Advertisement

Cellular and Molecular Bioengineering

, Volume 11, Issue 5, pp 367–382 | Cite as

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

  • Daniel D. Lewis
  • Rosario Vanella
  • Christopher Vo
  • Lesilee Rose
  • Michael Nash
  • Cheemeng TanEmail author
Article

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.

Keywords

Synthetic biology Adhesion Adhesin 

Notes

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.

Supplementary material

12195_2018_552_MOESM1_ESM.docx (678 kb)
Supplementary material 1 (DOCX 678 kb)

References

  1. 1.
    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.CrossRefGoogle Scholar
  2. 2.
    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.CrossRefGoogle Scholar
  3. 3.
    Artyukhin, A. B., F. C. Schroeder, and L. Avery. Density dependence in Caenorhabditis larval starvation. Sci Rep 3:2777, 2013.CrossRefGoogle Scholar
  4. 4.
    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.CrossRefGoogle Scholar
  5. 5.
    Bateman, A., et al. UniProt: the universal protein knowledgebase. Nucl. Acids Res. 45:D158–D169, 2017.CrossRefGoogle Scholar
  6. 6.
    Baugh, L. R. To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest. Genetics 194:539–555, 2013.CrossRefGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    Boder, E. T., and K. D. Wittrup. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15:553–557, 1997.CrossRefGoogle Scholar
  9. 9.
    Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77:71–94, 1974.Google Scholar
  10. 10.
    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.CrossRefGoogle Scholar
  11. 11.
    Chhatwal, G. S. Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors. Trends Microbiol. 10:205–208, 2002.CrossRefGoogle Scholar
  12. 12.
    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.Google Scholar
  13. 13.
    Cucarella, C., et al. Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun 72:2177–2185, 2004.CrossRefGoogle Scholar
  14. 14.
    Cullen, T. W., et al. Gut microbiota. Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation. Science 347:170–175, 2015.CrossRefGoogle Scholar
  15. 15.
    Dodds, M. G., K. J. Grobe, and P. S. Stewart. Modeling biofilm antimicrobial resistance. Biotechnol. Bioeng. 68:456–465, 2000.CrossRefGoogle Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    El-Kirat-Chatel, S., et al. Forces in yeast flocculation. Nanoscale 7:1760–1767, 2015.CrossRefGoogle Scholar
  18. 18.
    El-Kirat-Chatel, S., et al. Force nanoscopy of hydrophobic interactions in the fungal pathogen Candida glabrata. ACS Nano. 9:1648–1655, 2015.CrossRefGoogle Scholar
  19. 19.
    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.CrossRefGoogle Scholar
  20. 20.
    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.CrossRefGoogle Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    Hamberg, Y., et al. Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin–dockerin interactions. PeerJ 2:e636, 2014.CrossRefGoogle Scholar
  23. 23.
    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.CrossRefGoogle Scholar
  24. 24.
    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.CrossRefGoogle Scholar
  25. 25.
    Inhulsen, S., et al. Identification of functions linking quorum sensing with biofilm formation in Burkholderia cenocepacia H111. Microbiologyopen 1:225–242, 2012.CrossRefGoogle Scholar
  26. 26.
    Jobst, M. A., et al. Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy. Elife 4:e10319, 2015.CrossRefGoogle Scholar
  27. 27.
    Kos, B., et al. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J. Appl. Microbiol. 94:981–987, 2003.CrossRefGoogle Scholar
  28. 28.
    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.CrossRefGoogle Scholar
  29. 29.
    Kumar Shukla, S., and T. S. Rao. Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K. J. Antibiot. 66:55–60, 2013.CrossRefGoogle Scholar
  30. 30.
    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.Google Scholar
  31. 31.
    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.CrossRefGoogle Scholar
  32. 32.
    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.CrossRefGoogle Scholar
  33. 33.
    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.Google Scholar
  34. 34.
    Mechaly, A., et al. Cohesin-dockerin recognition in cellulosome assembly: experiment versus hypothesis. Proteins 39:170–177, 2000.CrossRefGoogle Scholar
  35. 35.
    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.CrossRefGoogle Scholar
  36. 36.
    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.CrossRefGoogle Scholar
  37. 37.
    Parsek, M. R., and E. P. Greenberg. Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol. 13:27–33, 2005.CrossRefGoogle Scholar
  38. 38.
    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.CrossRefGoogle Scholar
  39. 39.
    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.CrossRefGoogle Scholar
  40. 40.
    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.CrossRefGoogle Scholar
  41. 41.
    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.CrossRefGoogle Scholar
  42. 42.
    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.CrossRefGoogle Scholar
  43. 43.
    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.Google Scholar
  44. 44.
    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.CrossRefGoogle Scholar
  45. 45.
    Sadamoto, R., et al. Control of bacteria adhesion by cell-wall engineering. J. Am. Chem. Soc. 126:3755–3761, 2004.CrossRefGoogle Scholar
  46. 46.
    Schoeler, C., et al. Ultrastable cellulosome-adhesion complex tightens under load. Nat. Commun. 5:5635, 2014.CrossRefGoogle Scholar
  47. 47.
    Spinelli, S., et al. Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition. J. Mol. Biol. 304:189–200, 2000.CrossRefGoogle Scholar
  48. 48.
    Stahl, S. W., et al. Single-molecule dissection of the high-affinity cohesin-dockerin complex. Proc. Natl. Acad. Sci. USA 109:20431–20436, 2012.CrossRefGoogle Scholar
  49. 49.
    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.CrossRefGoogle Scholar
  50. 50.
    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.CrossRefGoogle Scholar
  51. 51.
    Tripathi, P., et al. Adhesion and nanomechanics of pili from the probiotic Lactobacillus rhamnosus GG. ACS Nano 7:3685–3697, 2013.CrossRefGoogle Scholar
  52. 52.
    Verstrepen, K. J., and F. M. Klis. Flocculation, adhesion and biofilm formation in yeasts. Mol. Microbiol. 60:5–15, 2006.CrossRefGoogle Scholar
  53. 53.
    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.CrossRefGoogle Scholar
  54. 54.
    Xu, Q., et al. A novel Acetivibrio cellulolyticus anchoring scaffoldin that bears divergent cohesins. J. Bacteriol. 186:5782–5789, 2004.CrossRefGoogle Scholar
  55. 55.
    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.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2018

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of California DavisDavisUSA
  2. 2.Department of Molecular and Cellular BiologyUniversity of California DavisDavisUSA
  3. 3.Integrative Genetics and GenomicsUniversity of California DavisDavisUSA
  4. 4.Department of ChemistryUniversity of BaselBaselSwitzerland
  5. 5.Department of Biosystems Science and EngineeringETH ZurichBaselSwitzerland

Personalised recommendations