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Large-scale production and isolation of Candida biofilm extracellular matrix

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Abstract

The extracellular matrix of biofilm is unique to the biofilm lifestyle, and it has key roles in community survival. A complete understanding of the biochemical nature of the matrix is integral to the understanding of the roles of matrix components. This knowledge is a first step toward the development of novel therapeutics and diagnostics to address persistent biofilm infections. Many of the assay methods needed for refined matrix composition analysis require milligram amounts of material that is separated from the cellular components of these complex communities. The protocol described here explains the large-scale production and isolation of the Candida biofilm extracellular matrix. To our knowledge, the proposed procedure is the only currently available approach in the field that yields milligram amounts of biofilm matrix. This procedure first requires biofilms to be seeded in large-surface-area roller bottles, followed by cell adhesion and biofilm maturation during continuous movement of the medium across the surface of the rotating bottle. The formed matrix is then separated from the entire biomass using sonication, which efficiently removes the matrix without perturbing the fungal cell wall. Subsequent filtration, dialysis and lyophilization steps result in a purified matrix product sufficient for biochemical, structural and functional assays. The overall protocol takes ∼11 d to complete. This protocol has been used for Candida species, but, using the troubleshooting guide provided, it could be adapted for other fungi or bacteria.

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Figure 1: Large-scale biofilm matrix production and isolation protocol.
Figure 2: Impact of the matrix isolation process on Candida biofilm and planktonic cells.
Figure 3

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References

  1. Kojic, E.M. & Darouiche, R.O. Candida infections of medical devices. Clin. Microbiol. Rev. 17, 255–267 (2004).

    Article  Google Scholar 

  2. O'Toole, G., Kaplan, H.B. & Kolter, R. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54, 49–79 (2000).

    Article  CAS  Google Scholar 

  3. Costerton, J.W., Stewart, P.S. & Greenberg, E.P. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999).

    Article  CAS  Google Scholar 

  4. Fanning, S. & Mitchell, A.P. Fungal biofilms. PLoS Pathog. 8, e1002585 (2012).

    Article  CAS  Google Scholar 

  5. Flemming, H.C. & Wingender, J. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633 (2010).

    Article  CAS  Google Scholar 

  6. Branda, S.S., Vik, S., Friedman, L. & Kolter, R. Biofilms: the matrix revisited. Trends Microbiol. 13, 20–26 (2005).

    Article  CAS  Google Scholar 

  7. Nett, J.E . et al. Host contributions to construction of three device-associated Candida albicans biofilms. Infect. Immun. 83, 4630–4638 (2015).

    Article  CAS  Google Scholar 

  8. Jabra-Rizk, M.A., Falkler, W.A. & Meiller, T.F. Fungal biofilms and drug resistance. Emerg. Infect. Dis. 10, 14–19 (2004).

    Article  CAS  Google Scholar 

  9. Vediyappan, G., Rossignol, T. & d'Enfert, C. Interaction of Candida albicans biofilms with antifungals: transcriptional response and binding of antifungals to beta-glucans. Antimicrob. Agents Chemother. 54, 2096–2111 (2010).

    Article  CAS  Google Scholar 

  10. Baillie, G.S. & Douglas, L.J. Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents. Antimicrob. Agents Chemother. 42, 1900–1905 (1998).

    Article  CAS  Google Scholar 

  11. Baillie, G.S. & Douglas, L.J. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J. Antimicrob. Chemother. 46, 397–403 (2000).

    Article  CAS  Google Scholar 

  12. Beauvais, A . et al. An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus. Cell. Microbiol. 9, 1588–1600 (2007).

    Article  CAS  Google Scholar 

  13. Branda, S.S., Chu, F., Kearns, D.B., Losick, R. & Kolter, R. A major protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 59, 1229–1238 (2006).

    Article  CAS  Google Scholar 

  14. Zarnowski, R . et al. Novel entries in a fungal biofilm matrix encyclopedia. MBio 5, e01333–01314 (2014).

    Article  CAS  Google Scholar 

  15. O'Toole, G.A. To build a biofilm. J. Bacteriol. 185, 2687–2689 (2003).

    Article  CAS  Google Scholar 

  16. Al-Fattani, M.A. & Douglas, L.J. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J. Med. Microbiol. 55, 999–1008 (2006).

    Article  CAS  Google Scholar 

  17. Thomas, D.P., Bachmann, S.P. & Lopez-Ribot, J.L. Proteomics for the analysis of the Candida albicans biofilm lifestyle. Proteomics 6, 5795–5804 (2006).

    Article  CAS  Google Scholar 

  18. Pierce, C.G . et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat. Protoc. 3, 1494–1500 (2008).

    Article  CAS  Google Scholar 

  19. Lattif, A.A . et al. Proteomics and pathway mapping analyses reveal phase-dependent over-expression of proteins associated with carbohydrate metabolic pathways in Candida albicans biofilms. Open Proteom. J. 1, 5–26 (2008).

    Article  CAS  Google Scholar 

  20. Beauvais, A., Loussert, C., Prevost, M.C., Verstrepen, K. & Latge, J.P. Characterization of a biofilm-like extracellular matrix in FLO1-expressing Saccharomyces cerevisiae cells. FEMS Yeast Res. 9, 411–419 (2009).

    Article  CAS  Google Scholar 

  21. Loussert, C . et al. In vivo biofilm composition of Aspergillus fumigatus. Cell. Microbiol. 12, 405–410 (2010).

    Article  CAS  Google Scholar 

  22. Gey, G.O. An improved technic for massive tissue culture. Am. J. Cancer 17, 752–756 (1933).

    Google Scholar 

  23. Dominguez, E . et al. 13th ASM Conference on Candida and Candidiasis Abstract no. 148 (American Society for Microbiology, 2016).

  24. Nachlas, M.M., Margulies, S.I., Goldberg, J.D. & Seligman, A.M. The determination of lactic dehydrogenase with a tetrazolium salt. Anal. Biochem. 1, 317–326 (1960).

    Article  CAS  Google Scholar 

  25. DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356 (1956).

    Article  CAS  Google Scholar 

  26. Smith, P.K . et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985).

    Article  CAS  Google Scholar 

  27. Taff, H.T . et al. A Candida biofilm-induced pathway for matrix glucan delivery: implications for drug resistance. PLoS Pathog. 8, e1002848 (2012).

    Article  CAS  Google Scholar 

  28. Mitchell, K.F . et al. Community participation in biofilm matrix assembly and function. Proc. Natl. Acad. Sci. USA 112, 4092–4097 (2015).

    Article  CAS  Google Scholar 

  29. Chandra, J., Mukherjee, P.K. & Ghannoum, M.A. In vitro growth and analysis of Candida biofilms. Nat. Protoc. 3, 1909–1924 (2008).

    Article  CAS  Google Scholar 

  30. Anonymous. A guide to freeze drying for the laboratory. An industry service publication. 1–12 (Labconco, 2010).

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Acknowledgements

This work was funded by NIH R01 AI073289 (to D.R.A.). The C. albicans SN250, C. tropicalis CAY2597, C. parapsilosis CLib21, and C. glabrata ATCC2001 strains were generous gifts kindly provided by S. Noble (University of California–San Francisco), R. Bennett (Brown University), G. Butler (University College Dublin) and B. Cormack (John Hopkins University), respectively.

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Authors and Affiliations

  1. Department of Medicine, Section of Infectious Diseases, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Robert Zarnowski, Hiram Sanchez & David R Andes

  2. Department of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Robert Zarnowski, Hiram Sanchez & David R Andes

Authors
  1. Robert Zarnowski
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  2. Hiram Sanchez
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  3. David R Andes
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Contributions

R.Z., H.S. and D.R.A. designed the research. R.Z. and H.S. performed experiments, and validated and optimized the protocol. R.Z. and D.R.A. wrote and edited the manuscript.

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Correspondence to David R Andes.

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The authors declare no competing financial interests.

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Zarnowski, R., Sanchez, H. & Andes, D. Large-scale production and isolation of Candida biofilm extracellular matrix. Nat Protoc 11, 2320–2327 (2016). https://doi.org/10.1038/nprot.2016.132

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