The type III secretory system is conserved in a large number of Gram-negative pathogenic bacteria. This system is dedicated to the export of virulence-related effector proteins that have a variety of targets in mammalian cells. The bacterial pathogen Yersinia pseudotuberculosis translocates the effector protein YopE, along with several other proteins, into macrophages upon host cell contact. The 23 kD YopE is required for virulence and acts as a Rho-specific GTPase activating protein (GAP) to interrupt actin polymerization in the macrophage. Rho proteins are key regulators of the actin cytoskeleton and also play a role in transcriptional regulation. The activity of YopE prevents Y. pseudotuberculosis from being phagocytosed and allows the bacterium to survive and proliferate within the lymphatic spaces. The goal of my research is to gain structural and biochemical insight into how YopE is delivered directly into the host cell cytosol.

I have discovered that YopE is rescued from aggregation by the co-expression of its specific bacterial chaperone, SycE. YopE and SycE are tightly associated and the complex was co-purified from protein over-expressed in E. coli. The complex was determined to be a heterotrimer of two chaperone molecules bound to one molecule of YopE using gel filtration, chemical cross-linking and analytical ultracentrifugation techniques. We have also found that YopE retains its GAP activity while in complex with SycE as determined by Rho GAP activity assays performed with the purified complex. I have solved the X-ray crystal structure of a proteolyzed YopE-SycE complex as well as the structure of the chaperone by itself with the aim of understanding the structural relationship between the two proteins. It has been shown previously that SycE stays behind in the bacterial cytosol upon secretion of YopE. It remains to be determined how the effector and chaperone proteins are separated and how YopE is stabilized within the host cell to retain its GAP activity.

PUBLICATIONS (resulting from this training)

Birtalan S, Ghosh P. (2001) Structure of the Yersinia type III secretory system chaperone SycE. Nat Struct Biol. 8:974-8.

Birtalan SC, Phillips RM, Ghosh P. (2002) Three-dimensional secretion signals in chaperone-effector complexes of bacterial pathogens. Mol Cell. 9:971-80.