RESUMO
Listeria monocytogenes is a gram-positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon ß (IFNß) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria-derived cyclic-di-AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNß expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic-di-AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNß expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.
Assuntos
Interações Hospedeiro-Patógeno , Interferon beta/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Animais , Células Cultivadas , Citosol/metabolismo , DNA Bacteriano/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeriose/metabolismo , Listeriose/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-ß), which, surprisingly, promote L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFN-ß. We have identified the L. monocytogenes strain LO28 as able to hyperinduce IFN-ß production in infected cells â¼30-fold more than the common laboratory clone L. monocytogenes strain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-ß, it results in significant attenuation in multiple mouse models of infection. While type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo by an unknown mechanism.
Assuntos
Proteínas de Bactérias/metabolismo , Genes MDR , Interferon beta/biossíntese , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Listeria monocytogenes/patogenicidade , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de DNARESUMO
Mammalian bile has potent anti-microbial activity, yet bacterial pathogens of the gastrointestinal tract and hepatobiliary system nonetheless persist and replicate within bile-rich environments. Listeria monocytogenes, a Gram-positive pathogen, encounters bile at three stages throughout its infectious cycle in vivo: in the gut during initial infection, in the liver where it replicates robustly and in the gallbladder, from which it can return to the intestine and thence to the environment. The mechanisms by which L. monocytogenes senses mammalian bile and counteracts its bactericidal effects are not fully understood. In this report, we have determined the L. monocytogenes bile-induced transcriptome, finding that many critical virulence factors are regulated by bile. Among these, the multidrug efflux pumps MdrM and MdrT, previously shown to be critical for the bacterial provocation of a pathogenesis-promoting host innate immune response, are robustly and specifically induced by the bile component cholic acid. This induction is mediated by BrtA, the first identified L. monocytogenes sensor of bile, which loses the ability to bind to and repress the mdrT promoter in the presence of cholic acid. We show that MdrT can export cholic acid, and that ΔmdrT bacteria are significantly attenuated both in vitro when exposed to cholic acid or bile, and in vivo in the gallbladders and livers of infected mice.
Assuntos
Ácido Cólico/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Repressoras/metabolismo , Animais , Transporte Biológico Ativo , DNA Bacteriano/metabolismo , Modelos Animais de Doenças , Vesícula Biliar/microbiologia , Deleção de Genes , Perfilação da Expressão Gênica , Listeria monocytogenes/genética , Listeriose/microbiologia , Listeriose/patologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Doenças dos Roedores/microbiologia , Doenças dos Roedores/patologia , VirulênciaRESUMO
Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Ciclofilina A/metabolismo , Elementos de DNA Transponíveis , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/genética , Mutagênese Insercional/métodos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Ciclofilina A/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Hemólise , Humanos , Listeria monocytogenes/enzimologia , Listeriose/microbiologia , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Fenótipo , Dobramento de Proteína , Ovinos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNbeta. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNbeta was reduced in nod2-/- macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNbeta induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens.
Assuntos
Regulação da Expressão Gênica/fisiologia , Imunidade Celular/genética , Listeria monocytogenes/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteína Adaptadora de Sinalização NOD2/genética , Receptores Toll-Like/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína Adaptadora de Sinalização NOD2/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Transcrição GênicaRESUMO
Adaptive immune responses require clonal expansion and differentiation of naive T cells into cytokine-secreting effector cells. After priming via signals through the T cell receptor, naive T helper cells express cytokine mRNA but do not secrete cytokine protein without additional T cell receptor stimulation. Here we show that primed T cells demonstrated phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha), a 'collapsed' polysome profile, increased expression of stress-response genes and accumulation of cytoplasmic granules associated with RNA-binding proteins, all features of the integrated stress response. Restimulation of the cells resulted in rapid eIF2alpha dephosphorylation, ribosomal mRNA loading and cytokine secretion. Interference with the function of granule-associated proteins or accumulation of phosphorylated eIF2alpha enhanced release of interleukin 4 during T helper type 2 priming. Therefore, T lymphocytes require components of the integrated stress response to uncouple differentiation from the execution of effector functions.
Assuntos
Citocinas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária , Células Th2/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Citocinas/genética , Interleucina-4/genética , Camundongos , Fenótipo , Fosforilação , Biossíntese de Proteínas , Proteína Fosfatase 1 , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Deleção de Sequência , Antígeno-1 Intracelular de Células TRESUMO
Nonconventional splicing of the gene encoding the Hac1p transcription activator regulates the unfolded protein response (UPR) in Saccharomyces cerevisiae. This simple on/off switch contrasts with a more complex circuitry in higher eukaryotes. Here we show that a heretofore unrecognized pathway operates in yeast to regulate the transcription of HAC1. The resulting increase in Hac1p production, combined with the production or activation of a putative UPR modulatory factor, is necessary to qualitatively modify the cellular response in order to survive the inducing conditions. This parallel endoplasmic reticulum-to-nucleus signaling pathway thereby serves to modify the UPR-driven transcriptional program. The results suggest a surprising conservation among all eukaryotes of the ways by which the elements of the UPR signaling circuit are connected. We show that by adding an additional signaling element to the basic UPR circuit, a simple switch is transformed into a complex response.