27As an obligate intracellular pathogenic bacterium, C. trachomatis develops within a membrane-28 bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion 29 membrane proteins (Incs), which act as the mediators of host-pathogen interactions. An in vivo 30 understanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to 31 overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-32 hybrid studies established that certain Incs have the propensity to bind other Incs while others 33 have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane 34 whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this 35 hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels 36 proximal proteins with biotin in vivo, and chose to analyze Inc proteins with varying Inc-binding 37 propensities. We inducibly expressed these Incs fused to APEX2 in Chlamydia trachomatis L2, 38 verified their localization and labeling activities by transmission electron microscopy, and used 39 affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass 40 spectrometry results for statistical significance, we used Significance Analysis of INTeractome 41 (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as 42 known and previously unreported eukaryotic proteins that localize to the inclusion. Our results 43 broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, 44 LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously 45 published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution 46 microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial 47 two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting 48 protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of 49 APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion. 50 Author summary 51 Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, 52 grow within a membrane-bound "bacteria containing vacuole" (BCV) that, in most cases, prevents 53 association with the lysosome. Secreted cytosolic effectors modulate host activity, but an 54understanding of the host-pathogen interactions that occur at the BCV membrane is limited by 55 the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate 56 peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo,
57to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The 58 inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins 59 (Incs), ...