Abstract
Coxiella burnetii, the agent of human Q fever, is a zoonotic bacterial pathogen with a worldwide distribution. Owing to an historic lack of methods for genetic manipulation, virulence factors deployed by this bacterium for disease pathogenesis are poorly understood. However, the recent advance of host cell-free (axenic) growth of C. burnetii has coincided with development of several new genetic technologies including site-specific and random transposon systems, shuttle vectors, and an inducible gene expression system. We have recently added two methods for targeted gene inactivation to the expanding C. burnetii genetics toolbox. Here, we describe a “loop in/loop out” gene inactivation system for C. burnetii. This procedure allows for generation of site-directed mutants in approximately 10 weeks and has been used by our laboratory to generate more than 50 individual C. burnetii mutants. The collection of C. burnetii genetic tools now allows for conventional mutation and complementation strategies to define virulence factors.
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References
Maurin M, Raoult D (1999) Q fever. Clin Microbiol Rev 12:518–553
Voth DE, Heinzen RA (2007) Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii. Cell Microbiol 9:829–840
Howe D, Shannon JG, Winfree S, Dorward DW, Heinzen RA (2010) Coxiella burnetii phase I and II variants replicate with similar kinetics in degradative phagolysosome-like compartments of human macrophages. Infect Immun 78: 3465–3474
Voth DE, Howe D, Heinzen RA (2007) Coxiella burnetii inhibits apoptosis in human THP-1 cells and monkey primary alveolar macrophages. Infect Immun 75: 4263–4271
Luhrmann A, Roy CR (2007) Coxiella burnetii inhibits activation of host cell apoptosis through a mechanism that involves preventing cytochrome c release from mitochondria. Infect Immun 75:5282–5289
Voth DE, Broederdorf LJ, Graham JG (2012) Bacterial type IV secretion systems: versatile virulence machines. Future Microbiol 7: 241–257
Voth DE, Howe D, Beare PA, Vogel JP, Unsworth N, Samuel JE, Heinzen RA (2009) The Coxiella burnetii ankyrin repeat domain-containing protein family is heterogeneous, with C-terminal truncations that influence Dot/Icm-mediated secretion. J Bacteriol 191: 4232–4242
Chen C, Banga S, Mertens K, Weber MM, Gorbaslieva I, Tan Y, Luo ZQ, Samuel JE (2010) Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii. Proc Natl Acad Sci U S A 107:21755–21760
Carey KL, Newton HJ, Luhrmann A, Roy CR (2011) The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication. PLoS Pathog 7: e1002056
Voth DE, Beare PA, Howe D, Sharma UM, Samoilis G, Cockrell DC, Omsland A, Heinzen RA (2011) The Coxiella burnetii cryptic plasmid is enriched in genes encoding type IV secretion system substrates. J Bacteriol 193: 1493–1503
Luhrmann A, Nogueira CV, Carey KL, Roy CR (2010) Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein. Proc Natl Acad Sci U S A 107: 18997–19001
Klingenbeck L, Eckart RA, Berens C, Luhrmann A (2012) The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level. Cell Microbiol 15:675–687
Beare PA, Gilk SD, Larson CL, Hill J, Stead CM, Omsland A, Cockrell DC, Howe D, Voth DE, Heinzen RA (2011) Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages. Mbio 2:e00175–11
Beare PA, Howe D, Cockrell DC, Omsland A, Hansen B, Heinzen RA (2009) Characterization of a Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis. J Bacteriol 191:1369–1381
Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA (2009) Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A 106:4430–4434
Omsland A, Beare PA, Hill J, Cockrell DC, Howe D, Hansen B, Samuel JE, Heinzen RA (2011) Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium. Appl Environ Microbiol 77:3720–3725
Beare PA, Sandoz KM, Omsland A, Rockey DD, Heinzen RA (2011) Advances in genetic manipulation of obligate intracellular bacterial pathogens. Front Microbiol 2:97
Beare PA, Larson CL, Gilk SD, Heinzen RA (2012) Two systems for targeted gene deletion in Coxiella burnetii. Appl Environ Microbiol 78:4580–4589
Hackstadt T (1996) Biosafety concerns and Coxiella burnetii. Trends Microbiol 4:341–342
Acknowledgement
This study was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
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Beare, P.A., Heinzen, R.A. (2014). Gene Inactivation in Coxiella burnetii . In: Vergunst, A., O'Callaghan, D. (eds) Host-Bacteria Interactions. Methods in Molecular Biology, vol 1197. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1261-2_19
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DOI: https://doi.org/10.1007/978-1-4939-1261-2_19
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