Team:Manchester/Design
Introduction
The aim of the project of the iGEM Manchester 2021 team is to create a living therapeutic hydrogel coating for urinary catheters with the potential to:
- Become acceptable to users from both low and high income countries,
- Be developed from sustainable sources to allow the scaling up of production in the long-term
- Actively prevent urinary tract infections and counteract pathogenic bacterial virulence factors
We have designed a therapeutic hydrogel coating for urinary catheters that focuses on the prevention of Catheter-Associated Urinary Tract infections (CAUTIs). We have chosen to create a cellulose-based hydrogel coating in order to safely bind our chassis to the coating without imposing a risk of our chassis colonizing the urinary tract. We have anchored our chassis to the hydrogel coating by expressing a Cellulose Binding Domain (CBD). Our system secretes a biofilm degrading enzyme, Dispersin B upon sensing the AI-2 autoinducer commonly present during bacterial colonization. Additionally, our system will secrete a urease inhibitor to limit the breakdown of urea into ammonia and carbamate by pathogenic ureases. Our chassis of choice was the non-pathogenic Lactobacillus plantarum as Lactobacillus spp. are widely present in the microbiome of the urinary tract and help maintain a slightly acidic urinary pH. However, as a proof-of-concept, our wet lab and modelling work was carried out assuming the system would be used in a non-pathogenic E. coli strain such as common lab strain DH5a.
Hydrogel Design
There are several goals that need to be achieved for the matrix material to house the engineered bacteria:
- excellent biocompatibility and sufficient structural integrity to allow the integration of engineered bacteria
- sustainable, abundant, and cheap enough material to allow scalability
Initially, we chose chitosan to be the material for the hydrogel. However, after some research and discussion with Dr. Ahu Parry and Prof. Christopher Blanford at the University of Manchester, we realised several significant drawbacks of chitosan that are relevant to our design:
- chitosan extraction often requires harsh, corrosive chemical conditions that are harmful to the environment
- chitosan extracted from animal sources has significant allergic risk to patients
We explored numerous alternative biopolymer options and eventually settled on a blend of sodium carboxymethylcellulose (NaCMC) and hydroxypropyl methylcellulose (HPMC) that is crosslinked with citric acid. There are several application-related advantages of this design:
- all three components are literally edible, meaning the risk of cytotoxity is minimal
- NaCMC is highly hydrophilic thanks to the carboxymethyl group on the polymer chains, reducing the protein adsorption and providing a certain degree of anti-fouling property
- The NaCMC/HPMC mix is chosen as the self-sensing smart material for the catheter application. Since the carboxymethyl (CMC) group is charge neutral in acidic environment, the antimicrobial property of NaCMC does not function under normal design conditions (urease inhibitor prevents bacteria from raising the pH of the medium). However, in the case of pathogenic bacteria colonization of our hydrogel, the increase in pH creates negative charges in the hydrogel. The anionic polymer network exhibits antimicrobial properties, as well as swelling.
These mechanisms will enable the detection of infection and allow prompt removal of the infected catheter with minimum spread of bacteria.
How to imagine the different sizes of things in this system:
Our engineered bacteria has a size of micrometer scale, polymer chains have a radius of sub-nanometer scale, Dispersin B protein has radius of nanometer scale, mesh size has a size of 10s of nanometers. What does this mean? Imagine a big swimming pool (Olympic sized or larger) filled with cooked spaghetti noodles. The spaghetti noodles represent the polymer chains. The noodles are glued together, these glue points are the crosslinks. In this swimming pool there is a few family cars embedded in this forest of spaghetti noodles. These cars represent our bacteria. In addition to cars, there are table tennis balls floating around the cars. These table tennis balls are the AI-2 molecules. Inside the cars, we have some scuba divers. When they catch a table tennis ball, they throw out a basketball from the car. The basketball represent the Dispersin B molecule. This is how big things relatively are in our designed system.
For more detailed information about what the technical considerations for the hydrogel design, please check out our hydrogel modelling page
Binding our chassis to the Hydrogel
The binding mechanism of our chassis to hydrogel matrix is designed to be the expression of a cellulose-binding domain. (CBD-cex) Since the mesh size is significantly smaller than the dimension of our bacteria, it was assumed that there will be sufficient number density of CBD-cex expressed to anchor our bacteria down in place.
Preventing Biofilm Formation
Pathogenic bacteria produce biofilm which protects them from environmental stressors such as toxins, mechanical stress and host immune systems. In order to make pathogens in UTIs more susceptible to cell death we decided to engineer a mechanism which would break down these biofilms. We arrived upon dispersin B (DspB) a protein which can cleave the glyosidic bonds found in molecules that make up the biofilm. Previously iGEM team Oxford 2015 used a similar mechanism and had created a dspA-dspB protein (BBa_K1659201) which was secreted and maintained dispersin B activity. By secreting this protein, our bacteria would be able to reduce the fitness of pathogenic bacteria making the formation of pathogenic colonies less likely.
Rather than expressing this protein constitutively which would use up valuable resources and could have a negative impact on the growth of our engineered bacteria, we decided to only produce dispersin B when pathongenic bacteria are present in the catheter. E. coli are the most common bacteria found in UTIs. A molecule which E. coli secrete in order to communicate (i.e. a quorum signal) is autoinducer-2 (AI-2). We found the LsrA promoter (BBa_K117002) which is indirectly activated by AI-2. With this mechanism our engineered bacteria will secrete dispersin B when pathogenic bacteria are present, and a result reduce the formation of UTIs in the catheter.
We designed experiments to test the effectiveness of this mechanism at reducing biofilm formation which unfortunately we could not carry out due to lack of laboratory access due to SARS-COV-2 restrictions. Nevertheless, we have prepared the laboratory protocols, which can can be seen in our Wet-lab page .
Urease Inhibitor
While considering potential mechanisms that could be employed to prevent CAUTIs, we found that bacterial ureases are important virulence factors during pathogenesis. Ureases are a superfamily of enzymes produced by bacteria, fungi, invertebrates, and plants that catalyze the breakdown of urea into carbonic acid and ammonia. These enzymes play a significant role in the nitrogen and carbon metabolism and are considered as essential for the survival of pathogens in the urinary tract where nitrogen sources are limited[2]. A number of ureolytic bacteria (organisms that catalyze the breakdown of urea into ammonia) such as Klebsiella aerogenes or Helicobacter pylori produce urease, which are associated with the occurrence of CAUTIs, gastritis and even hepatic coma[2,3]. Ureases are di-nickel-containing enzymes that hydrolyze urea to carbonic acid and ammonia through the intermediate formation of carbamic acid and in the urinary tract result in the rise of urinary pH[3].
A healthy urinary tract typically maintains a slightly acidic pH ranging from 4.5 to 7.0[3]. The patient is considered to have urinary alkalosis above pH 7.0 and urinary acidosis below pH 4.5 [3]. An elevated urinary pH promotes the formation of urinary stones made from the precipitation of calcium crystals (apatite) and magnesium ammonium phosphate precipitates (struvite), which can lead to catheter blockage [2].
Urinary stones can block the urethra or catheters and lead to bacteremia. Consequently, researchers have turned their attention to the discovery of urease inhibitors as therapeutic agents for gastric and urinary tract infections[8]. The most common and only licensed urease inhibitor currently is the 1983 FDA-approved acetohydroxamic acid (AHA)[4]. This inhibitor induces severe side effects such as hemolytic anaemia, palpitations, or even phlebitis, thus there is growing research into alternative nurease inhibitors with a lower toxicity [2].
We were advised to research multiple types of urease inhibitors and we created a table of the most potent and biosafe types within the alkaloid, flavonoid, terpenoid categories as there is information available on the metabolic pathways of these compounds. After evaluating the safety of a number of compounds and after performing molecular docking analysis, we settled on Quercetin as our urease inhibitor.
Quercetin
Quercetin is a polyphenolic flavonoid present ubiquitously in plant foods with potent antioxidant activity[7]. Extracts from quercetin have been used to treat and prevent conditions ranging from cardiovascular disease, hypercholesterolemia, rheumatic diseases, cancer, and bacterial infections, but has not shown promising results in clinical trials[7]. However, currently quercetin is mostly used as a nutritional supplement rather than a therapeutic agent.
Figure 4.- Chemical Structure of Quercetin from (PubChem CID 5280343). Quercetin has five hydroxy groups at positions 3-, 3'-, 4'-, 5- and 7. It is a widely available flavonoid and antioxidant in edible vegetables.
Due to a lack of lab access we were not able to metabolically engineer our chassis to secrete quercetin. However, we have performed Molecular Docking simulations with the Heliobacter pylori and Klebsiella aerogenes ureases to test their binding affinity for a number of ligands. Of the compounds we chose for docking, we evaluated the biosafety and toxicity based on information available from literature on toxicity assays, and finally, we settled on Quercetin and structural derivatives Quercetin 3,4'-diglucoside and Quercetin 7-O-glucoside as our chosen urease inhibitors. In our envisioned design, our urease inhibitor would be co-secreted with our Dispersin B mechanism upon pathogen detection by quorum sensing.
References:
- [1]Flores-Mireles, A., Walker, J., Caparon, M. et al. (2015). Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 13, 269–284
- [2]Hassan, S.T.S. and Žemlička, M. (2016). Plant-Derived Urease Inhibitors as Alternative Chemotherapeutic Agents. Archiv der Pharmazie (Weinheim), 349(7), pp.507–522.
- [3]Lai,H., Chang, S., Lin,H. et al. (2021).Association between urine pH and common uropathogens in children with urinary tract infections, Journal of Microbiology, Immunology and Infection, 54(2). pp.290-298
- [4]Milo, S, Heylen, R.A., Glancy, J., Williams, G.T., Patenall, B..L., Hathaway, H.J., Thet, N, T., Allinson, S.L., Laabei, M., Jenkins, A.T.A. (2021). A small-molecular inhibitor against Proteus mirabilis urease to treat catheter-associated urinary tract infections. Scientific reports, 11(1), pp.3726–3726.
- [5]Protein Data Bank, R., 2021. RCSB PDB - 1E9Y: Crystal structure of Helicobacter pylori urease in complex with acetohydroxamic acid. [online] Rcsb.org. Available at:
- [6]Pubchem.ncbi.nlm.nih.gov. 2021. Quercetin. [online] Available at:
- [7]Salehi B. et al (2020). Therapeutic Potential of Quercetin: New Insights and Perspectives for Human Health,ACS Omega,5(20): pp.11849–11872, doi: 10.1021/acsomega.0c01818
- [8]Svane, S., Sigurdarson, J.J., Finkenwirth, F., Eitinger, T., Karring, H. (2020). Inhibition of urease activity by different compounds provides insight into the modulation and association of bacterial nickel import and ureolysis. Scientific reports, 10(1), pp.8503–8503.
- [9]Timothy K. Lu, James J. Collins. (2007). Dispersing biofilms with engineered enzymatic bacteriophage.Proceedings of the National Academy of Sciences, 104 (27).pp. 11197-11202; DOI: 10.1073/pnas.0704624104
- [10]Yan,Z., Huang, M. Melander, C. and Kjelle, B.V. (2019). Dispersal and inhibition of biofilms associated with infections. Applied Microbiology, 1364-5072, pp.1279-1288; DOI: :10.1111/jam.14491