Widner, R. E. | Search bioRxiv

bioRxiv2024

Effect of tryptophan starvation on inclusion membrane composition and chlamydial-host interactions

Chlamydia is an obligate intracellular bacterial pathogen that develops within a membrane-bound vacuole called an inclusion. Throughout its developmental cycle, Chlamydia modifies the inclusion membrane (IM) with type III secreted (T3S) membrane proteins, known as inclusion membrane proteins (Incs). Via the IM, Chlamydia manipulates the host cell to acquire lipids and nutrients necessary for its growth. One key nutrient is tryptophan (Trp). As a Trp auxotroph, Chlamydia is very sensitive to Trp starvation and, in response to low Trp levels induced by the immune response, enters a viable but nonreplicating state called persistence. To maintain viability during persistence, Chlamydia must necessarily maintain both the integrity of the IM and its ability to modify host cell responses, but how Trp starvation affects IM composition and subsequent interactions with the host cell remains poorly understood. We hypothesize that, under Trp starvation conditions, Inc expression/stability or T3S function during persistence alters IM composition but that key host-Chlamydia interactions will be preserved. To examine host-Chlamydia interactions during persistence, we examined sphingomyelin, cholesterol, and transferrin trafficking to the inclusion, as well as localization of host proteins that bind to specific Incs. We identified IM composition changes during persistence by monitoring endogenous Inc abundance at the IM. Chlamydial T3S is generally functional during persistence. Specific changes in Inc composition in the IM can be linked to Trp content of a specific Inc or effector-specific defects in chlamydial T3S. Overall, our findings reveal that critical host-Chlamydia interactions are maintained during persistence mediated by Trp starvation.

microbiology↗

bioRxiv2019

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

As an obligate intracellular pathogenic bacterium, C. trachomatis develops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. An in vivo understanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 in Chlamydia trachomatis L2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.\n\nAuthor summaryMany intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound \"bacteria containing vacuole\" (BCV) that, in most cases, prevents association with the lysosome. 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. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane site in vivo in the context of infection.

microbiology↗