Proximity labeling to map host-pathogen interactions at the membrane of a bacterium-containing vacuole in chlamydia trachomatis-infected human cells

Macy G. Olson, Ray E. Widner, Lisa M. Jorgenson, Alyssa Lawrence, Dragana Lagundzin, Nicholas T. Woods, Scot P. Ouellette, Elizabeth A. Rucks

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Article numbere00537-19
JournalInfection and immunity
Volume87
Issue number11
DOIs
StatePublished - Jan 1 2019

Fingerprint

Host-Pathogen Interactions
Chlamydia trachomatis
Vacuoles
Bacteria
Membranes
Proteins
Chlamydia
Ascorbate Peroxidases
Two-Hybrid System Techniques
Biotin
Mass Spectrometry
Membrane Proteins

Keywords

  • APEX2
  • Chlamydia trachomatis
  • Host-pathogen interactions
  • Inc proteins
  • Inclusion membrane
  • LRRF1
  • Proximity labeling

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Cite this

Proximity labeling to map host-pathogen interactions at the membrane of a bacterium-containing vacuole in chlamydia trachomatis-infected human cells. / Olson, Macy G.; Widner, Ray E.; Jorgenson, Lisa M.; Lawrence, Alyssa; Lagundzin, Dragana; Woods, Nicholas T.; Ouellette, Scot P.; Rucks, Elizabeth A.

In: Infection and immunity, Vol. 87, No. 11, e00537-19, 01.01.2019.

Research output: Contribution to journalArticle

@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.}",
year = "2019",
month = "1",
day = "1",
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",

}

TY - JOUR

T1 - Proximity labeling to map host-pathogen interactions at the membrane of a bacterium-containing vacuole in chlamydia trachomatis-infected human cells

AU - Olson, Macy G.

AU - Widner, Ray E.

AU - Jorgenson, Lisa M.

AU - Lawrence, Alyssa

AU - Lagundzin, Dragana

AU - Woods, Nicholas T.

AU - Ouellette, Scot P.

AU - Rucks, Elizabeth A.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - APEX2

KW - Chlamydia trachomatis

KW - Host-pathogen interactions

KW - Inc proteins

KW - Inclusion membrane

KW - LRRF1

KW - Proximity labeling

UR - http://www.scopus.com/inward/record.url?scp=85073583614&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85073583614&partnerID=8YFLogxK

U2 - 10.1128/IAI.00537-19

DO - 10.1128/IAI.00537-19

M3 - Article

C2 - 31405957

AN - SCOPUS:85073583614

VL - 87

JO - Infection and Immunity

JF - Infection and Immunity

SN - 0019-9567

IS - 11

M1 - e00537-19

ER -