Team:Leiden/Description

iGEM Leiden | 2019

S.P.L.A.S.H.

Suckerin Polymer Layer to Achieve Sustainable Health

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Description & Inspiration

Burn wounds as a leading cause of morbidity

Severe burn wounds are the cause of 300,000 deaths annually [1]. Several studies over the last decade have shown that approximately 42%-65% of fatalities are caused by bacterial infections [2-5]. In addition, 11 million victims a year are faced with major psychological, social and economic problems due to prolonged hospitalization, disfigurement and disability [6, 7]. Treatment is challenging because of significant fluid loss and increased risk of wound infections, further impairing vital functions performed by the skin [8]. Current treatment consists of donor skin application, which focuses on the prevention of excessive fluid loss and recovery from circulatory shock. As a result heart rate and blood flow to the tissues are restored. However, the availability of suitable donor skin is limited due to highly specific requirements and multiple necessary processing steps [9]. Moreover, skin grafts give rise to a high risk of disease transmission and graft rejection [10].


The WHO states that various materials are used to make dressings for donor skin substitution, including hydrocolloids, alginates, hydrogels, collagen, and chitosan [11]. Hydrogels are emerging as the most promising substitute, since they keep the wound moist, prevent surrounding bacteria from reaching the wound, and have soft tissue-like properties [12]. In addition, hydrogels are non-adhesive to cells due to their hydrophilic nature, subsequently minimizing pain and discomfort by removal. Moreover, hydrogels have the ability to absorb and retain wound exudate, thereby promoting fibroblast proliferation and keratinocyte migration [13-15], and require less frequent dressing replacement [16]. Nonetheless, materials currently used for hydrogels are costly and have poor mechanical properties leading to faster contraction of the graft [17, 18]. Proteins as a biomaterial for hydrogel formation provide additional advantages due to non-immunogenicity, long shelf-life, and easy degradation into non-toxic byproducts [16]. In the past decades, research has focused on the bio-inspired material spider silk in the use of medical applications [9]. Silk fibroins show an astonishing combination of toughness, strength, and ductility, whilst being a biocompatible material [19]. The modular mechanical properties and silk’s ability to self-assemble by the formation of β-sheets are ideal for hydrogel construction. It has been shown that silk fibroin hydrogels support the proliferation of primary human dermal fibroblasts and the migration of keratinocytes in vitro and provide an instructive and supportive matrix to the full-thickness third-degree burn wound in vivo for the regeneration of skin tissue with neo-dermis and neo-epithelium [20]. However, due to the high molecular weight (300-450 kDa) and genetic complexity, recombinant gene expression is difficult and large scale production costs are high [21]. Therefore, we directed our search on novel biomaterials with comparable properties as spider silk.

"Two years ago, I burned 55% of my body and I still require interventions to regain mobility and flexibility of my scars" -- A burn wound victim

Suckerin as a novel protein for medical applications

The suckerin protein family, originally isolated from the sucker ring teeth (SRT) of the Humboldt squid Dosidicus gigas, shows great analogy with silk fibroins on a molecular level [22]. Suckerins have a molecular weight ranging from 5 to 57 kDa, thereby increasing the efficiency of production by molecular engineered microorganisms [23]. The unique genetical composition of the suckerins, constituting two main alternating modules, provides SRTs with beneficial features, including flexibility and strength (Fig. 2) [22, 24-26]. The first module (M1) is predominated by alanine (Ala) residues and through stabilization by hydrogen bonding, these regions form rigid β-sheet nanocrystals. This module is reminiscent of poly-Ala β-sheet forming domains found in spider silk [27, 28]. The second module (M2) comprises flexible chains rich in glycine (Gly), tyrosine (Tyr) and leucine (Leu) residues forming an amorphous matrix, which resembles the amorphous-forming sequences found in silk proteins [29]. Through hydrogen bond interactions suckerins assemble into a robust supramolecular network rich in mechanical properties [8]. The characteristics of suckerins enable the proteins to self-assemble, which is an improvement over existing hydrogels [30]. For medical applications, biocompatibility is of utmost importance. Biocompatibility assays for suckerin-19 have thus far shown negligible in vitro cytotoxicity against various cell lines, including dermal fibroblast cells (HDF) [24, 30]. These studies also describe faster cell proliferation than under classical culture plate conditions, suggesting accelerated wound healing. Solubilized suckerin allows for the formation of mechanically robust, enzymatically cross-linked hydrogels [31]. Altogether, a suckerin-based hydrogel is more beneficial than existing hydrogels and can be used to improve skin regeneration of superficial wounds by promoting wound healing. In addition, it can serve as a substitution of donor skin for severe burn wound treatment.


Suckerin synthesis using microorganisms empowers fast and high-yield production. Additionally, this biomaterial enables genetic modification, thereby opening up the possibility to generate a gene construct with different ratios of modules 1 and 2. Besides this, a suckerin-based hydrogel enables easy introduction of a linker system to which supportive compounds, such as antimicrobial peptides, can be coupled.

Figure 2. genetic composition of a suckerin protein with the proposed protein structure.

Suckerin production in various microorganisms

For the realization of our project, we considered several suckerins, including suckerin-19, which has been most extensively studied, since this protein is most abundant in nature and has a classical representation of the suckerin protein family [24]. Besides this, we focus on the production of suckerin-12, as it has previously been studied in relation to hydrogel formation [31], as well as suckerin-8 and -9, which have a higher abundance in module 1 or 2, respectively, thereby representing different flexibility and rigidity [23, 24]. Instead of damaging the environment and harming animals by harvesting the proteins from the Humboldt squid, we aimed to produce them using genetically engineered microorganisms. Genetically modified Escherichia coli (E. coli) is the best-established host for industrial-scale production due to its well-understood genetics, ease of manipulation, and wide range of commercially available expression vectors [32, 33]. Since suckerin is naturally produced by cephalopods, we also assessed whether production is more efficient in a eukaryote using Saccharomyces cerevisiae (S. cerevisiae) to account for post-translational modification, which is absent in E. coli.


Genes encoding suckerin-8, -9, and -12 were synthesized and codon-optimized for production in E. coli. Genes were amplified with standard RFC10 prefix and suffix primers. These constructs were subsequently inserted into pBS1A3, succeeded by insertion of the PLac expression cassette. IPTG-induced production of suckerin-12 by E. coli Rosetta resulted in a significant yield. Besides this, suckerin-19 linked to a His6-tag and inserted into pQE80-L was provided by Miserez et al., and successfully produced by E. coli Rosetta. Following this, the production of both suckerin-12 and -19 was upscaled by growing E. coli Rosetta in one-liter fermenters. Both suckerins were purified based on inclusion body formation. Production yield was optimized based on the predictions made by our model, which suggested the use of optimized Studier Phosphate Glucose (SPG) medium and provided metabolic pathways that can serve as bottlenecks of production. These pathways constitute potential targets for future genetic intervention.


With regard to our project, we designed the iGEMized “iPET” plasmid, which allows for the inducible production of a desired protein by placing the gene under control of the T7/lac promoter. In addition, the plasmid allows for the insertion of any protein gene with automatic linkage to a His-tag, thereby enabling quick and easy His-tag protein purification.

Figure 3. schematic overview of the challenges of burn wound treatment

Future prospects

The ratio of M1 and M2 modules determines the rigidity and flexibility of the protein, making suckerin applicable to mimic a wide range of human tissues [30]. For this reason, we synthesized customized sequences of the M1 and M2 modules compatible with RFC25 systems. Inclusion of NgoMIV and AgeI sites allows for the addition of multiple modules. These sequences were used to create M1-M1, M2-M2, and M1-M2 constructs. This system enables the production of adjustable, on-demand artificial suckerins, that possess all desired characteristic. 


As previously mentioned, open wounds increase the risk of incurring bacterial infections and most dressings do not provide a mechanism that prevents these infections. Therefore, we decided to introduce a linker system onto which antimicrobial peptides (AMPs) can be coupled. These AMPs will be cleaved in the presence of pathogenic activity. To achieve this, we used substrates of the proteases V8 [34] and SplB [35–37] to function as cleavable linkers. V8 and SplB are secreted by Staphylococcus aureus, which is a frequent cause of infections encountered in hospitals [38, 39]. For the future, we envision the synthesis of a gene construct of suckerin + linker + GFP. This construct would ideally be coupled to streptavidin monomers to obtain a high-affinity biotin linker system to AMPs. In the case of a pathogenic infection caused by S. aureus, the activity of the V8 and SplB proteases will result in cleavage of the linkers, thereby releasing the antimicrobial peptides that eliminate the pathogen. To ensure safety and biocompatibility, FDA-approved or human-derived antimicrobial peptides, such as CAP18 can be used [40]. This linker system provides future opportunities for the coupling of other beneficial compounds, including wound healing promoters, such as proinflammatory cytokines and growth factors (Fig. 3). Besides this, to decrease disfigurement and disability caused by scarring, compounds can be added to improve the process of wound repair and thus a reduction of scar formation.


In conclusion, our suckerin-based hydrogel improves the prognosis and aftercare for burn wound victims by providing a system that minimizes complications during recovery, prevents bacterial infections and supports wound healing.

References

  1. Peck M., Molnar J., & Swart D. (2009). A global plan for burn prevention and care. Bulletin of the World Health Organization, 87(10), 802-803.
  2. Krishnan P., Frew Q., Green A., Martin R., & Dziewulski P. (2013). Cause of death and correlation with autopsy findings in burns patients. Burns, 39(4), 583-588.
  3. Keen E., Robinson B., Hospenthal D., Aldous W., Wolf S., Chung K., & Murray C. (2010). Incidence and bacteriology of burn infections at a military burn center. Burns, 36(4), 461-468.
  4. Bloemsma G., Dokter J., Boxma H., & Oen I. (2008). Mortality and causes of death in a burn centre. Burns, 34(8), 1103-1107.
  5. Sharma B., Harish D., Singh V., & Bangar S. (2006). Septicemia as a cause of death in burns: An autopsy study. Burns, 32(5), 545-549.
  6. https://www.who.int/en/news-room/fact-sheets/detail/burns, “visited on May 16, 2019.” .
  7. https://www.who.int/violence_injury_prevention/other_injury/burns/en/, “visited on September 9, 2019.” .
  8. Ramzy P. I., Barret J. P., & Herndon D. N. (1999). Thermal injury. Crit. Care Clin., 15(2), 333-352.
  9. Wang Y., Beekman J., Hew J., Jackson S., Issler-Fisher A., Parungao R., . . . Maitz P. (2018). Burn injury: Challenges and advances in burn wound healing, infection, pain and scarring. Advanced Drug Delivery Reviews, 123, 3-17.
  10. Atiyeh B., Gunn S. & Hayek W. (2005). State of the Art in Burn Treatment. World Journal of Surgery, 29(2), 131-148.
  11. World Health Organization: Burns injuries. (2014).
  12. Madaghiele M., Demitri C., Sannino A. & Ambrosio L. (2014). Polymeric hydrogels for burn wound care: Advanced skin wound dressings and regenerative templates. Burns & Trauma, 2(4), 153-161.
  13. Winter G. D. (1962). Formation of the Scab and the Rate of Epithelization of Superficial Wounds in the Skin of the Young Domestic Pig. Nature, 193(4812), 293-294.
  14. Bullock A. J., Pickavance P., Haddow D. B., Rimmer S., MacNeil S. (2010). Development of a calcium-chelating hydrogel for treatment of superficial burns and scalds. Regen. Med., 5(1), 55-64.
  15. Ribeiro M. P., Espiga A., Silva D., Baptista P., Henriques J., Ferreira C., . . . Correia I. J. (2009). Development of a new chitosan hydrogel for wound dressing. Wound Repair and Regeneration, 17(6), 817-824.
  16. Dhaliwal K., & Lopez N. (2018). Hydrogel dressings and their application in burn wound care. Br J Community Nurs, 1;23(Sup9):S24-S27.
  17. Bell E., Ivarsson B., & Merrill C. (1979). Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proceedings of the National Academy of Sciences of the United States of America, 76(3), 1274-1278.
  18. Ng K., Khor H., & Hutmacher D. (2004). In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts. Biomaterials, 25(14), 2807-2818.
  19. Gosline J., Guerette P., Ortlepp C., & Savage K. (1999). The mechanical design of spider silks: From fibroin sequence to mechanical function. The Journal of Experimental Biology, 202(23), 3295-3303.
  20. Chouhan D., Lohe T., Samudrala P., & Mandal B. (2018). In Situ Forming Injectable Silk Fibroin Hydrogel Promotes Skin Regeneration in Full Thickness Burn Wounds. Advanced Healthcare Materials, 7(24), E1801092.
  21. Rising A., Widhe M., Johansson J., & Hedhammar M. (2011). Spider silk proteins: Recent advances in recombinant production, structure–function relationships and biomedical applications. Cellular and Molecular Life Sciences, 68(2), 169-184.
  22. Ding D., Guerette P., Hoon S., Kong K., Cornvik T., Nilsson M., . . . Miserez A. (2014). Biomimetic production of silk-like recombinant squid sucker ring teeth proteins. Biomacromolecules, 15(9), 3278-3289.
  23. Guerette P., Hoon S., Ding D., Amini S., Masic A., Ravi V., . . . Miserez A. (2014). Nanoconfined beta-Sheets Mechanically Reinforce the Supra-Biomolecular Network of Robust Squid Sucker Ring Teeth. Acs Nano, 8(7), 7170-7179.
  24. Hiew S., & Miserez A. (2017). Squid Sucker Ring Teeth: Multiscale Structure-Property Relationships, Sequencing, and Protein Engineering of a Thermoplastic Biopolymer. Acs Biomaterials Science & Engineering, 3(5), 680-693.
  25. Hiew S., Sánchez-Ferrer A., Amini S., Zhou F., Adamcik J., Guerette P., . . . Miserez A. (2017). Squid Suckerin Biomimetic Peptides Form Amyloid-like Crystals with Robust Mechanical Properties. Biomacromolecules, 18(12), 4240-4248.
  26. Pena-Francesch A., & Demirel M. (2019). Squid-Inspired Tandem Repeat Proteins: Functional Fibers and Films. Frontiers in Chemistry, 7, 69.
  27. Guerette P., Ginzinger D., Weber B., & Gosline J. (1996). Silk properties determined by gland-specific expression of a spider fibroin gene family. Science, 272(5258), 112-115.
  28. Gatesy J., Hayashi C., Motriuk D., Woods J., & Lewis R.  (2001). Extreme diversity, conservation, and convergence of spider silk fibroin sequences. Science, 291(5513), 2603-2605.
  29. Gosline J., Guerette P., Ortlepp C., & Savage K. (1999). The mechanical design of spider silks: From fibroin sequence to mechanical function. The Journal of Experimental Biology, 202(23), 3295-3303.
  30. Ding D., Guerette P., Fu J., Zhang L., Irvine S., & Miserez A. (2015). From Soft Self‐Healing Gels to Stiff Films in Suckerin‐Based Materials Through Modulation of Crosslink Density and β‐Sheet Content. Advanced Materials, 27(26), 3953-3961.
  31. Buck C., Dennis P., Gupta M., Grant M., Crosby M., Slocik J., . . . Naik R. (2019). Anion‐Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels. Macromolecular Bioscience, 19(3), N/a.
  32. Schmidt F. (2004). Recombinant expression systems in the pharmaceutical industry. Applied Microbiology and Biotechnology, 65(4), 363-372.
  33. Terpe K. (2006). Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 72(2), 211-222.
  34. Prasad L., Leduc Y., Hayakawa K., & Delbaere L. (2004). The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus. Acta Crystallographica Section D, 60(2), 256-259.
  35. Reed S. B., Wesson C. A., Liou L. E., Trumble W. R., Schlievert P. M., Bohach G. A., & Bayles K. W. (2001). Molecular Characterization of a Novel Staphylococcus aureus Serine Protease Operon. Infection and Immunity, 69(3), 1521-1527.
  36. Popowicz G. M., Dubin G., Stec-Niemczyk J., Czarny A., Dubin A., Potempa J., & Holak T. A. (2006). Functional and Structural Characterization of Spl Proteases from Staphylococcus aureus. Journal of Molecular Biology, 358(1), 270-279.
  37. Dubin G., Stec-Niemczyk J., Kisielewska M., Pustelny K., Popowicz G. M., Bista M., . . . Potempa J. (2008). Enzymatic Activity of the Staphylococcus aureus SplB Serine Protease is Induced by Substrates Containing the Sequence Trp-Glu-Leu-Gln. Journal of Molecular Biology, 379(2), 343-356.
  38. Morin C., & Hadler J. (2001). Population-based incidence and characteristics of community-onset Staphylococcus aureus infections with bactermia in 4 metropolitan connecticut areas, 1998. The Journal of Infectious Diseases, 184(8), 1029-34.
  39. Lowy F. D. (1998). Staphylococcus aureus Infections. N. Engl. J. Med., 339(8), 520-532.
  40. Larrick J. W., Hirata M., Balint R. F., Lee J., Zhong J., & Wright S. C. (1995). Human CAP18: A novel antimicrobial lipopolysaccharide-binding protein. Infection and Immunity, 63(4), 1291-1297.