title: Quantitative analysis of VirB8-VirB9-VirB10 interactions provides a dynamic model of type IV secretion system core complex assembly.
abstract: Type IV secretion systems are multiprotein complexes that translocate macromolecules across the bacterial cell envelope. The type IV secretion system in Brucella species encodes 12 VirB proteins that permit this pathogen to translocate effectors into mammalian cells, where they contribute to its survival inside the host. The "core" complex proteins are conserved in all type IV secretion systems, and they are believed to form the channel for substrate translocation. We have investigated the in vitro interactions between the soluble periplasmic domains of three of these VirB components, VirB8, VirB9, and VirB10, using enzyme-linked immunosorbent assays, circular dichroism, and surface plasmon resonance techniques. The in vitro experiments helped in the quantification of the self-association and binary interactions of VirB8, VirB9, and VirB10. Individually, distinct binding properties were revealed that may explain their biological functions, and collectively, we provide direct evidence of the in vitro formation of the VirB8-VirB9-VirB10 ternary complex. To assess the dynamics of these interactions in a simplified in vivo model of complex assembly, we applied the bacterial two-hybrid system in studying interactions between the full-length proteins. This approach demonstrated that VirB9 stimulates the self-association of VirB8 but inhibits VirB10-VirB10 and VirB8-VirB10 interaction. Analysis of a dimerization site variant of VirB8 (VirB8(M102R)) suggested that the interactions with VirB9 and VirB10 are independent of its self-association, which stabilizes VirB8 in this model assay. We propose a dynamic model for secretion system assembly in which VirB8 plays a role as an assembly factor that is not closely associated with the functional core complex comprising VirB9 and VirB10.
authors.affiliation: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada.
authors.forename: Durga
authors.initials: D
authors.lastname: Sivanesan
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hancock
authors.forename: Ana María
authors.initials: AM
authors.lastname: Villamil Giraldo
authors.forename: Christian
authors.initials: C
authors.lastname: Baron
mesh_terms: D041021:Brucella suis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay