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The Extracellular Polymeric Substances of Legionella pneumophila Biofilms Contain Amyloid Structures

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Abstract

Human infection by bacteria of the genus Legionella most often result in the pneumonia known as Legionnaires Disease. Legionella is found as a resident of adherent biofilms in man-made water systems. Disinfection efforts to prevent Legionella infections require a better understanding of the structures that promote Legionella surface attachment and biofilm colonization. Various enzymatic treatments, including multiple carbohydrate-targeting mixtures, failed to disrupt Legionella biofilms, despite the presence of carbohydrates in the biofilms as shown by biochemical methods and concanavalin-A lectin staining. Moreover, Legionella biofilms contained amyloids as detected by three microscopic staining methods (congo red, thioflavin T, and the amyloid-specific antibody WO2). Amyloid structures were seen in biofilms of both L. pneumophila and L. longbeachae, the two Legionella species most associated with human infection. Inhibition of amyloid assembly by congo red and thioflavin T limited both self-aggregation and surface attachment of L. pneumophila, indicating that functional amyloid structures have a key role in initial biofilm formation by these pathogenic bacteria.

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Acknowledgements

The authors thank the SUNY Cortland Undergraduate Research Council for funding this research and NSF grant 1337695. We would like to thank Ronald Wetzel (WO2 antibody) and Matthew Chapman (Curli-deficient and WT E. coli strains) for reagents. We kindly thank Kessler McCoy-Simandle for assistance in preparation of this manuscript.

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    1. Department of Biological Sciences, SUNY Cortland, Cortland, NY, USA

      Casey P. Peterson, Cassidy Sauer & Christa H. Chatfield

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    Correspondence to Christa H. Chatfield.

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    Casey P. Peterson and Christa H. Chatfield have contributed equally to this work.

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    Supplemental figure 1: Functional amyloid staining controls in

    Escherichia coli curli-forming strain MC4100 and the curli- deficient csgA mutant. In each row, the first image shows DAPI staining for E. coli MC4100 (a, b and c) or csgA mutant (d) cultures. The second image in each row shows amyloid staining by (a) congo red (b) thioflavin T and (c) WO2 antibody in MC4100 biofilm. Row (d) shows thioflavin T results for the csgA mutant. The third image in each row is the overlay of the two images. Each image was obtained at 40x magnification and is representative of at least two independent replicates. (TIF 32795 KB)

    Supplemental figure 2: Functional amyloid are in

    L. longbeachae MSBB biofilms. In each row, the first image shows DAPI staining for Llo in MSBB biofilms at day 3 post inoculation. The second image in each row shows amyloid staining by (a) congo red (b) thioflavin T and (c) WO2 antibody (scale bar = 20 µm). The third image in each row is the overlay of the two images. Each image was obtained at 40x magnification and is representative of at least two independent replicates. (TIF 37736 KB)

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    Peterson, C.P., Sauer, C. & Chatfield, C.H. The Extracellular Polymeric Substances of Legionella pneumophila Biofilms Contain Amyloid Structures. Curr Microbiol 75, 736–744 (2018). https://doi.org/10.1007/s00284-018-1440-1

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