RESUMEN
Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe-like nanomachine for delivery of protein toxins from different species. In addition, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the probable size limitation, the charge of the cargo also influences the efficiency of packing and transport into mammalian cells. Our data show that the PTC constitutes a powerful system to inject recombinant proteins, peptides, and potentially, other molecules into mammalian cells. In addition, in contrast to other protein transporters based on pore formation, the closed, compact structure of the PTC may protect cargo from degradation.
Asunto(s)
Proteínas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Cisteína Endopeptidasas/administración & dosificación , Photorhabdus/metabolismo , Ingeniería de Proteínas/métodos , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clonación Molecular , Copépodos/genética , Copépodos/metabolismo , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Inyecciones , Luciferasas/genética , Luciferasas/metabolismo , Nanopartículas , Photorhabdus/genéticaRESUMEN
The human disease fibrodysplasia ossificans progressiva (FOP) is a rare and highly disabling disorder of extensive heterotopic bone growth that is caused by a point mutation (R206H) in the activation domain of Alk2, a BMP (bone morphogenic protein) type 1 receptor. The mutation leads to extensive BMP-signaling induced by Activin A, which is normally an antagonist for wildtype receptors, resulting in excessive and uncontrolled bone formation. Here, we studied the effects of Pasteurella multocida toxin (PMT), which activates osteoclasts and inhibits osteoblast activity, in C2C12 myoblasts expressing the mutant Alk2(R206H) receptor as model of FOP. In our study, we mainly used alkaline phosphatase (ALP) activity as marker to determine osteoblast differentiation. BMP-4 stimulated an increase in ALP activity in C2C12-Alk2wt and C2C12-Alk2(R206H) cells. By contrast, Activin A only induced ALP activity in C2C12-Alk2(R206H) cells. In both cases, PMT acted as a potent inhibitor of ALP activity. PMT-induced inhibition of ALP activity was paralleled by a constitutive activation of the heterotrimeric Gq protein. Expression of a permanently active Gαq blocked Activin A/Alk2(R206H)-dependent increase in ALP activity. Inactivation of Gq by specific inhibitor FR900359 blocked the PMT effect. Similarly, canonical second messengers and effectors of Gαq (e.g. ionophore A23187-induced increase in intracellular Ca2+ and activation of PKC by PMA (phorbol 12-myristate 13-acetate)) inhibited Alk2(R206H)-mediated induction of ALP activity. Notably, Activin A-induced increase in ALP activity in C2C12-Alk2(R206H) cells was also inhibited by stimulation of the α1A-adrenoceptor, which couples to Gαq, by phenylephrine. PMT did not alter tail phosphorylation of the major downstream effectors of the Alk2 receptor, Smad1/5/9; neither did the toxin affect nuclear translocation of the Smad-complex. However, PMT diminished BMP responsive element-induced gene expression. The data indicate that PMT potently inhibits the induction of osteoblast markers in a FOP model via activation of G proteins. Moreover, our findings indicate that activation of G protein-coupled receptors and of G protein signaling might be a rationale for pharmacological therapy of FOP.
Asunto(s)
Activinas/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Modelos Biológicos , Mioblastos/metabolismo , Miositis Osificante/patología , Osteoblastos/metabolismo , Transducción de Señal , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Señalización del Calcio , Línea Celular , Ratones , Proteínas Smad/metabolismoRESUMEN
Salmonella enterica serotype Typhimurium (S. Typhimurium) is one of the most frequent causes of food-borne illness in humans and usually associated with acute self-limiting gastroenteritis. However, in immunocompromised patients, the pathogen can disseminate and lead to severe systemic diseases. S. Typhimurium are facultative intracellular bacteria. For uptake and intracellular life, Salmonella translocate numerous effector proteins into host cells using two type-III secretion systems (T3SS), which are encoded within Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). While SPI-1 effectors mainly promote initial invasion, SPI-2 effectors control intracellular survival and proliferation. Here, we elucidate the mode of action of Salmonella SPI-2 effector SseI, which is involved in control of systemic dissemination of S. Typhimurium. SseI deamidates a specific glutamine residue of heterotrimeric G proteins of the Gαi family, resulting in persistent activation of the G protein. Gi activation inhibits cAMP production and stimulates PI3-kinase γ by Gαi-released Gßγ subunits, resulting in activation of survival pathways by phosphorylation of Akt and mTOR. Moreover, SseI-induced deamidation leads to non-polarized activation of Gαi and, thereby, to loss of directed migration of dendritic cells.
Asunto(s)
Proteínas Bacterianas/fisiología , Quimiotaxis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Salmonella typhimurium , Sistemas de Secreción Tipo III/fisiología , Animales , Proteínas Bacterianas/genética , Supervivencia Celular/genética , Quimiotaxis/genética , Desaminación/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Multimerización de Proteína/genética , Procesamiento Proteico-Postraduccional/genética , Células RAW 264.7 , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/patología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Pasteurella multocida toxin (PMT) causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. It has been reported that the toxin deamidates and activates heterotrimeric G proteins, resulting in increased differentiation of osteoclasts and blockade of osteoblast differentiation. So far, the action of PMT on osteocytes, which is the most abundant cell type in bone tissue, is not known. In MLO-Y4 osteocytes, PMT deamidated heterotrimeric G proteins, resulting in loss of osteocyte dendritic processes, stress fiber formation, cell spreading and activation of RhoC but not of RhoA. Moreover, the toxin caused processing of membrane-bound receptor activator of NF-κB ligand (RANKL) to release soluble RANKL and enhanced the secretion of osteoclastogenic TNF-α. In a co-culture model of osteocytes and bone marrow cells, PMT-induced osteoclastogenesis was largely increased as compared to the mono-culture model. The enhancement of osteoclastogenesis observed in the co-culture was blocked by sequestering RANKL with osteoprotegerin and by an antibody against TNF-α indicating involvement of release of the osteoclastogenic factors from osteocytes. Data support the crucial role of osteocytes in bone metabolism and osteoclastogenesis and identify osteocytes as important target cells of PMT in progressive atrophic rhinitis.