Team:TU Darmstadt/project


The Project: ChiTUcare

Efficient and cheap treatment of wounds is a challenging task. Especially burns and diabetic wounds are difficult to handle. Since the wound dressing should not be removed before the healing process is completed, pathogenic bacteria present in the wound cannot be detected and therefore, there is a high risk of infection and possible scar formation. With ChiTUcare we aim to manufacture a smart band-aid which enables detection of bacteria while simultaneously having positive effects on the healing process. For detection of bacteria present in the wound, we make use of the fact that the protease level in wounds increases significantly during bacterial infections. We exploit this high protease level to visualize infections by immobilizing a quenched fluorophore via a peptide linker onto a hydrogel. Due to protease activity, the fluorophore lights up and gives an easy read-out for the infection status of the wound. As substrate for the hydrogel we use chitosan, a biopolymer with intrinsic antibacterial and wound-healing properties. Since chitosan is biodegradable, bandages do not have to be removed from the wound for analysis.
Chitosan shows biological activities, depending on its chemical properties, such as chain length and deacetylation pattern. For example, its antimicrobial properties increase with decreasing chain length, while the deacetylation pattern is responsible for support of scar-free wound healing.
Currently, chitosan is produced chemically using chitin from marine sources like crab shells. However, the chemical deacetylation leads to randomly deacetylated chitosans, which are unsuitable for medical applications. To enable the precise production of defined chain length and deacetylation pattern oligomers, we aim to synthesize chitin and chitosan in E. coli. By using a chitin synthase and two chitin deacetylases that differ in their regioselectivity, generation of specific deacetylation patterns is possible. Then, processing of chitosans with chitinase gives defined chain lengths in the final product. With the combination of fermenter-based designer chitosans and an easy to visualize detection system for pathogenic bacteria we will provide a band-aid enhancing wound healing and preventing formation of scars with minimal need for physician support.

What is Chitosan?

Chitosan is the N-deacetylated form of chitin composed of N-acetylglucosamine and glucosamine units, which can naturally occur in fungi cell walls. Different types of chitosan can be distinguished by degree and pattern of acetylation as well as the polymers length, which also results in different chemical and physical properties. It is water-insoluble and highly viscous in diluted acidic solutions. In contrast, as the hydrolysed products of chitosan, oligosaccharides have better solubility and lower viscosity under physiological conditions because of shorter chain lengths and free amino groups in D-glucosamine units. Formerly described physico-chemical properties also have a large impact on the biological activity of chitosan, creating a large field of possible applications based on the molecules specific composition. For example, it was discovered that longer polymers with medium acetylation degrees can be utilized in agriculture for plant strengthening, as they strongly increase plant growth and promote disease resistance.
Additionally, chitosans properties enable the production of hydrogels and other smart materials. However, short to midrange chitosans with low degrees of acetylation on the other hand have strong antimicrobial properties and are thus usable for many medical practices, like wound dressings.

Chitin Synthase

We set out to produce chitosan hydrogels that use chitin as a source material. Originally, this N-acetylglucosamine oligosaccharide (chitin) is extracted chemically from crustacean shells, which uses a lot of chemicals and produces chitin oligosaccharides of unspecified length. To avoid the usage of chemicals, one aim of this project was to produce chitin in E. coli by insertion of a chitin synthase (CHS) in E. coli, using the BioBrick system. The CHS can produce chitin in E. coli in an enviromentally friendly way. The enzyme that was employed in this project is the CHS NodC from the bacteria Rhizobium leguminosarum. NodC is an N-acetylglucosaminyl transferase which catalyzes the formation of chitin tetramers and pentamers using activated N-acetylglucosamine monomers. In addition, NodC reliably produces short oligosaccharides of certain lengths which can be processed in vitro further.
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The chitinase is an enzyme, whose ability to break down glycosidic bonds in chitin, brings more variability into the molecules. Since not just the grade and pattern of deacetylation, but also the amount of connected chitin monomers influences the entire molecule´s behavior, there is a great limitation of the properties and the bioactivity of the products. Its possible implementation in the project shows the future prospects of how chitins and chitosans with all kind of properties can be produced in E. coli.
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Chitin Deacetylase

Chitosan is a polymeric product of deacetylated chitin, which exists in a wide variety of patterns differing in their degree of deacetylation. Our goal is to design chitosan oligomers with a specific pattern of deacetylation. It can then be used for the production of our hydrogels.
We implemented chitin deacetylases originating from the organisms Sinorhizobium meliloti (NodB) and Vibrio cholerae (COD) into our E. coli cells. These enzymes deacetylate chitin individually. NodB targets the first position of the non-reducing end, while COD works similarly on the second unit. By implementing an orthogonal expression system to regulate the patterns, designer chitosan could be adjusted to the respective task. This would allow the expression of each enzyme separately, creating a defined deacetylation pattern.
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Hydrogels are three-dimensional networks made out of synthetic or natural polymers containing a high water content, therefore they receive an increasing attention in various fields. We were focused on not using any expensive or toxic compounds in combination with chitosan to manufacture a hydrogel. It could be formed at any shape with the perfect alignment to the surrounding tissue. The aim was to produce such a hydrogel with basic lab equipment and modify it to detect pathogenic bacteria visually in wounds. To evaluate an ideal hydrogel, various compounds like agarose, agar or alginate were tested. During this work various promising hydrogels were produced.
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Chitosan can be modified at the amino group with succinic anhydride and a variable peptide with a fluorogenic substrate to form a reliable structure to detect proteases. In this study, we reproduce the findings of the paper “Enzyme-Sensing Chitosan Hydrogels” by Ebrahimi and Prof. Dr. Schönherr from the university of Siegen and use the fluorogenic substrate alanyl-alanyl-phenylalanine-7-amido-4-methylcoumarin (Ala-Ala-Phe-AMC) to detect α-chymotrypsin. This protease is secreted by Staphylococcus aureus or Pseudomonas aeruginosa that are examples of pathogenic bacteria that can infect wounds.
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