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.
Highlights
Chlamydia trachomatis is an obligate intracellular bacterium that infects mucosal epithelial cells of the endocervix and conjunctiva
We applied this system to identify by mass spectrometry the major host and chlamydial proteins recruited to early, mid, and late stage inclusion membranes of C. trachomatis infected cells
Functional testing through protein knockdown and pharmacological inhibition demonstrated that interactions between early C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES) are critical for promoting the developmental growth of Chlamydia
Summary
Chlamydia trachomatis is an obligate intracellular bacterium that infects mucosal epithelial cells of the endocervix and conjunctiva. It infects millions of people every year and is the etiological agent of ocular trachoma [1,2]. Chlamydial growth within host cells is critically dependent on recruitment of host proteins to the inclusion membrane early during infection, and on extracting nutrients from the host cell. The streamlined genome of Chlamydia necessitates a dependency on the host cell for many nutrients, yet some of these molecules cannot freely permeate the inclusion membrane and import systems have not been identified [7]. While much is known about chlamydial manipulation of host signaling, very little is known about the molecular processes necessary for Chlamydia to obtain nutrients from the host [6,8]
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