RESUMEN
The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Anticuerpos de Dominio Único/inmunología , ADP Ribosa Transferasas/inmunología , ADP Ribosa Transferasas/toxicidad , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/toxicidad , Supervivencia Celular/efectos de los fármacos , Clostridioides difficile/patogenicidad , Perros , Mapeo Epitopo , Epítopos/inmunología , Células HT29 , Humanos , Células de Riñón Canino Madin Darby , Microscopía de Interferencia , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/toxicidad , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genéticaRESUMEN
The NALP3 inflammasome is activated by low intracellular potassium concentrations [K(+)](i), leading to the secretion of the proinflammatory cytokine IL-1ß. However, the mechanism of [K(+)](i) lowering after phagocytosis of monosodium urate crystals is still elusive. Here, we propose that endosomes containing monosodium urate crystals fuse with acidic lysosomes. The low pH in the phagolysosome causes a massive release of sodium and raises the intracellular osmolarity. This process is balanced by passive water influx through aquaporins leading to cell swelling. This process dilutes [K(+)](i) to values below the threshold of 90 mm known to activate NALP3 inflammasomes without net loss of cytoplasmic potassium ions. In vitro, the inhibitors of lysosomal acidification (ammonium chloride, chloroquine) and of aquaporins (mercury chloride, phloretin) all significantly decreased the production of IL-1ß. In vivo, only the pharmacological inhibitor of lysosome acidification chloroquine could be used which again significantly reduced the IL-1ß production. As a translational aspect one may consider the use of chloroquine for the anti-inflammatory treatment of refractory gout.
Asunto(s)
Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Sodio/metabolismo , Agua/metabolismo , Animales , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Fagocitos/citología , Fagocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Potasio/metabolismo , Ácido Úrico/química , Ácido Úrico/metabolismoRESUMEN
Accumulation in tissues of post-apoptotic cells is a feature frequently observed in patients with systemic lupus erythematosus and in murine models of systemic autoimmune diseases. One of the endogenous danger molecules released by secondarily necrotic cells is monosodium urate (MSU), which is already established to be the causative agent of gout. Here, we show that MSU is taken up by eosinophils, neutrophils and monocytes in a process involving (a) heat-labile serum factor(s) and divalent cations. The uptake induces the release of the pro-inflammatory cytokines IL-1beta/IL-18/TNFalpha and IL-6/IL-8 by monocytes and PMN, respectively.
Asunto(s)
Citocinas/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Fagocitos/inmunología , Ácido Úrico/sangre , Cationes Bivalentes/sangre , Cationes Bivalentes/inmunología , Gota/sangre , Gota/inmunología , Humanos , Fagocitos/metabolismo , Ácido Úrico/inmunologíaRESUMEN
Uric acid (UA) is identified as a danger signal released from dying cells. It precipitates in sodium-rich extracellular fluid and in potassium-rich intracellular fluid as monosodium urate (MSU) and monopotassium urate (MPU), respectively. Here, we examined the structural and functional features of these crystals. In contrast to MPU MSU crystals induced reactive oxygen species production and release of pro-inflammatory cytokines in whole blood ex vivo assays. These results show that the cation of urate crystals determines the response of innate immune cells, indicating that the micromilieu at the site of crystal formation is important for their inflammatory potential.