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 are 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 interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.
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Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Paris Cedex 15, France.
Fromont-Racine
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Sansonetti
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Legrain
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Parsot
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D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D018832:Molecular Chaperones / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012763:Shigella flexneri / Q000737:chemistry / Q000378:metabolism* / Q000472:pathogenicity; D020798:Two-Hybrid System Techniques
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C063664:IpaA protein, Shigella flexneri; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C087447:ipaB protein, Shigella; C092342:IpaC protein, Shigella