Riboswitches for intracellular study of genes involved in francisella pathogenesis

Colleen M.K. Reynoso, Mark Miller, James E. Bina, Justin P. Gallivan, David S. Weiss

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish (US)
JournalmBio
Volume3
Issue number6
DOIs
StatePublished - Nov 1 2012

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Francisella
Riboswitch
Genes
Macrophages
Theophylline
Virulence
Ehrlichia chaffeensis
Growth
Infection
Orientia tsutsugamushi
Anaplasma phagocytophilum
Galactosidases
Bacteria
Gene Expression
Bacterial Genes
Virulence Factors
Green Fluorescent Proteins
Proteins
RNA
Ligands

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Virology

Cite this

Riboswitches for intracellular study of genes involved in francisella pathogenesis. / Reynoso, Colleen M.K.; Miller, Mark; Bina, James E.; Gallivan, Justin P.; Weiss, David S.

In: mBio, Vol. 3, No. 6, 01.11.2012.

Research output: Contribution to journalArticle

Reynoso, Colleen M.K. ; Miller, Mark ; Bina, James E. ; Gallivan, Justin P. ; Weiss, David S. / Riboswitches for intracellular study of genes involved in francisella pathogenesis. In: mBio. 2012 ; Vol. 3, No. 6.
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