle , Serviço Hospitalar de Engenharia e Manutenção/métodos , Óxidos , Microbiologia da Água , Purificação da Água/métodos , Centros Médicos Acadêmicos , Baltimore/epidemiologia , Lavagem Broncoalveolar , Compostos Clorados/análise , Compostos Clorados/normas , Corrosão , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Desinfecção/normas , Humanos , Legionella pneumophila/isolamento & purificação , Legionella pneumophila/patogenicidade , Doença dos Legionários/diagnóstico , Doença dos Legionários/transmissão , Óxidos/análise , Óxidos/normas , Vigilância de Evento Sentinela , Purificação da Água/normas , Abastecimento de Água/análiseRESUMO
A fundamental question regarding any immune system is how it can discriminate between pathogens and non-pathogens. Here, we discuss how this discrimination can be mediated by a surveillance system distinct from pattern-recognition receptors that recognize conserved microbial patterns. It can be based instead on the ability of the host to sense perturbations in host cells induced by bacterial toxins or 'effectors' that are encoded by pathogenic microorganisms. Such 'effector-triggered immunity' was previously demonstrated mainly in plants, but recent data confirm that animals can also use this strategy.