RESUMO
Innate immune pattern recognition receptors, such as the Toll-like receptors (TLRs), are key mediators of the immune response to infection and central to our understanding of health and disease1. After microbial detection, these receptors activate inflammatory signal transduction pathways that involve IκB kinases, mitogen-activated protein kinases, ubiquitin ligases and other adaptor proteins. The mechanisms that connect the proteins in the TLR pathways are poorly defined. To delineate TLR pathway activities, we engineered macrophages to enable microscopy and proteomic analysis of the endogenous myddosome constituent MyD88. We found that myddosomes form transient contacts with activated TLRs and that TLR-free myddosomes are dynamic in size, number and composition over the course of 24 h. Analysis using super-resolution microscopy revealed that, within most myddosomes, MyD88 forms barrel-like structures that function as scaffolds for effector protein recruitment. Proteomic analysis demonstrated that myddosomes contain proteins that act at all stages and regulate all effector responses of the TLR pathways, and genetic analysis defined the epistatic relationship between these effector modules. Myddosome assembly was evident in cells infected with Listeria monocytogenes, but these bacteria evaded myddosome assembly and TLR signalling during cell-to-cell spread. On the basis of these findings, we propose that the entire TLR signalling pathway is executed from within the myddosome.
Assuntos
Macrófagos , Transdução de Sinais , Receptores Toll-Like , Animais , Humanos , Camundongos , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Listeriose/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Fator 88 de Diferenciação Mieloide/metabolismo , Proteômica , Receptores Toll-Like/metabolismo , Microscopia , Imunidade InataRESUMO
The NOX2 NADPH oxidase (NOX2) produces reactive oxygen species to kill phagosome-confined bacteria. However, we previously showed that Listeria monocytogenes is able to avoid the NOX2 activity in phagosomes and escape to the cytosol. Thus, despite the established role of NOX2 limiting L. monocytogenes infection in mice, the underlying mechanisms of this antibacterial activity remain unclear. In this article, we report that NOX2 controls systemic L. monocytogenes spread through modulation of the type I IFN response, which is known to be exploited by L. monocytogenes during infection. NOX2 deficiency results in increased expression of IFN-stimulated genes in response to type I IFN and leads to 1) promotion of cell-to-cell spread by L. monocytogenes, 2) defective leukocyte recruitment to infection foci, and 3) production of anti-inflammatory effectors IL-10 and thioredoxin 1. Our findings report a novel antimicrobial role for NOX2 through modulation of type I IFN responses to control bacterial dissemination.
Assuntos
Inflamação/imunologia , Interferon Tipo I/metabolismo , Leucócitos/imunologia , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Macrófagos/metabolismo , NADPH Oxidase 2/metabolismo , Animais , Movimento Celular , Células Cultivadas , Interleucina-10/metabolismo , Listeriose/transmissão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/genética , TiorredoxinasRESUMO
Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We hypothesized that a stressful environmental milieu in a rapidly proliferating tissue might instigate a proinflammatory response. We report that Xbp1 deletion in intestinal epithelial cells (IECs) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell dysfunction and an epithelium that is overly reactive to inducers of IBD such as bacterial products (flagellin) and TNFalpha. An association of XBP1 variants with both forms of human IBD (Crohn's disease and ulcerative colitis) was identified and replicated (rs35873774; p value 1.6 x 10(-5)) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IECs, thus linking cell-specific ER stress to the induction of organ-specific inflammation.
Assuntos
Proteínas de Ligação a DNA/imunologia , Retículo Endoplasmático/imunologia , Doenças Inflamatórias Intestinais/genética , Fatores de Transcrição/imunologia , Animais , Apoptose , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/patologia , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Listeria monocytogenes/imunologia , Camundongos , Camundongos Transgênicos , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Fatores de Transcrição de Fator Regulador X , Proteína 1 de Ligação a X-BoxRESUMO
Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens.
Assuntos
Antígeno Carcinoembrionário/imunologia , Imunidade nas Mucosas/imunologia , Intestinos/imunologia , Linfócitos T/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Animais , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Citoplasma/genética , Citoplasma/imunologia , Citoplasma/metabolismo , Homeostase , Imunidade nas Mucosas/genética , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Mucosa Intestinal/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária , Metagenoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Isoformas de Proteínas , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Tirosina/genética , Tirosina/imunologia , Tirosina/metabolismoRESUMO
Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes, can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes. Expression of the cell surface protein ActA allows L. monocytogenes to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. Here we show that protrusion formation is associated with plasma membrane damage due to LLO's pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4(-/-) mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during infection.
Assuntos
Extensões da Superfície Celular/microbiologia , Listeria monocytogenes/fisiologia , Fagocitose , Actinas/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Membrana Celular/patologia , Extensões da Superfície Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/microbiologia , Feminino , Células HeLa , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Listeria monocytogenes/patogenicidade , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Camundongos , Fagossomos/metabolismo , Fagossomos/microbiologia , Fosfatidilserinas/metabolismo , Fosfolipases Tipo C/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
Listeria monocytogenes is a common food-borne pathogen that can disseminate from the intestine and infect multiple organs. Here, we used sequence tag-based analysis of microbial populations (STAMP) to investigate Lmonocytogenes population dynamics during infection. We created a genetically barcoded library of murinized Lmonocytogenes and then used deep sequencing to track the pathogen's dissemination routes and quantify its founding population (Nb) sizes in different organs. We found that the pathogen disseminates from the gastrointestinal tract to distal sites through multiple independent routes and that Nb sizes vary greatly among tissues, indicative of diverse host barriers to infection. Unexpectedly, comparative analyses of sequence tags revealed that fecally excreted organisms are largely derived from the very small number of L. monocytogenes cells that colonize the gallbladder. Immune depletion studies suggest that distinct innate immune cells restrict the pathogen's capacity to establish replicative niches in the spleen and liver. Finally, studies in germ-free mice suggest that the microbiota plays a critical role in the development of the splenic, but not the hepatic, barriers that prevent L. monocytogenes from seeding these organs. Collectively, these observations illustrate the potency of the STAMP approach to decipher the impact of host factors on population dynamics of pathogens during infection.
Assuntos
Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Animais , Carga Bacteriana , Código de Barras de DNA Taxonômico , Feminino , Vesícula Biliar/imunologia , Vesícula Biliar/microbiologia , Microbioma Gastrointestinal , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Vida Livre de Germes , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Listeriose/microbiologia , Fígado/imunologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Baço/microbiologiaRESUMO
Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Anticâncer/imunologia , Venenos de Cnidários/administração & dosagem , Neoplasias Experimentais/patologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Venenos de Cnidários/imunologia , Feminino , Citometria de Fluxo , Lipossomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologiaRESUMO
Listeria monocytogenes is a Gram-positive intracellular pathogen that causes a severe invasive disease. Upon infecting a host cell, L. monocytogenes upregulates the transcription of numerous factors necessary for productive infection. VirR is the response regulator component of a two-component regulatory system in L. monocytogenes In this report, we have identified the putative ABC transporter encoded by genes lmo1746-lmo1747 as necessary for VirR function. We have designated lmo1746-lmo1747 virAB We constructed an in-frame deletion of virAB and determined that the ΔvirAB mutant exhibited reduced transcription of VirR-regulated genes. The ΔvirAB mutant also showed defects in in vitro plaque formation and in vivo virulence that were similar to those of a ΔvirR deletion mutant. Since VirR is important for innate resistance to antimicrobial agents, we determined the MICs of nisin and bacitracin for ΔvirAB bacteria. We found that VirAB expression was necessary for nisin resistance but was dispensable for resistance to bacitracin. This result suggested a VirAB-independent mechanism of VirR regulation in response to bacitracin. Lastly, we found that the ΔvirR and ΔvirAB mutants had no deficiency in growth in broth culture, intracellular replication, or production of the ActA surface protein, which facilitates actin-based motility and cell-to-cell spread. However, the ΔvirR and ΔvirAB mutants produced shorter actin tails during intracellular infection, which suggested that these mutants have a reduced ability to move and spread via actin-based motility. These findings have demonstrated that L. monocytogenes VirAB functions in a pathway with VirR to regulate the expression of genes necessary for virulence and resistance to antimicrobial agents.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Nisina/farmacologia , Fatores de Transcrição/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Regulon , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1-/- ) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell-to-cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin-based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.
Assuntos
Interações Hospedeiro-Patógeno , Interferon Tipo I/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Listeriose/microbiologia , Listeriose/patologia , Fígado/microbiologia , Fígado/patologia , Macrófagos/microbiologia , Camundongos , Receptor de Interferon alfa e beta/metabolismoRESUMO
The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymerization factors, such as formins, may participate in protrusion formation. Here, we show that Arp2/3 localized to only a minor portion of the protrusion. Moreover, treatment of L. monocytogenes-infected HeLa cells with a formin FH2-domain inhibitor significantly reduced protrusion length. In addition, the Diaphanous-related formins 1-3 (mDia1-3) localized to protrusions, and knockdown of mDia1, mDia2, and mDia3 substantially decreased cell-to-cell spread of L. monocytogenes. Rho GTPases are known to be involved in formin activation. Our studies also show that knockdown of several Rho family members significantly influenced bacterial cell-to-cell spread. Collectively, these findings identify a Rho GTPase-formin network that is critically involved in the cell-to-cell spread of L. monocytogenes.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Extensões da Superfície Celular/metabolismo , Listeria monocytogenes/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/ultraestrutura , Forminas , Técnicas de Silenciamento de Genes , Genes Reporter , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/patogenicidade , Modelos Biológicos , Estrutura Terciária de Proteína , Tionas/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Listeria monocytogenes is an intracellular bacterial pathogen that replicates rapidly in the cytosol of host cells during acute infection. Surprisingly, these bacteria were found to occupy vacuoles in liver granuloma macrophages during persistent infection of severe combined immunodeficient (SCID) mice. Here we show that L. monocytogenes can replicate in vacuoles within macrophages. In livers of SCID mice infected for 21 days, we observed bacteria in large LAMP1(+) compartments that we termed spacious Listeria-containing phagosomes (SLAPs). SLAPs were also observed in vitro, and were found to be non-acidic and non-degradative compartments that are generated in an autophagy-dependent manner. The replication rate of bacteria in SLAPs was found to be reduced compared to the rate of those in the cytosol. Listeriolysin O (LLO, encoded by hly), a pore-forming toxin essential for L. monocytogenes virulence, was necessary and sufficient for SLAP formation. A L. monocytogenes mutant with low LLO expression was impaired for phagosome escape but replicated slowly in SLAPs over a 72 h period. Therefore, our studies reveal a role for LLO in promoting L. monocytogenes replication in vacuoles and suggest a mechanism by which this pathogen can establish persistent infection in host macrophages.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Macrófagos/citologia , Macrófagos/microbiologia , Vacúolos/microbiologia , Animais , Autofagia , Toxinas Bacterianas/genética , Doença Crônica , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/metabolismo , Listeriose/microbiologia , Listeriose/patologia , Fígado/microbiologia , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Camundongos SCID , Fagossomos/metabolismo , Fagossomos/microbiologia , Vacúolos/metabolismo , VirulênciaRESUMO
Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.
Assuntos
Citosol/microbiologia , Genética Microbiana/métodos , Listeria monocytogenes/patogenicidade , Biologia Molecular/métodos , Fatores de Virulência/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/microbiologia , Citometria de Fluxo , Corantes Fluorescentes/análise , Deleção de Genes , Teste de Complementação Genética , Testes Genéticos , Ensaios de Triagem em Larga Escala , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/microbiologia , Listeriose/patologia , Macrófagos/microbiologia , Camundongos , Microscopia de Fluorescência , Coloração e Rotulagem/métodos , VirulênciaRESUMO
Facultative bacterial pathogens must adapt to multiple stimuli to persist in the environment or establish infection within a host. Temperature is often utilized as a signal to control expression of virulence genes necessary for infection or genes required for persistence in the environment. However, very little is known about the molecular mechanisms that allow bacteria to adapt and respond to temperature fluctuations. Listeria monocytogenes (Lm) is a food-borne, facultative intracellular pathogen that uses flagellar motility to survive in the extracellular environment and to enhance initial invasion of host cells during infection. Upon entering the host, Lm represses transcription of flagellar motility genes in response to mammalian physiological temperature (37°C) with a concomitant temperature-dependent up-regulation of virulence genes. We previously determined that down-regulation of flagellar motility is required for virulence and is governed by the reciprocal activities of the MogR transcriptional repressor and the bifunctional flagellar anti-repressor/glycosyltransferase, GmaR. In this study, we determined that GmaR is also a protein thermometer that controls temperature-dependent transcription of flagellar motility genes. Two-hybrid and gel mobility shift analyses indicated that the interaction between MogR and GmaR is temperature sensitive. Using circular dichroism and limited proteolysis, we determined that GmaR undergoes a temperature-dependent conformational change as temperature is elevated. Quantitative analysis of GmaR in Lm revealed that GmaR is degraded in the absence of MogR and at 37°C (when the MogR:GmaR complex is less stable). Since MogR represses transcription of all flagellar motility genes, including transcription of gmaR, changes in the stability of the MogR:GmaR anti-repression complex, due to conformational changes in GmaR, mediates repression or de-repression of flagellar motility genes in Lm. Thus, GmaR functions as a thermo-sensing anti-repressor that incorporates temperature signals into transcriptional control of flagellar motility. To our knowledge, this is the first example of a protein thermometer that functions as an anti-repressor to control a developmental process in bacteria.
Assuntos
Proteínas de Bactérias/biossíntese , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Listeria monocytogenes/metabolismo , Transdução de Sinais/fisiologia , Fatores de Virulência/biossíntese , Proteínas de Bactérias/genética , Flagelos/genética , Temperatura Alta , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Fatores de Virulência/genéticaRESUMO
An estimated eight million people are infected each year with the pathogen Mycobacterium tuberculosis, and more than two million die annually. Yet only about 10% of those infected develop tuberculosis. Genetic variation within host populations is known to be significant in humans and animals, but the nature of genetic control of host resistance to tuberculosis remains poorly understood. Previously we mapped a new genetic locus on mouse chromosome 1, designated sst1 (for supersusceptibility to tuberculosis 1). Here we show that this locus mediates innate immunity in sst1 congenic mouse strains and identify a candidate gene, Intracellular pathogen resistance 1 (Ipr1), within the sst1 locus. The Ipr1 gene is upregulated in the sst1 resistant macrophages after activation and infection, but it is not expressed in the sst1 susceptible macrophages. Expression of the Ipr1 transgene in the sst1 susceptible macrophages limits the multiplication not only of M. tuberculosis but also of Listeria monocytogenes and switches a cell death pathway of the infected macrophages from necrosis to apoptosis. Our data indicate that the Ipr1 gene product might have a previously undocumented function in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death and pathogenesis.
Assuntos
Imunidade Inata/genética , Imunidade Inata/imunologia , Macrófagos/imunologia , Transativadores/genética , Transativadores/metabolismo , Tuberculose/genética , Tuberculose/imunologia , Animais , Animais Congênicos , Apoptose , Transplante de Medula Óssea , Progressão da Doença , Predisposição Genética para Doença/genética , Humanos , Interferon gama/imunologia , Listeria monocytogenes/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Necrose , Proteínas Nucleares/química , Proteínas Nucleares/genética , Polimorfismo Genético/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Transativadores/química , Transgenes/genética , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a "crossover" binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.
Assuntos
Sequência Rica em At , Proteínas de Bactérias/química , Proteínas Repressoras/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Flagelina/química , Flagelina/genética , Flagelina/metabolismo , Sequências Hélice-Volta-Hélice , Listeria monocytogenes/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Conformação Proteica , Proteínas Repressoras/metabolismoRESUMO
The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3-/- mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.
Assuntos
Antibacterianos/farmacologia , Listeria/efeitos dos fármacos , Listeriose/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Fagócitos/imunologia , Fagócitos/microbiologia , Animais , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Interferon Tipo I/metabolismo , Listeria monocytogenes/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/imunologia , Células RAW 264.7 , Transcriptoma , Fatores de Virulência , Internalização do Vírus/efeitos dos fármacosRESUMO
Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.
Assuntos
Venenos de Cnidários/metabolismo , Células Dendríticas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Compostos Orgânicos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Flagellar motility in Listeria monocytogenes (Lm) is restricted to temperatures below 37 degrees C due to the opposing activities of the MogR transcriptional repressor and the GmaR antirepressor. Previous studies have suggested that both the DegU response regulator and MogR regulate expression of GmaR. In this report, we further define the role of DegU for GmaR production and flagellar motility. We demonstrate that deletion of the receiver domain of DegU has no effect on flagellar motility in Lm. Using transcriptional reporter fusions, we determined that gmaR is cotranscribed within an operon initiating with fliN. Furthermore, the fliN-gmaR promoter (p(fliN-gmaR)) is transcriptionally activated by DegU and is also MogR-repressed. DNA affinity purification, gel mobility shift and footprinting analyses revealed that both DegU and MogR directly bind fliN-gmaR promoter region DNA and that the binding sites do not overlap. Quantitative analysis of gmaR transcripts in Delta mogR bacteria indicated that transcriptional activation of p(fliN-gmaR) by DegU is not inherently temperature-dependent. However, GmaR protein was not detectable at 37 degrees C in Delta mogR bacteria, indicating that a temperature-dependent, post-transcriptional mechanism limits GmaR production to temperatures below 37 degrees C. Our findings reveal that flagellar motility in Lm is governed by both temperature-dependent transcriptional and post-transcriptional regulation of the GmaR antirepressor.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Flagelos/fisiologia , Listeria monocytogenes/genética , Temperatura , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Pegada de DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Flagelos/genética , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/metabolismo , Listeria monocytogenes/fisiologia , Dados de Sequência Molecular , Óperon , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Ativação TranscricionalRESUMO
Listeria monocytogenes is a bacterial pathogen that replicates within the cytosol of infected host cells. The ability to rapidly escape the phagocytic vacuole is essential for efficient intracellular replication. In the murine model of infection, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for vacuolar dissolution, as LLO-deficient (DeltaLLO) mutants remain trapped within vacuoles. In contrast, in many human cell types DeltaLLO L. monocytogenes are capable of vacuolar escape at moderate to high frequencies. To better characterize the mechanism of LLO-independent vacuolar escape in human cells, we conducted an RNA interference screen to identify vesicular trafficking factors that play a role in altering vacuolar escape efficiency of DeltaLLO L. monocytogenes. RNA interference knockdown of 18 vesicular trafficking factors resulted in increased LLO-independent vacuolar escape. Our results suggest that knockdown of one factor, RABEP1 (rabaptin-5), decreased the maturation of vacuoles containing DeltaLLO L. monocytogenes. Thus, we provide evidence that increased vacuolar escape of DeltaLLO L. monocytogenes in human cells correlates with slower vacuolar maturation. We also determined that increased LLO-independent dissolution of vacuoles during RABEP1 knockdown required the bacterial broad-range phospholipase C (PC-PLC). We hypothesize that slowing the kinetics of vacuolar maturation generates an environment conducive for vacuolar escape mediated by the bacterial phospholipases.
Assuntos
Toxinas Bacterianas/toxicidade , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Vacúolos/microbiologia , Vacúolos/fisiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , Proteínas de Transporte Vesicular/fisiologiaRESUMO
Listeria monocytogenes is an intracellular bacterial pathogen that causes life-threatening disease. The mechanisms used by L. monocytogenes to invade non-professional phagocytic cells are not fully understood. In addition to the requirement of bacterial determinants, host cell conditions profoundly influence infection. Here, we have shown that inhibition of the RhoA/ROCK pathway by pharmacological inhibitors or RNA interference results in increased L. monocytogenes invasion of murine fibroblasts and hepatocytes. InlF, a member of the internalin multigene family with no known function, was identified as a L. monocytogenes-specific factor mediating increased host cell binding and entry. Conversely, activation of RhoA/ROCK activity resulted in decreased L. monocytogenes adhesion and invasion. Furthermore, virulence of wild-type bacteria during infection of mice was significantly increased upon inhibition of ROCK activity, whereas colonization and virulence of an inlF deletion mutant was not affected, thus supporting a role for InlF as a functional virulence determinant in vivo under specific conditions. In addition, inhibition of ROCK activity in human-derived cells enhanced either bacterial adhesion or adhesion and entry in an InlF-independent manner, further suggesting a host species or cell type-specific role for InlF and that additional bacterial determinants are involved in mediating ROCK-regulated invasion of human cells.