RESUMEN
The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.
Asunto(s)
Autoinmunidad , Activación de Linfocitos , Glicoproteínas de Membrana/inmunología , ARN/inmunología , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 7/inmunología , Animales , Ligandos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 7/genética , Uridina/genética , Uridina/inmunologíaRESUMEN
Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.
Asunto(s)
Membrana Celular/metabolismo , Proteína Ligando Fas/metabolismo , Glaucoma/metabolismo , Glaucoma/patología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Animales , Muerte Celular , Membrana Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Proteína Ligando Fas/farmacología , Glaucoma/complicaciones , Inyecciones , Ratones , Ratones Mutantes , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Unión Proteica/efectos de los fármacos , Degeneración Retiniana/complicaciones , Degeneración Retiniana/patología , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Solubilidad/efectos de los fármacos , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/metabolismoRESUMEN
Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine-uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB-GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB-GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB-GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH-GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB-GFP and IunH-GFP resides in the region between the coat and the exosporium, called the interspace.
Asunto(s)
Bacillus anthracis/fisiología , Bacillus anthracis/ultraestructura , Proteínas Bacterianas/metabolismo , Animales , Carbunco/microbiología , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Proteínas Bacterianas/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cobayas , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mutación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Esporas Bacterianas/ultraestructura , Fracciones Subcelulares/metabolismoRESUMEN
Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis, the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called Cotbeta, which is encoded only in the genomes of the Bacillus cereus group. We found that Cotbeta is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotbeta null mutant in the Sterne strain reveals that Cotbeta has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotbeta null mutation has no effect on virulence in a murine model of B. anthracis infection.
Asunto(s)
Carbunco/microbiología , Bacillus anthracis/patogenicidad , Proteínas Bacterianas , Esporas Bacterianas , Secuencia de Aminoácidos , Animales , Carbunco/mortalidad , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Bacillus anthracis/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Mutación , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Esporas Bacterianas/ultraestructuraRESUMEN
Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.