RESUMO
Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61% of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response.
Assuntos
Epigênese Genética , Código das Histonas , Imunidade Inata , Macrófagos/microbiologia , Yersiniose/microbiologia , Yersinia enterocolitica/patogenicidade , Proteínas rho de Ligação ao GTP/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Yersiniose/genética , Yersiniose/imunologia , Yersiniose/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The virulence strategy of pathogenic Yersinia spp. involves cell-invasive as well as phagocytosis-preventing tactics to enable efficient colonisation of the host organism. Enteropathogenic yersiniae display an invasive phenotype in early infection stages, which facilitates penetration of the intestinal mucosa. Here we show that invasion of epithelial cells by Yersinia enterocolitica is followed by intracellular survival and multiplication of a subset of ingested bacteria. The replicating bacteria were enclosed in vacuoles with autophagy-related characteristics, showing phagophore formation, xenophagy, and recruitment of cytoplasmic autophagosomes to the bacteria-containing compartments. The subsequent fusion of these vacuoles with lysosomes and concomitant vesicle acidification were actively blocked by Yersinia. This resulted in increased intracellular proliferation and detectable egress of yersiniae from infected cells. Notably, deficiency of the core autophagy machinery component FIP200 impaired the development of autophagic features at Yersinia-containing vacuoles as well as intracellular replication and release of bacteria to the extracellular environment. These results suggest that Y. enterocolitica may take advantage of the macroautophagy pathway in epithelial cells to create an autophagosomal niche that supports intracellular bacterial survival, replication, and, eventually, spread of the bacteria from infected cells.
Assuntos
Autofagossomos/microbiologia , Células Epiteliais/microbiologia , Yersinia enterocolitica/patogenicidade , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Morte Celular , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Células HeLa , Interações entre Hospedeiro e Microrganismos , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , Lisossomos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Vacúolos/ultraestrutura , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/metabolismoRESUMO
Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/biossíntese , Yersiniose/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/imunologia , Imunofluorescência , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Parasita/fisiologia , Humanos , Tolerância Imunológica/fisiologia , Imunoprecipitação , Macrófagos/microbiologia , Espectrometria de Massas , Microscopia Confocal , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Yersiniose/metabolismo , Yersinia enterocoliticaRESUMO
Many pathogenic microorganisms have evolved tactics to modulate host cell death or survival pathways for establishing infection. The enteropathogenic bacterium Yersinia enterocolitica deactivates TLR-induced signaling pathways, which triggers apoptosis in macrophages. In this article, we show that Yersinia-induced apoptosis of human macrophages involves caspase-dependent cleavage of the TLR adapter protein MyD88. MyD88 was also cleaved when apoptosis was mediated by overexpression of the Toll-IL-1R domain-containing adapter inducing IFN-ß in epithelial cells. The caspase-processing site was mapped to aspartate-135 in the central region of MyD88. MyD88 is consequently split by caspases in two fragments, one harboring the death domain and the other the Toll-IL-1R domain. Caspase-3 was identified as the protease that conferred the cleavage of MyD88 in in vitro caspase assays. In line with a broad role of caspase-3 in the execution of apoptosis, the processing of MyD88 was not restricted to Yersinia infection and to proapoptotic Toll-IL-1R domain-containing adapter inducing IFN-ß signaling, but was also triggered by staurosporine treatment. The cleavage of MyD88 therefore seems to be a common event in the advanced stages of apoptosis, when caspase-3 is active. We propose that the processing of MyD88 disrupts its scaffolding function and uncouples the activation of TLR and IL-1Rs from the initiation of proinflammatory signaling events. The disruption of MyD88 may consequently render dying cells less sensitive to proinflammatory stimuli in the execution phase of apoptosis. The cleavage of MyD88 could therefore be a means of conferring immunogenic tolerance to apoptotic cells to ensure silent, noninflammatory cell demise.
Assuntos
Apoptose/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Macrófagos/microbiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Yersinia enterocolitica/fisiologia , Sequência de Aminoácidos , Animais , Caspase 3/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Interferon beta/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Macrófagos/imunologia , Glicoproteínas de Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/deficiência , NF-kappa B/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores de Interleucina-1/fisiologia , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Especificidade da Espécie , Receptores Toll-Like/fisiologiaRESUMO
Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/metabolismo , Neuropeptídeos/metabolismo , Yersiniose/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Toxinas Bacterianas/genética , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Transporte Proteico/genética , Yersiniose/genética , Yersiniose/patologia , Yersinia enterocolitica/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ras/genética , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoCRESUMO
The multifunctional Yersinia effector YopM inhibits effector triggered immunity and increases production of the anti-inflammatory cytokine Interleukin-10 (IL-10) to suppress the host immune response. Previously it was shown that YopM induces IL-10 gene expression by elevating phosphorylation of the serine-threonine kinase RSK1 in the nucleus of human macrophages. Using transcriptomics, we found that YopM strongly affects expression of genes belonging to the JAK-STAT signaling pathway. Further analysis revealed that YopM mediates nuclear translocation of the transcription factor Stat3 in Y. enterocolitica infected macrophages and that knockdown of Stat3 inhibited YopM-induced IL-10 gene expression. YopM-induced Stat3 translocation did not depend on autocrine IL-10, activation of RSK1 or tyrosine phosphorylation of Stat3. Thus, besides activation of RSK1, stimulation of nuclear translocation of Stat3 is another mechanism by which YopM increases IL-10 gene expression in macrophages.
Assuntos
Proteínas de Bactérias , Interleucina-10 , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Macrófagos/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , FosforilaçãoRESUMO
Assembly of adherent biofilms is the key mechanism involved in Staphylococcus epidermidis virulence during device-associated infections. Aside from polysaccharide intercellular adhesin (PIA), the accumulation-associated protein Aap and the extracellular matrix binding protein Embp act as intercellular adhesins, mediating S. epidermidis cell aggregation and biofilm accumulation. The aim of this study was to investigate structural features of PIA-, Aap-, and Embp-mediated S. epidermidis biofilms in more detail and to evaluate their specific contributions to biofilm-related S. epidermidis immune escape. PIA-, Embp-, and Aap-mediated biofilms exhibited substantial morphological differences. Basically, PIA synthesis induced formation of macroscopically visible, rough cell clusters, whereas Aap- and Embp-dependent biofilms preferentially displayed a smooth layer of aggregated bacteria. On the microscopic level, PIA was found to form a string-like organized extracellular matrix connecting the bacteria, while Embp produced small deposits of intercellular matrix and Aap was strictly localized to the bacterial surface. Despite marked differences, S. epidermidis strains using PIA, Aap, or Embp for biofilm formation were protected from uptake by J774A.1 macrophages, with similarly efficiencies. In addition, compared to biofilm-negative S. epidermidis strains, isogenic biofilm-forming S. epidermidis induced only a diminished inflammatory J774A.1 macrophage response, leading to significantly (88.2 to 88.7%) reduced NF-κB activation and 68.8 to 83% reduced interleukin-1ß (IL-1ß) production. Mechanical biofilm dispersal partially restored induction of NF-κB activation, although bacterial cell surfaces remained decorated with the respective intercellular adhesins. Our results demonstrate that distinct S. epidermidis biofilm morphotypes are similarly effective at protecting S. epidermidis from phagocytic uptake and at counteracting macrophage activation, providing novel insights into mechanisms that could contribute to the chronic and persistent course of biofilm-related S. epidermidis foreign material infections.
Assuntos
Biofilmes/crescimento & desenvolvimento , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus epidermidis/fisiologia , Adesinas Bacterianas/fisiologia , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Interleucina-1beta/metabolismo , Camundongos , Microscopia Confocal , NF-kappa B/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/imunologiaRESUMO
Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Tecido Linfoide/microbiologia , Transdução de Sinais/fisiologia , Yersiniose/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Linhagem Celular , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Virulência/fisiologia , Yersiniose/genética , Yersinia enterocolitica/genéticaRESUMO
Autophagy is a central lysosomal degradation process that is essential for the maintenance of cellular homeostasis. Autophagy has furthermore emerged as integral part of the host immune response. Autophagic processes promote the separation and degradation of intracellular microorganisms which contributes to the development of innate and adaptive immunity. Some pathogenic microbes have therefore evolved mechanisms to evade or impede autophagy. We analyzed the effects of the enteropathogenic bacterium Yersinia enterocolitica on autophagy in macrophages. Yersiniae use a number of defined adhesins and secreted proteins to manipulate host immune responses. Our results showed that Y. enterocolitica defective in type III protein secretion efficiently activated autophagy in macrophages. Autophagy was mediated by the Yersinia adhesins invasin and YadA and particularly depended on the engagement of beta(1) integrin receptors. Several autophagy-related events followed beta(1) integrin-mediated engulfment of the bacteria including the formation of autophagosomes, processing of the marker protein LC3, redistribution of GFP-LC3 to bacteria-containing vacuoles, and the segregation of intracellular bacteria by autophagosomal compartments. These results provide direct evidence for the linkage of beta(1) integrin-mediated phagocytosis and autophagy induction. Multiple microbes signal through integrin receptors, and our results suggest a general principle by which the sensing of an extracellular microbe triggers autophagy. Owing to the importance of autophagy as host defense response, wild-type Y. enterocolitica suppressed autophagy by mobilizing type III protein secretion. The subversion of autophagy may be part of the Y. enterocolitica virulence strategy that supports bacterial survival when beta(1) integrin-dependent internalization and autophagy activation by macrophages are deleterious for the pathogen.
Assuntos
Autofagia/imunologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Integrina beta1/fisiologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Yersinia enterocolitica/imunologia , Adesinas Bacterianas/fisiologia , Animais , Autofagia/genética , Proteínas de Bactérias/genética , Biomarcadores/metabolismo , Linhagem Celular , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Integrina beta1/biossíntese , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/fisiologia , Plasmídeos/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Virulência/genética , Virulência/imunologia , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidadeRESUMO
RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S > D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice.
Assuntos
Apoptose , Modelos Imunológicos , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Caspase 8/genética , Caspase 8/metabolismo , Caspase 8/fisiologia , Linhagem Celular , Quinase I-kappa B/metabolismo , Quinase I-kappa B/fisiologia , Imunidade/fisiologia , Camundongos , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serina/química , Serina/metabolismo , Yersinia , Yersiniose/imunologiaRESUMO
The interaction of microbial pathogens with host cells critically determines the genesis of infectious diseases. Gram-negative, pathogenic bacteria from the genus Yersinia deliver a set of virulence proteins, the so-called Yersinia outer proteins (Yops), inside the eukaryotic cell where the Yops perturb key cellular functions of innate immunity. In our past work, we used Yersinia enterocolitica as a tool to explore the crosstalk between the bacterial pathogen and its host cell. Yersiniae counteract phagocytosis, suppress proinflammatory signalling and trigger apoptosis in macrophages. Macrophage cell death results from the deregulation of Toll-like receptors-dependent conserved signalling pathways by Yersinia infection. We summarize our current understanding about the signals and reactions elicited on both the bacterial and host cell sides that determine the fate of the infected cell along with the innate immune response.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Imunidade Inata , Macrófagos/microbiologia , Yersiniose/microbiologia , Yersinia enterocolitica/metabolismo , Yersinia/fisiologia , Animais , Apoptose , Proteínas de Bactérias/metabolismo , Humanos , Macrófagos/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Ubiquitina/metabolismo , Yersinia/metabolismoRESUMO
Complex posttranslational modifications determine the effects of receptor-interacting protein kinase-1 (RIPK1) on cell survival and death. Studies from us and others have revealed a p38MAPK/MK2-dependent checkpoint in RIPK1 signaling. MAPKAP kinase 2 (MK2) phosphorylates RIPK1 to suppress RIPK1-mediated apoptosis and necroptosis in response to diverse stimuli relevant to inflammation, infection, genotoxic stress and chemotherapy.
RESUMO
Pathogenic species of the genus Yersinia suppress and reorient the immune system to infect lymphatic tissues, inner organs and at times also the vasculature. For this purpose yersiniae employ a type III secretion system to translocate effector proteins (Yersinia outer proteins; Yops) into immune cells. Yops often exert unique biochemical activities for modulating the activity of Rho GTP-binding proteins, focal adhesion proteins, inflammatory pathways and cell survival/apoptosis. In this review we will put emphasis on the biochemistry, cell- and infection biology of Yersinia effector Yops.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Translocação Bacteriana , Sepse/metabolismo , Transdução de Sinais , Yersiniose/metabolismo , Yersinia/patogenicidade , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Adesões Focais/metabolismo , Humanos , Viabilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sepse/enzimologia , Sepse/microbiologia , Terpenos/metabolismo , Virulência , Yersinia/enzimologia , Yersinia/crescimento & desenvolvimento , Yersinia/metabolismo , Yersiniose/enzimologia , Yersiniose/microbiologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Receptor-interacting protein kinase-1 (RIPK1), a master regulator of cell fate decisions, was identified as a direct substrate of MAPKAP kinase-2 (MK2) by phosphoproteomic screens using LPS-treated macrophages and stress-stimulated embryonic fibroblasts. p38MAPK/MK2 interact with RIPK1 in a cytoplasmic complex and MK2 phosphorylates mouse RIPK1 at Ser321/336 in response to pro-inflammatory stimuli, such as TNF and LPS, and infection with the pathogen Yersinia enterocolitica. MK2 phosphorylation inhibits RIPK1 autophosphorylation, curtails RIPK1 integration into cytoplasmic cytotoxic complexes, and suppresses RIPK1-dependent apoptosis and necroptosis. In Yersinia-infected macrophages, RIPK1 phosphorylation by MK2 protects against infection-induced apoptosis, a process targeted by Yersinia outer protein P (YopP). YopP suppresses p38MAPK/MK2 activation to increase Yersinia-driven apoptosis. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection that determines the outcome of bacteria-host cell interaction.
Assuntos
Apoptose , Inflamação/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Yersiniose/enzimologia , Yersinia enterocolitica/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Citosol/enzimologia , Citosol/microbiologia , Feminino , Genótipo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Quinase I-kappa B/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Knockout , Necrose , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Serina , Transdução de Sinais , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/toxicidade , Yersiniose/microbiologia , Yersiniose/patologia , Yersinia enterocolitica/metabolismoRESUMO
The interaction between macrophages and bacterial pathogens plays a crucial role in the pathogenesis of infectious diseases. Pathogenic species of the Gram-negative bacterium Yersinia deploy complex strategies to disarm macrophages and to disrupt their response to infection. For this purpose, Yersinia sp. engage a type III protein secretion system that mediates the polarized translocation of Yersinia virulence factors, the so-called Yops (Yersinia outer proteins), into the host cell cytoplasm. There, the Yops act on different cellular levels to neutralize a sequence of programmed phagocyte effector functions. Yersiniae initially impair the phagocytic machinery and block the generation of the bactericidal oxidative burst. Furthermore, yersiniae uncouple an array of fine-tuned signals of innate immunity, which leads to suppression of macrophage TNF-alpha production and to macrophage apoptosis. The impairment of cellular functions results in a scenario by which Yersinia efficiently resists the attack of the macrophage and finally kills the macrophage by activating its intrinsic cell suicide mechanism. This review highlights the aspects of Yersinia-macrophage interaction that determine the fate of the infected cell.
Assuntos
Macrófagos/imunologia , Macrófagos/microbiologia , Yersinia/patogenicidade , Animais , Apoptose , Proteínas da Membrana Bacteriana Externa/fisiologia , Humanos , Técnicas In Vitro , Macrófagos/patologia , Macrófagos/fisiologia , Modelos Biológicos , Fagocitose , Transdução de Sinais , Virulência/imunologia , Yersinia/imunologiaRESUMO
The Yersinia enterocolitica Rho GTPase Activating Protein (Rho GAP) YopE belongs to a group of bacterial virulence factors that is translocated into infected target cells by a type three secretion system. Structurally and biochemically YopE resembles eukaryotic Rho GAPs which control various cellular functions by modulating the activity of Rho GTP binding proteins. Here we summarise the published information on cellular effects, Rho protein substrates, compartmentalisation and turnover of YopE. A fascinating picture evolves of how this virulence factor integrates in host cellular regulatory mechanisms to fine tune bacterial pathogenicity.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/química , Toxinas Bacterianas/metabolismo , Proteínas Ativadoras de GTPase/química , Humanos , UbiquitinaçãoRESUMO
BACKGROUND: Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells. METHODOLOGY/PRINCIPAL FINDINGS: In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221. CONCLUSIONS/SIGNIFICANCE: Our study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Yersinia/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Sequência de Bases , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Primers do DNA , Ativação Enzimática , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Pathogenic bacteria of the genus Yersinia (Y. pestis, Y. enterocolitica and Y. pseudotuberculosis) have evolved numerous virulence factors (termed a stratagem) to manipulate the activity of Rho GTPases. Here, we show that Y. enterocolitica modulates RhoG, an upstream regulator of other Rho GTPases. At the contact site of virulent Y. enterocolitica and host cells, we could visualise spatiotemporally organised activation and deactivation of RhoG. On the one hand, the beta1-integrin clustering protein Invasin on the bacterial surface was found to activate RhoG and this promoted cell invasion. On the other hand, active RhoG was downregulated by the type III secretion system effector YopE acting as a GTPase-activating protein (GAP). YopE localised to Golgi and endoplasmic reticulum, and this determined its specificity for RhoG and other selected Rho GTPases. RhoG and its downstream effector module Elmo/Dock180 controlled both Rac1 activation by Invasin and Rac1 deactivation by YopE. We propose that RhoG is a central target of the Yersinia stratagem and a major upstream regulator of Rac1 during different phases of the Yersinia infection cycle.
Assuntos
Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidade , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Yersinia enterocolitica/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/fisiologia , Yersinia enterocolitica/classificação , Yersinia enterocolitica/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Linhagem Celular , Estabilidade Enzimática/genética , Humanos , Lisina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sorotipagem , Transdução de Sinais/genética , Especificidade da Espécie , Yersinia enterocolitica/genéticaRESUMO
Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIPL degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55-/- mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.