Tonsillitis is when the tonsils are infected, in which symptoms of having tonsillitis include having sore throat, fever, fatigue, or trouble swallowing. Some extreme symptoms even include difficulty breathing. Moreover, tonsillitis is very common in children and many have to undergo tonsillectomy, the surgical removal of the tonsils.
Streptococcus pyogenes is the cause of strep throat and tonsillitis! S. pyogenes is responsible for up to 30% of the pharyngitis cases in children and up to 20% in adults. On top of that, it is the main cause of tonsillitis, a bacterial infection on the tonsils.
We confirmed the need for our project by surveying over 200 people in the public and interviewing Dr Thiwan Wirawan, a pediatrician at Thainakarin Hospital, who specializes on the topic. She provided us with valuable information on strep throat, and how penicillin is not commonly used anymore due to S. pyogenes’s antibiotic resistance. Furthermore, antibiotic resistance is very common, leading to patients having to come back to check up and either change medication or increase the dosage. Tonsillitis also poses a large problem for patients, with antibiotic resistance becoming more and more of a relevant subject when treating tonsillitis. In fact, Dr. Wirawan believes that within the next few decades, antibiotics will not be as reliable. She supports the notion of phage therapy as long as it is tested to be safe and reliable, as phage therapy is a promising alternative due to it’s high specificity. This means that it will only target S. pyogenes without killing other bacteria.
We hope to create alternate ways to treat tonsillitis due to the current limitations of antibiotics– that is, antibiotic-resistant strains of bacteria. We hope to utilize phage therapy to target Streptococcus pyogenes in the throat and tonsils. Our ultimate goal is to create a mouthwash that contains Lactococcus lactis that would release a phage that could bind to as well as deactivate S. pyogenes, thus reducing its harmful effects in the cases of strep throat and tonsillitis.
The first step into making our idea of the probiotic mouthwash become a reality was to engineer a phage that could infect a probiotic while being able to bind to S. pyogenes. This would not be a simple feat, and we decided on Phage P2 as our phage candidate. Phage P2 is a good candidate as it binds to a macromolecule on its natural host, Lactococcus lactis. S. pyogenes’s natural phage also binds to a macromolecule, though in this case, it is peptidoglycan. This means that we are likely to be able to mutate a Phage P2 that can bind to S. pyogenes. Moreover, Lactococcus lactis is a common bacteria naturally found in saliva, so it can play the role as our probiotic.
Our approach was to sequence different computational models of Phage P2 that we mutated that could bind with S. pyogenes in hopes to compare them with the structure of mutated antibodies on a phage that binds to S. pyogenes in the wet lab. This will allow us to determine which properties allow bacteriophages to bind to S. pyogenes so we could further engineer the Phage P2 to be able to bind with S. pyogenes.
Hence, we decided to take both a computational approach and a wet lab approach, meaning that the Phage P2 that binds with S. pyogenes best in the computational simulation will be compared with the phage that binds with S. pyogenes best in the wet lab.
For the computational approach, we mutated the Phage P2 into numerous different forms and docked it with S. pyogenes to determine which version of Phage P2 has the best binding affinity with S. pyogenes.
For the wet lab approach, we hoped to use biopanning on S. pyogenes, a common screening method, in which phage clones are enriched by binding to the antigen for the isolation of specific ligands. However, due to unforeseen circumstances with the advent of COVID-19, we were unable to receive S. pyogenes in a timely manner and could not receive blood agar for S. pyogenes. Therefore, for this year’s project, we decided to use our limited amount of time allowed in the lab to validate the process of biopanning by using Phage KM13 with Staphylococcus aureus instead. In future projects, we hope to complete our goal of sequencing the mutated Phage KM13 that binds to S. pyogenes best.
WHY “PROJECT TROJAN HORSE”?
Our project name is called Project Trojan Horse inspired from the well known story of the Trojan War. A long, long time ago, the Greeks devised a plan to take over the city of Troy. They did this by constructing a wooden horse and giving it as a gift to the people of Troy. However, it was no ordinary gift. The Greeks hid an army of men inside the horse, making them able to enter and destroy the city of Troy. Thus, winning the war.
Thanks to the Greeks, we were able to make up our own plan as well! You see, instead of the Trojan horse, we used probiotics as a carrier for phage therapy as it contains the hidden army or bacteriophage. Once the probiotic enters the city, so to speak, it then will release this bacteriophage and kill our target.