title: Cell-free propagation of Coxiella burnetii does not affect its relative virulence.
abstract: Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.
authors: Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands; Host Microbe Interactomics, Wageningen University, Wageningen, The Netherlands.
authors: Runa
authors: R
authors: Kuley
authors: Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands.
authors: Hilde E
authors: HE
authors: Smith
authors: Bundeswehr Institute of Microbiology, Munich, Germany.
authors: Dimitrios
authors: D
authors: Frangoulidis
authors: Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands; Host Microbe Interactomics, Wageningen University, Wageningen, The Netherlands.
authors: Mari A
authors: MA
authors: Smits
authors: Department of Bacteriology and TSEs, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands.
authors: Hendrik I
authors: HI
authors: Jan Roest
authors: Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands.
authors: Alex
authors: A
authors: Bossers
mesh_terms: D000818:Animals; D001386:Azides / Q000378:metabolism; D001431:Bacteriological Techniques / Q000379:methods*; D001681:Biological Assay; D016997:Coxiella burnetii / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D017353:Gene Deletion; D018628:Gene Dosage; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008070:Lipopolysaccharides / Q000235:genetics; D051379:Mice; D050296:Microbial Viability / Q000235:genetics; D011419:Propidium / Q000031:analogs & derivatives / Q000378:metabolism; D011778:Q Fever / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014774:Virulence / Q000235:genetics
chemical_list: D001386:Azides; D008070:Lipopolysaccharides; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium