RESUMEN
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
The AB-type protein toxin from Pasteurella multocida (PMT) contains a functionally important disulfide bond within its catalytic domain, which must be cleaved in the host cell cytosol to render the catalytic domain of PMT into its active conformation. Here, we found that the reductive potential of the cytosol of target cells, and more specifically, the activity of the thioredoxin reductase (TrxR) is crucial for this process. This was demonstrated by the strong inhibitory effect of the pharmacological TrxR inhibitor auranofin, which inhibited the intoxication of target cells with PMT, as determined by analyzing the PMT-catalyzed deamidation of GTP-binding proteins (G-proteins) in the cytosol of cells. The amount of endogenous substrate levels modified by PMT in cells pretreated with auranofin was reduced compared to cells treated with PMT alone. Auranofin had no inhibitory effect on the activity of the catalytic domain of constitutively active PMT in vitro, demonstrating that auranofin did not directly inhibit PMT activity, but interferes with the mode of action of PMT in cells. In conclusion, the results show that TrxR is crucial for the mode of action of PMT in mammalian cells, and that the drug auranofin can serve as an efficient inhibitor, which might be a starting point for novel therapeutic options against toxin-associated diseases.