A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence determinant. Transcription of the T3SS is highly regulated and intimately coupled to the activity of the type III secretion channel. The secretion channel is generally closed, and transcription is repressed. Inducing signals such as calcium depletion, however, open the secretion channel and derepress transcription of the USS. The coupling of transcription with secretion requires three previously identified cytoplasmic regulatory proteins. ExsA is a DNA-binding protein required for transcriptional activation of the entire T3SS. The second regulatory protein, ExsD, functions as anti-activator by directly binding to ExsA. Finally, ExsC functions as an anti-anti-activator by directly binding to and inhibiting ExsD. Although the regulatory roles of ExsC, ExsD, and ExsA were defined through these previous studies, the mechanism of coupling transcription to secretion was unclear. We now report the identification of ExsE as a secreted regulator of the T3SS and provide evidence that ExsE functions as a direct inhibitor of ExsC. When the secretion channel is closed, ExsE is complexed with ExsC in the cytoplasm, and transcription of the T3SS is repressed by sequestration of ExsA by ExsD. We propose that the secretion of ExsE provides an initiating signal that results in an equilibrium shift whereby ExsC becomes preferentially bound to ExsD, thus allowing liberated ExsA to activate transcription of the T3SS. The presence of ExsE homologs in the T3SSs of other bacterial species suggests that this mechanism of coupling transcription to secretion may be commonly used.