Engineered Stochastic Adhesion Between Microbes as a Protection Mechanism Against Environmental Stress
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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.
Construction of synthetic proteins that mediate aggregation between microbes to enhance the survival of cells delivered to stressful environments.
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.
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.
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.
KeywordsSynthetic biology Adhesion Adhesin
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.
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.
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.
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.
This article does not contain any studies with human participants or animals performed by any of the authors.
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