Mode of action of Yersinia enterocolitica "outer protein" T (Yop T)

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The pathogenicity of Yersinia enterocolitica and Yersina pseudotuberculosis depends on a 70 kb virulence plasmid pYV, which encodes a complex type-III secretion machinery (YsC) as well as several Yop (Yersinia outer proteins) effector proteins. One of the effector proteins is YopT, which is directly delivered into the target cell by type-III secretion. In eukaryotic cells, YopT causes modification of Rho GTPases and thereby redistribution of the actin cytoskeleton. YopT is a cysteine protease, which cleaves Rho proteins in front of its C-terminal cysteine together with the bound isoprene groups. Thus the GTPase is released from membranes. The overall objective of this research is to study the kinetics of YopT and to analyze the substrate specificity and structural requirements for recognition by YopT. Moreover, we want to give a precise structure-function analyses of the protease. To achieve this goal, it will be necessary (i) to characterize the protease activity of YopT biochemically and functionally, and to understand how the protease activity is regulated. Therefore, we plan to establish a quantitative in vitro assay using isoprenylated and methylated peptides and HPLC analysis. (ii) to study the substrate specificity and the resulting biological consequences. In a first step we want to incubate peptides with YopT and analyze the cleavage by MALDI-TOF mass spectrometry. Proceeding from these data, we plan to study identified substrates in vivo and to analyzed the pathways affected. (iii) to determine crucial structural features of YopT and to identify the minimal essentiel structure for protease activity in vitro and in vivo, (iv) to clarify localization and interactions of YopT subsequent translocation into the target cells. (v) it is planned to study the influence of YopT on Rho GTPase dependent activation of transcription.