Evaluation of Protein Translocation into Eukaryotic Cells by Yersinia pestis
Protein–protein interactions are important during bacterial pathogenesis. Bacterial proteins, whether surface bound or secreted, are crucial for mediating bacterial virulence. Many Gram-negative bacteria, including pathogenic Yersinia spp., employ type III secretion systems to translocate effector proteins into eukaryotic target cells (Du Z, Tan Y, Yang H, Qiu J, Qin L, Wang T: Int J Med Microbiol 299(5):355–66, 2009; Perry RD, Fetherston JD: Clin Microbiol Rev 10:35–66, 1997). When yersiniae are attached to host cells, virulent effectors termed Yersinia outer-membrane proteins (Yops) are delivered into the cytosol of eukaryotic cells, where they modulate the host cellular immune response (Bleves S, Marenne MN, Detry G, Cornelis GR: J Bacteriol 184(12):3214–23, 2002; Cornelis GR: Philos Trans R Soc Lond B Biol Sci 355(1397):681–93, 2000; Brubaker RR: Infect Immun 73(8):4743–4752, 2005). Thus, to elucidate the molecular mechanisms behind bacterial pathogenesis, it is important to study whether these effector proteins are translocated into eukaryotic target cells. It is also important for researchers to identify and characterize new effector proteins. The classic method for validating the cytosolic localization of secreted effector proteins is based on lysing infected cells and then using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting to identify translocated effectors. More recently, some enzymatic tags have been used to determine the cytosolic localization of secreted effector proteins. The use of β-lactamase translational fusions has proven to be a convenient and sensitive tool for detecting bacterial protein secretion. Here, we present a fluorescence resonance energy transfer-based method that can be used to identify the localization of bacterial proteins in the eukaryotic cell cytosol.
Key wordsFluorescence resonance energy transfer Interaction Protein Yersinia pestis Yops
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