title: Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems.
abstract: Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium.
  
  
  OBJECTIVE
  Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA JGillespie@som.umaryland.edu arto.pulliainen@utu.fi.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Gillespie
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease, Seattle, Washington, USA The Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), Seattle, Washington, USA.
authors.forename: Isabelle Q H
authors.initials: IQ
authors.lastname: Phan
authors.affiliation: Venom Evolution Lab, School of Biological Sciences, University of Queensland, St. Lucia, Queensland, Australia.
authors.forename: Holger
authors.initials: H
authors.lastname: Scheib
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease, Seattle, Washington, USA The Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), Seattle, Washington, USA.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Subramanian
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease, Seattle, Washington, USA Beryllium Discovery Corp., Bainbridge Island, Washington, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Edwards
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Lehman
authors.affiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Hanna
authors.initials: H
authors.lastname: Piitulainen
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: M Sayeedur
authors.initials: MS
authors.lastname: Rahman
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Rennoll-Bankert
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease, Seattle, Washington, USA The Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), Seattle, Washington, USA.
authors.forename: Bart L
authors.initials: BL
authors.lastname: Staker
authors.affiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Suvi
authors.initials: S
authors.lastname: Taira
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease, Seattle, Washington, USA The Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), Seattle, Washington, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Stacy
authors.affiliation: Seattle Structural Genomics Center for Infectious Disease, Seattle, Washington, USA The Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), Seattle, Washington, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Myler
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
authors.forename: Abdu F
authors.initials: AF
authors.lastname: Azad
authors.affiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland Institute of Biomedicine, University of Turku, Turku, Finland JGillespie@som.umaryland.edu arto.pulliainen@utu.fi.
authors.forename: Arto T
authors.initials: AT
authors.lastname: Pulliainen
mesh_terms: D001473:Bartonella / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012286:Rickettsia typhi / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D000069319:Type IV Secretion Systems / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000069319:Type IV Secretion Systems