A plasmid-encoded Type III Secretion System (T3SS) is employed by human pathogenic yersiniae to inject effector proteins, termed Yops, directly into host cells. The secretion of Yops is tightly regulated, and occurs only upon contact with a eukaryotic cell in vivo or in media devoid of calcium in vitro. A complex containing the secreted protein YopN, its heterodimeric chaperone SycN/YscB, and TyeA is required to prevent secretion of effector Yops until the appropriate secretion-triggering signals are encountered. The mechanism by which these proteins regulate the T3S process is unknown. A mutational analysis of YopN and TyeA was performed to identify regions and residues of these proteins that are required to regulate Yop secretion. Amino-acid residues of TyeA were identified that were specifically required for the interaction of TyeA with YopN, confirming that the YopN/TyeA interaction is essential for the regulation of Yop secretion. Furthermore, analysis of TyeA mutants identified a surface-exposed region that was critical for the regulation of Yop secretion, but not required for interaction with YopN. YopN residues critical for the regulation of secretion clustered within the N- and C-terminal regions of YopN that were required to interact with the SycN/YscB chaperone and TyeA, respectively. No residues critical for the regulation of secretion were identified in the central region of YopN, suggesting that this region acts primarily to maintain proper positioning of the functional N- and C-terminal regions of this complex. A novel role for the chaperone binding domain (CBD) of YopN in the regulation of Yop secretion was identified. This role was separate from its role in binding the SycN/YscB chaperone and targeting YopN for secretion. Finally, it was demonstrated that the SycN/YscB chaperone is dispensable for the regulation of secretion if the expression of both YopN and TyeA is increased, indicating that these chaperones have no direct role in the regulation of Yop secretion. These results indicate that the YopN secretion signal and SycN/YscB chaperone function to efficiently target the YopN/TyeA complex to the T3S apparatus, whereas the YopN CBD and C-terminal region of YopN complexed with TyeA mediate the block in Yop secretion.
| Identifer | oai:union.ndltd.org:UMIAMI/oai:scholarlyrepository.miami.edu:oa_dissertations-1311 |
| Date | 19 November 2009 |
| Creators | Joseph, Sabrina S. |
| Publisher | Scholarly Repository |
| Source Sets | University of Miami |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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