Riboswitches for intracellular study of genes involved in Francisella pathogenesis.
Riboswitches for intracellular study of genes involved in Francisella pathogenesis.
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DOI：
10.1128/mbio.00253-12
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发表时间：
2012-11-20
期刊：
影响因子：
6.4
通讯作者：
Weiss DS
中科院分区：
文献类型：
--
作者：
Reynoso CM;Miller MA;Bina JE;Gallivan JP;Weiss DS
许多重要的细胞内细菌病原体的研究需要了解特定的毒力因子如何在宿主细胞感染过程中促进发病机制。这需要工具来剖析基因功能，但不幸的是，缺乏这样的工具来研究许多难以研究或研究不足的细胞内病原体。核糖开关是基于RNA的遗传控制元件，其在配体结合时直接调节基因表达。在这里，我们报告的应用茶碱敏感的合成核糖开关诱导蛋白质表达的细胞内病原体弗朗西斯。我们表明，该系统可用于激活细菌在丰富培养基中生长期间报告基因β-半乳糖苷酶的表达。此外，我们应用该系统控制绿色荧光蛋白的表达，在细胞内感染的茶碱直接感染的巨噬细胞。重要的是，我们可以控制一种新的内源性蛋白质的表达，这种蛋白质是在营养限制条件下生长和在巨噬细胞中复制所必需的，即FTN_0818。核糖开关介导的FTN_0818控制挽救了基本培养基中和巨噬细胞感染期间FTN_0818突变体的生长。这是首次证明使用合成核糖开关来控制细胞内细菌中毒力性状所需的内源基因。由于该系统可以适应不同的细菌，使用核糖开关来调节细胞内细菌基因表达的能力将可能有助于深入研究许多难以研究的细胞内病原体，如查菲埃里希体，嗜吞噬细胞无形体和恙虫病东方体，以及未来新兴的病原体的毒力机制。确定特定的细菌基因如何在宿主细胞感染期间促成毒力对于理解病原体如何引起疾病至关重要。这对于许多难以研究的细胞内病原体来说尤其具有挑战性。核糖开关是基于RNA的遗传控制元件，可用于帮助剖析基因功能，特别是因为它们可用于广泛的细菌。我们证明了核糖开关的效用，并首次表明，核糖开关可用于功能控制细菌基因，这是至关重要的病原体的能力，引起疾病，在细胞内感染。由于这种系统可以适应不同的细菌，核糖开关将可能促进深入研究许多难以研究的细胞内病原体的毒力机制，以及未来新兴的病原体。
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. 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.