title: Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii.
abstract: Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based β-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.
authors.affiliation: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College Station, TX 77843, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Simran
authors.initials: S
authors.lastname: Banga
authors.forename: Katja
authors.initials: K
authors.lastname: Mertens
authors.forename: Mary M
authors.initials: MM
authors.lastname: Weber
authors.forename: Ivana
authors.initials: I
authors.lastname: Gorbaslieva
authors.forename: Yunhao
authors.initials: Y
authors.lastname: Tan
authors.forename: Zhao-Qing
authors.initials: ZQ
authors.lastname: Luo
authors.forename: James E
authors.initials: JE
authors.lastname: Samuel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000502:physiology*; D019295:Computational Biology; D016997:Coxiella burnetii / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D016680:Genome, Bacterial; D016952:Legionella pneumophila / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D021381:Protein Transport / Q000502:physiology*; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems