Dataset Information

Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

ABSTRACT: Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound “bacteria 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 proximity labeling system (APEX2), 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-chlamydial interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-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 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 that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc, CT226, using bacterial two-hybrid and co-immunoprecipitation assays. 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Chlamydia Trachomatis

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Macy Olson

LAB HEAD: Elizabeth A. Rucks and Scot P. Ouellette

PROVIDER: PXD015890 | Pride | 2019-10-25

REPOSITORIES: Pride

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			Action	DRS
	14Sep18_Olson_S01.raw	Raw
	14Sep18_Olson_S01.raw (F001327).mzid_14Sep18_Olson_S01.raw_(F001327).pride.mgf.gz	Mzid
	14Sep18_Olson_S01.raw (F001327).pride.mztab.gz	Mztab
	14Sep18_Olson_S01.rawF001327.mzid.gz	Mzid
	14Sep18_Olson_S01.rawF001327.mzid_14Sep18_Olson_S01.raw_F001327.MGF	Mzid

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Proximity Labeling To Map Host-Pathogen Interactions at the Membrane of a Bacterium-Containing Vacuole in Chlamydia trachomatis-Infected Human Cells.

Olson Macy G MG Widner Ray E RE Jorgenson Lisa M LM Lawrence Alyssa A Lagundzin Dragana D Woods Nicholas T NT Ouellette Scot P SP Rucks Elizabeth A EA

Infection and immunity 20191018 11

Many intracellular bacteria, including the obligate intracellular pathogen <i>Chlamydia trachomatis</i>, 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 ...[more]

PMID: 31405957

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Project description:Introduction Chlamydia trachomatis (C. trachomatis) is a Gram-negative bacterium and a common human pathogen. The World Health Organization (WHO) estimates that over 130 million people are infected with C. trachomatis globally each year and with increasing incidence. C. trachomatis causes long-lasting and recurrent infections that over time induce severe tissue damage in the female genital tract that can lead to ectopic pregnancy and infertility. Thus, the human immune system fails to control and eradicate C. trachomatis during primary infection and fails to develop protective immunity against secondary infections. In vivo infection models, using complement knock out mice, suggest that the complement system is critically involved in both anti-chlamydial immunity and infection-induced pathology. To increase our understanding of complement-mediated immunity against C. trachomatis we analyzed global complement deposition on serum-incubated C. trachomatis by mass spectrometry. Methods Purified C. trachomatis was incubated in seronegative normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) for 30 min, thoroughly washed, and processed for mass spectrometry. All samples were lysed, reduced and alkylated and digested with trypsin. Some samples were chemically modified to acetylate free amino groups (N-terminal and lysine amino groups) before trypsin digestion. Peptides were analyzed on a UltimateTM 3500 RSLCnano coupled to a Q Exactive HF-X mass spectrometer. Raw data files were searched against the Uniprot human reference proteome using MaxQuant. Results We demonstrate that C. trachomatis elicits potent complement activation demonstrated by deposition of both early and late complement factors together with several complement regulators. We further demonstrate proteolytically processing of complement C3b to “inactive” C3 cleavage fragments. Conclusion We demonstrate the deposition of several novel complement-associated proteins and -cleavage fragments on the surface of C. trachomatis.