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 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
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
Listeria monocytogenes is an intracellular pathogen responsible for listeriosis, a foodborne disease that can lead to life-threatening meningitis. The 2011 L. monocytogenes cantaloupe outbreak was among the deadliest foodborne outbreaks in the United States. We conducted in vitro and in vivo infection analyses to determine whether strains LS741 and LS743, two clinical isolates from the cantaloupe outbreak, differ significantly from the common laboratory strain 10403S. We showed that LS741 and LS743 exhibited increased virulence, characterized by higher colonization of the brain and other organs in mice. Assessment of cellular immune responses to known CD8+ T cell antigens was comparable between all strains. However, pre-existing immunity to 10403S did not confer protection in the brain against challenge with LS741. These studies provide insights into the pathogenesis of clinical isolates linked to the 2011 cantaloupe outbreak and also indicate that currently utilized laboratory strains are imperfect models for studying L. monocytogenes pathogenesis.
Assuntos
Surtos de Doenças , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/microbiologia , Animais , Toxinas Bacterianas , Linfócitos T CD8-Positivos/citologia , Cucumis melo/microbiologia , Feminino , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos , Células HeLa , Proteínas de Choque Térmico , Proteínas Hemolisinas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estados Unidos , VirulênciaRESUMO
Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.
Assuntos
Venenos de Cnidários/metabolismo , Células Dendríticas/fisiologia , Macrófagos/fisiologia , Linfócitos T Citotóxicos/imunologia , Vacúolos/metabolismo , Animais , Antígenos/imunologia , Antígenos CD8/metabolismo , Células Cultivadas , Venenos de Cnidários/química , Apresentação Cruzada , Feminino , Leupeptinas/farmacologia , Lipossomos/química , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaRESUMO
Plasma membrane integrity is essential for the viability of eukaryotic cells. In response to bacterial pore-forming toxins, disrupted regions of the membrane are rapidly repaired. However, the pathways that mediate plasma membrane repair are unclear. Here we show that autophagy-related (ATG) protein ATG16L1 and its binding partners ATG5 and ATG12 are required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote membrane repair. ATG16L1 deficiency causes accumulation of cholesterol in lysosomes that contributes to defective membrane repair. Cell-to-cell spread by Listeria monocytogenes requires membrane damage by the bacterial toxin listeriolysin O, which is restricted by ATG16L1-dependent membrane repair. Cells harbouring the ATG16L1 T300A allele associated with inflammatory bowel disease were also found to accumulate cholesterol and be defective in repair, linking a common inflammatory disease to plasma membrane integrity. Thus, plasma membrane repair could be an important therapeutic target for the treatment of bacterial infections and inflammatory disorders.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Animais , Autofagia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Toxinas Bacterianas/toxicidade , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Colesterol/metabolismo , Modelos Animais de Doenças , Exocitose , Células HeLa , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Humanos , Listeria monocytogenes/metabolismo , Lisossomos , Masculino , CamundongosRESUMO
Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.IMPORTANCEListeria monocytogenes is an intracellular bacterial pathogen that is capable of invading numerous host cells during infection. L. monocytogenes can cross the blood-brain barrier, leading to life-threatening meningitis. Here we show that an L. monocytogenes surface protein, InlF, is necessary for optimal colonization of the brain in mice. Furthermore, in the absence of vimentin, a cytosolic intermediate filament protein that is also present on the surface of brain endothelial cells, colonization of the brain was significantly impaired. We further show that InlF binds vimentin to mediate invasion of host cells. This work identifies InlF as a bacterial surface protein with specific relevance for infection of the brain and underscores the significance of host cell surface vimentin interactions in microbial pathogenesis.
Assuntos
Encéfalo/parasitologia , Endocitose , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/parasitologia , Vimentina/metabolismo , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Listeriose/patologia , Camundongos , RatosRESUMO
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
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
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
Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted infection, the total burden of which is underestimated due to the asymptomatic nature of the infection. Untreated C. trachomatis infections can cause significant morbidities, including pelvic inflammatory disease and tubal factor infertility (TFI). The human immune response against C. trachomatis, an obligate intracellular bacterium, is poorly characterized but is thought to rely on cell-mediated immunity, with CD4(+) and CD8(+) T cells implicated in protection. In this report, we present immune profiling data of subjects enrolled in a multicenter study of C. trachomatis genital infection. CD4(+) and CD8(+) T cells from subjects grouped into disease-specific cohorts were screened using a C. trachomatis proteomic library to identify the antigen specificities of recall T cell responses after natural exposure by measuring interferon gamma (IFN-γ) levels. We identified specific T cell responses associated with the resolution of infection, including unique antigens identified in subjects who spontaneously cleared infection and different antigens associated with C. trachomatis-related sequelae, such as TFI. These data suggest that novel and unique C. trachomatis T cell antigens identified in individuals with effective immune responses can be considered as targets for vaccine development, and by excluding antigens associated with deleterious sequelae, immune-mediated pathologies may be circumvented.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Infecções do Sistema Genital/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imunidade Celular , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Proteômica , Infecções do Sistema Genital/microbiologia , Adulto JovemRESUMO
Listeria monocytogenes is an intracellular bacterial pathogen that can replicate in the cytosol of host cells. These bacteria undergo actin-based motility in the cytosol via expression of ActA, which recruits host actin-regulatory proteins to the bacterial surface. L. monocytogenes is thought to evade killing by autophagy using ActA-dependent mechanisms. ActA-independent mechanisms of autophagy evasion have also been proposed, but remain poorly understood. Here we examined autophagy of non-motile (ΔactA) mutants of L. monocytogenes strains 10403S and EGD-e, two commonly studied strains of this pathogen. The ΔactA mutants displayed accumulation of ubiquitinated proteins and p62/SQSTM1 on their surface. However, only strain EGD-e ΔactA displayed colocalization with the autophagy marker LC3 at 8 hours post infection. A bacteriostatic agent (chloramphenicol) was required for LC3 recruitment to 10403S ΔactA, suggesting that these bacteria produce a factor for autophagy evasion. Internalin K was proposed to block autophagy of L. monocytogenes in the cytosol of host cells. However, deletion of inlK in either the wild-type or ΔactA background of strain 10403S had no impact on autophagy evasion by bacteria, indicating it does not play an essential role in evading autophagy. Replication of ΔactA mutants of strain EGD-e and 10403S was comparable to their parent wild-type strain in macrophages. Thus, ΔactA mutants of L. monocytogenes can block killing by autophagy at a step downstream of protein ubiquitination and, in the case of strain EGD-e, downstream of LC3 recruitment to bacteria. Our findings highlight the strain-specific differences in the mechanisms that L. monocytogenes uses to evade killing by autophagy in host cells.
Assuntos
Autofagia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cães , Proteínas de Choque Térmico/metabolismo , Cinética , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico , Proteína Sequestossoma-1 , Proteínas Ubiquitinadas/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 a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.
Assuntos
Biofilmes/crescimento & desenvolvimento , Listeria monocytogenes/fisiologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis/genética , Espaço Extracelular/metabolismo , Loci Gênicos/genética , Listeria monocytogenes/citologia , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Mutagênese , Mutação , Fosfatos/metabolismo , Propriedades de SuperfícieRESUMO
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
Vaccines are the most cost-effective means of preventing infectious diseases and have the potential to be used in a therapeutic capacity for the treatment of numerous chronic diseases and cancer. The majority of available vaccines function by eliciting antibodies that can neutralize toxins or opsonize the pathogen leading to elimination by professional phagocytes. However, there are many infectious and non-infectious diseases for which there are no available vaccines or the current antibody-mediated vaccines offer insufficient protection. There is emerging evidence that successful protection for these conditions requires the stimulation of T cell responses in addition to antibody. Genome/proteome-wide screening of pathogens to identify appropriate antibody targets for inclusion in vaccines has become widely used in recent years. However, the application of high-throughput proteomic screening approaches to identify T cell antigens has substantially lagged behind, primarily due to the lack of methods to identify full protein targets of T cell immunity across a broad human population. In this review, we will discuss some of the significant advances that have been made in high-throughput identification of T cell antigens for the development of novel efficacious vaccines.
Assuntos
Antígenos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Proteínas/análise , Proteínas/imunologia , Linfócitos T/imunologia , Animais , Ensaios de Triagem em Larga Escala/economia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Biblioteca de PeptídeosRESUMO
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
Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3(+) bacteria were present within single-membrane phagosomes that are LAMP1(+). Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3(+) bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.