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, 14-3-3beta, was identified that interacted specifically with IncG. The interaction between 14-3-3beta and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP-14-3-3beta 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 14-3-3beta via a conserved 14-3-3-binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14-3-3beta interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14-3-3beta is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.
Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, National Institutes of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT 59840, USA.
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Scidmore
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Hackstadt
D048948:14-3-3 Proteins; D001665:Binding Sites; D002692:Chlamydia trachomatis / Q000378:metabolism*; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013045:Species Specificity; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C482982:YWHAB protein, human; D012694:Serine; D014446:Tyrosine 3-Monooxygenase