Pathogenesis Francisella Involved in Riboswitches for Intracellular Study of Genes 2012

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 -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. Received 26 July 2012 Accepted 19 October 2012 Published 20 November 2012 Citation Reynoso CMK, Miller MA, Bina JE, Gallivan JP, Weiss DS. 2012. Riboswitches for intracellular study of genes involved in Francisella pathogenesis. mBio 3(6):e00253-12. doi:10.1128/mBio.00253-12. Editor Philippe Sansonetti, Institut Pasteur Copyright © 2012 Reynoso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to David S. Weiss, david.weiss@emory.edu. The detailed study of virulence proteins is critical for understanding how bacterial pathogens cause disease. The use of inducible genetic control elements has facilitated the study and understanding of gene function in many species of bacteria. Many genetic tools exist for such studies of common model organisms such as Escherichia coli and Bacillus subtilis (1–3). However, the same is not true for less genetically tractable organisms, including many intracellular pathogens such as Francisella tularensis. Riboswitches represent a potential solution, since they can be used to induce protein expression in diverse bacteria (4). Riboswitches are RNA regulatory elements usually located in the 5= untranslated region of mRNA. They possess an aptamer domain that binds to a specific ligand and an expression platform located downstream that undergoes a conformational change upon ligand binding, leading to an alteration of protein expression (5). Since riboswitches do not require the presence of accessory proteins (6) and can function independently of promoter identity, they can bypass pitfalls, such as promoter incompatibility or improper folding of accessory proteins, often encountered with exogenous expression of genetic control systems. In addition, riboswitches can be coupled with native promoters to drive expression (4), avoiding the incompatibility of foreign genetic regulation systems. Unlike natural riboswitches, synthetic riboswitches do not require binding to a cellular metabolite, which allows for orthogonal genetic regulation that minimizes interruption of normal cellular function. Theophylline, a purine base structurally similar to caffeine and previously used as an antiasthmatic medication (7), has RESEARCH ARTICLE November/December 2012 Volume 3 Issue 6 e00253-12 ® mbio.asm. org 1 been used as a ligand for synthetic riboswitches. A number of theophylline-sensitive synthetic riboswitches have been reported as robust, broad-host-range genetic control elements (4, 8, 9). We thus anticipated that these riboswitches would be useful tools for studying the mechanisms of virulence in the Gram-negative bacterium F. tularensis. F. tularensis is an intracellular bacterial pathogen that is the causative agent of the zoonotic disease tularemia, which is characterized by flu-like symptoms and exhibits a mortality rate of 30% (10) to 60% for the pneumonic form of the disease (11). Francisella novicida is closely related to F. tularensis, although it is rarely pathogenic to immunocompetent humans. F. novicida causes a tularemia-like disease in mice (12), possesses a genome that is 98% genetically identical to that of F. tularensis (13, 14), and has many virulence genes in common with F. tularensis. Additionally, F. novicida, like F. tularensis, exhibits the important virulence trait of replicating within host cells such as macrophages (12, 15). It is therefore often used as a model system to study F. tularensis biology. Relatively little is known about the virulence factors required for Francisella pathogenesis, and the lack of genetic tools to modulate protein expression has hampered the investigation of protein functions (16). Many attempts to employ pre-existing tools from other organisms in Francisella have not been fruitful (17). We therefore set out to determine if riboswitches could be used to induce protein expression in Francisella and whether this system could be employed to modulate the expression of endogenous proteins during intracellular infection. In this study, we show the efficacy of theophylline-sensitive synthetic riboswitches in inducing protein expression in F. novicida and highly virulent F. tularensis by using riboswitchcontrolled -galactosidase as a reporter. We also employed this system to control green fluorescent protein (GFP) expression during F. novicida infection of macrophages. We adapted this system to regulate the expression of an endogenous novel protein, FTN_0818, and showed that such control could functionally rescue the growth defect of an FTN_0818 mutant in minimal medium. Furthermore, riboswitch-mediated induction of FTN_0818 during macrophage infection restored the ability of the mutant strain to replicate intracellularly. This is the first demonstration that a synthetic riboswitch can be used to efficiently control an endogenous virulence factor and rescue a virulence trait in an intracellular pathogen. We anticipate that synthetic riboswitches will prove useful in numerous applications of gene expression control to study the biology of intracellular bacteria.