Structural Studies of Chaperones and Chaperone-Tip interactions from the type III secretion systems of Yersinia and Pseudomonas

Abstract

Many Gram-negative bacteria assemble a complex nanomachine, the type III secretion system (T3SS) to inject virulence proteins directly into eukaryotic cells to initiate infection. The T3SS is composed of structural and non-structural proteins. The structural component of the T3SS is a needle apparatus composed of a membrane embedded basal structure, an external needle, a tip complex that caps the needle and a translocon. Chaperones are small cytoplasmic proteins that assist in the assembly and proper operation of the T3SS. They serve as important regulators of secretion and sequester effector proteins and other structural components while inside the bacterial cytosol. Yersinia pestis is the causative agent of plague that in the past has caused worldwide pandemics, and is considered a potential agent of bioterrorism. Pseudomonas aeruginosa is an opportunistic pathogen that is most frequently associated with nosocomial infections of immuno-compromised patients. A key factor in the pathogenesis of Y. pestis and P. aeruginosa is that they harbor the T3SS, which enables them to inject virulent effector proteins that hijack the host cellular machinery and facilitate the survival of these bacteria inside the host cells. An important component of the Y. pestis and the P. aeruginosa T3SS is the LcrG/PcrG family of chaperone proteins, which form a complex with the T3SS tip proteins LcrV and PcrV. They also function as important regulators of effector secretion and facilitate the translocation of effector proteins into the targeted host cells. The atomic structures of members of the LcrG/PcrG family of chaperone proteins are currently unknown. Further, how they interact with their cognate tip proteins is poorly understood. Here, I show by CD and NMR spectroscopy that the chaperones, LcrG and PcrG, lack compact tertiary structures. However, they are not completely disordered but contain secondary structure dominated by at least two major α-helices from residues Val-16 to Gly-41 and Glu-55 to Ser-76 in PcrG, and residues Asp-7 to Gly-38 and Ala-59 to Arg-73 in LcrG. NMR backbone dynamics studies show that the helices in the chaperones have semi-rigid flexibility and they tumble as a single entity with similar backbone dynamics. Thus the LcrG/PcrG family of T3SS chaperone proteins is partially folded. The conformational dynamics perhaps contribute to their functional versatility, which is a characteristic signature of many flexible proteins. Overall, the LcrG/PcrG family of proteins can be added to a growing list of partially folded proteins that play important roles in the bacterial type III secretion systems. The NMR titration data indicate that the tip proteins induce a global change in the overall structure of the chaperones upon complex formation. Further, the C-terminus of LcrG (Ser-52 to Ile-67) was identified to be important in blocking the secretion of Yop effectors in the Y. pestis T3SS. Also described here is a crystal structure of LcrV, at a higher resolution (1.65 Å) than the previously described structure 1R6F. This current structure contains native residues in the N-terminal domain of LcrV as opposed to the original crystal structure, 1R6F, which was solved using triple mutations (K40A/D41A/K42A) in the N-terminal domain. The current structure should prove useful in future studies of the LcrV-translocon interaction. The work presented in this dissertation provides the first structural insight into the conformation and dynamics of the LcrG/PcrG family of chaperones in solution and upon complex formation. This has enhanced our understanding of the mechanism by which these chaperones perform their multiple roles in regulating the type III secretion system.