Riboswitches for intracellular study of genes involved in Francisella pathogenesis

Abstract

The study of many important intracellular bacterial pathogens requires an understanding of how specific virulence factors contribute to pathogenesis during the infection of host cells. This requires tools to dissect gene function, but unfortunately, there is a lack of such tools for research on many difficult-to-study, or understudied, intracellular pathogens. Riboswitches are RNA-based genetic control elements that directly modulate gene expression upon ligand binding. Here we report the application of theophylline-sensitive synthetic riboswitches to induce protein expression in the intracellular pathogen Francisella. We show that this system can be used to activate the bacterial expression of the reporter β-galactosidase during growth in rich medium. Furthermore, we applied this system to control the expression of green fluorescent protein during intracellular infection by the addition of theophylline directly to infected macrophages. Importantly, we could control the expression of a novel endogenous protein required for growth under nutrient-limiting conditions and replication in macrophages, FTN_0818. Riboswitch-mediated control of FTN_0818 rescued the growth of an FTN_0818 mutant in minimal medium and during macrophage infection. This is the first demonstration of the use of a synthetic riboswitch to control an endogenous gene required for a virulence trait in an intracellular bacterium. Since this system can be adapted to diverse bacteria, the ability to use riboswitches to regulate intracellular bacterial gene expression will likely facilitate the in-depth study of the virulence mechanisms of numerous difficult-to-study intracellular pathogens such as Ehrlichia chaffeensis, Anaplasma phagocytophilum, and Orientia tsutsugamushi, as well as future emerging pathogens.

Importance: Determining how specific bacterial genes contribute to virulence during the infection of host cells is critical to understanding how pathogens cause disease. This can be especially challenging with many difficult-to-study intracellular pathogens. Riboswitches are RNA-based genetic control elements that can be used to help dissect gene function, especially since they can be used in a broad range of bacteria. We demonstrate the utility of riboswitches, and for the first time show that riboswitches can be used to functionally control a bacterial gene that is critical to the ability of a pathogen to cause disease, during intracellular infection. Since this system can be adapted to diverse bacteria, riboswitches will likely facilitate the in-depth study of the virulence mechanisms of numerous difficult-to-study intracellular pathogens, as well as future emerging pathogens.

FIG 1

Riboswitch-mediated control of β-galactosidase in F. novicida. Strains encoding riboswitch E (E-Rs-βgal) or F (F-Rs-βgal) upstream of lacZ were grown in the presence (●) or absence (○) of 1 mM theophylline to an OD₆₀₀ between 0.7 and 0.8. β-galactosidase activity was measured in Miller units (left axis), and the activation ratio (green bar, right axis) was calculated by dividing the number of Miller units in the presence of theophylline by the number of Miller units in the absence of theophylline. The standard deviations of triplicate samples lie within the symbols. The data are representative of three independent experiments performed in triplicate.

FIG 2

Dose-dependent riboswitch-mediated induction of β-galactosidase. Strains E-Rs-βgal and F-Rs-βgal were subcultured and grown to exponential phase (OD₆₀₀ between 0.7 and 0.8) in medium containing 0, 0.1, 0.5, 1, or 2 mM theophylline. β-Galactosidase activity was measured in Miller units. The data are representative of two independent experiments performed in triplicate.

FIG 3

Riboswitch-mediated control of GFP (GFP-neg) in F. novicida during macrophage infection. RAW264.7 murine macrophages were infected at an MOI of 50:1 with F. novicida harboring gfp lacking a promoter (GFP-neg; panel A) or constitutively expressing gfp (GFP-pos; panel B) or the E-Rs-GFP riboswitch construct (panels C and D). At 30 min postinfection, medium without theophylline (panels A to C) or with 1 mM theophylline (panel D) was added and the macrophages were incubated at 37°C for 24 h and fixed. GFP (green) and DAPI-stained macrophage nuclei (blue) are shown. The magnification is ×100, and the scale bar represents 10 µm. The data are representative of three independent experiments in which at least 10 fields of view were analyzed for each condition.

FIG 4

Theophylline-dependent rescue of bacterial replication. Growth rates are represented by cell density measured as OD₆₀₀ every hour for 18 h. The wild-type (WT) and ΔFTN_0818 and F-Rs-FTN_0818 mutant strains were grown overnight (~18 h) in TSB. Cultures were washed and then diluted in Chamberlain’s minimal medium to an OD₆₀₀ of 0.03 in the presence or absence of 1 mM theophylline. The standard deviations of triplicate samples lie within the areas of the symbols.

FIG 5

Riboswitch-mediated control of FTN_0818 facilitates intracellular replication. RAW264.7 murine macrophages were infected with the indicated F. novicida strains at an MOI of 20:1 in the presence of 0, 1, or 2 mM theophylline. Macrophages were lysed at 30 min and 24 h postinfection and plated to enumerate the intracellular CFU. Fold replication was calculated by dividing the number of CFU at 24 h postinfection by the number of CFU at 30 min. Bars represent the average fold replication of the strains, and error bars depict the standard deviations. The data are representative of four independent experiments. Asterisks indicate a significant difference in fold replication from that of the ΔFTN_0818 mutant strain in the presence of the indicated concentration of theophylline (***, P < 0.0005). WT, wild type.

FIG 6

Riboswitch E controlling β-galactosidase activity in F. novicida and F. tularensis. F. novicida strain E-Rs-βgal and F. tularensis strain E-Rs-βgal were subcultured and grown to exponential phase (OD₆₀₀ of ~0.7 to 0.8) in medium containing 0, 0.5, 1, or 2 mM theophylline. β-galactosidase activity was measured in Miller units. The data are representative of two independent experiments performed in triplicate.