Team:Warwick/Description
Contents
Description
The colibactin detection kit
The COVID-19 pandemic's consequences run far deeper than one might think - the added strain on healthcare systems worldwide has caused a disruption in programs unrelated to respiratory disease, one major victim being cancer screening programs. More specifically, colorectal cancer (CRC), being a leading cause of death in the UK even before the onset of the pandemic, is at risk of going unnoticed in many patients due to resources for screening being diverted, both in terms of materials and manpower.
3D model of one of the more stable conformers of the N-myristoyl-D-asparagine molecule.
In 2006, the presence of the clb gene cluster in E.coli and other Enterobacteriaceae was identified to induce double-stranded breaks in the DNA of eukaryotic cells[1]. This gene cluster allows for the production of colibactin (Clb), a substance which has been proven to be a very potent genotoxin - among others, it can alkylate and induce double-stranded breaks in the DNA of the colon's epithelial cells[2]. This has been very closely linked with the occurrence of CRC - as many as two thirds of CRC patients were found to harbour clb+ E. coli[3]. Fluorescence-based mechanisms for detection of colibactin have already been proposed[4] but we intend to take this one step further.
We have endeavoured to create a non-invasive, reliable, cheap, and easy-to-use testing kit for the presence of the clb+ E. coli strain in the human gut, based on a modified transcription factor which can sense the presence of one of the more stable byproducts of colibactin's biosynthetic pathway - a compound called N-myristoyl-D-asparagine (N-myr-D-Asn).
Project design - how it works
As a design template, we used a natural biosensor named MmfR, identified in Streptomyces coelicolor as a receptor for a family of bacterial hormones known as MMFs, which regulate the production of bacterial antibiotics. This protein is made up of a DNA-binding domain (DBD) and a hormone-binding domain (HBD), generally encountered in a homodimeric form. We chose this protein due to the fact that the DBD binds to specific 18bp sequences in the DNA, which eliminates the need for modification of the gene whose expression will be determined by the presence of N-myr-D-Asn.
In addition, the structure of the MMFs and the structure of N-myr-D-Asn are somewhat analogous; both are made up of a polar group (a complex functionality made up of a furan ring, a carboxyl functionality and a hydroxyl functionality and an asparagine residue, respectively) and an alkyl chain[5]. Hence, the structure of the HBD would theoretically be a great candidate for modification.
Structure of the MmfR homodimer with one of its native ligands, MMF2, displayed in the binding pocket. Used with permission from [5]
As a cloning vector, we planned to use the pET151 TOPO plasmid, into which we will insert the sequence encoding the mutated MmfR. This will be expressed in cultures of non-pathogenic strains of E. coli.
Plasmid map of our chosen vector, the pET TOPO 151 plasmid, containing the gene encoding one of our modified proteins - an N-acetyl-D-asparagine sensor.
The N-myr-D-Asn sensor would be part of a testing kit - as it binds to a short motif in the DNA which can be placed before any gene, it would be used in regulating the production of a fluorescent protein such as GFP.
Our testing kit would ideally be used by the screening patients themselves, as the GFP produced in the event of a positive result is easily visible with the naked eye when exposed to blue light. The distribution of this screening kit would be handled much in the same way as with current screening methods - healthcare systems would send these kits to people they deem at risk of developing CRC and assess whether further investigation is warranted.
References
- ↑ Nougayrede, J. P., Homburg, S., Taieb, F., Boury, M., Brzuszkiewicz, E., Gottschalk, G., Buchrieser, C., Hacker, J., Dobrindt, U., Oswald, E., Escherichia coli Induces Double-Stranded Breaks in Eukaryotic Cells, Science, 2016, 313, 848-850
- ↑ Engel, P., Vizcaino, M. I., Crawford, J. M., Gut Symbionts from Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways, Appl. Environ. Microbiol., 2015, 81, 1502-1512
- ↑ Arthur, J. C., Perez-Chanona, E., Muhlbauer, M., Tomkovich, S., Uronis, J. M., Fan, T-J., Campbell, B. J., Abujamel, T., Dogan, B., Rogers, A., B., Rhodes, J. M., Stintzi, A., Simpson, K. W., Hansen, J. J., Keku, T. O., Fodor, A. A., Jobin, C., Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota, Science, 2012, 338, 120−123
- ↑ Hirayama, Y., Tsunematsu, Y., Yoshikawa, Y., Tamafune, R., Matsuzaki, N., Iwashita, Y., Ohnishi, I., Tanioka, F., Sato, M., Miyoshi, N., Mutoh, M., Ishikawa, H., Sugimura, H., Wakabayashi, K., Watanabe, K., Activity-Based Probe for Screening of High-Colibactin Producers from Clinical Samples, Org. Lett. 2019, 21, 4490-4494
- ↑ 5.0 5.1 Zhou, S., Bhukya, H., Malet, N., Harrison, P. J., Rea, D., Belousoff, M. J., Venugopal, H., Sydor, P. K., Styles, K. M., Song, L., Cryle, M. J., Alkhalaf, L. M., Fulop, V., Challis., G. L., Corre, C., Structural basis for control of antibiotic production by bacterial hormones, BioRxiv, 2020, doi:10.1101/2020.05.02.073981