Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri

The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that a re secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the inter action partners of the Shigella flexneri effectors and chaperones. Librarie s of preys containing random fusions with fragments of the TTS proteins wer e screened using effectors and chaperones as baits. Interactions between th e effectors IpaB and lpaC and their chaperone IpgC were detected by this me thod, and interaction domains were identified. Using a His-tagged IpgC prot ein to co-purify truncated IpaB and lpaC proteins, we showed that the chape rone-binding domain was unique and located in the N-terminus of these prote ins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of lpaC and instability of IpaB. Homotyp ic interactions were identified with the baits IpaA, IpaB and IpaC. Interac tions between effectors and components of the TTS machinery were also selec ted that might give insights into regulation of the TTS process.