Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 91
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
País de afiliación
Intervalo de año de publicación
1.
Nat Immunol ; 21(8): 857-867, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32601469

RESUMEN

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1ß suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1ß specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.


Asunto(s)
Resistencia a la Enfermedad/genética , Fiebre Mediterránea Familiar/genética , Peste , Pirina/genética , Selección Genética/genética , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Resistencia a la Enfermedad/inmunología , Haplotipos , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Peste/inmunología , Peste/metabolismo , Pirina/inmunología , Pirina/metabolismo , Turquía , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Yersinia pestis
2.
Immunity ; 47(6): 1154-1168.e6, 2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-29221731

RESUMEN

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.


Asunto(s)
Tejido Adiposo Blanco/trasplante , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Toxoplasmosis/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Tejido Adiposo Blanco/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/parasitología , Expresión Génica , Genes Reporteros , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-5/genética , Interleucina-5/inmunología , Metabolismo de los Lípidos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Supervivencia , Trasplante de Tejidos , Toxoplasma/inmunología , Toxoplasmosis/genética , Toxoplasmosis/mortalidad , Toxoplasmosis/parasitología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/microbiología , Infecciones por Yersinia pseudotuberculosis/mortalidad
4.
Immunol Rev ; 297(1): 96-107, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32721043

RESUMEN

Pyrin is a cytosolic pattern-recognition receptor that normally functions as a guard to trigger capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. The MEFV gene encoding human pyrin is preferentially expressed in phagocytes. Key domains in pyrin include a pyrin domain (PYD), a linker region, and a B30.2 domain. Binding of ASC to pyrin by a PYD-PYD interaction triggers inflammasome assembly. Pyrin is held in an inactive conformation by negative regulation mechanisms to avoid premature inflammasome assembly. One mechanism of negative regulation involves phosphorylation of the linker by PRK kinase which in turn is positively regulated by active RhoA. The B30.2 domain also negatively regulates pyrin. Gain of function mutations in MEFV responsible for the autoinflammatory disease Familial Mediterranean Fever (FMF) map to exon 10 encoding the B30.2 domain. Insights into pyrin regulation have come from studies of several Yersinia effectors, which are injected into phagocytes and interact with the RhoA-PRK-pyrin axis during infection. Two effectors, YopE and YopT, inactivate RhoA to disrupt phagocytic signaling. To counteract an effector-triggered immune response, a third effector, YopM, binds to and inhibits pyrin by hijacking PRK and RSK and directing linker phosphorylation. Inhibition of pyrin by YopM is required for virulence of Yersinia pestis, the agent of plague. Recent results from infection studies with human phagocytes and mice producing pyrin B30.2 FMF variants show that gain of function MEFV mutations bypass inhibition by YopM. Population genetic data suggest that MEFV mutations were selected for in individuals of Mediterranean decent during historic plague pandemics. This review discusses current concepts of pyrin regulation and its interaction with Yersinia effectors.


Asunto(s)
Toxinas Bacterianas , Fiebre Mediterránea Familiar , Animales , Inflamasomas , Ratones , Mutación , Pirina/genética , Yersinia
5.
PLoS Pathog ; 17(12): e1010103, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34871329

RESUMEN

Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity.


Asunto(s)
Proteínas Bacterianas/inmunología , Evasión Inmune/inmunología , Interferón gamma/biosíntesis , Linfocitos Intraepiteliales/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Animales , Humanos , Ratones , Yersinia pseudotuberculosis/inmunología
6.
Infect Immun ; 89(8): e0026521, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34031132

RESUMEN

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 epitope was inserted into YopE in Y. pseudotuberculosis and antigen-specific CD8+ T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE69-77 elicited significantly larger CD8+ T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen, and liver between 7 and 30 days postinfection, despite residing in the same protein and having an affinity for H-2Kb similar to that of OVA257-264. YopE-specific CD8+ T cell precursors were ∼4.6 times as abundant as OVA-specific precursors in the MLNs, spleens, and other lymph nodes of naive mice, explaining the dominance of YopE69-77 over OVA257-264 at early infection times. However, other factors contributed to this dominance, as the ratio of YopE-specific to OVA-specific CD8+ T cells increased between 7 and 30 days postinfection. We also compared the YopE-specific and OVA-specific CD8+ T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short-lived effectors, while higher percentages of OVA-specific cells were memory precursor effectors at day 30 postinfection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8+ T cells generated in response to the protective YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.


Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD8-positivos/inmunología , Interacciones Huésped-Patógeno/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/microbiología , Yersinia pseudotuberculosis/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Infecciones por Yersinia pseudotuberculosis/transmisión
7.
PLoS Pathog ; 15(7): e1007847, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31306468

RESUMEN

Salmonella exploit host-derived nitrate for growth in the lumen of the inflamed intestine. The generation of host-derived nitrate is dependent on Nos2, which encodes inducible nitric oxide synthase (iNOS), an enzyme that catalyzes nitric oxide (NO) production. However, the cellular sources of iNOS and, therefore, NO-derived nitrate used by Salmonella for growth in the lumen of the inflamed intestine remain unidentified. Here, we show that iNOS-producing inflammatory monocytes infiltrate ceca of mice infected with Salmonella. In addition, we show that inactivation of type-three secretion system (T3SS)-1 and T3SS-2 renders Salmonella unable to induce CC- chemokine receptor-2- and CC-chemokine ligand-2-dependent inflammatory monocyte recruitment. Furthermore, we show that the severity of the pathology of Salmonella- induced colitis as well as the nitrate-dependent growth of Salmonella in the lumen of the inflamed intestine are reduced in mice that lack Ccr2 and, therefore, inflammatory monocytes in the tissues. Thus, inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine.


Asunto(s)
Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Monocitos/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Animales , Quimiocina CCL2/deficiencia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Femenino , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/fisiología , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Monocitos/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores CCR2/deficiencia , Receptores CCR2/genética , Receptores CCR2/metabolismo , Salmonelosis Animal/metabolismo , Salmonelosis Animal/microbiología , Salmonelosis Animal/patología , Salmonella typhimurium/genética , Sistemas de Secreción Tipo III/metabolismo
8.
J Biol Chem ; 293(4): 1466-1479, 2018 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-29197826

RESUMEN

Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell ß1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δinv, ΔyadA, and ΔinvΔyadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The ΔinvΔyadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered ß1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis.


Asunto(s)
Adhesión Bacteriana , Deshidrocolesteroles/metabolismo , Integrina beta1/metabolismo , Infecciones por Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/metabolismo , Línea Celular Tumoral , Femenino , Eliminación de Gen , Humanos , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad , Infecciones por Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/patología
9.
Infect Immun ; 87(3)2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30602502

RESUMEN

Pathogenic Yersinia species deliver Yop effector proteins through a type III secretion system into host cells. Among these effectors, YopE and YopT are Rho-modifying toxins, which function to modulate host cell physiology and evade immune responses. YopE is a GTPase-activating protein (GAP) while YopT is a protease, and they inhibit RhoA by different modes of action. Modifications to RhoA are sensed by pyrin, which, once activated, assembles a caspase-1 inflammasome, which generates cytokines such as interleukin-1ß (IL-1ß) and cell death by pyroptosis. In Yersinia-infected macrophages, YopE or YopT triggers inflammasome assembly only in the absence of another effector, YopM, which counteracts pyrin by keeping it inactive. The glucosyltransferase TcdB from Clostridium difficile, a well-studied RhoA-inactivating toxin, triggers activation of murine pyrin by dephosphorylation of Ser205 and Ser241. To determine if YopE or YopT triggers pyrin dephosphorylation, we infected lipopolysaccharide (LPS)-primed murine macrophages with ΔyopMYersinia pseudotuberculosis strains expressing wild-type (wt) or YopE mutant variants or YopT. By immunoblotting pyrin after infection, we observed that wt YopE triggered dephosphorylation of Ser205 and inflammasome activation. Pyrin dephosphorylation was reduced if a YopE variant had a defect in stability or RhoA specificity but not membrane localization. We also observed that wt YopT triggered pyrin dephosphorylation but more slowly than YopE, suggesting that YopE is dominant in this process. Our findings provide evidence that RhoA-modifying toxins trigger activation of pyrin by a conserved dephosphorylation mechanism. In addition, by characterization of YopE and YopT, we show that different features of effectors, such as RhoA specificity, affect the efficiency of pyrin dephosphorylation.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Cisteína Endopeptidasas/inmunología , Inflamasomas/metabolismo , Macrófagos/metabolismo , Pirina/metabolismo , Yersinia/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Macrófagos/inmunología , Fosforilación
10.
J Bacteriol ; 200(9)2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29440252

RESUMEN

Yersinia pestis, the causative agent of plague, evolved from the closely related pathogen Yersinia pseudotuberculosis During its emergence, Y. pestis is believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system. Y. pseudotuberculosis and the basal human-avirulent strains of Y. pestis harbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation of Y. pestis to stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability of Y. pestis to survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele of phoP (phoP-S215), a derivative carrying the basal allele (phoP-G215) exhibited slightly defective growth under a low-Mg2+ condition and decreased transcription of a PhoP target gene, ugd, as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. The phoP-G215 strain showed no apparent defect in flea colonization, although a phoP-null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence in Y. pestisIMPORTANCEY. pestis acquired a single nucleotide polymorphism (SNP) in phoP when the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show that Y. pestis carrying the modern phoP allele has an increased ability to induce the PhoP-regulated ugd gene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence in Y. pestis Additionally, we present the first evidence that phoP confers a survival fitness advantage to Y. pestis inside the flea midgut.


Asunto(s)
Sustitución de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Polimixina B/farmacología , Yersinia pestis/efectos de los fármacos , Yersinia pestis/genética , Animales , Evolución Molecular , Glicina/metabolismo , Macrófagos/microbiología , Ratones , Mutación , Serina/metabolismo , Siphonaptera/microbiología , Transcripción Genética , Virulencia , Yersinia pestis/patogenicidad
11.
Infect Immun ; 86(3)2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29263104

RESUMEN

Murine Ly6Chi inflammatory monocytes (IMs) require CCR2 to leave the bone marrow and enter mesenteric lymph nodes (MLNs) and other organs in response to Yersinia pseudotuberculosis infection. We are investigating how IMs, which can differentiate into CD11c+ dendritic cells (DCs), contribute to innate and adaptive immunity to Y. pseudotuberculosis Previously, we obtained evidence that IMs are important for a dominant CD8+ T cell response to the epitope YopE69-77 and host survival using intravenous infections with attenuated Y. pseudotuberculosis Here we challenged CCR2+/+ or CCR2-/- mice orally with wild-type Y. pseudotuberculosis to investigate how IMs contribute to immune responses during intestinal infection. Unexpectedly, CCR2-/- mice did not have reduced survival but retained body weight better and their MLNs cleared Y. pseudotuberculosis faster and with reduced lymphadenopathy compared to controls. Enhanced bacterial clearance in CCR2-/- mice correlated with reduced numbers of IMs in spleens and increased numbers of neutrophils in livers. In situ imaging of MLNs and spleens from CCR2-GFP mice showed that green fluorescent protein-positive (GFP+) IMs accumulated at the periphery of neutrophil-rich Yersinia-containing pyogranulomas. GFP+ IMs colocalized with CD11c+ cells and YopE69-77-specific CD8+ T cells in MLNs, suggesting that IM-derived DCs prime adaptive responses in Yersinia pyogranulomas. Consistently, CCR2-/- mice had reduced numbers of splenic DCs, YopE69-77-specific CD8+ T cells, CD4+ T cells, and B cells in organs and lower levels of serum antibodies to Y. pseudotuberculosis antigens. Our data suggest that IMs differentiate into DCs in MLN pyogranulomas and direct adaptive responses in T cells at the expense of innate immunity during oral Y. pseudotuberculosis infection.


Asunto(s)
Inmunidad Adaptativa , Inmunidad Innata , Monocitos/inmunología , Boca/microbiología , Receptores CCR2/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/inmunología , Animales , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores CCR2/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/fisiología , Infecciones por Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/microbiología
12.
Infect Immun ; 85(10)2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28784930

RESUMEN

Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (GbpChr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Unión al GTP/genética , Inflamasomas/inmunología , Sistemas de Secreción Tipo III/metabolismo , Yersinia pseudotuberculosis/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Caspasa 1/metabolismo , Línea Celular , Citocinas/biosíntesis , Citocinas/inmunología , Proteínas de Unión al GTP/metabolismo , Galectina 3/metabolismo , Inflamasomas/genética , Inflamasomas/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Transporte de Proteínas , Virulencia , Yersinia pseudotuberculosis/fisiología
13.
PLoS Pathog ; 11(10): e1005167, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26468944

RESUMEN

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8+ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8+ T cells recognize the epitope YopE69-77. The features of the interaction between pathogen and host that result in this large CD8+ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE69-77-specific CD8+ T cells generated during the large response are polyclonal and are produced by a "translocation-dependent" pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE69-77-specific CD8+ T cell response (~10% of the large expansion) can be generated in a "translocation-independent" pathway in which CD8α+ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE69-77-specific CD8+ T cell expansion because this response was significantly reduced in Ccr2-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE69-77-specific CD8+ T cells ex vivo and promoted the expansion of YopE69-77-specific CD8+ T cells in infected Ccr2-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8+ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE69-77-specific CD8+ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Sistemas de Secreción Tipo III/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Citometría de Flujo , Immunoblotting , Inflamación/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Transporte de Proteínas/inmunología , Receptores CCR2/inmunología , Factores de Virulencia/inmunología , Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/patogenicidad
14.
Infect Immun ; 84(4): 1062-1072, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26810037

RESUMEN

Pathogenic Yersinia species utilize a type III secretion system to translocate Yop effectors into infected host cells. Yop effectors inhibit innate immune responses in infected macrophages to promote Yersinia pathogenesis. In turn,Yersinia-infected macrophages respond to translocation of Yops by activating caspase-1, but different mechanisms of caspase-1 activation occur, depending on the bacterial genotype and the state of phagocyte activation. In macrophages activated with lipopolysaccharide (LPS) prior to Yersinia pseudotuberculosis infection, caspase-1 is activated by a rapid inflammasome-dependent mechanism that is inhibited by translocated YopM. The possibility that other effectors cooperate with YopM to inhibit caspase-1 activation in LPS-activated macrophages has not been investigated. Toward this aim, epistasis analysis was carried out in which the phenotype of aY. pseudotuberculosis yopM mutant was compared to that of a yopJ yopM, yopE yopM, yopH yopM, yopT yopM, or ypkA yopM mutant. Activation of caspase-1 was measured by cleavage of the enzyme, release of interleukin-1ß (IL-1ß), and pyroptosis in LPS-activated macrophages infected with wild-type or mutant Y. pseudotuberculosis strains. Results show enhanced activation of caspase-1 after infection with the yopJ yopM mutant relative to infection by any other single or double mutant. Similar results were obtained with the yopJ, yopM, and yopJ yopM mutants ofY ersinia pestis Following intravenous infection of mice, theY. pseudotuberculosis yopJ mutant was as virulent as the wild type, while the yopJ yopM mutant was significantly more attenuated than the yopM mutant. In summary, through epistasis analysis this work uncovered an important role for YopJ in inhibiting caspase-1 in activated macrophages and in promoting Yersinia virulence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Caspasa 1/metabolismo , Activación de Macrófagos/fisiología , Macrófagos/fisiología , Yersinia pseudotuberculosis/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Caspasa 1/genética , Células Cultivadas , Epistasis Genética , Femenino , Regulación Enzimológica de la Expresión Génica/fisiología , Inmunidad Innata , Lipopolisacáridos/toxicidad , Macrófagos/microbiología , Ratones , Mutación , Piroptosis , Virulencia
15.
PLoS Pathog ; 10(8): e1004346, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25165815

RESUMEN

The mammalian immune system has the ability to discriminate between pathogens and innocuous microbes by detecting conserved molecular patterns. In addition to conserved microbial patterns, the mammalian immune system may recognize distinct pathogen-induced processes through a mechanism which is poorly understood. Previous studies have shown that a type III secretion system (T3SS) in Yersinia pseudotuberculosis leads to decreased survival of this bacterium in primary murine macrophages by unknown mechanisms. Here, we use colony forming unit assays and fluorescence microscopy to investigate how the T3SS triggers killing of Yersinia in macrophages. We present evidence that Yersinia outer protein E (YopE) delivered by the T3SS triggers intracellular killing response against Yersinia. YopE mimics eukaryotic GTPase activating proteins (GAPs) and inactivates Rho GTPases in host cells. Unlike wild-type YopE, catalytically dead YopER144A is impaired in restricting Yersinia intracellular survival, highlighting that the GAP activity of YopE is detected as a danger signal. Additionally, a second translocated effector, YopT, counteracts the YopE triggered killing effect by decreasing the translocation level of YopE and possibly by competing for the same pool of Rho GTPase targets. Moreover, inactivation of Rho GTPases by Clostridium difficile Toxin B mimics the effect of YopE and promotes increased killing of Yersinia in macrophages. Using a Rac inhibitor NSC23766 and a Rho inhibitor TAT-C3, we show that macrophages restrict Yersinia intracellular survival in response to Rac1 inhibition, but not Rho inhibition. In summary, our findings reveal that primary macrophages sense manipulation of Rho GTPases by Yersinia YopE and actively counteract pathogenic infection by restricting intracellular bacterial survival. Our results uncover a new mode of innate immune recognition in response to pathogenic infection.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Sistemas de Secreción Bacterianos/inmunología , Proteínas Activadoras de GTPasa/inmunología , Inmunidad Innata/inmunología , Macrófagos/inmunología , Animales , Immunoblotting , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Yersinia pseudotuberculosis/inmunología
16.
PLoS Pathog ; 10(5): e1004142, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24854422

RESUMEN

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Celular/genética , Interferón gamma/fisiología , Peste/inmunología , Neumonía Bacteriana/inmunología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Yersinia pestis/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peste/complicaciones , Peste/genética , Neumonía Bacteriana/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Factor de Necrosis Tumoral alfa/genética
17.
Infect Immun ; 82(7): 3033-44, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24799630

RESUMEN

YopE is a virulence factor that is secreted into host cells infected by Yersinia species. The YopE C-terminal domain has GTPase-activating protein (GAP) activity. The YopE N-terminal domain contains an epitope that is an immunodominant CD8(+) T cell antigen during primary infection of C57BL/6 mice with Yersinia pseudotuberculosis. The characteristics of the CD8(+) T cells generated in response to the epitope, which comprises YopE amino acid residues 69 to 77 (YopE(69-77)), and the features of YopE that are important for antigenicity during primary infection, are unknown. Following intravenous infection of naïve C57BL/6 mice with a yopE GAP mutant (the R144A mutant), flow cytometry analysis of splenocytes by tetramer and intracellular cytokine staining over a time course showed that YopE69-77-specific CD8(+) T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) were generated by day 7, with a peak at day 14. In addition, ∼80% of YopE(69-77)-specific CD8(+) T cells were positive for KLRG1, a memory phenotype marker, at day 21. To determine if residues that regulate YopE activity by ubiquitination or membrane localization affect the antigenicity of YopE(69-77), mice were infected with a yopE ubiquitination or membrane localization mutant (the R62K or L55N I59N L63N mutant, respectively). These mutants elicited YopE(69-77)-specific CD8(+) T cells producing IFN-γ and TNF-α with kinetics and magnitudes similar to those of the parental R144A strain, indicating that primary infection primes effector CD8(+) T cells independently of the ubiquitination or membrane localization of YopE. Additionally, at day 7, there was an unexpected positive correlation between the numbers of YopE(69-77)-specific CD8(+) T cells and CD11b(+) cells, but not between the numbers of YopE(69-77)-specific CD8(+) T cells and bacterial cells, in spleens, suggesting that the innate immune response contributes to the immunodominance of YopE(69-77).


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Linfocitos T CD8-positivos/fisiología , Epítopos Inmunodominantes/inmunología , Yersiniosis/inmunología , Yersinia pseudotuberculosis/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Regulación Bacteriana de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Mutación , Virulencia , Yersiniosis/microbiología , Yersinia pseudotuberculosis/patogenicidad
18.
Infect Immun ; 82(6): 2606-14, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24711563

RESUMEN

Immature myeloid cells in bone marrow are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cell types such as granulocytes and macrophages. Under certain pathological conditions, myeloid cell homeostasis is altered and immature forms of these cells appear in tissues. Murine immature myeloid cells that express CD11b and Ly6C or Ly6G (two isoforms of Gr-1) have been associated with immunosuppression in cancer (in the form of myeloid-derived suppressor cells) and, more recently, infection. Here, we found that CD11b(+) Ly6C(hi) Ly6G(-) and CD11b(+) Ly6C(int) Ly6G(+) cells accumulated and persisted in tissues of mice infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Recruitment of CD11b(+) Ly6C(hi) Ly6G(-) but not CD11b(+) Ly6C(int) Ly6G(+) cells from bone marrow into infected tissues depended on chemokine receptor CCR2. The CD11b(+) Ly6C(hi) Ly6G(-) cells exhibited a mononuclear morphology, whereas the CD11b(+) Ly6C(int) Ly6G(+) cells exhibited a polymorphonuclear or band-shaped nuclear morphology. The CD11b(+) Ly6C(hi) Ly6G(-) cells differentiated into macrophage-like cells following ex vivo culture and could present antigen to T cells in vitro. However, significant proliferation of T cells was observed only when the ability of the CD11b(+) Ly6C(hi) Ly6G(-) cells to produce nitric oxide was blocked. CD11b(+) Ly6C(hi) Ly6G(-) cells recruited in response to S. Typhimurium infection could also present antigen to T cells in vivo, but increasing their numbers by adoptive transfer did not cause a corresponding increase in T cell response. Thus, CD11b(+) Ly6C(hi) Ly6G(-) immature myeloid cells recruited in response to S. Typhimurium infection exhibit protective and immunosuppressive properties that may influence the outcome of infection.


Asunto(s)
Antígenos Ly/inmunología , Antígeno CD11b/inmunología , Células Mieloides/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Análisis de Varianza , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Inmunidad Innata/fisiología , Terapia de Inmunosupresión , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CCR2/fisiología , Bazo/citología , Linfocitos T/inmunología
19.
Int J Med Microbiol ; 304(3-4): 444-51, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24636859

RESUMEN

The Yersinia outer protein M (YopM) is a type 3 secretion system (T3SS)-dependent effector protein of Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. Although YopM is indispensable for full virulence, its molecular functions still remain largely elusive. Recently, we could identify the recombinant YopM (rYopM) protein derived from the Y. enterocolitica strain 8081 (JB580) as a cell-penetrating protein, which down-regulates the expression of various pro-inflammatory cytokines including TNFα. In this study, we have generated rabbit monoclonal anti-YopM antibodies (RabMabs). RabMabs were characterized by SDS-PAGE and Western blotting using various truncated versions of rYopM to identify epitope-containing domains. RabMabs recognizing either the N- or C-terminus of YopM were characterized further and validated using a collection of 61 pathogenic and non-pathogenic Yersinia strains as well as exemplary strains of major intestinal bacterial pathogens such as Salmonella enterica ssp. enterica, Shigella flexneri and intestinal pathogenic Escherichia coli. RabMab 41.3 directed at the N-terminus of YopM of Y. enterocolitica strain 8081 recognized all YopM-expressing pathogenic Yersinia strains analyzed in this study but failed to recognize non-pathogenic isolates. Thus, RabMab 41.3 might be applicable for the detection of pathogenic Yersinia strains.


Asunto(s)
Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/inmunología , Yersinia enterocolitica/aislamiento & purificación , Yersinia pestis/aislamiento & purificación , Yersinia pseudotuberculosis/aislamiento & purificación , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Femenino , Conejos , Sensibilidad y Especificidad , Yersinia enterocolitica/inmunología , Yersinia pestis/inmunología , Yersinia pseudotuberculosis/inmunología
20.
Cell Microbiol ; 15(10): 1622-31, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23834311

RESUMEN

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called 'pathogen-associated molecular patterns' (PAMPs). Pathogens use virulence factors to counteract PAMP-directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram-negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP-directed responses and are critical for infection. A plasmid-encoded T3SS in the human-pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences.


Asunto(s)
Sistemas de Secreción Bacterianos , Evasión Inmune , Inmunidad Innata , Factores Inmunológicos/metabolismo , Factores de Virulencia/metabolismo , Yersinia/fisiología , Animales , Humanos , Mamíferos , Plásmidos , Factores de Virulencia/genética , Yersinia/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA