Team:TAU Israel/Description
BACKGROUND
Inspiration and Project Choosing Process
When tasked with choosing this year’s iGEM TAU project, we’ve decided that each team member research past iGEM projects, come up with an idea and present it to the entire team and PIs. Out of those suggestions, and with guidance provided by our PIs, we’ve narrowed the options to 4 possible projects that are relevant to pressing issues the world is currently facing and will suit the team’s various specialties (biology, computer science and engineering), while still presenting a challenge.
Eventually we’ve decided to pursue our current project, dealing with antibiotic resistant bacteria, a problem which was declared in 2016 by the World Health Organization (WHO) as one of the biggest threats to global health, food security and development today.
Our project attempts to assist in providing a solution to this issue by creating a novel modifiable bactericide based on R-type pyocins. In our approach, we attempted to form a modular, easily distributed and easily controlled pyocin system that targets numerous different bacteria, determined by introduction of various induction agents.
Eventually we’ve decided to pursue our current project, dealing with antibiotic resistant bacteria, a problem which was declared in 2016 by the World Health Organization (WHO) as one of the biggest threats to global health, food security and development today.
Our project attempts to assist in providing a solution to this issue by creating a novel modifiable bactericide based on R-type pyocins. In our approach, we attempted to form a modular, easily distributed and easily controlled pyocin system that targets numerous different bacteria, determined by introduction of various induction agents.
Antibiotic Resistant Bacteria
Ever since antibiotics were first discovered, they have been used to save countless lives and became the go-to medicine in cases of bacterial infection. In recent years, and mainly due to the wide usage of antibiotics, there has been a sharp increase in the amount of antibiotic resistant bacteria. Meaning, bacteria that used to be sensitive to a certain type of antibiotic have developed resistance and can no longer be killed by it.
When infections can no longer be treated by first-line antibiotics, more expensive medicines must be used. That, along with a longer duration of illness and treatment, increases health care costs as well as the economic burden on families and societies[1].
According to the UN Ad hoc Interagency Coordinating Group on Antimicrobial Resistance, if actions against antibiotic resistance will not be proceeded, the number of annual deaths might rise from 1 million to 10 million by 2050, leading to a damage to the economy as catastrophic as the 2008-2009 global financial crisis. By 2030, antimicrobial resistance could force up to 24 million people into extreme poverty2.
When infections can no longer be treated by first-line antibiotics, more expensive medicines must be used. That, along with a longer duration of illness and treatment, increases health care costs as well as the economic burden on families and societies[1].
According to the UN Ad hoc Interagency Coordinating Group on Antimicrobial Resistance, if actions against antibiotic resistance will not be proceeded, the number of annual deaths might rise from 1 million to 10 million by 2050, leading to a damage to the economy as catastrophic as the 2008-2009 global financial crisis. By 2030, antimicrobial resistance could force up to 24 million people into extreme poverty2.
R-Type Pyocins
Bacteriocins are high molecular weight bactericidal protein complexes produced by different bacteria species and evolutionary related to bacteriophage tails. Bacteriocins differ from bacteriophages mainly because they lack DNA. Although bacteriocins have been known for a long time, their potential as a weapon against different bacteria species wasn't fully realized.
The genes that encode the bacteriocins are arranged in a cluster. This cluster contains the structural and regulatory genes that control the expression, release, and function of the assembled bacteriocins. The resulting bacteriocins function to kill competing bacteria, and have a narrow killing spectrum-usually a subset of strains from the same species as the bacterium that produces the bacteriocin. The bacteriocins-producing bacteria are often resistant to the bacteriocin that they produce.
R-type pyocins are bacteriocins evolutionary related to the Myoviridae phage tails that are produced by Pseudomonas aeruginosa. Five different R-type pyocins have been identified, and each type has a specific killing spectrum. R-type pyocins consist of a core that is shaped like a cylinder, surrounded by a sheath. In one end of the core, there is a complex baseplate structure, and attached to this baseplate are the tail spikes that can identify and bind a receptor-binding protein on the target bacteria's membrane.
In our project, we chose to re-target the R2-type pyocins of P. aeruginosa (PAO1 strain) and transfer this pyocin gene cluster to E. coli by synthetic biology tools.
This pyocin gene cluster has been shown to be under the regulation of the bacteria's SOS response. The SOS response triggers the activation of RecA, which in turn degrades the prtN gene repressor called prtR. Following prtR degredation, prtN gene is expressed and then enhances the R2-type pyocin gene cluster expression[4].
The genes that encode the bacteriocins are arranged in a cluster. This cluster contains the structural and regulatory genes that control the expression, release, and function of the assembled bacteriocins. The resulting bacteriocins function to kill competing bacteria, and have a narrow killing spectrum-usually a subset of strains from the same species as the bacterium that produces the bacteriocin. The bacteriocins-producing bacteria are often resistant to the bacteriocin that they produce.
R-type pyocins are bacteriocins evolutionary related to the Myoviridae phage tails that are produced by Pseudomonas aeruginosa. Five different R-type pyocins have been identified, and each type has a specific killing spectrum. R-type pyocins consist of a core that is shaped like a cylinder, surrounded by a sheath. In one end of the core, there is a complex baseplate structure, and attached to this baseplate are the tail spikes that can identify and bind a receptor-binding protein on the target bacteria's membrane.
In our project, we chose to re-target the R2-type pyocins of P. aeruginosa (PAO1 strain) and transfer this pyocin gene cluster to E. coli by synthetic biology tools.
This pyocin gene cluster has been shown to be under the regulation of the bacteria's SOS response. The SOS response triggers the activation of RecA, which in turn degrades the prtN gene repressor called prtR. Following prtR degredation, prtN gene is expressed and then enhances the R2-type pyocin gene cluster expression[4].
OUR PROJECT
The Idea - Pyo Pyo
As we decided to use pyocins as an efficient alternative to antibiotics, we came to the conclusion that two main steps must be taken into consideration:
The first is to allow the pyocin to target other bacteria and not only P. aeruginosa. Due to the fact that the pyocins in nature only target specific strains of P. aeruginosa, this step is not trivial at all. Luckily, the relatively short evolutionary distance between pyocins and certain bacteriophages enables us to use the latter's tails as the new tails of the pyocins in our system. In order to discover suitable bactriophage tails, which can be used as our new pyocins tails, we have created a software that uses bioinformatics algorithms – Tail-or Swift.
The second step is to produce the pyocins in a non-pathogenic strain of E. coli. Other than the fact the P. aeruginosa produces pyocins, it is also a dangerous pathogenic bacterium, and therefore not the ideal candidate for the production of our pyocins. However, E. coli is a well-studied and a safer bacterium, and therefore it was chosen to be the vessel of our system.
While considering the idea of producing our pyocins in an E. coli, we reached the understanding that E. coli already exist in our body. This lead us to consider inserting (in the far future) the engineered E. coli itself to the body, while including genes for many different tails in it. Meaning, we thought about designing an E. coli that senses the harmful bacteria in its surroundings and produces a tailor-made pyocin, which targets the specific harmful bacteria that the E. coli encountered. This engineered E. coli could serve as an in-body drug factory, which fights different kinds of bacteria without the need of lab diagnosis and without causing collateral damage to the microbiome - as antibiotics causes.
The first is to allow the pyocin to target other bacteria and not only P. aeruginosa. Due to the fact that the pyocins in nature only target specific strains of P. aeruginosa, this step is not trivial at all. Luckily, the relatively short evolutionary distance between pyocins and certain bacteriophages enables us to use the latter's tails as the new tails of the pyocins in our system. In order to discover suitable bactriophage tails, which can be used as our new pyocins tails, we have created a software that uses bioinformatics algorithms – Tail-or Swift.
The second step is to produce the pyocins in a non-pathogenic strain of E. coli. Other than the fact the P. aeruginosa produces pyocins, it is also a dangerous pathogenic bacterium, and therefore not the ideal candidate for the production of our pyocins. However, E. coli is a well-studied and a safer bacterium, and therefore it was chosen to be the vessel of our system.
While considering the idea of producing our pyocins in an E. coli, we reached the understanding that E. coli already exist in our body. This lead us to consider inserting (in the far future) the engineered E. coli itself to the body, while including genes for many different tails in it. Meaning, we thought about designing an E. coli that senses the harmful bacteria in its surroundings and produces a tailor-made pyocin, which targets the specific harmful bacteria that the E. coli encountered. This engineered E. coli could serve as an in-body drug factory, which fights different kinds of bacteria without the need of lab diagnosis and without causing collateral damage to the microbiome - as antibiotics causes.
Proof Of Concept
In order to get closer to achieving this dream of creating a drug factory inside an E. coli, we first wanted to reach a simple Proof Of Concept (POC). The process included planning three different plasmids – each with a different purpose in the E. coli and, naturally, each under different regulation.
In our project, we decided to create two regulations for the gene cluster expression. The first regulation is similar to the WT regulation that PAO1 exhibits. RecA+ E. coli were transformed with the gene cluster including prtR, prtN and the other essential gene for cluster assembly and release named prf5 until prf24 as a previous study showed[5].
The second regulation is based on our idea to separate prtN, the cluster activator, from the negative regulation of prtR, and therefore separate it from the rest of the gene cluster.
We decided to insert the prtN gene into a Marionette plasmid under the induction of vanillic acid[6], whereas the gene cluster was inserted to another vector, relying on the induction of prtN.
The two systems we constructed share a deletion mutation in the prf15 gene. Prf15 encodes for the tail fiber protein, hence it is held responsible for the targeting of the assembled pyocin proteins complex. By creating this mutation and introducing another plasmid containing a different tail fiber from the original prf15, re-targeting the R2-type pyocins towards novel bacteria has been shown to be successful[5][7][8].
We decided to create another plasmid containing the different tail fibers, while each tail fiber was cloned separately inside the vector. The tail fiber is now induced by IPTG. We started our project by creating tail fiber plasmids that target EDL293 (originate in V10 bacteriophage), lab common E. coli (originate in P2 bacteriophage), and the WT prf15 that targets S13 for comparison. All of these were shown to effectively kill its target bacteria[5][7][8]. Later, based on bioinformatic work, we identified another tail fiber candidate that targets LT2 Salmonella (originate in FELS2 bacteriophage).
In our project, we decided to create two regulations for the gene cluster expression. The first regulation is similar to the WT regulation that PAO1 exhibits. RecA+ E. coli were transformed with the gene cluster including prtR, prtN and the other essential gene for cluster assembly and release named prf5 until prf24 as a previous study showed[5].
The second regulation is based on our idea to separate prtN, the cluster activator, from the negative regulation of prtR, and therefore separate it from the rest of the gene cluster.
We decided to insert the prtN gene into a Marionette plasmid under the induction of vanillic acid[6], whereas the gene cluster was inserted to another vector, relying on the induction of prtN.
The two systems we constructed share a deletion mutation in the prf15 gene. Prf15 encodes for the tail fiber protein, hence it is held responsible for the targeting of the assembled pyocin proteins complex. By creating this mutation and introducing another plasmid containing a different tail fiber from the original prf15, re-targeting the R2-type pyocins towards novel bacteria has been shown to be successful[5][7][8].
We decided to create another plasmid containing the different tail fibers, while each tail fiber was cloned separately inside the vector. The tail fiber is now induced by IPTG. We started our project by creating tail fiber plasmids that target EDL293 (originate in V10 bacteriophage), lab common E. coli (originate in P2 bacteriophage), and the WT prf15 that targets S13 for comparison. All of these were shown to effectively kill its target bacteria[5][7][8]. Later, based on bioinformatic work, we identified another tail fiber candidate that targets LT2 Salmonella (originate in FELS2 bacteriophage).
FUTURE PLANS
Drug Delivery System - We have already taken the first step into the world of drug delivery by creating a theoretical diffusion-based prey-predator model (see our Model Page).
This model provides a glimpse to the challenging world of delivering a protein drug to its target location, without creating collateral damage or losing its functionality in the process. We would like to suggest another alternative for the delivery issue, an outer-body delivery method, which can also be implemented with plants, water sources etc. We believe that storing the purified pyocins as a spray, as performed in the field of phage therapy, can be an optional solution for the delivery problem, and if found effective, can even replace the inner-body method.
Immunogenic response - The immunogenicity of protein therapeutics has so far proven to be difficult to predict in patients, with many biologics inducing undesirable immune responses directed towards the therapeutic, resulting in reduced efficacy or damage to the patient [9]. This is also the case when regarding pyocins as a matter with therapeutic potential. As a result, any further exploration in the field of pyocins and their therapeutic potential should be done with notation to this matter.
Quorum Sensing - In nature, quorum sensing is the use of an autoinducer, usually a small secreted molecule, to regulate gene expression according to local population density. Modern science has managed to harness this ability of bacteria and engineer quorum sensing systems, and we believe that the next step for our solution could be the use of such system. We wish that in the future, the pyocin production would be induced by the presence of the target bacteria, thus improving the accuracy, while narrowing down the risks of causing collateral damage.
References
[1] https://www.who.int/en/news-room/fact-sheets/detail/antibiotic-resistance
[2] https://www.who.int/news-room/detail/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis
[3] Scholl, Dean. "Phage tail–like bacteriocins." Annual review of virology 4 (2017): 453-467
[4] Matsui, Hidenori, et al. "Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes." Journal of bacteriology 175.5 (1993): 1257-1263
[5] Williams, Steven R., et al. "Retargeting R-type pyocins to generate novel bactericidal protein complexes." Appl. Environ. Microbiol. 74.12 (2008): 3868-3876.
[6] Meyer, Adam J., et al. "Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors." Nature chemical biology 15.2 (2019): 196
[7] Scholl, Dean, et al. "An engineered R-type pyocin is a highly specific and sensitive bactericidal agent for the food-borne pathogen Escherichia coli O157: H7." Antimicrobial agents and chemotherapy 53.7 (2009): 3074-3080
[8] Ritchie, Jennifer M., et al. "An Escherichia coli O157-specific engineered pyocin prevents and ameliorates infection by E. coli O157: H7 in an animal model of diarrheal disease." Antimicrobial agents and chemotherapy 55.12 (2011): 5469-5474
[9] Matthew P Baker, Helen M Reynolds, Brooke Lumicisi, Christine J Bryson, "Immunogenicity of protein therapeutics: The key causes, consequences and challenges". Self Nonself. 2010 Oct-Dec; 1(4): 314–322. doi: 10.4161/self.1.4.13904.
[2] https://www.who.int/news-room/detail/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis
[3] Scholl, Dean. "Phage tail–like bacteriocins." Annual review of virology 4 (2017): 453-467
[4] Matsui, Hidenori, et al. "Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes." Journal of bacteriology 175.5 (1993): 1257-1263
[5] Williams, Steven R., et al. "Retargeting R-type pyocins to generate novel bactericidal protein complexes." Appl. Environ. Microbiol. 74.12 (2008): 3868-3876.
[6] Meyer, Adam J., et al. "Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors." Nature chemical biology 15.2 (2019): 196
[7] Scholl, Dean, et al. "An engineered R-type pyocin is a highly specific and sensitive bactericidal agent for the food-borne pathogen Escherichia coli O157: H7." Antimicrobial agents and chemotherapy 53.7 (2009): 3074-3080
[8] Ritchie, Jennifer M., et al. "An Escherichia coli O157-specific engineered pyocin prevents and ameliorates infection by E. coli O157: H7 in an animal model of diarrheal disease." Antimicrobial agents and chemotherapy 55.12 (2011): 5469-5474
[9] Matthew P Baker, Helen M Reynolds, Brooke Lumicisi, Christine J Bryson, "Immunogenicity of protein therapeutics: The key causes, consequences and challenges". Self Nonself. 2010 Oct-Dec; 1(4): 314–322. doi: 10.4161/self.1.4.13904.