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.
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Adams, J. J., B. A. Webb, H. L. Spencer, and S. P. Smith. Structural characterization of type II dockerin module from the cellulosome of Clostridium thermocellum: Calcium-induced effects on conformation and target recognition. Biochemistry 44:2173–2182, 2005.
An, Y. H., and R. J. Friedman. Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. J. Biomed. Mater. Res. 43:338–348, 1998.
Artyukhin, A. B., F. C. Schroeder, and L. Avery. Density dependence in Caenorhabditis larval starvation. Sci Rep 3:2777, 2013.
Ashby, M. J., J. E. Neale, S. J. Knott, and I. A. Critchley. Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli. J. Antimicrob. Chemother. 33:443–452, 1994.
Bateman, A., et al. UniProt: the universal protein knowledgebase. Nucl. Acids Res. 45:D158–D169, 2017.
Baugh, L. R. To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest. Genetics 194:539–555, 2013.
Bayer, E. A., J. P. Belaich, Y. Shoham, and R. Lamed. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58:521–554, 2004.
Boder, E. T., and K. D. Wittrup. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15:553–557, 1997.
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77:71–94, 1974.
Brooun, A., S. Liu, and K. Lewis. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 44:640–646, 2000.
Chhatwal, G. S. Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors. Trends Microbiol. 10:205–208, 2002.
Church, D. L., K. L. Guan, and E. J. Lambie. Three genes of the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans. Development 121:2525–2535, 1995.
Cucarella, C., et al. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun 72:2177–2185, 2004.
Cullen, T. W., et al. Gut microbiota. Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation. Science 347:170–175, 2015.
Dodds, M. G., K. J. Grobe, and P. S. Stewart. Modeling biofilm antimicrobial resistance. Biotechnol. Bioeng. 68:456–465, 2000.
Dwyer, D. J., D. M. Camacho, M. A. Kohanski, J. M. Callura, and J. J. Collins. Antibiotic-induced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis. Mol Cell 46:561–572, 2012.
El-Kirat-Chatel, S., et al. Forces in yeast flocculation. Nanoscale 7:1760–1767, 2015.
El-Kirat-Chatel, S., et al. Force nanoscopy of hydrophobic interactions in the fungal pathogen Candida glabrata. ACS Nano. 9:1648–1655, 2015.
George, R. A., and J. Heringa. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. 15:871–879, 2002.
Gietz, R. D., and R. A. Woods. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350:87–96, 2002.
Goh, Y. J., and T. R. Klaenhammer. Functional roles of aggregation-promoting-like factor in stress tolerance and adherence of Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol. 76:5005–5012, 2010.
Hamberg, Y., et al. Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin–dockerin interactions. PeerJ 2:e636, 2014.
Henzler Wildman, K. A., D. K. Lee, and A. Ramamoorthy. Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37. Biochemistry 42:6545–6558, 2003.
Hwang, I. Y., et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat. Commun. 8:15028, 2017.
Inhulsen, S., et al. Identification of functions linking quorum sensing with biofilm formation in Burkholderia cenocepacia H111. Microbiologyopen 1:225–242, 2012.
Jobst, M. A., et al. Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy. Elife 4:e10319, 2015.
Kos, B., et al. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J. Appl. Microbiol. 94:981–987, 2003.
Kotzamanidis, C., A. Kourelis, E. Litopoulou-Tzanetaki, N. Tzanetakis, and M. Yiangou. Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains. Int. J. Food Microbiol. 140:154–163, 2010.
Kumar Shukla, S., and T. S. Rao. Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K. J. Antibiot. 66:55–60, 2013.
Kumar, A. et al. Adhesion and formation of microbial biofilms in complex microfluidic devices. In: Proceedings of the Asme Micro/Nanoscale Heat and Mass Transfer International Conference, 2012, pp. 79–84.
Latasa, C., et al. BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enterica serovar Enteritidis. Mol. Microbiol. 58:1322–1339, 2005.
Maiques, E., et al. Beta-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus. J. Bacteriol. 188:2726–2729, 2006.
McIntire, F. C., A. E. Vatter, J. Baros, and J. Arnold. Mechanism of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34. Infect. Immun. 21:978–988, 1978.
Mechaly, A., et al. Cohesin-dockerin recognition in cellulosome assembly: experiment versus hypothesis. Proteins 39:170–177, 2000.
Nash, M. A., S. P. Smith, C. M. Fontes, and E. A. Bayer. Single versus dual-binding conformations in cellulosomal cohesin-dockerin complexes. Curr. Opin. Struct. Biol. 40:89–96, 2016.
Pamp, S. J., M. Gjermansen, H. K. Johansen, and T. Tolker-Nielsen. Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes. Mol. Microbiol. 68:223–240, 2008.
Parsek, M. R., and E. P. Greenberg. Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol. 13:27–33, 2005.
Pinero-Lambea, C., et al. Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins. ACS Synth. Biol. 4:463–473, 2015.
Portal-Celhay, C., and M. J. Blaser. Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut. Infect. Immun. 80:1288–1299, 2012.
Rice, J. J., and P. S. Daugherty. Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Eng. Des. Sel. 21:435–442, 2008.
Rickard, A. H., P. Gilbert, N. J. High, P. E. Kolenbrander, and P. S. Handley. Bacterial coaggregation: an integral process in the development of multi-species biofilms. Trends Microbiol. 11:94–100, 2003.
Rickard, A. H., S. A. Leach, C. M. Buswell, N. J. High, and P. S. Handley. Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectin–saccharide interactions. Appl. Environ. Microb. 66:431–434, 2000.
Rickard, A. H., A. J. Underwood, and W. Nance. A holistic view of interspecies bacterial interactions within human dental plaque. Oral Microb. Ecol. 97–110, 2013.
Robertson, B. R., and D. K. Button. Characterizing aquatic bacteria according to population, cell-size, and apparent DNA content by flow-cytometry. Cytometry 10:70–76, 1989.
Sadamoto, R., et al. Control of bacteria adhesion by cell-wall engineering. J. Am. Chem. Soc. 126:3755–3761, 2004.
Schoeler, C., et al. Ultrastable cellulosome-adhesion complex tightens under load. Nat. Commun. 5:5635, 2014.
Spinelli, S., et al. Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition. J. Mol. Biol. 304:189–200, 2000.
Stahl, S. W., et al. Single-molecule dissection of the high-affinity cohesin-dockerin complex. Proc. Natl. Acad. Sci. USA 109:20431–20436, 2012.
Stoodley, P., I. Dodds, J. D. Boyle, and H. M. Lappin-Scott. Influence of hydrodynamics and nutrients on biofilm structure. J. Appl. Microbiol. 85(Suppl 1):19S–28S, 1998.
Tavares, G. A., P. Beguin, and P. M. Alzari. The crystal structure of a type I cohesin domain at 1.7 angstrom resolution. J. Mol. Biol. 273:701–713, 1997.
Tripathi, P., et al. Adhesion and nanomechanics of pili from the probiotic Lactobacillus rhamnosus GG. ACS Nano 7:3685–3697, 2013.
Verstrepen, K. J., and F. M. Klis. Flocculation, adhesion and biofilm formation in yeasts. Mol. Microbiol. 60:5–15, 2006.
Xu, H., H. S. Jeong, H. Y. Lee, and J. Ahn. Assessment of cell surface properties and adhesion potential of selected probiotic strains. Lett. Appl. Microbiol. 49:434–442, 2009.
Xu, Q., et al. A novel Acetivibrio cellulolyticus anchoring scaffoldin that bears divergent cohesins. J. Bacteriol. 186:5782–5789, 2004.
Yoshida, K., M. Toyofuku, N. Obana, and N. Nomura. Biofilm formation by Paracoccus denitrificans requires a type I secretion system-dependent adhesin BapA. FEMS Microbiol. Lett. 2017. https://doi.org/10.1093/femsle/fnx029.
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.
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.
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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