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Engineered Stochastic Adhesion Between Microbes as a Protection Mechanism Against Environmental Stress

Abstract

Introduction

Microbes aggregate when they display adhesive proteins on their outer membrane surfaces, which then form bridges between microbes. Aggregation protects the inner microbes from harsh environmental conditions such as high concentrations of antibiotics, high salt conditions, and fluctuations in pH. The protective effects of microbial aggregation make it an attractive target for improving the ability of probiotic strains to persist in the gut environment. However, it remains challenging to achieve synthetic microbial aggregation using natural adhesive proteins because these proteins frequently mediate microbial virulence.

Objectives

Construction of synthetic proteins that mediate aggregation between microbes to enhance the survival of cells delivered to stressful environments.

Methods

We construct synthetic adhesins by fusing adhesive protein domains to surface display peptides. The resulting aggregated populations of bacteria are characterized using immunofluorescence, microscopy, flow cytometry, and quantification of colony forming units.

Results

We assemble a series of synthetic adhesins, demonstrate their display on the outer membrane of Escherichia coli, and show that they mediate bacterial aggregation. Further engineering of the size and motif composition of the adhesive domain shows that principles from natural adhesins can be applied to our synthetic adhesins. Finally, we show that aggregation allows E. coli cells to resist treatment with antimicrobial peptides and survive inside the gut of Caenorhabditis elegans.

Conclusions

Our results demonstrate that synthetic aggregation can allow bacteria to resist biocidal environmental conditions. Synthetic adhesins may be used to facilitate microbial colonization of previously inaccessible environmental niches, either in remote natural environments or inside living organisms.

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Acknowledgments

We thank the Tan Lab members, especially Fan Wu for his help with CFU and antimicrobial peptide assays. We also thank Riley Allen and Prof. Jamal Lewis for their help with flow cytometry. Prof. John Yoder gave valuable suggestions concerning the dissociation experiments. Adam Miltner assisted us in the initial characterization of one adhesin pair under different expression conditions.

Author contributions

DL and CT designed the study and wrote the manuscript. DL performed all wet lab experiments involving bacteria, RV performed all wet lab experiments involving yeast. CV assisted with Hoescht staining and proteinase K experiments. MN and RV provided cohesin and dockerin components as well as advice for adhesin design. LR helped design C. elegans experiments and analyzed the results. MN, RV, LR all helped edit the manuscript.

Funding

The work was supported by Human Frontier Science Programs (RGY0080/2015), the Branco Weiss Fellowship Collaborative Grants Program, and an industry/campus supported fellowship under the Training Program in Biomolecular Technology (T32-GM008799) at the University of California, Davis. Lesilee Rose is supported by NIFA CA-D* -MCB-6239-H.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon request.

Conflict of interests

All authors, including D. Lewis, R. Vanella, C. Vo, L. Rose, M. Nash, and C. Tan, declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Correspondence to Cheemeng Tan.

Additional information

Dr. Cheemeng Tan is an assistant professor in the Department of Biomedical Engineering at University of California, Davis. He received a bachelor’s degree (first class honors) from National University of Singapore and an M.S. degree in High Performance Computing from Singapore-MIT Alliance. In 2010, he obtained a doctorate in Biomedical Engineering from Duke University. After his Ph.D., he worked in the Lane Center for Computational Biology at Carnegie Mellon University as a Lane Postdoctoral Fellow. He has received several awards, including the Medtronic Fellowship, the Society-in-Science: Branco Weiss Fellowship, a young investigator grant from the Human Frontier Science Program, and the Scialog Fellow. His research group at UC Davis aims to understand the regulatory principles of protein synthesis in cell-free systems, artificial cells, and microbes, for biomedical applications.

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This article is part of the 2018 CMBE Young Innovators special issue.

Associate Editor Michael R. King oversaw the review of this article.

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Lewis, D.D., Vanella, R., Vo, C. et al. Engineered Stochastic Adhesion Between Microbes as a Protection Mechanism Against Environmental Stress. Cel. Mol. Bioeng. 11, 367–382 (2018). https://doi.org/10.1007/s12195-018-0552-9

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Keywords

  • Synthetic biology
  • Adhesion
  • Adhesin