RESUMEN
The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.
Asunto(s)
Borrelia burgdorferi/efectos de los fármacos , Borrelia burgdorferi/fisiología , Dermis/efectos de los fármacos , Dermis/microbiología , Gelatina/farmacología , Enfermedad de Lyme/microbiología , Animales , Adhesión Bacteriana/efectos de los fármacos , Cinética , Metilcelulosa/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Movimiento/efectos de los fármacos , Reología/efectos de los fármacos , Soluciones , Imagen de Lapso de TiempoRESUMEN
While the roles of rpoS(Bb) and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable.
Asunto(s)
Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Enfermedad de Lyme/genética , Enfermedad de Lyme/transmisión , Factor sigma/genética , Animales , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/metabolismo , Vectores de Enfermedades , Ixodes , Microscopía Confocal , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor sigma/metabolismoRESUMEN
Phagocytosed Borrelia burgdorferi (Bb) induces inflammatory signals that differ both quantitatively and qualitatively from those generated by spirochetal lipoproteins interacting with Toll-like receptor (TLR) 1/2 on the surface of human monocytes. Of particular significance, and in contrast to lipoproteins, internalized spirochetes induce transcription of IFN-ß. Using inhibitory immunoregulatory DNA sequences (IRSs) specific to TLR7, TLR8, and TLR9, we show that the TLR8 inhibitor IRS957 significantly diminishes production of TNF-α, IL-6, and IL-10 and completely abrogates transcription of IFN-ß in Bb-stimulated monocytes. We demonstrate that live Bb induces transcription of TLR2 and TLR8, whereas IRS957 interferes with their transcriptional regulation. Using confocal and epifluorescence microscopy, we show that baseline TLR expression in unstimulated monocytes is greater for TLR2 than for TLR8, whereas expression of both TLRs increases significantly upon stimulation with live spirochetes. By confocal microscopy, we show that TLR2 colocalization with Bb coincides with binding, uptake, and formation of the phagosomal vacuole, whereas recruitment of both TLR2 and TLR8 overlaps with degradation of the spirochete. We provide evidence that IFN regulatory factor (IRF) 7 is translocated into the nucleus of Bb-infected monocytes, suggesting its activation through phosphorylation. Taken together, these findings indicate that the phagosome is an efficient platform for the recognition of diverse ligands; in the case of Bb, phagosomal signaling involves a cooperative interaction between TLR2 and TLR8 in pro- and antiinflammatory cytokine responses, whereas TLR8 is solely responsible for IRF7-mediated induction of IFN-ß.
Asunto(s)
Borrelia burgdorferi/fisiología , Interferón beta/genética , Monocitos/microbiología , Fagosomas/metabolismo , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 8/metabolismo , Núcleo Celular/metabolismo , Citocinas/biosíntesis , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Viabilidad Microbiana , Monocitos/metabolismo , Fagocitosis , Fagosomas/microbiología , Unión Proteica , Transporte de Proteínas , Transcripción Genética , Vacuolas/metabolismo , Vacuolas/microbiologíaRESUMEN
Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic ß-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the ß-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its ß-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and ß-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/microbiología , Sífilis/microbiología , Treponema pallidum/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Sífilis/metabolismo , Treponema pallidum/química , Treponema pallidum/genéticaRESUMEN
Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) "nuclear pathway," where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) "membrane pathway," in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1.
Asunto(s)
Citoplasma/metabolismo , Biosíntesis de Proteínas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Animales , Brefeldino A/farmacología , Bovinos , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Recuperación de Fluorescencia tras Fotoblanqueo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Modelos Biológicos , Transporte de Proteínas/efectos de los fármacos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/análisis , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/análisisRESUMEN
Lyme disease is caused by transmission of the spirochete Borrelia burgdorferi from ticks to humans. Although much is known about B. burgdorferi replication, the routes and mechanisms by which it disseminates within the tick remain unclear. To better understand this process, we imaged live, infectious B. burgdorferi expressing a stably integrated, constitutively expressed GFP reporter. Using isolated tick midguts and salivary glands, we observed B. burgdorferi progress through the feeding tick via what we believe to be a novel, biphasic mode of dissemination. In the first phase, replicating spirochetes, positioned at varying depths throughout the midgut at the onset of feeding, formed networks of nonmotile organisms that advanced toward the basolateral surface of the epithelium while adhering to differentiating, hypertrophying, and detaching epithelial cells. In the second phase of dissemination, the nonmotile spirochetes transitioned into motile organisms that penetrated the basement membrane and entered the hemocoel, then migrated to and entered the salivary glands. We designated the first phase of dissemination "adherence-mediated migration" and provided evidence that it involves the inhibition of spirochete motility by one or more diffusible factors elaborated by the feeding tick midgut. Our studies, which we believe are the first to relate the transmission dynamics of spirochetes to the complex morphological and developmental changes that the midgut and salivary glands undergo during engorgement, challenge the conventional viewpoint that dissemination of Lyme disease-causing spirochetes within ticks is exclusively motility driven.
Asunto(s)
Borrelia burgdorferi/patogenicidad , Ixodes/microbiología , Animales , Borrelia burgdorferi/genética , Borrelia burgdorferi/aislamiento & purificación , Borrelia burgdorferi/fisiología , Sistema Digestivo/microbiología , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Ixodes/anatomía & histología , Ixodes/crecimiento & desarrollo , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/transmisión , Ratones , Ratones Endogámicos C3H , Proteínas Recombinantes/genética , Glándulas Salivales/microbiología , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Nuclear FGFR1 acts as a developmental gene regulator in cooperation with FGF-2, RSK1, and CREB-binding protein (CBP). FRAP analysis revealed three nuclear FGFR1 populations: i) a fast mobile, ii) a slower mobile population reflecting chromatin-bound FGFR1, and iii) an immobile FGFR1 population associated with the nuclear matrix. Factors (cAMP, CBP) that induce FGFR1-mediated gene activation shifted FGFR1 from the nuclear matrix (immobile) to chromatin (slow) and reduced the movement rate of the chromatin-bound population. Transcription inhibitors accelerated FGFR1 movement; the content of the chromatin-bound slow FGFR1 decreased, whereas the fast population increased. The transcriptional activation appears to involve conversion of the immobile matrix-bound and the fast nuclear FGFR1 into a slow chromatin-binding population through FGFR1's interaction with CBP, RSK1, and the high-molecular-weight form of FGF-2. Our findings support a general mechanism in which gene activation is governed by protein movement and collisions with other proteins and nuclear structures.
Asunto(s)
Núcleo Celular/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transcripción Genética , Animales , Proteína de Unión a CREB/metabolismo , Compartimento Celular , Línea Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Modelos Biológicos , Peso Molecular , Matriz Nuclear/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Activación Transcripcional/genética , TransfecciónRESUMEN
In integrative nuclear fibroblast growth factor receptor-1 (FGFR1) signaling a newly synthesized FGFR1 translocates to the nucleus to stimulate cell differentiation and associated gene activities. The present study shows that FGFR1 accumulates and interacts with the transcriptional co-activator CREB-binding protein (CBP) in nuclear speckle domains in the developing brain and in neural progenitor-like cells in vitro, which accompanies differentiation and postmitotic growth. Cell differentiation and gene activation by nuclear FGFR1 do not require tyrosine kinase activity. Instead, FGFR1 stimulates transcription in cooperation with CBP by increasing recruitment of RNA polymerase II and histone acetylation at the active gene promoter. FGFR1 is a multifactorial protein whose N terminus interacts with CBP and C terminus with ribosomal S6 kinase 1 (RSK1). Nuclear FGFR1 augments CBP-mediated transcription by 1) releasing the CBP C-terminal domain from RSK1 inhibition and 2) activating the CBP N-terminal domain. The interaction of FGFR1 with CBP and RSK1 allows activation of gene transcription and may play a role in cell differentiation.