title: A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis.
abstract: Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.
authors: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America; Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.
authors: Hayley J
authors: HJ
authors: Newton
authors: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Lara J
authors: LJ
authors: Kohler
authors: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Justin A
authors: JA
authors: McDonough
authors: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Morayma
authors: M
authors: Temoche-Diaz
authors: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Emerson
authors: E
authors: Crabill
authors: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.
authors: Elizabeth L
authors: EL
authors: Hartland
authors: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Craig R
authors: CR
authors: Roy
mesh_terms: D001343:Autophagy; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000235:genetics; D016997:Coxiella burnetii / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D004251:DNA Transposable Elements / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006367:HeLa Cells; D054884:Host-Pathogen Interactions / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D009154:Mutation / Q000235:genetics*; D010588:Phagosomes / Q000378:metabolism; D011778:Q Fever / Q000378:metabolism* / Q000382:microbiology; D014617:Vacuoles / Q000378:metabolism* / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D004251:DNA Transposable Elements