Mammalian 14‐3‐3β associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG

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Abstract
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.