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Int J Mol Sci ; 21(9)2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32366039


Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

Front Microbiol ; 6: 396, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25972862


Salmonella enterica expresses two virulence-related type III secretion systems (T3SSs) encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. SseK1 is a poorly characterized substrate of the SPI2-encoded T3SS. Here, we show that this effector is essential to get full virulence both in oral and intraperitoneal mice infections, in spite of not having a role in invasion or intracellular proliferation in cultured mammalian cells. In vitro, expression of sseK1 was higher in media mimicking intracellular conditions, when SPI2 was induced, but it was also significant under SPI1 inducing conditions. A detailed analysis of translocation of SseK1 into host cells unveiled that it was a substrate of both, T3SS1 and T3SS2, although with different patterns and kinetics depending on the specific host cell type (epithelial, macrophages, or fibroblasts). The regulation of the expression of sseK1 was examined using lacZ and bioluminescent lux fusions. The two-component system PhoQ/PhoP is a positive regulator of this gene. A combination of sequence analysis, directed mutagenesis and electrophoretic mobility shift assays showed that phosphorylated PhoP binds directly to the promoter region of sseK1 and revealed a PhoP binding site located upstream of the predicted -35 hexamer of this promoter.

Methods Mol Biol ; 1225: 93-104, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25253250


CyaA from Bordetella pertussis is a calmodulin-dependent adenylate cyclase. Fusions to the catalytic domain of CyaA (CyaA') are useful tools to detect translocation of type III secretion system effectors from gram-negative pathogens like Salmonella enterica. These fusions are usually generated using plasmids with strong promoters. Here, we describe a protocol to insert the CyaA'-encoding sequence in a specific site in the bacterial chromosome in order to get a monocopy fusion whose expression is driven by the native promoter. We also describe the procedure to detect translocation of a CyaA' fusion into mammalian cells.

Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Fusão Gênica Artificial/métodos , Cromossomos Bacterianos/genética , Biossíntese de Proteínas , Salmonella enterica/enzimologia , Salmonella enterica/genética , Animais , Bacteriófago lambda/genética , Células HeLa , Humanos , Camundongos
Biochem Biophys Res Commun ; 423(2): 240-6, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22640733


Salmonella harbors two type III secretion systems, T3SS1 and T3SS2, encoded on the pathogenicity islands SPI1 and SPI2, respectively. Several effector proteins are secreted through these systems into the eukaryotic host cells. PipB2 is a T3SS2 effector that contributes to the modulation of kinesin-1 motor complex activity. Here, we show that PipB2 is also a substrate of T3SS1. This result was obtained infecting human epithelial HeLa cells for 2 h and was confirmed in murine RAW264.7 macrophages, and rat NRK fibroblasts. Analysis at different time points after infection revealed that translocation of PipB2 is T3SS1-dependent in epithelial cells throughout the infection. In contrast, translocation into macrophages is T3SS1-dependent during invasion but T3SS2-dependent at later time points. The N-terminal 10 amino acid residues contain the signal necessary for translocation through both systems. These results confirm the functional overlap between these virulence-related secretion systems and suggest a new role for the effector PipB2.

Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Ilhas Genômicas , Proteínas de Membrana/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Sinais Direcionadores de Proteínas , Transporte Proteico , Ratos