Illuminating Targets of Bacterial Secretion

Roger D. Pechous, William E. Goldman, Deborah A. Hogan
2015 PLoS Pathogens  
The ability to secrete proteins is important to the pathogenesis of many bacteria. For gram-negative bacteria, the secretion system must deliver cargo through both an inner and outer membrane to reach a potential target. To date, there are six known gram-negative bacterial secretion systems, designated types I-VI secretion. For many highly pathogenic bacteria including Yersinia pestis and Salmonella typhimurium, secretion of protein effectors directly into target host cells is essential for
more » ... lence. Proteins secreted via the type III, IV, and VI pathways result in direct transfer of proteins across the host membrane and into the cytosol, and these systems will be the focus of the technology highlighted in this article. Although the function of effector proteins secreted by these systems varies among different pathogens, common virulence mechanisms are evident. One common function of many secreted virulence factors is the targeting of host cytoskeletal function in order to promote uptake or inhibit phagocytosis. Another is modulating host cell cytotoxicity by inhibiting or promoting cell death in order to suppress innate immune function or to establish a replicative niche. Finally, an important mechanism common to many secreted effectors is the manipulation of host immune signaling. Until recently, fully evaluating the functional targets of bacterial secretion in vivo during infection was extremely difficult. FRET-Based β-lactamase Substrates as a Molecular Biology Tool Förster (fluorescence) resonance energy transfer (FRET) has been used extensively as a cell biology tool to monitor the dynamics of intermolecular interactions within cells. FRET employs a donor fluorophore and an acceptor fluorophore in close proximity, and the emission spectra of the donor overlaps with the absorption spectrum of the acceptor. Excitation of the donor fluorophore results in resonance energy transfer to, and emission from, the acceptor. Disruption of the proximity between the two fluorophores results in strong emission from the donor upon excitation. The utility of FRET for measuring inter-and intramolecular interactions has been evident for some time. Using fluorescence as a measure of proximity, fluorophores exhibiting FRET can be incorporated to measure protein-folding dynamics in real time. Further, labeling separate molecules allows for measuring protein-protein interactions, the distance between molecules, and determining protein localization within a cell. In 1998, Zlokarnik et al. used the gene encoding a common β-lactamase along with a FRETbased substrate to isolate individual cells with defined transcriptional responses from within a population of mammalian cells [1]. Zlokarnik et al. designed and synthesized the membranepermeant ester CCF2/AM, which consists of a 7-hydroxycoumarin donor fluorophore and a fluorescein acceptor linked by a cephalosporin antibiotic. In the intact molecule, donor excitation at 405 nm (violet light) will result in fluorescein acceptor emission as green light at 520 nm that can be detected using flow cytometry or fluorescence microscopy. In the presence of PLOS Pathogens |
doi:10.1371/journal.ppat.1004981 pmid:26247771 pmcid:PMC4527701 fatcat:w6gikcmjpvcn5ofsiv646bstoe