Inclusion Membrane Growth and Composition Are Altered by Overexpression of Specific Inclusion Membrane Proteins in Chlamydia trachomatis L2.

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 laboratories 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 are 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.

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

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 (Stx10), 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*-Stx10 fusion constructs to use the BioID proximity labeling system. While we identified a novel eukaryotic protein-protein interaction between Stx10 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 Stx10 during infection with Chlamydia trachomatis serovars L2 and D and Coxiella 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 Stx10, it is not a reliable methodology to examine intracellular trafficking pathway dynamics during infection with intracellular pathogens.