Tracking bacterial pathogens with genetically‐encoded reporters

Campbell-Valois, François-Xavier; Sansonetti, Philippe J.

doi:10.1016/j.febslet.2014.05.022

Cited by 25 publications

(25 citation statements)

References 100 publications

(129 reference statements)

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“…Despite these recent advances in molecular tools, there is a need for improved in vivo analysis of bacterial pathogens (9). It is necessary to validate the expression of individual genes identified by next-generation sequencing applications, such as transposon insertion sequencing (Tn-seq) (10), or to complement in vivospecific genetic techniques, such as in vivo expression technology (IVET) (11).…”

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confidence: 99%

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Shivak

MacKenzie

Watson

et al. 2016

Appl Environ Microbiol

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Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin-and chloramphenicolresistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of 70 -dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10-to 30-fold boost in transposase gene expression and transposition efficiencies of 10 ؊8 to 10 ؊10 in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae. This technology will allow for improved in vivo analysis of bacterial pathogens.T he ability to generate recombinant strains of Escherichia coli and Salmonella enterica has facilitated insight into their ecology and pathogenic mechanisms. In recent years, strain collections consisting of single-gene knockouts of the entire genomes of E. coli K-12 (1) and S. enterica serovar Typhimurium 14028 (2) have been assembled. These collections can be used for finely tuned analysis of gene function and host-pathogen interactions, as well as for strain fitness and competition experiments (3). There are also a wide array of reporter systems available to analyze gene expression in detail, for the ordering of hierarchical gene circuits (4), a deeper understanding of metabolism (5), or the development of biosensors (6). The use of these systems, coupled with next-generation sequencing approaches that have facilitated functio...

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mentioning

confidence: 99%

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Shivak

MacKenzie

Watson

et al. 2016

Appl Environ Microbiol

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“…Biologically speaking, this environment is essential for the full understanding of a given pathogen's invasive strategies and the complex host response; however, this also places additional complexity and limitations in regards to imaging, particularly in maintaining cellular and molecular resolution. Novel tools and studies addressing bacterial infection in vivo are frequently reported in the literature (4,5). However, emphasis has been traditionally placed on qualitative observations at the expense of extensive quantitative efforts.…”

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confidence: 99%

Bioimage analysis of Shigella infection reveals targeting of colonic crypts

Arena

Campbell-Valois

Tinévez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent.Shigella flexneri | host-pathogen interactions | intestinal crypts | bioimage analysis | tissue microbiology

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“…Single-cell RNA-seq is useful to detect cell-to-cell variability in non-coding RNA expression. Physiology of intracellular bacteria located in defined subcellular locations can be also monitored with reporters based on fluorescent proteins expressed constitutively or promoters responding to the infection (Campbell-Valois and Sansonetti, 2014). Transcriptome and proteome quantification with single-molecule sensitivity was performed in single E. coli cells (Taniguchi et al, 2010).…”

Section: Isolating Intracellular Bacteria From Eukaryotic Cellsmentioning

confidence: 99%

Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

Ortega

Quereda

Pucciarelli

et al. 2014

Front. Cell. Infect. Microbiol.

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Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of “intact” intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

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Tracking bacterial pathogens with genetically‐encoded reporters

Cited by 25 publications

References 100 publications

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Bioimage analysis of Shigella infection reveals targeting of colonic crypts

Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

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