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Modified Bacteriophage Tail Fiber Proteins for Labeling, Immobilization, Capture, and Detection of Bacteria

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Foodborne Bacterial Pathogens

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1918))

Abstract

A critical component of bacterial detection assays is choosing a suitable affinity molecule that retains sensitivity and specificity for the target pathogen over a wide range of in situ applications. Bacteriophages (phages) are bacterial viruses that bind and infect their host cells with unmatched specificity. Phage host range is often determined by their long tail fibers (LTFs) that mediate adsorption of the virus particle to potential bacterial host cells, by binding to specific cell surface receptors. The inherent specificity of the LTFs for distinct bacterial species makes them ideal candidates for development into recombinant affinity molecules. In this chapter, we describe the development of the Salmonella phage S16 LTF (S16 LTF) into an affinity molecule as part of a novel assay to detect Salmonella cells. The enzyme-linked long tail fiber assay (ELLTA) involves two steps: (1) Immobilization and separation of Salmonella cells using S16 LTF-coated paramagnetic beads (LTF-MBs), and (2) Labeling of bead-captured Salmonella using horseradish peroxidase-conjugated S16 LTF (HRP-LTF). Rapid HRP-mediated conversion of a chromogenic substrate provides visual confirmation for the presence of Salmonella. Overall, the ELLTA assay requires as little as 2 h to detect as few as 102 cfu/ml Salmonella cells from liquid culture. The absorbance of the enzyme-generated color substrate is largely proportional to the present bacterial concentrations between 102 and 107 cfu/ml, providing semiquantitative determination of Salmonella cell counts. The methodology described in this chapter can be adapted for other phage receptor-binding proteins, to develop ELLTAs for the detection of other relevant bacterial pathogens.

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Acknowledgments

We thank Jenna M. Denyes for her significant contribution toward the development of the ELLTA methodology and for performing the LTF-MB pulldown assays and preliminary HRP-LTF detection tests. We are also grateful for the continuous support and ideas received from Jochen Klumpp. We thank Roger Stephan and Herbert Hächler (University of Zurich, Zurich, Switzerland), and Herbert Schmidt and Agnes Weiss (Hohenheim University, Germany) for valuable discussions regarding Salmonella detection, and finally Stefan Miller (Regensburg, Germany) for advice regarding the production and use of recombinant phage proteins. The project was funded by the AiF/FEI, Bundesministerium für Wirtschaft und Technologie, Berlin, Germany (Grant number 16756 N).

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Authors and Affiliations

  1. Laboratory of Food Microbiology, Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland

    Matthew Dunne & Martin J. Loessner

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  1. Matthew Dunne
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  2. Martin J. Loessner
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Correspondence to Martin J. Loessner .

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  1. Fougères Laboratory, ANSES, Fougères, France

    Arnaud Bridier

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Dunne, M., Loessner, M.J. (2019). Modified Bacteriophage Tail Fiber Proteins for Labeling, Immobilization, Capture, and Detection of Bacteria. In: Bridier, A. (eds) Foodborne Bacterial Pathogens. Methods in Molecular Biology, vol 1918. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9000-9_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9000-9_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8999-7

  • Online ISBN: 978-1-4939-9000-9

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