Project Description

What is Degratin and why does it matter?

The Problem

A build-up of keratin in the epidermis often results in the development of visible skin lesions that may cause pain, discomfort, and irritation.

figure 1: keratin structural composition ¹

Various types of skin lesions resulting in pain, discomfort, and irritation are characterized by buildup of keratin. Keratosis pilaris and seborrheic keratosis are two examples of such diseases, the former of which affects 50-80% of all adolescents and about 40% of all adults ¹. Common treatments include cryotherapy, laser surgery, and salicylic acid medications, however, the effectiveness of these therapies tends to vary between patients. Additionally, they are often associated with high costs and frequently go untreated. In severe cases, such as that of Actinic Keratosis, keratin-buildup on the skin has been associated with the development of skin cancer.

OUR SOLUTION: Engineer strains of E.coli to produce and secrete keratinases acting synergistically to degrade keratin. Encapsulate these modified bacteria in a hydrogel only permeable to the enzymes and essential nutrients for growth. Develop a prototype for a keratin-degrading patch to place on the afflicted area to mitigate the lesion, eliminating the need for conventional invasive treatments.

The Three Keratinases

Degradation of keratin structure is difficult but can be achieved with a combination of endo-acting, exo-acting, and oligopeptide-acting enzymes.

figure 2: keratin degradation by enco, exo, and oligo proteases ²

Keratin, known for being a tough, fibrous protein, is composed of long polypeptide chains, which are resistant to the activity of non-substrate-specific proteases. Huang et al. characterized enzymes relevant to keratin degradation using cultures obtained from the fungus Onygena corvina ². It was demonstrated that a minimum of three different classes of keratinases are required break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase.

We want to harness this synergistic activity by genetically engineering E.coli to produce keratinase enzymes from each of these classes.

The Csg Secretion Pathway

We hypothesized bacteria can be modified to express and secrete the above required keratinases via the Csg Curli Secretion Pathway.

figure 3: curli secretion pathway mechanism ⁴

The Csg curli secretion pathway allows Gram-negative bacteria to produce a biofilm-like extracellular matrix from “curli” amyloid fibers. The formation of this biofilm is important because it protects the cells from environmental stresses, thereby aiding colonization and persistence under stresses ³. The CsG consists of multiple subunits as depicted above. CsgA enters the periplasm via the SecYEG translocon wherein degradation is prevented by CsgC. Secretion tags located at the N-termini allow CsgA to be transported across the CsgG channel to the extracellular space (CsgE aids in this process). Finally, CsgA binds to CsgB to create the curli structure ⁴.

The Csg pathway is adept for the purposes of our project because we can utilize the N22 and SEC tags to secrete our keratinases instead of CsgA from our modified E.coli.

The Patch

Encapsulating the engineered bacteria within a biogel such as sodium alginate in the form of a patch will allow them to continuously secrete treatment without coming into contact with the skin.

figure 4: patch function ⁵

Biodegradable polymers are capable of providing an isolated environment conducive to cell growth. They provide a protective barrier against external stresses, maintain high cell viability, and allow for the transport of essential nutrients. Sodium alginate is a particularly popular polymer for cell encapsulation because it exhibits mild gelling and high biocompatibility while also allowing for the modification of pore size.

The pore size of the alginate biogel can be manipulated to allow for the secretion of our keratinases and administration of treatment while also providing a viable enviromnent for cell growth. Encapsulating our keratinase-secreting E.coli inside sodium alginate polymer will allow for the gradual and continuous supply of treatment, painless administration, and inexpensive cost of production.

The Future

The elucidation of keratinase and biogel behavior has implications for other fields of study.

figure 5: future applications

A continuation of our project can be applied for the treating of skin lesions and further, for hair removal. A keratin-degrading biogel would be a cost-effective and direct-to-consumer means of mitigating the qualms people have about their skin.
The study of keratinases and how they work will also be helpful in the understanding of cancer progression on the skin. Characterizing these proteins will enable us to better treat these related diseases such as actinic keratosis.

References

¹Keratosis Pilaris. (2018, June 12). Retrieved from https://emedicine.medscape.com/article/1070651-overview
²Lange, L., Huang, Y., & Busk, P. K. (2016). Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance. Applied Microbiology and Biotechnology, 100, 2083–2096. http://doi.org/10.1007/s00253-015-7262-1
³Van Gerven, Klein, Hultgren, & Remaut. (2015). Bacterial Amyloid Formation: Structural Insights into Curli Biogensis. Trends in Microbiology, 23(11), 693-706.
⁴Klein, & Hultgren. (2015). Chaos Controlled: Discovery of a Powerful Amyloid Inhibitor. Molecular Cell, 57(3), 391-393.
⁵Cell Encapsulation. (2016, April 05). Retrieved from http://www.novamatrix.biz/cell-encapsulation/