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.

Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H 2 O 2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncA TM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncA TM -APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.

Chlamydia trachomatis, an obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion. To facilitate interactions with the host cell, Chlamydia modifies the inclusion membrane with type III secreted proteins, called Incs. As with all chlamydial proteins, Incs are temporally expressed, modifying the chlamydial inclusion during the early- and mid-developmental cycle. VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion. Their recruitment requires de novo chlamydial protein synthesis during the mid-developmental cycle. Thus, we hypothesize that VAMPs 3 and 4 are recruited by Incs. In chlamydial infected cells, identifying Inc binding partners for SNARE proteins specifically has been elusive. To date, most studies examining chlamydial Inc-eukaryotic proteins have benefitted from stable interacting partners or a robust interaction at a specific time point post-infection. While these types of interactions are the predominant class that have been identified, they are likely the ‘exception’ to chlamydial-host interactions. Therefore, we applied two separate but complementary experimental systems to identify candidate chlamydial Inc binding partners for VAMPs. Based on these results, we created transformed strains of C. trachomatis serovar L2 to inducibly express a candidate Inc-FLAG protein. In chlamydial infected cells, we found that five Incs temporally and transiently interact with VAMP3. Further, loss of incA or ct813 expression altered VAMP3 localization to the inclusion. For the first time, our studies demonstrate the transient nature of certain host protein-Inc interactions that contribute to the chlamydial developmental cycle.

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), ...

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. This obligate intracellular bacterium develops within a membrane-bound vacuole called an inclusion, which sequesters the chlamydiae from the host cytoplasm. Host-pathogen interactions at this interface are mediated by chlamydial inclusion membrane proteins (Incs). However, the specific functions of most Incs are poorly characterized. Previous work from our labs indicated that expressing an IncF fusion protein at high levels in C. trachomatis L2 negatively impacted inclusion expansion and progeny production. We hypothesize that some Incs function in the structure and organization of the inclusion membrane and that overexpression of those Incs will alter the composition of endogenous Incs within the inclusion membrane. Consequently, inclusion biogenesis and chlamydial development is negatively impacted. To investigate this, C. trachomatis L2 was transformed with inducible expression plasmids encoding incF-, ct813-, or ct226-FLAG. Overexpression of IncF-FLAG or CT813-FLAG, but not CT226-FLAG, altered chlamydial development as demonstrated by smaller inclusions, fewer progeny, and increased plasmid loss. The overexpression of CT813-FLAG reduced the detectable levels of endogenous IncE and IncG in the inclusion membrane. Notably, recruitment of sorting nexin-6, a eukaryotic protein binding partner of IncE, was also reduced after CT813 overexpression. Gene expression studies and ultrastructural analysis of chlamydial organisms demonstrated that chlamydial development was altered when CT813-FLAG was overexpressed. Overall, these data indicate that disrupting the expression of specific Incs changed the composition of Incs within the inclusion membrane and the recruitment of associated host cell proteins, which negatively impacted C. trachomatis development.