RESUMO
Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-ß) and coregulated genes. We previously identified the IFN-ß stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-ß. One mutant that stimulated elevated levels of IFN-ß harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-ß expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.
Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/fisiologia , Fosfatos de Dinucleosídeos/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Feminino , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Homeostase , Listeria monocytogenes/enzimologia , Listeria monocytogenes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fatores de Virulência/genéticaRESUMO
Acquired cell-mediated immunity to Listeria monocytogenes is induced by infection with live, replicating bacteria that grow in the host cell cytosol, whereas killed bacteria, or those trapped in a phagosome, fail to induce protective immunity. In this chapter, we focus on how L. monocytogenes is sensed by the innate immune system, with the presumption that innate immunity affects the development of acquired immunity. Infection by L. monocytogenes induces three innate immune pathways: an MyD88-dependent pathway emanating from a phagosome leading to expression of inflammatory cytokines; a STING/IRF3-dependent pathway emanating from the cytosol leading to the expression of IFN-ß and coregulated genes; and very low levels of a Caspase-1-dependent, AIM2-dependent inflammasome pathway resulting in proteolytic activation and secretion of IL-1ß and IL-18 and pyroptotic cell death. Using a combination of genetics and biochemistry, we identified the listerial ligand that activates the STING/IRF3 pathway as secreted cyclic diadenosine monophosphate, a newly discovered conserved bacterial signaling molecule. We also identified L. monocytogenes mutants that caused robust inflammasome activation due to bacteriolysis in the cytosol, release of DNA, and activation of the AIM2 inflammasome. A strain was constructed that ectopically expressed and secreted a fusion protein containing Legionella pneumophila flagellin that robustly activated the Nlrc4-dependent inflammasome and was highly attenuated in mice, also in an Nlrc4-dependent manner. Surprisingly, this strain was a poor inducer of adaptive immunity, suggesting that inflammasome activation is not necessary to induce cell-mediated immunity and may even be detrimental under some conditions. To the best of our knowledge, no single innate immune pathway is necessary to mount a robust acquired immune response to L. monocytogenes infection.
Assuntos
Citocinas/imunologia , Imunidade Celular , Listeria monocytogenes/imunologia , Listeriose/imunologia , Animais , Humanos , Imunidade Inata , Inflamação , Camundongos , Transdução de SinaisRESUMO
A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogen's intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.
Assuntos
Morte Celular , Listeria monocytogenes/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas Nucleares/imunologia , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Bacteriólise , Bacteriófagos/genética , Citosol/microbiologia , Proteínas de Ligação a DNA , Camundongos , Mucoproteínas/genética , Mucoproteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.