Shifting proteomes: limitations in using the BioID proximity labeling system to study SNARE protein trafficking during infection with intracellular pathogens

Abstract

Abstract We hypothesize that intracellular trafficking pathways are altered in chlamydial infected cells to maximize the ability of Chlamydia to scavenge nutrients while not overtly stressing the host cell. Previous data demonstrated the importance of two eukaryotic SNARE proteins, VAMP4 and syntaxin 10 (St × 10), in chlamydial growth and development. Although, the mechanism for these effects is still unknown. To interrogate whether chlamydial infection altered these proteins' networks, we created BirA*-VAMP4 and BirA*-St × 10 fusion constructs to use the BioID proximity labeling system. While we identified a novel eukaryotic protein-protein interaction between St × 10 and VAPB, we also identified caveats in using the BioID system to study the impact of infection by an obligate intracellular pathogen on SNARE protein networks. The addition of the BirA* altered the localization of VAMP4 and St × 10 during infection with C. trachomatis serovars L2 and D and C. burnetii Nine Mile Phase II. We also discovered that BirA* traffics to and biotinylates Coxiella-containing vacuoles and, in general, has a propensity for labeling membrane or membrane-associated proteins. While the BioID system identified a novel association for St × 10, it is not a reliable methodology to examine intracellular trafficking pathway dynamics during infection with intracellular pathogens.