@article{ff8ad588b9b74dd9bf034d198b5b5790,
title = "Proximity labeling to map host-pathogen interactions at the membrane of a bacterium-containing vacuole in chlamydia trachomatis-infected human cells",
abstract = "Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase (APEX2) proximity labeling system, which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydia interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane, whereas other Incs bind eukaryotic proteins to promote chlamydia-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using significance analysis of the interactome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate, using bacterial two-hybrid and coimmunoprecipitation assays, that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc CT226. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.",
keywords = "APEX2, Chlamydia trachomatis, Host-pathogen interactions, Inc proteins, Inclusion membrane, LRRF1, Proximity labeling",
author = "Olson, {Macy G.} and Widner, {Ray E.} and Jorgenson, {Lisa M.} and Alyssa Lawrence and Dragana Lagundzin and Woods, {Nicholas T.} and Ouellette, {Scot P.} and Rucks, {Elizabeth A.}",
note = "Funding Information: This work was supported by UNMC start-up funds for E. A. Rucks and S. P. Ouellette and was partially supported by both grant R01AI114670-01A1, awarded to E. A. Rucks, and grant R35GM124798-01, awarded to S. P. Ouellette. N. T. Woods is supported by grant P20 GM121316. A. Lawrence is supported by The Sherwood Foundation. The transmission electron microscope used in this study is supported by an NIH Shared Instrument grant (NIH grant 1 S10 RR024650 01A1). This work was also supported by the UNMC Center Advanced Microscopy Core Facility, the UNMC Advanced Proteomics Core Facility, and the UNMC Electron Microscopy Core Facility. The University of Nebraska Medical Center Advanced Microscopy Core Facility receives partial support from National Institute for General Medical Sciences (NIGMS) grants INBRE-P20 GM103427 and COBRE-P30 GM106397, as well as support from the National Cancer Institute (NCI) for the Fred & Pamela Buffett Cancer Center (support grant P30 CA036727) and the Nebraska Research Initiative. Funding Information: This work was supported by UNMC start-up funds for E. A. Rucks and S. P. Ouellette and was partially supported by both grant R01AI114670-01A1, awarded to E. A. Rucks, and grant R35GM124798-01, awarded to S. P. Ouellette. N. T. Woods is supported by grant P20 GM121316. A. Lawrence is supported by The Sherwood Foundation. The transmission electron microscope used in this study is supported by an NIH Shared Instrument grant (NIH grant 1 S10 RR024650 01A1). This work was also supported by the UNMC Center Advanced Microscopy Core Facility, the UNMC Advanced Proteomics Core Facility, and the UNMC Electron Microscopy Core Facility. The University of Nebraska Medical Center Advanced Microscopy Core Facility receives partial support from National Institute for General Medical Sciences (NIGMS) grants INBRE-P20 GM103427 and COBRE-P30 GM106397, as well as support from the National Cancer Institute (NCI) for the Fred & Pamela Buffett Cancer Center (support grant P30 CA036727) and the Nebraska Research Initiative. We thank R. Suchland (University of Washington, WA) and D. Rockey (Oregon State University, OR) for the anti-CT223 antibody, Ted Hackstadt (NIAID, Rocky Mountain Laboratories, Hamilton, MT) for the anti-IncA antibody, R. Morrison (University of Arkansas for Medical Sciences, Little Rock, AR) for the anti-chlamydial Hsp60 antibody, and H. Caldwell (NIAID, Bethesda, MD) for the C. pneumoniae anti-MOMP antibody. We also thank Bob Heinzen (NIAID, Rocky Mountain Laboratories, Hamilton, MT) for the avirulent Coxiella burnetii Nine Mile phase II strain. We also thank Eric Troudt and Vikas Kumar for processing samples for mass spectrometry and their technical expertise and Tom Bargar for processing samples for electron microscopy. This publication's contents and interpretations are the sole responsibility of the authors. Publisher Copyright: Copyright {\textcopyright} 2019 Olson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.",
year = "2019",
doi = "10.1128/IAI.00537-19",
language = "English (US)",
volume = "87",
journal = "Infection and immunity",
issn = "0019-9567",
publisher = "American Society for Microbiology",
number = "11",
}