Abstract
The pathogenesis of brucellosis depends on the ability of bacteria from the genus Brucella to invade and replicate within animal cells. To understand the molecular pathways used by Brucella spp. to reach its intracellular niche, robust and reproducible bacteria purification protocols that provide enough material for biochemical and molecular biology studies are essential. Here, we describe a detailed methodology designed to extract and purify viable brucellae from mammalian host cells at different time periods of their intracellular cycle. The yield of proteins and nucleic acids is sufficient to perform immunochemical analysis, genetic studies, transcriptomics, and proteomics among others.
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Acknowledgements
Plasmid pJC45 encoding for GFP under the control of the tetracycline-inducible promoter tetA was kindly provided by Jean Celli (Rocky Mountain Laboratories, USA) and anti-Omp19 monoclonal antibodies were kindly provided by Axel Cloeckaert (Institut National de la Recherche Agronomique, France). We thank Reinaldo Pereira for his technical assistance. This work was supported by grants from the International Center for Genomic Engineering and Biotechnology (contract CRP/12/007) to E.C.O., Fondos de Recursos del Sistema FEES/CONARE, Netropica grant 8-R-2008 to CGV and Vicerrectoría de Investigación, Universidad de Costa Rica. PAS received a training fellowship from CIEMIC, Universidad de Costa Rica. This work was done as part of the UCR/DAAD Humboldt Fellow award 2012 to E.M.
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Chaves-Olarte, E., Altamirano-Silva, P., Guzmán-Verri, C., Moreno, E. (2014). Purification of Intracellular Bacteria: Isolation of Viable Brucella abortus from Host Cells. 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_14
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DOI: https://doi.org/10.1007/978-1-4939-1261-2_14
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