Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis
The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.
Source Type:Doctoral Dissertation
Keywords:NATURAL SCIENCES; Chemistry; Biochemistry; Molecular biology; Yersinia pseudotuberculosis; T3SS; YopD; translocation process; LcrH; class II chaperone; substrate secretion pathway specificity
Date of Publication:01/01/2007