Characterization of interactions of the Type IV secretion system core component VirB8

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

<p> Type IV secretion systems (T4SS) are essential for the virulence of many gram-negative \npathogens. The systems studied here comprise eleven VirB proteins in case of \nAgrobacterium tumefaciens and twelve in case of Brucella suis. The VirB proteins \nassociate in the cell envelope and form a complex that mediates the translocation of \nvirulence factors into host cells. In this report, VirB8, a core component of T4SS, is \ncharacterized with regards to its interaction with itself and with other VirB proteins. </p> <p> VirB8 was found to exist in monomer-dimer equilibrium and the self-association \nwas demonstrated by analytical ultracentrifugation, analytical gel filtration, surface \nplasmon resonance and bacterial two-hybrid assay. The above experiments demonstrated \nthat residues M102, Y105 and E214 o fVirB8 from B. suis are involved in self-association \nand mutagenesis of these residues led to the impairment of T4SS function in B. suis. \nFurthermore, this information was utilized to unravel the contribution of VirB8 self-association \ntowards T4SS assembly and function. To this end dimerization variants of \nVirB8 from Agrobacterium tumefaciens were created and the effects were assessed with \npurified proteins in vitro. Following this, the effects of VirB8 dimer site changes were \nassessed in vivo. Introduction of a cysteine residue at the predicted interface (V97C) \nsupported DNA transfer but not T-pilus formation. Variants that reduced the self-association \ndid not support T4SS functions and T-pilus formation. Moreover, VirB2- \nVirB5 co-fractionated with high molecular mass components from membranes of A. \ntumefaciens and VirB8 dimerization was shown to be necessary for VirB2 association \nwith the high molecular mass components. Using purified VirB8 and VirB5 it was shown that VirB5 interacts with VirB8 via its globular domain and this interaction dissociates \nVirB8 dimers. Taking these results together, a mechanistic contribution of VirB8 \ndimerization to T4SS assembly was proposed. </p> <p> Next, the interactions of VirB8 with other core components (VirB9 and VirBlO) \nwere analyzed by using various in vitro and in vivo experiments. Purified soluble \nperiplasmic domains of VirB8, VirB9 and VirB10 were used in enzyme-linked \nimmunosorbent assays, circular dichroism, and surface plasmon resonance experiments. \nThe pair-wise interactions and self-association of VirB8, VirB9 and VirB 10 were \ndemonstrated with the in vitro experiments. In addition, a ternary complex formation \nbetween VirB8, VirB9, and VirBlO was identified. Using the bacterial two-hybrid \nsystem, the dynamics of the interactions between VirB8-VirB9-VirB 10 full-length \nproteins were analyzed demonstrating that VirB9 stimulates VirB8 self-association, but \nthat it inhibits the VirB10-VirB10 as well as the VirB8-VirB10 interaction. Based on \nthese results, a dynamic model for secretion system assembly is proposed where VirB8 \nplays a role as an assembly factor that is not closely associated with the functional core \ncomplex comprising VirB9 and VirB10. </p> <p> The work reported in this thesis advances the understanding of VirB8 self-association \nand its contribution to T4SS assembly and function. Furthermore, the \nestablishment of the bacterial two-hybrid system to detect VirB interactions has helped \nidentify inhibitors for the VirB8 dimerization through collaboration with Dr. Athanasios \nPaschos. Moreover, techniques such as ELISA, analytical ultracentrifugation, circular dichroism and surface plasmon resonance will be utilized routinely to characterize other \nVirB-VirB interactions in future. </p>