Description and Inspiration
The goal of our project is to functionalize a cellulose wound dressing with antimicrobial agents to help fight infections. We will engineer E.coli and V.natriegens to produce antimicrobial peptides and enzymes linked to a carbohydrate binding domain (CBD). These fusion proteins will then be purified and attached to a cellulose bandage where they will be applied to a patient and help prevent infections. The CBD enables the attachment of antimicrobial agents to the cellulose and also inactivates the agents while being produced in E.coli and V.natriegens. Later, when the bandage comes in contact with thrombin in the blood, the antimicrobial agents will be activated. This is a result of thrombin cutting a linker between the agents and the carbohydrate binding domain.
Figure 1. Mechanism of action. The carbohydrate binding domain (CBD) is bound to a polysaccharide material (e.g. cellulose). The CBD has a linker (GS-linker) which ends in a thrombin cleavage site. On the end of the thrombin site is an antimicrobial agent. When the fusion protein is exposed to thrombin the linker will be cleaved, releasing the agent into the wound.
The inspiration for our project came from multiple sources. Linköping has a national burn care center (BRIVA) and disaster medicine center (KMC) which inspired us to focus on burn wounds. We learned that burn wounds often are infected and BRIVA, who is currently testing cellulose bandages, inspired us to focus on the material. We further learned that burn wounds are a type of wound that demands high doses of antibiotics and are generally expensive to treat. These interactions made us want to optimize burn care and also to develop a project that could tackle the difficulties in costs and using antibiotics. When researching alternatives to antibiotics we found out that antimicrobial peptides are currently being discussed as an alternative to antibiotics . This inspired us to use them in our project and our modeling. We further read that there are different kinds of antimicrobial enzymes which we also wanted to try as alternatives to antibiotics.
With the decision to focus on the cellulose material and alternatives to antibiotics, we decided to create an antimicrobial bandage. We then looked into how to functionalize cellulose with antimicrobial agents. In this process, we were inspired by Imperial 2014 who used a carbohydrate binding domain (CBD). Also, one of our team members were involved in a theoretical project about functionalizing cellulose were the CBD was presented as a solution. With this in mind, we decided to try to create fusion proteins composed of antimicrobial agents (both peptides and enzymes) linked to CBD:s which can then bind to cellulose in order to create an antimicrobial cellulose bandage. When deciding on which CBD to use we found other iGEM projects: Imperial 2014, Edinburgh 2015 and Ecuador 2018. Their work guided us in the CBD selection and these teams also helped us in deciding which terminal our linker should be.
Synthetic biology was a central part of this project because we expressed proteins in bacterial chassis. All our constructs are designed and originate from many different organisms. Our use of V.natriegens were inspired by Marburg 2018 who described V.natriegens as an alternative to E.coli due to their fast growth rate. This would further assist in decreasing the costs of producing our bandage.
The Antimicrobial Peptides and Enzymes
The antimicrobial agents we use are two enzymes: CHAP(K) and PlyF307 and five peptides: LL-37, Magainin 2, Pln1, Parseq α and Parseq β. The enzymes are bacteriophage lysins which are used for their specific targeting. The peptides used are instead focused on broad-spectrum activity and can be divided into two groups: naturally occurring peptides and novel synthetic peptides. The two Parseq peptides are the novel synthetic peptides which will be developed by the modeling team and then will their activity be tested.
|Antimicrobial Agent||Peptide or Enzyme||Source Organism||Active Against|
|LL-37||Peptide||Human||Gram positive & negative|
|Magainin 2||Peptide||African Clawed Frog||Gram positive & negative|
|Pln1||Peptide||Lactobacillus plantarum||Gram positive|
|Parseq-α||Model Peptide||Our Model||Gram positive & negative|
|Parseq-β||Model Peptide||Our Model||Gram positive & negative|
Carbohydrate Binding Domain CipA
Carbohydrate binding domains (CBD) are derived from the microorganisms need to degrade cellulose. This degradation is mediated by enzymes which are linked to the CBD. CBDs mediate the cell binding to cellulose when the cellulases or cellusomes are present on the cell surface.The cellulose degrading enzyme will bind to cellulose via its active site. This binding is further assisted and enhanced by the independent binding domain of the CBD. The CBD can be considered as an anchor to the enzyme [2, 3].
The CipA gene is from Clostridium Thermocellum. CBDCipA is a member of the “family 3 CBDs”. This family has been observed in several bacterial enzymes and non-hydrolytic proteins. They are responsible for the structural organization of cellulosomes in their host organism (2). CBDCipA is a part of scaffoldinCipA which is a large scaffold for the expressing bacteria to interact and degrade cellulose. The CBDCipA together with cellulose degrading enzymes are the interacting cellulose domains. CBDCipA is linked to several cohesins along a long polypeptide chain, around 1800 amino acids long, in which the chain branches off in several places where the enzymes are exposed to the cellulose - allowing cellulose degradation. Scaffoldins have a muddled amount of domains which interact with catalytic subunits to form cohesive cellulosome structures and a single CBD structure, known so far .
CBDCipA is composed of a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. It further contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of faces mainly contains planar strips of aromatic and polar residues which may be the cellulose binding part. Further aspects are unknown and unique with this CBD, such as the other conserved residues which are contained in a groove .
Antimicrobial peptides (AMP) are short oligopeptides which are are often cationic and amphiphilic. They vary from five to over a hundred amino acids. AMPs are utilized by almost all organisms and play an immense role in the innate immune system. They have a wide spectrum and can target any living organism and even viruses. However, AMPs are mainly effective against one class i.e. viruses or bacteria. Antibacterial AMPs mainly target the bacterial membrane, in which some lead to permeabilization via pore formation. Due to pore forming actions, AMPs are effective against antibiotic resistant strains following that the pore allows a higher dose of antibiotics to reach its target. AMPs are grouped by their secondary structure: β-sheet, α-helix, extended and loop, however some AMPs do not belong to these groups .
AMPs target cell membranes containing lipopolysaccharides. Eukaryotic cells which have a high level of cholesterol and a low anionic charge are most often safe from many AMPs .
Due to their high affinity to the bacterial cell membranes and the disintegration of the lipid bilayer; AMPs are often rapid in eliminating bacteria. The majority of antibacterial AMPs have a hydrophilic and hydrophobic domain. This structure allows the AMP to bind to the hydrophobic regions of the lipid components and the hydrophilic phospholipids .
AMPs are currently used today, such as in food preservation. Nisin is an AMP and is the only bacteriocin officially approved by the European Union (EU) as a protective food additive to prevent bacterial pathogens .
Many researchers are looking towards LL-37 due to it being endogenous to humans, such as Promore Pharma which has started a clinical phase IIb study for it . The innate immune system utilizes AMPs, they are evolutionary conserved and are a crucial defence against fungal and microbial infections. They are for expressed in several cells such as epithelial cells and immune cells such as macrophages and NK-cells. LL-37 is the only human cathelicidin member and is cationic. It is produced from the extracellular cleavage of the C-terminal end of hCAP18, this is done by serine proteases in keratinocytes and proteinases in neutrophils [4,8].
LL-37 can create aggregates in solution and lipid bilayers and is therefore protected from protease degradation. Due to LL-37’s positive charge, it can easily associate with negatively charged membranes containing phospholipids. LL-37’s 37 amino acids form an alpha helix when interacting with membranes. When the α-helix is formed the hydrophobic residues are segregated unilaterally which activates the membrane penetrating ability to take action. The penetration results in transmembrane pores and therefore lysis of the bacteria .
Magainin 2Magainin 2 is an amphiphilic (it has both a hydrophobic, positively charged domain and a hydrophilic, cationic domain.), α-helical AMP. The name magainin is derived from the Hebrew word for shield due to its shielding effect on its native host's skin, the African clawed frog (Xenopus laevis). It acts directly on the bacterial membrane and is mainly thought to create pores . Magainin 2 is also thought to have some effect of inhibiting viral action to Junin virus (JV) and herpes simplex virus (HSV) .
Pln1The plantaricin Pln1 is expressed by Lactobacillus plantarum to inhibit competing bacteria. The AMP has not been studied extensively but is thought to have an α-helical C-terminal and to be amphiphilic. The N-terminus consists probably of a random coil (therefore no stable structure outside the cell membrane) and has a more hydrophilic nature. With this overlay of Pln1 is has a capability to disrupt membrane bilayers and associate themselves in pore formation inside it, leading to bacterial leakage. Pln1 has a better activity to methicillin-resistant S. epidermidis (MRSE), M. luteus and same activity to S. aureus compared to nisin. Therefore, Pln1 in combination with nisin or by replacing it could be a lucrative candidate .
LysK-CHAPBacteriophage endolysins digest the peptidoglycan wall of bacteria. The LysK is an endolysin targeting gram positive bacteria. It has two catalytical domains known as CHAP (cysteine-and histidine dependent amidohydrolase/peptidase) and a central amidase-2 domain together with a cell binding domain. The CHAP domain which is the truncated part expressed in this year’s LiU iGEM (more specifically CHAP C1-C162) team still has lytic activity against staphylococcus. The C1 to C162 amino acids are required for full endopeptidase activity .
PlyF307PlyF307 is a prophage lysin specified for Acinetobacter baumannii, it’s also active generally for Gram-negative bacteria. The protein is composed of a single amidase domain spanning almost the whole protein (1-103). It also has an AMP at the C-terminal of the chain (104-147) which assists the protein in finding and binding to bacterial surfaces. A study of only the AMP with an addition to a hepatitis B peptide observed an increased activity compared to only the native PlyF307 AMP . We chose to integrate this peptide to our PlyF307 construct in hope of creating a more efficient lysin construct. The combination of the native PlyF307 in addition to the fusion of the hepatitis B peptide to the C-terminal native AMP has not been done before. A. baumannii is an unsettling bacteria to have in wounds  and BRIVA has expressed that it is a problematic bacteria. It is also a nosocomial bacteria. During our visit one patient was suffering from a large infection due to A. baumannii.
Silver and Cellulose BandagesBurn wounds are dressed with several types of dressings, one of the more common treatments for burns is the use of silver. The antimicrobial dressings which contains silver often contain nanocrystalline silver which is slowly released into the wound. There are also hydrocolloid dressings which releases silver ions and hydrofiber dressings as well as dressings containing silver bound to activated charcoal. There has however, been numerous debates on how effective silver bandages actually are . Our team discovered this to also be the case in the Swedish wound care system. When speaking to the Linköping University Burn Care Centre (BRIVA) we discovered that they still used silver bandages. BRIVA serves half of the Swedish population and is one of the two national burn centres, in which the other is in Uppsala. We also had the opportunity to view wound dressing of patients and talk to the wound care unit at the University Hospital at Linköping. The staff explained that they often use silver and had a very positive view on it. However, they also said that the silver bandages can be very expensive, limiting their use. To get both views of burn care we also contacted the other burn care center in Uppsala (University Hospital). The staff there described that they did not use silver and seemed to instead have a negative view on silver usage.
Data has been published on the toxic effect of silver on regenerating keratinocytes - something that can cause a delay wound healing. Silver is also considered environmentally damaging and the use of silver bandages are controlled in swedish hospitals. The Swedish Agency for Health Technology Assessment and Assessment of Social Services (SBU) is an independent government agency which assesses methods used in i.e. healthcare and they have analyzed silver wound dressings. They concluded that there is no clear evidence that silver wound dressings are more effective than non-silver dressings. This pertains not only to preventing infection, but also to pain, wound healing and other negative effects. Alternative bandages are therefore needed .
Cellulose has recently been discussed as an alternative wound dressing, including both biocellulose and microbial cellulose. Cellulose has been proposed as an alternative both due to its safe properties and to ease pain which is a major factor interfering with patient health. A recent study suggest that using cellulose bandages provides better results than using silver sulphadiazine cream. The use of cellulose provides fewer wound dressings changes and lower pain scores which is beneficial not only for the patients and staff, but also for the health care system. No irritation or allergy towards cellulose has been reported either .
Burn Wounds and Care
The major cause of burns are scalds, followed by fire. Burns damages the mechanical barrier of our defense against microorganisms, the skin. Burns can damage the skin at different depths. The first burn depth is the epidermal, followed by dermal and lastly “full thickness” burn. Full thickness burns can even destroy tissues beneath the skin such as fat, muscle, and bone. Burn wounds are unique in the way that a large area of the skin is damaged, leading to a large exposed area for microorganisms to infect. The healing procedure is therefore also different. The blood vessels in deeper flame burn wounds are burnt closed, leading to insufficient amounts of blood reaching the area. The healing process is also long due to the larger area of a burn wound in which it takes a longer time for skin cells and other types to proliferate inwards towards the wound center .
An estimated figure of 300 000 deaths are caused by burns and 95% of fatal burns take care in low-income to middle-income countries each year according to the world health organization (WHO). However, millions of survivors lives with permanent disabilities and disfigurements as a result of burns . In high income countries, in-hospital mortality has decreased significantly. This effect is due to several factors such as advances in intensive care which includes fluid resuscitation and antibiotics but also early excision of the burnt tissue. It is important to prevent burn injuries not only for survival but also better wound healing, both for wounds self-healing but also for xeno-and autographs to heal [17,18].
The Bacterial ParadoxToday, along with a greater understanding of the microbiome, questions of how infections are treated have been raised. Numerous meta-analysis has described how the microbiome is needed to protect its host from invading microorganisms. One way to deal with this is to target certain bacteria or bacterial groups . By choosing a certain combination of AMPs and/or lysins a Novosite bandage can be customized for the patients infection. Other opinions believe in targeting all bacteria and therefore a broad spectrum bandage with a combination of AMPs and/or lysins that compliment each other can be used to disinfect the entire area. This can create an optimal bandage either for one patient or a group of patients facing a similar infection.
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