Mammalian 14-3-3beta associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG.

Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles. We have recently identified four chlamydial inclusion membrane proteins, IncD–G, whose expression is detected within the first 2 h after internalization. To gain a better understanding of how these Inc proteins function, a yeast two-hybrid screen was employed to identify interacting host proteins. One protein, 14-3-3β, was identified that interacted specifically with IncG. The interaction between 14-3-3β and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP-14-3-3β fusion protein. 14-3-3 proteins are phosphoserine-binding proteins. Immunoprecipitation studies with [32P]-orthophosphate-labelled cells demonstrated that IncG is phosphorylated in both chlamydia-infected HeLa cells and in yeast cells expressing IncG. Site-directed mutagenesis of predicted 14-3-3 phosphorylation sites demonstrated that IncG binds to 14-3-3β via a conserved 14-3-3-binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14-3-3β interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14-3-3β is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.