Team:OhioState/Description
Description
Our Beginning
Our first in depth encounter with sepsis came through research and one of our members’ secondhand experiences. Before this research, only some of us were aware of the condition, but not the extent at which it impacted the world and how hard it was to treat. As an iGEM team we thought there had to be a way to combat this devastating condition using our knowledge of molecular genetics. To start, we focused on what sepsis is, what causes it, and what are the statistics. Additionally we researched what the recovery is like, and what happens biologically during sepsis.
A Life Threatening Complication
Sepsis is a life threatening complication that occurs during an infection. This infection can be caused by a virus, fungi, parasite, or bacteria. Many patients make a full recovery from sepsis, but some are left with lifelong detriments including fatigue, hair loss, depression, hallucinations, and post-traumatic stress disorder1. 1.7 million people in the United States each year are diagnosed with sepsis leading to at least 250,000 deaths. On the global scale, 48.9 million cases of sepsis are diagnosed in a year. 1 in 3 patients who died in a hospital died from sepsis resulting in sepsis as one of the leading causes of death in the hospital setting. These numbers were recorded before the 2019 coronavirus pandemic, which has contributed to the rise of viral induced sepsis cases.
Sepsis occurs when the immune system’s response to an infection damages the host. This hyper immune response can cause a cascade that can result in multiple organ failure and eventually death. Gram negative bacteria, one cause of sepsis, have a membrane containing lipopolysaccharide (LPS). The immune system detects the hydrophobic region of LPS, endotoxin lipid A. When the bacteria is lysed by the immune system or antibiotics, they release lipid A throughout the body which can result in a hyper immune response and thus sepsis.
The primary treatment for sepsis is antibiotics; however they lead to the lysing of bacteria. This results in the release of lipid A which further triggers an immune response, creating a cytokine storm, and potentially fatally damaging itself in the process. Other forms of treatment involve corticosteroids and vasopressors. Corticosteriods are used to suppress the immune system to prevent the cytokine storm that occurs during sepsis. However, since they do suppress the immune system, often these drugs worsen the bacterial infection. Vasopressors combat the low blood pressure that is a side effect of sepsis.
An idea
Antibiotics, corticosteriods, and vasopressors do not address the build up of excessive amounts of lipid A. Neutralizing lipid A would help reduce the immune response and avoid cytokine storms. Thus our goal became creating a novel therapy to treat sepsis that avoids the large release of lipid A and therefore a cytokine storm.
The therapy would address gram negative bacteria induced sepsis, and help reduce the risk of drug resistant microbes by relying less on antibiotics. We needed something to kill the bacteria but than also neutralize the released lipid A. We could use proteins to neutralize lipid A, but we needed a way to transport the proteins to the bacteria cells. Simply putting the proteins into the bloodstream would have a negligible effect because of the low probability of finding enough lipid A molecules to delay the immune system reaction.
Previous Research into Phage Therapy
Phage therapy for sepsis has been explored previously in murine or mice models. Natural bacteriophage, or phage for short, have been previously shown to infect sepsis-causing bacteria and have resulted in a 50%-66% rescue rate in mice models with severe sepsis23. Phage have also been shown to have strong anti-inflammatory proteins that are independent of their antibacterial activity. These properties might be due to the LPS-binding ability of phage, inhibition of reactive oxygen production and the induction of IL-10 production, an anti-inflammatory cytokine. This helps with the inflammatory reaction that is often caused by sepsis4.
Unmodified phage have already shown potential in treating sepsis and have had success in murine models of sepsis. In March 2016, the first ever intravenous bacteriophage therapy was successfully administered to Dr. Tom Patterson to combat a multi drug resistant strain of Acinetobacter baumannii. That was a compassionate use scenario and no phage therapy has been approved by the FDA as of yet; however it holds great promise in clinical applications4.
Defining Our Solution
We quickly came to realize that synthetic biology could be used to further improve the efficacy of phage in treating sepsis! Phages are a natural predator for bacteria. Similar to viruses that infect eukaryotic cells, the phages target specific bacteria cells. This fact could be used to our advantage as we can pick phages that will target the specific bacteria causing sepsis in a patient. If we insert lipid A neutralizing proteins into a phage, the proteins could be transported to our desired bacteria. Phage naturally use the host bacteria cell’s machinery against them by either inserting their genome into the hosts, or using the cytoplasm as a location to make millions more phage particles and lyse the bacteria as a result. Our phage will be able to insert its genome into the host cell and produce our anti-lipid A proteins.
But what if those proteins are lethal to the bacteria we clone them in? How can we mass produce phage to work with in lab? To bypass this uncertainty, we designed a regulatory system for our phage where the proteins are repressed when a host cell contains the appropriate sequence. This stems from a natural system used in salmonella.
Overall Scope
Our designed bacteriophage infects bacteria in a way that will prevent the cytokine storm that will cause sepsis. Our phage will infect the bacteria that cause sepsis in patients and inject DNA that will code for the production of anti-Lipid A proteins. One of the primary benefits from using a phage to combat sepsis is that the phage will not immediately lyse the cell like how antibiotics function. The proteins that are going to be expressed by the phage genome will bind or modify the lipid A in the bacterial cell wall. In doing so they will make lipid A less immunogenic and thus prevent the overreaction of the body to the bacterial infection. Therefore, when the body begins to combat and kill the bacteria causing the infection, the bound or modified lipid A will trigger the immune system much less so when compared to unmodified Lipid A. The modifying proteins we chose consist of LpxF, LpxE, LpxR, PagL, and PagP while the binding proteins we chose were LF and LptA. The modifying proteins are naturally found in bacteria where the bacteria uses them to change its membrane to avoid detection by the immune system5. We realized we could use these already existing bacterial systems to our benefit in the context of sepsis. If these proteins are so powerful, why not just make them as a drug? Being membrane proteins they do not solubilize well in water and are hard to isolate. Because of this it would be very challenging to make a concentrated drug of these proteins; however, this is not an issue when they are created inside the bacteria as in our solution.
Sources
(1) - “Post-Sepsis Syndrome.” Sepsis Alliance, https://www.sepsis.org/sepsis-basics/post-sepsis-syndrome/. Accessed 4 Oct. 2021.
(2) - Pouillot, Flavie, et al. "Efficacy of bacteriophage therapy in experimental sepsis and meningitis caused by a clone O25b: H4-ST131 Escherichia coli strain producing CTX-M-15." Antimicrobial agents and chemotherapy 56.7 (2012): 3568-3575.
(3) - Watanabe, Ryohei, et al. "Efficacy of bacteriophage therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice." Antimicrobial agents and chemotherapy 51.2 (2007): 446-452.
(4) - Górski, Andrzej, et al. "The potential of phage therapy in sepsis." Frontiers in immunology 8 (2017): 1783.
(5) - Raetz, Christian RH, et al. "Lipid A modification systems in gram-negative bacteria." Annu. Rev. Biochem. 76 (2007): 295-329.
