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Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane

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Chlamydia trachomatis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2042))

Abstract

In the study of intracellular bacteria that reside within a membrane-bound vacuole, there are many questions related to how prokaryotic or eukaryotic transmembrane or membrane-associated proteins are organized and function within the membranes of these pathogen-containing vacuoles. Yet this host–pathogen interaction interface has proven difficult to experimentally resolve. For example, one method to begin to understand protein function is to determine the protein-binding partners; however, examining protein–protein interactions of hydrophobic transmembrane proteins is not widely successful using standard immunoprecipitation or coimmunoprecipitation techniques. In these scenarios, the lysis conditions that maintain protein–protein interactions are not compatible with solubilizing hydrophobic membrane proteins. In this chapter, we outline two proximity labeling systems to circumvent these issues to study (1) eukaryotic proteins that localize to the membrane-bound inclusion formed by Chlamydia trachomatis using BioID, and (2) chlamydial proteins that are inserted into the inclusion membrane using APEX2. BioID is a promiscuous biotin ligase to tag proximal proteins with biotin. APEX2 is an ascorbate peroxidase that creates biotin-phenoxyl radicals to label proximal proteins with biotin or 3,3′-diaminobenzidine intermediates for examination of APEX2 labeling of subcellular structures using transmission electron microscopy. We present how these methods were originally conceptualized and developed, so that the user can understand the strengths and limitations of each proximity labeling system. We discuss important considerations regarding experimental design, which include careful consideration of background conditions and statistical analysis of mass spectrometry results. When applied in the appropriate context with adequate controls, these methods can be powerful tools toward understanding membrane interfaces between intracellular pathogens and their hosts.

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Acknowledgments

We would like to thank Scot Ouellette for critical reading of the manuscript. We would also like to thank Tom Bargar and Nicholas Conoan of the Electron Microscopy Core Facility (EMCF) at the University of Nebraska Medical Center for technical assistance. The EMCF is supported by state funds from the Nebraska Research Initiative (NRI) and the University of Nebraska Foundation, and institutionally by the Office of the Vice Chancellor for Research. This work is supported by NIH/NIAID R01 AI114670 (E. Rucks) and UNMC Department of Pathology and Microbiology start-up funds (E. Rucks).

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  1. Macy G. Olson and Lisa M. Jorgenson contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA

    Macy G. Olson, Lisa M. Jorgenson, Ray E. Widner & Elizabeth A. Rucks

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  1. Macy G. Olson
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  2. Lisa M. Jorgenson
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Correspondence to Elizabeth A. Rucks .

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  1. Department of Microbiology and Immunology, Vet School, Cornell University, Ithaca, NY, USA

    Amanda Claire Brown

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Olson, M.G., Jorgenson, L.M., Widner, R.E., Rucks, E.A. (2019). Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane. In: Brown, A. (eds) Chlamydia trachomatis. Methods in Molecular Biology, vol 2042. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9694-0_17

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  • DOI: https://doi.org/10.1007/978-1-4939-9694-0_17

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9693-3

  • Online ISBN: 978-1-4939-9694-0

  • eBook Packages: Springer Protocols

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