Monitoring gene expression of Salmonella inside mammalian cells: comparison of luciferase and β-galactosidase fusion systems

Abstract

Gene expression of bacteria can be studied through the fusion of bacterial genes with reporter genes encoding assayable enzymes such as luciferase and β-galactosidase. However, quantitative measurement of gene expression from intracellular bacteria (bacteria inside eukaryotic host cells) can be difficult, due to problems such as low bacterial numbers or the presence of endogenous enzymes that mimic reporter enzyme activity. In this paper, we determined the efficacy of two reporter systems, luxAB (encoding luciferase from Vibrio harveyi) and lacZ (encoding β-galactosidase), to measure gene expression from intracellular Salmonella. One set of genes shown previously to have increased expression by intracellular Salmonella are the Salmonella plasmid virulence genes ( spvRAB). The spv operon is known to be involved in Salmonella virulence in animal models of infection. Therefore, in this study the spvRAB-lacZ reporter system was compared with an spvRAB-luxAB reporter system. Although both spv reporter systems showed comparable sensitivity, the luciferase assay provided several advantages. First, it was faster and easier to perform than the β-galactosidase assay. It was not necessary to lyse bacteria containing luxAB fusions for measurement of luciferase activity, whereas it was necessary to lyse bacteria containing lacZ fusions for the measurement of β-galactosidase activity. Furthermore, mammalian host cells and Salmonella did not contain endogenous luciferase, so that any light produced was a result of expression from the luxAB constructs. Collectively, these results demonstrate that bacterial luciferase is a favorable alternative to β-galactosidase for determining gene expression of bacterial pathogens that reside within mammalian host cells.