Project Description and Inspiration
We present our project PHANGEL for the iGEM competition 2020. PHANGEL is a genetically engineered bacteriophage that not only presents an attractive alternative to traditional antibiotic therapy but also tackles the adverse effects arising from bacterial lysis. In our Promotion Video we already gave a little sneak peek into our project idea. If you have not seen it you can watch it here again:
Estimations suggest that more than 11 million people die of sepsis every year. That is one in five deaths around the world and makes sepsis the most prevalent cause of death, even more deadly than cancer1. Sepsis is caused by pathogenic infections, during which endotoxins are released from the bacterial membrane that cause a dysregulated systemic immune reaction in which the immune system attacks endogenous tissues.2
This topic is especially urgent in time like ours when an increasing number of pathogens cannot be combated with well-known strategies such as antibiotics anymore. Studies show that it can take less than a year for a resistance against a newly discovered antibiotic to be evolved and spread.3 Thus, alternative therapies to common antibiotic treatment are desperately needed.
One very promising branch of research in the search for new ways to combat bacterial infections are bacteriophages (phages). Even though they have been discovered as early as 1915 (and 1917), they were mostly disregarded as therapeutic in western countries. Only in the last few decades, they came into scientific focus due to their ability to kill bacteria. Since then a lot of new discoveries have been made: we now know e.g. that bacteriophages are the most abundant organism in the world, with a total number of 1031, approximately ten times higher than bacteria, and a big part of the human microbiome. This should not scare us! Bacteriophages are small viruses that can only infect bacteria and therefore naturally pose hardly any harm to humans or other animals. In fact, their habitation of our mucosal surfaces can reduce the colonization of pathogens and through this actually act as a protective layer on us!4 Regardless of additional therapeutic aspirations, this alone makes bacteriophages very useful organisms to us.
Furthermore, most bacteriophages are highly selective and have one or few specific bacterial hosts that they infect. This specificity is defined by host surface receptors and antiviral defense mechanisms of bacteria. Through this, they are less prone to adverse effects as common antibiotics when used in a clinical setting, since antibiotics often kill bacteria unspecifically and also often attack the desirable bacteria in our floras. Furthermore, they are so small and simple in their composition, oftentimes they only consist of nucleic acid and proteins, that they have a very low physiological toxicity.5
As phages are viruses that use their respective host as a replication factory, they are relatively simple to dose. In an active dosing regime, only a low concentration of bacteriophages (in relation to the bacteria concentration) needs to be administered to an infection because the phages will be replicated until there are no more hosts to infect.
There are also studies that demonstrate an interaction between bacteriophages and their host pathogens that make the pathogen more susceptible to antibiotics and may even reverse the resistance.6
Even though western countries do not have a long history in phage research and therapy that doesn't mean no one has. Eastern European countries, mainly Georgia, Poland and Russia have successfully employed bacteriophages for decades. In fact, it is even considered a routine use in these regions and research is very advanced.7
All of this makes phage therapy a very hot topic in the search for new antibiotic strategies.
However, phages alone do not tackle the problem with sepsis. The opposite might even be true. When a phage kills a bacterium in the so called “lytic cycle”, the membrane of the bacterium is destroyed, releasing all its endotoxic components. In gram-negative bacteria such as E.coli up to 75% of the outer cell membrane can consist of lipopolysaccharides, short LPS. The endotoxin LPS is heat- and pH stable and often involved in the origin of a sepsis as they elicit strong immune responses and weaken the immune system.8
Simplified, this immune response is mediated in the following way: LPS is opsonized by the LPS-binding protein (LBP). This complex is recognised by glycoprotein CD14 which, together with Toll-like receptor 4, forms the LPS receptor on macrophages. If the LPS-LBP complex binds to the LPS receptor, a signal cascade is induced that leads to the release of proinflammatory factors such as cytokines, TNF‐α, IL‐1β and IL‐6 which causes the systemic inflammation.9
This is a potentially dangerous side effect of bacterial degradation and already a matter of research among phage scientists: several studies have been conducted to impair or inhibit the lysis of host bacteria in order to keep the endotoxin contained. In several different studies genetically engineered phages were created to kill the bacteria not through lysis but through degradation of their genome keeping the membrane mostly intact.10 However, we know of no approach like PHANGEL.
Our Vision
Our goal for this project was to create a phage that reliably contained the gene human plasma gelsolin, a peptide of the LPS-binding region (aa 160-169) of gelsolin called GSN 160-169 or the antimicrobial peptide 19-2.5. All of these proteins or peptides possess the ability to bind and inactivate LPS.11,12,13We aimed to engineer a bacteriophage that would not only kill the host bacterium but would force it to express one of these LPS binding substrates in order to prevent the uncontrolled release of LPS into the system. We planned to replace the gene for the Major Capsid protein 10 of the T7 phage with these proteins and peptides which at the same time disables the uncontrolled replication of the engineered phages.
To make changes in the genome of lytic phages, few relatively easy and effective methods exist. One of the best described ones is the BRED system.14 BRED stands for Bacteriophage Recombineering of Electroporated DNA and was first used to modify difficult to engineer mycophages.15 The principle is simple: the purified linear phage DNA and the double-stranded linear DNA to insert/delete/replace in the phage (both are called the recombineering substrates) are co-electroporated into electrocompetent bacterial cells which carry a recombination plasmid, i.e., a plasmid which expresses proteins that promote a high degree of homologous recombination. For our project, we decided on the well studied Lambda RED system16, consisting of the exo, beta and gam. Exo is the lambda exonuclease that binds and degrades dsDNA non-sequence-specific from 5’ to 3’ , reforming the 5’-end into a 3’overhang17,18 and leaving ssDNA and promoting the annealing or loading of Beta.
Beta binds to the single-stranded version made by Exo and facilitates the recombination of the homologous sequences.17,19 This means that the insert needs to have at least 80bp of homologous sequences to the desired place of insertion in the T7 genome. These homologous sequences are called recombination sites.
Gam protects the ssDNA produced by Exo from endogen E. coli nucleases, specifically RecBCD and SbcCD.19
Due to the nature of the system, promoting a high level of recombination, it is quite toxic to the bacterial cell, and therefore needs a very tight promoter/terminator system.
After electroporation, the electroporated bacterial cells are mixed with new host bacteria, often the wild type of the species. In our project, both bacteria strains would be our so called “production cells”, an especially engineered E.coli strain that carries the plasmid expressing the T7 major capsid protein10. Since the major capsid protein 10 is replaced by a LPS binding protein or peptide, the phages are only able to proliferate in these producer cells.
To achieve this goal and create the phage, we first needed to clone the Lambda RED Recombination system into a single plasmid and engineer the producer strain containing the plasmid with the Major Capsid Protein10. The assembly of both these plasmids is described in the tab Engineering success and Results.
The next step would have been the phage recombination and several assays to prove the functionality of our approach. Unfortunately, we were unable to complete our experiments in the scope of our time in the lab, hence the next steps remain theoretical for now. Read more about it in the tab Engineering success.
The impact of COVID-19 on our project
To show the impact (positive as well as negative) COVID-19 had on our project we created two mind maps. One showing the obstacles we had to face, the other showing the advantages the adapted version of the iGEM competition had on our project.
1 https://www.bbc.com/news/health-51138859#:~:text=One%20in%20five%20deaths%20around,than%20are%20killed%20by%20cancer. (last seen on: 26.10.2020).
2 https://flexikon.doccheck.com/de/Sepsis (last seen on: 26.10.2020)
3 https://www.cdc.gov/drugresistance/about.html (last seen on: 26.10.2020)
4 Keen E. C. (2015). A century of phage research: bacteriophages and the shaping of modern biology. BioEssays : news and reviews in molecular, cellular and developmental biology, 37(1), 6–9. doi: 10.1002/bies.201400152
5 Romero-Calle, D., Guimarães Benevides, R., Góes-Neto, A., & Billington, C. (2019). Bacteriophages as Alternatives to Antibiotics in Clinical Care. Antibiotics (Basel, Switzerland), 8(3), 138. doi: 10.3390/antibiotics8030138
6 Chan, B., Sistrom, M., Wertz, J. et al. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. Sci Rep 6, 26717 (2016). doi: 10.1038/srep26717
7 Tom Parfitt, Georgia: an unlikely stronghold for bacteriophage therapy, The Lancet, Volume 365, Issue 9478, 2005, Pages 2166-2167, ISSN 0140-6736, doi: 10.1016/S0140-6736(05)66759-1.
8 https://www2.biomin.net/at/artikel/the-hidden-dangers-of-lipopolysaccharides/ (last seen on: 26.10.2020)
9 Cohen, J. The immunopathogenesis of sepsis. Nature 420, 885–891 (2002). doi: 10.1038/nature01326
10 Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J., & Lu, T. K. (2016). Genetically Engineered Phages: a Review of Advances over the Last Decade. Microbiology and molecular biology reviews : MMBR, 80(3), 523–543. doi: 10.1128/MMBR.00069-15
11 Schuerholz, T., Doemming, S., Hornef, M., Martin, L., Simon, T. P., Heinbockel, L., Brandenburg, K., & Marx, G. (2013). The anti-inflammatory effect of the synthetic antimicrobial peptide 19-2.5 in a murine sepsis model: a prospective randomized study. Critical care (London, England), 17(1), R3. doi: 10.1186/cc11920
12 Cheng Y, Hu X, Liu C, Chen M, Wang J, Wang M, Gao F, Han J, Zhang C, Sun D, Min R: Gelsolin Inhibits the Inflammatory Process Induced by LPS. Cell Physiol Biochem 2017;41:205-212. doi: 10.1159/000456043
13 Bucki R, Georges PC, Espinassous Q, Funaki M, Pastore JJ, Chaby R, Janmey PA. Inactivation of endotoxin by human plasma gelsolin. Biochemistry. 2005 Jul 19;44(28):9590-7. doi: 10.1021/bi0503504. PMID: 16008344.
14 Marinelli LJ, Hatfull GF, Piuri M. 2012. Recombineering: a powerful tool for modification of bacteriophage genomes. Bacteriophage 2:5–14. doi: 10.4161/bact.18778.
15 Marinelli LJ, Piuri M, Swigonová Z, et al. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One. 2008;3(12):e3957. doi: 10.1371/journal.pone.0003957
16 Christopher R. T. Hillyar ;Genetic recombination in bacteriophage lambda;
2012; Bioscience Horizons: The International Journal of Student Research, Volume 5, 2012, hzs001, doi: 10.1093/biohorizons/hzs001
17 Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate J. A. Mosberg,*†,1,2 M. J. Lajoie,*†,1,2 and G. M. Church*
Genetics. 2010 Nov; 186(3): 791–799.
18 Szczepanska, A. K., 2009. Bacteriophage-encoded functions engaged in initiation of homologous recombination events. Crit. Rev. Microbiol. 35 197–220.
19 Sawitzke, J. A., L. C. Thomason, N. Costantino, M. Bubunenko, S. Datta et al., 2007. Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421 171–199.
