RESUMEN
Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. Although laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability of these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here, we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models-C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi was able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle.IMPORTANCEA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host-usually rodents or birds-and ticks. In order to be successful in that endeavor, the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work, we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appears to colonize its natural host less successfully than conventional laboratory mouse models, which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.
Asunto(s)
Borrelia burgdorferi , Reservorios de Enfermedades , Enfermedad de Lyme , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Peromyscus , Animales , Peromyscus/microbiología , Ratones , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/transmisión , Enfermedad de Lyme/veterinaria , Borrelia burgdorferi/fisiología , Borrelia burgdorferi/genética , Reservorios de Enfermedades/microbiología , Modelos Animales de Enfermedad , Ixodes/microbiologíaRESUMEN
Borrelia burgdorferi (Bb), the causative agent of Lyme disease, establishes a long-term infection and leads to disease manifestations that are the result of host immune responses to the pathogen. Inflammatory manifestations resolve spontaneously despite continued bacterial presence, suggesting inflammatory cells become less responsive over time. This is mimicked by in vitro repeated stimulations, resulting in tolerance, a phenotypic subset of innate immune memory. We performed comparative transcriptional analysis of macrophages in acute and memory states and identified sets of Tolerized, Hyper-Induced, Secondary-Induced and Hyper-Suppressed genes resulting from memory induction, revealing previously unexplored networks of genes affected by cellular re-programming. Tolerized gene families included inflammatory mediators and interferon related genes as would be predicted by the attenuation of inflammation over time. To better understand how cells mediate inflammatory hypo-responsiveness, we focused on genes that could mediate maintenance of suppression, such as Hyper-Induced genes which are up-regulated in memory states. These genes were notably enriched in stress pathways regulated by anti-inflammatory modulators. We examined one of the most highly expressed negative regulators of immune pathways during primary stimulation, Aconitate decarboxylase 1 (Acod1), and tested its effects during in vivo infection with Bb. As predicted by our in vitro model, we show its inflammation-suppressive downstream effects are sustained during in vivo long-term infection with Bb, with a specific role in Lyme carditis.
Asunto(s)
Borrelia burgdorferi , Enfermedad de Lyme , Humanos , Inflamación , Enfermedad de Lyme/microbiología , Macrófagos , AntiinflamatoriosRESUMEN
Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.
Asunto(s)
Borrelia burgdorferi , Enfermedad de Lyme , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Borrelia burgdorferi/metabolismo , Ligandos , Enfermedad de Lyme/genética , Enfermedad de Lyme/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Fagosomas/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Proteínas de Unión al GTP rab , Animales , RatonesRESUMEN
Phagosomal compartments are critical in microbial defense as vesicles that degrade invading organisms. In a broader context, vesicular trafficking plays an important role in shuttling many different types of cargo that are critical for proper function of the cell. Endosomal and phagosomal vesicles are thus important locations for the assembly of intracellular signaling platforms that mediate host responses to invasive pathogens such as Borrelia burgdorferi. Isolation of phagosomes from cells is an important technique that allows for a detailed study of phagosomal components and signaling complex assembly. However, purification of phagosomes had previously been challenging and it has been difficult to obtain sufficient purity of the phagosomal fractions. Here, we modify a new magnetic isolation technique that greatly simplifies purification of phagosomes and isolates vesicles with sufficient purity for analysis.
Asunto(s)
Borrelia burgdorferi/aislamiento & purificación , Fraccionamiento Celular/métodos , Enfermedad de Lyme/inmunología , Macrófagos/microbiología , Fagosomas/microbiología , Receptor Toll-Like 2/inmunología , Animales , Western Blotting/métodos , Borrelia burgdorferi/inmunología , Línea Celular , Humanos , Ligandos , Lipopéptidos/química , Lipopéptidos/inmunología , Enfermedad de Lyme/microbiología , Macrófagos/inmunología , Magnetismo/métodos , Imanes/química , Fagocitosis , Fagosomas/inmunología , Transducción de SeñalRESUMEN
Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3ß1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin ß1 play a role in the activation of the Src kinase pathway. However, only integrin ß1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.
Asunto(s)
Borrelia burgdorferi/inmunología , Cadenas beta de Integrinas/metabolismo , Fagocitosis , Receptores Inmunológicos/metabolismo , Transducción de Señal , Quinasa Syk/metabolismo , Familia-src Quinasas/metabolismo , Animales , Borrelia burgdorferi/fisiología , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Ratones , Receptores Inmunológicos/inmunología , Receptores Depuradores/metabolismoRESUMEN
Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo.
Asunto(s)
Complejo 3 de Proteína Adaptadora/inmunología , Citocinas/inmunología , Mediadores de Inflamación/inmunología , Receptor Toll-Like 2/inmunología , Complejo 3 de Proteína Adaptadora/genética , Complejo 3 de Proteína Adaptadora/metabolismo , Animales , Borrelia burgdorferi/inmunología , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/fisiología , Células Cultivadas , Citocinas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Mediadores de Inflamación/metabolismo , Células L , Lipopéptidos/inmunología , Lipopéptidos/metabolismo , Lipopéptidos/farmacología , Lisosomas/inmunología , Lisosomas/metabolismo , Lisosomas/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Fagosomas/inmunología , Fagosomas/metabolismo , Fagosomas/microbiología , Transporte de Proteínas/inmunología , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Interleucina/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/metabolismoAsunto(s)
Borrelia burgdorferi/fisiología , Enfermedad de Lyme/microbiología , Animales , Vectores Arácnidos/microbiología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Borrelia burgdorferi/genética , Humanos , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/transmisión , Garrapatas/microbiologíaRESUMEN
Lyme disease is a long-term infection whose most severe pathology is characterized by inflammatory arthritis of the lower bearing joints, carditis, and neuropathy. The inflammatory cascades are initiated through the early recognition of invading Borrelia burgdorferi spirochetes by cells of the innate immune response, such as neutrophils and macrophage. B. burgdorferi does not have an intracellular niche and thus much research has focused on immune pathways activated by pathogen recognition molecules at the cell surface, such as the Toll-like receptors (TLRs). However, in recent years, studies have shown that internalization of the bacterium by host cells is an important component of the defense machinery in response to B. burgdorferi. Upon internalization, B. burgdorferi is trafficked through an endo/lysosomal pathway resulting in the activation of a number of intracellular pathogen recognition receptors including TLRs and Nod-like receptors (NLRs). Here we will review the innate immune molecules that participate in both cell surface and intracellular immune activation by B. burgdorferi.
Asunto(s)
Borrelia burgdorferi/fisiología , Inmunidad Innata , Enfermedad de Lyme/inmunología , Animales , Borrelia burgdorferi/genética , Borrelia burgdorferi/inmunología , Interacciones Huésped-Patógeno , Humanos , Enfermedad de Lyme/microbiologíaRESUMEN
TRIF is an adaptor molecule important in transducing signals from intracellularly signaling Toll-like receptor 3 (TLR3) and TLR4. Recently, TLR2 was found to signal from intracellular compartments. Using a synthetic ligand for TLR2/1 heterodimers, as well as Borrelia burgdorferi, which is a strong activator of TLR2/1, we found that TLR2 signaling can utilize TRIF. Unlike TRIF signaling by other TLRs, TLR2-mediated TRIF signaling is dependent on the presence of another adaptor molecule, MyD88. However, unlike MyD88 deficiency, TRIF deficiency does not result in diminished control of infection with B. burgdorferi in a murine model of disease. This appears to be due to the effects of MyD88 on phagocytosis via scavenger receptors, such as MARCO, which are not affected by the loss of TRIF. In mice, TRIF deficiency did have an effect on the production of inflammatory cytokines, suggesting that regulation of inflammatory cytokines and control of bacterial growth may be uncoupled, in part through transduction of TLR2 signaling through TRIF.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Borrelia burgdorferi/inmunología , Inflamación/inmunología , Enfermedad de Lyme/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Inflamación/metabolismo , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Ligandos , Enfermedad de Lyme/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Fagocitosis/inmunología , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunologíaRESUMEN
The human intestine harbors a large number of bacteria that are constantly interacting with the intestinal immune system, eliciting non-pathological basal level immune responses. Increasing evidence points to dysbiosis of microbiota in the intestine as an underlying factor in inflammatory bowel disease susceptibility. Loss-of-function mutations in NOD2 are among the stronger genetic factors linked to ileal Crohn's disease. Indeed, Nod2 is a key regulator of microbiota in the intestine, as microflora in the terminal ileum is dysregulated in Nod2-deficient mice. Nod2 is highly expressed in Paneth cells, which are responsible for the regulation of ileal microflora by anti-microbial compounds, and Nod2-deficient ileal intestinal epithelia are unable to kill bacteria efficiently. It is therefore likely that NOD2 mutations in Crohn's disease may increase disease susceptibility by altering interactions between ileal microbiota and mucosal immunity.
Asunto(s)
Inmunidad Mucosa , Intestinos/inmunología , Intestinos/microbiología , Metagenoma , Proteína Adaptadora de Señalización NOD2/metabolismo , Animales , Bacterias/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Humanos , Inmunidad Mucosa/genética , Mucosa Intestinal/metabolismo , Ratones , Modelos Biológicos , Mutación , Proteína Adaptadora de Señalización NOD2/genética , Transducción de SeñalRESUMEN
The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.
Asunto(s)
Borrelia burgdorferi/inmunología , Borrelia burgdorferi/fisiología , Tolerancia Inmunológica/genética , Enfermedad de Lyme/prevención & control , Miocarditis/prevención & control , Proteína Adaptadora de Señalización NOD2/fisiología , Enfermedad Aguda , Animales , Caspasa 1/metabolismo , Tolerancia Inmunológica/fisiología , Inflamación/complicaciones , Inflamación/genética , Mediadores de Inflamación/metabolismo , Enfermedad de Lyme/etiología , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocarditis/etiología , Miocarditis/genética , Miocarditis/inmunología , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismoRESUMEN
BACKGROUND: Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered. METHODOLOGY/PRINCIPAL FINDINGS: Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam(3)CSK(4), are dependent upon an integrin, α(3)ß(1). The mechanism for integrin α(3)ß(1) involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam(3)CSK(4) is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane. CONCLUSION/SIGNIFICANCE: Here we identify integrin α(3)ß(1) as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α(3)ß(1)-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α(3)ß(1) serves as a mechanism for modulating inflammatory responses.
Asunto(s)
Borrelia burgdorferi/metabolismo , Endosomas/metabolismo , Integrina alfa3beta1/metabolismo , Lipopéptidos/metabolismo , Enfermedad de Lyme/metabolismo , Receptor Toll-Like 2/metabolismo , Borrelia burgdorferi/inmunología , Endocitosis , Endosomas/genética , Humanos , Integrina alfa3beta1/genética , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Macrófagos/inmunología , Macrófagos/microbiología , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunologíaRESUMEN
Mutations in the Nod2 gene are among the strongest genetic risk factors in the pathogenesis of ileal Crohn's disease, but the exact contributions of Nod2 to intestinal mucosal homeostasis are not understood. Here we show that Nod2 plays an essential role in controlling commensal bacterial flora in the intestine. Analysis of intestinal bacteria from the terminal ilea of Nod2-deficient mice showed that they harbor an increased load of commensal resident bacteria. Furthermore, Nod2-deficient mice had a diminished ability to prevent intestinal colonization of pathogenic bacteria. In vitro, intestinal crypts isolated from terminal ilea of Nod2-deficient mice were unable to kill bacteria effectively, suggesting an important role of Nod2 signaling in crypt function. Interestingly, the expression of Nod2 is dependent on the presence of commensal bacteria, because mice re-derived into germ-free conditions expressed significantly less Nod2 in their terminal ilea, and complementation of commensal bacteria into germ-free mice induced Nod2 expression. Therefore, Nod2 and intestinal commensal bacterial flora maintain a balance by regulating each other through a feedback mechanism. Dysfunction of Nod2 results in a break-down of this homeostasis.
Asunto(s)
Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Proteína Adaptadora de Señalización NOD2/inmunología , Animales , Bacterias/genética , Bacterias/inmunología , Bacterias/patogenicidad , Secuencia de Bases , Enfermedad de Crohn/etiología , Cartilla de ADN/genética , Expresión Génica , Vida Libre de Gérmenes , Humanos , Íleon/inmunología , Íleon/microbiología , Inmunidad Innata , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Adaptadora de Señalización NOD2/deficiencia , Proteína Adaptadora de Señalización NOD2/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunologíaRESUMEN
The innate immune system is the first line of defense against microorganisms and is conserved in plants and animals. The nucleotide-binding domain, leucine rich containing (NLR) protein family is a recent addition to the members of innate immunity effector molecules. These proteins are characterized by a central oligomerization domain, termed nucleotide-binding domain (NBD) and a protein interaction domain, leucine-rich repeats (LRRs) at the C terminus. It has been shown that NLR proteins are localized to the cytoplasm and recognize microbial products. To date, it is known that Nod1 and Nod2 detect bacterial cell wall components, whereas Ipaf and Naip detect bacterial flagellin, and NACHT/LRR/Pyrin 1 has been shown to detect anthrax lethal toxin. NLR proteins comprise a diverse protein family (over 20 in humans), indicating that NLRs have evolved to acquire specificity to various pathogenic microorganisms, thereby controlling host-pathogen interactions. Activation of NLR proteins results in inflammatory responses mediated by NF-kappaB, MAPK, or Caspase-1 activation, accompanied by subsequent secretion of proinflammatory cytokines. Mutations in several members of the NLR protein family have been linked to inflammatory diseases, suggesting these molecules play important roles in maintaining host-pathogen interactions and inflammatory responses. Therefore, understanding NLR signaling is important for the therapeutic intervention of various infectious and inflammatory diseases.
Asunto(s)
Enfermedades del Sistema Inmune/inmunología , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/inmunología , Animales , Humanos , Enfermedades del Sistema Inmune/genética , Inflamación , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Inmunológicos , Transducción de Señal/inmunologíaRESUMEN
Pseudomonas syringae is a gram-negative bacterial plant pathogen that is dependent on a type III protein secretion system (TTSS) and the effector proteins it translocates into plant cells for pathogenicity. The P. syringae TTSS is encoded by hrp-hrc genes that reside in a central region of a pathogenicity island (Pai). Flanking one side of this Pai is the exchangeable effector locus (EEL). We characterized the transcriptional expression of the open reading frames (ORFs) within the EEL of P. syringae pv. tomato DC3000. One of these ORFs, PSPTO1406 (hopB1) is expressed in the same transcriptional unit as hrpK. Both HopB1 and HrpK were secreted in culture and translocated into plant cells via the TTSS. However, the translocation of HrpK required its C-terminal half. HrpK shares low similarity with a putative translocator, HrpF, from Xanthomonas campestris pv. vesicatoria. DC3000 mutants lacking HrpK were significantly reduced in disease symptoms and multiplication in planta, whereas DC3000 hopB1 mutants produced phenotypes similar to the wild type. Additionally, hrpK mutants were reduced in their ability to elicit the hypersensitive response (HR), a programmed cell death associated with plant defense. The reduced HR phenotype exhibited by hrpK mutants was complemented by hrpK expressed in bacteria but not by HrpK transgenically expressed in tobacco, suggesting that HrpK does not function inside plant cells. Further experiments identified a C-terminal transmembrane domain within HrpK that is required for HrpK translocation. Taken together, HopB1 is a type III effector and HrpK plays an important role in the TTSS and is a putative type III translocator.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Operón , Pseudomonas syringae/genética , Secuencia de Aminoácidos , Apoptosis , Genes Bacterianos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Enfermedades de las Plantas/microbiología , Estructura Terciaria de Proteína , Transporte de Proteínas , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidad , ARN Bacteriano/análisis , ARN Mensajero/análisis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Xanthomonas campestris/genéticaRESUMEN
The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.
Asunto(s)
Apoptosis/fisiología , Proteínas Bacterianas/genética , Nicotiana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Pseudomonas syringae/patogenicidad , Levaduras/fisiología , Apoptosis/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Inmunidad Innata/genética , Datos de Secuencia Molecular , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Plásmidos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Rhizobium/genética , Rhizobium/crecimiento & desarrollo , Eliminación de Secuencia , Transducción de Señal/genética , Transducción de Señal/fisiología , Nicotiana/genética , Nicotiana/microbiología , Virulencia , Levaduras/genética , Proteína X Asociada a bcl-2RESUMEN
Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos , Pseudomonas/metabolismo , Virulencia/genética , Arabidopsis/microbiología , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Genoma Bacteriano , Solanum lycopersicum/microbiología , Regiones Promotoras Genéticas , Pseudomonas/genética , Pseudomonas/patogenicidadRESUMEN
Pseudomonas syringae uses a type III protein secretion system encoded by the Hrp pathogenicity island (Pai) to translocate effector proteins into plant cells. One of these effector proteins is HopPsyA. A small open reading frame (ORF), named shcA, precedes the hopPsyA gene in the Hrp Pai of P. s. syringae 61. The predicted amino acid sequence of shcA shares general characteristics with chaperones used in type III protein secretion systems of animal pathogens. A functionally non-polar deletion of shcA in P. s. syringae 61 resulted in the loss of detectable HopPsyA in supernatant fractions, consistent with ShcA acting as a chaperone for HopPsyA. Cosmid pHIR11 carries a functional set of type III genes from P. s. syringae 61 and confers upon saprophytes the ability to secrete HopPsyA in culture and to elicit a HopPsyA-dependent hypersensitive response (HR) on tobacco. P. fluorescens carrying a pHIR11 derivative lacking shcA failed to secrete HopPsyA in culture, but maintained the ability to secrete another type III-secreted protein, HrpZ. This pHIR11 derivative was also greatly reduced in its ability to elicit an HR, indicating that the ability to translocate HopPsyA into plant cells was compromised. Using affinity chromatography, we showed that ShcA binds directly to HopPsyA and that the ShcA binding site must reside within the first 166 amino acids of HopPsyA. Thus, ShcA represents the first demonstrated chaperone used in a type III secretion system of a bacterial plant pathogen. We searched known P. syringae type III-related genes for neighbouring ORFs that shared the general characteristics of type III chaperones and identified five additional candidate type III chaperones. Therefore, it is likely that chaperones are as prevalent in bacterial plant pathogen type III systems as they are in their animal pathogenic counterparts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Nicotiana/microbiología , Pseudomonas/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico/fisiología , Genoma Bacteriano , Immunoblotting , Chaperonas Moleculares/genética , Enfermedades de las Plantas/microbiología , Pseudomonas/genéticaRESUMEN
The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and/or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the N-terminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADP-ribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.