Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites

Abstract

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia –host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX2 system of proximity-dependent biotinylation. APEX2 is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C . trachomatis to express the enzyme APEX2 fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX2 labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C . trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX2 is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

A renewed tool kit to explore Chlamydia pathogenesis: from molecular genetics to new infection models

“…The ability to express exogenous proteins, epitope tags, and fluorescent and other reporter proteins in Chlamydia has also expanded the repertoire of possible technologies that can be applied to study the Chlamydia –host interface 26, 27 . For instance, Incs fused to enzymes that enable the biotinylation of proteins identified host factors that are in proximity to the inclusion membrane by affinity capture of biotinylated proteins coupled with tandem mass spectrometry ( Figure 1E) 28, 29 .…”

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

“…trachomatis infection (30). APEX2 has also recently been utilized by others in the field (with noted differences described in Discussion [31]). APEX2, a mutated soybean peroxidase (32, 33), can be fused to a protein of interest and activated during a short (1-min) reaction to covalently modify proximal proteins with a biotin molecule (33).…”

Contributions of Mass Spectrometry-Based Proteomics to Understanding Salmonella-Host Interactions

“…Lately, the Ting group further enhanced the labeling efficiency of BioID via directed evolution of the biotin ligase (now named TurboID) [ 56 ]. Thus far, proximity-labeling methods have contributed the discovery of several novel protein-protein interactions at the host-pathogen interface [ 57 , 58 , 59 ]. Last but not least, chemical crosslinking in principle can stabilize protein complexes by introducing covalent interactions.…”