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1.
PLoS Pathog ; 14(8): e1007248, 2018 08.
Article in English | MEDLINE | ID: mdl-30102745

ABSTRACT

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.


Subject(s)
Bacterial Proteins/physiology , Chemotaxis , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Salmonella typhimurium , Type III Secretion Systems/physiology , Animals , Bacterial Proteins/genetics , Cell Survival/genetics , Chemotaxis/genetics , Deamination/genetics , Female , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Multimerization/genetics , Protein Processing, Post-Translational/genetics , RAW 264.7 Cells , Salmonella Infections/metabolism , Salmonella Infections/pathology , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Type III Secretion Systems/genetics , Type III Secretion Systems/metabolism
2.
Toxins (Basel) ; 9(1)2017 01 13.
Article in English | MEDLINE | ID: mdl-28098782

ABSTRACT

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.


Subject(s)
Auranofin/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Toxins/antagonists & inhibitors , Pasteurella multocida/enzymology , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Catalytic Domain , Cell Culture Techniques , Cytosol/metabolism , HeLa Cells , Heterotrimeric GTP-Binding Proteins/metabolism , Host-Pathogen Interactions , Humans , Pasteurella multocida/pathogenicity , Virulence
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