From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors.
Canonical reporters such as green fluorescent protein (GFP) and luciferase have assisted researchers in probing cellular pathways and processes. Prior research in pathogenesis depended on sensitivity of biochemical and biophysical techniques to identify effectors and elucidate entry mechanisms. Recently, the β-lactamase (βlac) reporter system has advanced toxin and effector reporting by permitting measurement of βlac delivery into the cytosol or host βlac expression in intact cells. βlac measurement in cells was facilitated by the development of the fluorogenic substrate, CCF2-AM, to identify novel effectors, target cells, and domains involved in bacterial pathogenesis. The assay is also adaptable for high-throughput screening of small molecule inhibitors against toxins, providing information on mechanism and potential therapeutic agents. The versatility and limitations of the βlac reporter system as applied to toxins and effectors are discussed in this review.
Medical College of Wisconsin, Microbiology and Molecular Genetics, Milwaukee, WI 53226, USA.
Madison
M
Zuverink
Medical College of Wisconsin, Microbiology and Molecular Genetics, Milwaukee, WI 53226, USA jtb01@mcw.edu.
Joseph T
JT
Barbieri
D003584:Cytological Techniques / Q000379:methods*; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D061848:Optical Imaging / Q000379:methods*; D001618:beta-Lactamases / Q000032:analysis
D049452:Green Fluorescent Proteins; D001618:beta-Lactamases
fluorescent reporters; protein toxins; protein translocation; β-lactamase