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Identification of host-induced pathogen genes by differential fluorescence induction reporter systems

Abstract

The ability to monitor a pathogen's gene expression program in response to the host environment is central to understanding host–microbe interactions. This protocol describes the application of a fluorescence-based promoter trap strategy, termed differential fluorescence induction (DFI), to identify and characterize bacterial genes that are preferentially expressed in infected tissues. In this approach, animals are infected with a library of bacteria expressing random GFP transcriptional gene fusions, and fluorescent bacteria are recovered directly from host tissues using fluorescence-activated cell sorting (FACS). This methodology allows for the identification of bacterial promoters induced in distinct anatomical sites and at different stages of infection. Furthermore, unlike other methodologies, the use of the GFP reporter allows for single cell, temporal and spatial monitoring of pathogen gene expression in infected animals. Library construction, promoter identification and analysis can be done in 4–8 weeks.

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Figure 1: Identification of bacterial genes expressed in animal tissues.
Figure 2: Construction of promoter trap libraries in Salmonella enterica.
Figure 3: Gating strategy for FACS of GFP-expressing Salmonella from infected mouse tissues.
Figure 4: Analysis of in vivo induced bacterial promoters.

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Acknowledgements

We thank Meike Soerensen for excellent technical assistance in developing the procedure, and Claudia Rollenhagen and Nguyen Le Quang for help in preparing the figures. D.B. is supported by the Deutsche Forschungsgemeinschaft, and R.H.V. is supported by grants from the NIH/NIAD, the WhiteHead Foundation and the Pew Charitable Trust. We also acknowledge Stanley Falkow for intellectual input during the development of DFI.

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Correspondence to Raphael H Valdivia.

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R.H. Valdivia holds a US patent for DFI technology (U.S. 5,994,077) and D. Bumann has filed patents for some of the in-vivo activated promoters identified in S. enterica.

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Bumann, D., Valdivia, R. Identification of host-induced pathogen genes by differential fluorescence induction reporter systems. Nat Protoc 2, 770–777 (2007). https://doi.org/10.1038/nprot.2007.78

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