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Large-scale production and isolation of Candida biofilm extracellular matrix

Nature Protocols volume 11, pages 2320–2327 (2016)Cite this article

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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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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

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  1. Robert Zarnowski
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  2. Hiram Sanchez
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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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