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 protein toxin Pasteurella multocida toxin (PMT) is the causative agent of atrophic rhinitis in pigs, leading to atrophy of the nasal turbinate bones by affecting osteoblasts and osteoclasts. The mechanism of PMT-induced intoxication is a deamidation of α-subunits of heterotrimeric G proteins, including Gαq, Gα13, and Gαi, thereby causing persistent activation of the G proteins. Here we utilized PMT as a transporter of the non-cell-permeating A domain of diphtheria toxin (DTa). Fusion proteins of PMT and DTa ADP-ribosylated elongation factor 2, the natural target of diphtheria toxin, leading to cell toxicity. PMT-DTa effects were competed by PMT, indicating binding to the same cell surface receptor. Fluorescently labeled PMT-DTa and PMT colocalized with specific markers of early and late endosomes. Bafilomycin A, which inhibits vacuolar H(+)-ATPase, blocked PMT-DTa-induced intoxication of HEK-293 cells. By constructing various PMT-DTa chimeras, we identified a minimal region of PMT necessary for uptake of DTa. The data suggest that PMT is able to transport cargo proteins into eukaryotic cells by utilizing the PMT-specific uptake route.