Enzyme-driven bacillus spore coat degradation leading to spore killing

Authors

  • Ruchir V. Mundra,

    1. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
    2. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
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  • Krunal K. Mehta,

    1. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
    2. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
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  • Xia Wu,

    1. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
    2. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
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  • Elena E. Paskaleva,

    1. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
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  • Ravi S. Kane,

    Corresponding author
    1. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
    2. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
    • Correspondence to: J.S. Dordick

      Correspondence to: R.S. Kane

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  • Jonathan S. Dordick

    Corresponding author
    1. Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York
    2. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York
    3. Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, New York
    4. Department of Biology, Rensselaer Polytechnic Institute, Troy, New York
    5. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
    • Correspondence to: J.S. Dordick

      Correspondence to: R.S. Kane

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ABSTRACT

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. Biotechnol. Bioeng. 2014;111: 654–663. © 2013 Wiley Periodicals, Inc.

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