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.