As a continuation of the 2015 WLC iGEM team's project, we have taken the idea of phage therapy for treatment of bacterial infection to a new environment: the coral reefs. Because WLC is a Christian school, we want our projects to always be rooted in our faith, which is why we chose a project involving good stewardship of the Earth and its resources. Last year's WLC team designed a way to screen environmental sources for bacteriophages (viruses that infect bacteria), and we continue to use these methods. Read on for a more detailed explanation of the premise and goals of this year's project.
Elkhorn Coral (Acropora palmata)
Elkhorn coral (Acropora palmata) is a vital reef-building coral native to the Caribbean. This name is given because of its resemblance to elk antlers, in both shape and the brown color given by the photosynthetic protozoans living symbiotically inside the coral’s tissue. The complicated structure of Elkhorn contains many large branches with a full-grown coral reaching up to twelve feet in diameter(1).
Part of the reason Elkhorn is such an important component of reef building is its ability to grow very rapidly. Many branches grow four or more inches in a single year. The quickly expanding branches provide reef structure but also a habitat for an immense variety of reef fish and crustaceans. Because of this, the importance of Elkhorn goes far beyond just reef building capabilities.
Formerly, Elkhorn was a very common structural coral throughout the Caribbean and Gulf of Mexico. More recently, due to a number of factors, Elkhorn coral population has been in severe decline. In 2005 it was added to the endangered species list and remains there today despite many efforts to restore and revive the population. The reasons for the low population in recent years is due to a wide variety of pressures including predation and bleaching. The most significant cause of this scarcity, however, is rampant disease, largely caused by the human pathogen Serratia marcescens.
Serratia marcescens, a pathogenic species belonging to the family Enterobacteriaceae, is known to infect humans, as well as other animals.(2) The bacteria are able to thrive in a wide variety of environments including soil, water, and the digestive tract of their host.(3) Because of this capacity, S. marcescens has become particularly dangerous to marine life. Specifically, the infection of Elkhorn coral by S. marcescens or “white pox” can be attributed to immense destruction of the coral reefs(4).
Elkhorn coral has been subject to infection by S. Marcescens since the first outbreak of white pox in 1996. At least one of the strains causing the infection (PDR60) can be traced back to human sewage. This makes the infection the only known marine reverse zoonotic disease. In other words, S. marcescens, which is a human pathogen, was transferred to a nonhuman host: A. palmata. However, certain studies based on bacterial isolates of A. palmata lesions suggest that there may be additional unknown causes of white pox disease separate from Serratia infection.
The pathology of S. marcescens in coral involves the mucus membranes of A. palmata. Because of the high efficiency of S. marcescens to utilize glycoprotein components in the mucus, it is able to completely outgrow any other bacteria present. This includes A. palmata’s own microphytes. Because of this the coral are, in effect, stripped of their immune defenses, and their nutrient providers.
If the S. marcescens infection could be wiped out with bacteriophage therapy (see our team’s previous article on phage therapy below), the coral would have the potential for rehabilitation. This is the idea central to our project. The outer membrane protein common to many gram negative bacteria: TolC (see our team’s pervious article on TolC below), can be cloned into a plasmid and function in E. coli. This way, phages specific to the Serratia tolC gene can be isolated without having to deal with any harmful bacteria in the lab. Once a phage is isolated, it could potentially be used to treat infected coral.
Sutherland, Kaitlyn. "Human Pathogen Shown to Cause Disease in the Threatened Eklhorn Coral Acropora Palmata." PLOS ONE:. N.p., 11 Aug. 2011. Web. 24 Sept. 2016.
Mahlen SD. Serratia infections: from military experiments to current practice. Clin Microbiol Rev. 2011 Oct. 24(4): 755-91.
Petersen LM, Tisa LS. Friend or foe? A review of the mechanisms that drive Serratia towards diverse lifestyles. Can J Microbiol. 2013 Sep. 59(9):627-40.
Gossard, Kimberly A., "Genetic Profiling of the White Pox Disease Coral Pathogen Serratia marcescens from the Florida Keys" (2014).Honors Program Theses. Paper 5.
Importance of Reef Building Coral and Associated Symbionts
The importance of hard corals to the global ecosystem as we know it cannot be overestimated. Without the coral reefs and the solid structure they provide, erosion of beaches and shorelines would proceed at significantly higher rates due to the lack of wave dissipation that reefs provide. Without reefs, many larval fish species would lack an environment in which to develop or would lack a vital link (a food source for example) in the food chain they rely upon. Coral skeleton formation (calcification) and energy production also, and perhaps most importantly, plays a large role in carbon fixation in tropical and subtropical environments (1). Most coral species rely heavily on close symbiotic relationships with unicellular microalgae called zooxanthellae (1). These zooxanthellae provide up to 90% of the coral’s organic molecule, amino acid, and carbon requirements (2) by fixing dissolved carbon dioxide into glucose via the process of photosynthesis. Without zooxanthellae, corals would bleach (lose color) and die due to a lack of adequate nutrient uptake (3).
Hard coral species also use the fixed carbon during the production of their Calcium Carbonate skeleton (3). These two processes for the fixing of carbon into usable molecules (such as glucose) and stable calcareous molecules are large contributors to the global carbon cycle. These numerous traits of our reefs, that affect so many global interactions, highlight just a few of the many benefits coral reefs provide for the earth we live on. But our reefs are threatened now as never before in a myriad of ways, from pollution, dredging and destruction of the physical reef, to disease and an increasingly acidic environment affecting many aspects of our lives and placing the future of our oceans in danger. One such pressing concern for the coastal reefs of the southern United States (such as those located along the Floridian coast) has been a decimation and massive decrease in the populations of the primary reef building coral of these reefs, Acropora palmata, otherwise known as Elkhorn coral.
 Falkowski PG, Dubinsky Z, Muscatine L, and James W. Porter: “Light and the Bioenergetics of a Symbiotic Coral.” BioScience: Vol. 34, No. 11 (Dec., 1984), pp. 705-709
 Berkelmans R, and Madeleine J.H Van Oppen: "The Role of Zooxanthellae in the Thermal Tolerance of Corals: A 'nugget of Hope' for Coral Reefs in an Era of Climate Change." Home. N.p., n.d. Web. 2 Aug. 2016.
Steffen P, and Bruce Moravchik. "Corals." NOAA National Ocean Service Education:. N.p., n.d. Web. 2 Aug. 2016.
Note: this article was written by last year's WLC iGEM team, we are using it as it still applies to this year's project.
TolC is a protein comprised of 471 amino acids to compose a homotrimer that forms a tapered hollow cylinder 140Å in length: a 40Å long outer membrane barrel [the channel domain] that anchors a 100Å long helical barrel that projects across the periplasmic space . In review, the trimer forms a single beta barrel for the passing of proteins. Each monomer gives four antiparal-lel beta strands and four antiparallel alpha helical strands. This forms the channel and tunnel do-mains, respectively . There are also small loops present on the sides of TolC that allow for phage binding or colicin attachment. It is thought that the tunnel assembly is supported by hydrogen bonds and salt bridges.
TolC is interesting because unlike other efflux pumps, TolC bypasses the periplasm. To bypass the periplasm, instead of using a multiprotein assembly to span the membrane, TolC only re-quires the outer membrane protein. This protein interacts with the inner membrane translocases traffic ATPase and an adaptor protein. There is not just one TolC and interacting translocase, but one cell may have more than one TolC homolog with multiple compatible translocases. This contributes to antibiotic resistance: multidrug-resistant Pseudomonas aeruginosa has four major efflux systems.
TolC is known as an efflux pump; it provides an exit for large proteins. TolC is used for protein export for proteins such as “heat-stable enterotoxin, cationic antimicrobial peptides, and micro-cins” . For interest of our project it also effluxes antibiotics, contributing to antibiotic resistance. The protein is exported through translocase, such as AcrAB, then through the TolC periplasmic entrance .
In Antibiotic Efflux
TolC’s role in antibiotic efflux is what interests us as a target for fighting multidrug-resistant bacterial infections. Koronakas expressed interest in finding a way to block the TolC pump. Our iGEM team aimed at finding bacteriophage that use this pump to bind and kill the bacterium. This year, we extend last year's project by adding a new tolC gene, that of S. marcescens. The goal being that a phage can be isolated that will bind to the S. marcescens TolC protein.
 Koronakis, V., Eswaran, J. & Hughes, C. STRUCTURE AND FUNCTION OF TOLC: The Bacterial Exit Duct for Proteins and Drugs. Annu. Rev. Biochem. 73,467–489 .
Note: Images also from Koronakis paper
Note: this article was written by last year's WLC iGEM team, we are using it as it still applies to this year's project.
According to the Phage Therapy Center, bacteriophage therapy is defined as “the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infection.” (1) And while this technique was discovered prior to antibiotic treatments, inconsistent results and a lack of understanding caused it to be almost completely overshadowed by antibiotics. (2) The ever increasing issue of antibiotic resistance has led to a phage research revival. Modern knowledge of bacteriophages gives hope that phage therapy will not only work with but surpass antibiotic treatment.
Bacteriophages are viruses that target bacteria to reproduce. The phages do this by injecting their DNA into the host. The bacteria can then enter one of two phases, the first of which is the lysogenic phase. In the lysogenic phase, the viral genome integrates into the bacterial DNA and goes into a hibernation allowing the cell to replicate on its own until it enters the lytic cycle. In the lytic cycle, the bacterium is used to create parts of the phage which are then assembled. (3) The phages create two enzymes, hollins, which perforate the plasma membrane, and lysins, which attack the cell wall. The enzymes ultimately cause the bacteria to lyse. (2) Phage therapy works based on the bacteriophages’ ability to lyse specific bacteria while not harming eukaryotic cells.
The early founders of phage therapy made many mistakes that ultimately led to antibiotic treatments being favored over phage therapy. The main issue was a lack of information about bacteriophages. While some at the time believed that they were viruses, many of the studies hinted at phages being an enzyme that was created by the bacteria. (4) The lack of knowledge also led to very inconsistent reports causing Eaton, Bayne, and Jones to write a report which deemed the technique no better than prior treatments.(4) Also, phages are very specific, making it difficult to kill the correct bacteria with the correct phages using the technology of the time. (5) The lack of research and understanding also led to impurities being injected into patients, further infecting them, and denaturing the bacteriophages during isolation leaving them inactive. Finally, the researchers faced the issue of the phages being rapidly eliminated by the body and were unable to prevent it.
Due to the overuse of antibiotics, alternative treatments like phage therapy have been sought. Fortunately, modern technology has had more success with phages than the discoverers. Thanks to scientific advances, many of the original problems have been solved, and phages are now even superior to antibiotics in many ways. First, phages can be very specific and can be programmed to target only the virulent bacteria so as to not damage the microbiomes or probiotics. (1) Modern technology has also allowed for the easy and effective isolation of phages without disrupting their function. In addition, while the body metabolizes antibiotics, lytic bacteriophages are constantly replicating in the host bacteria and thus grow exponentially in number. (1) Phages have also been identified with coat proteins that are harder for the body to identify as foreign. This allows the phages to stay in circulation longer and until they reach the lytic cycle and quickly kill the bacteria. (5) There is also much less risk of resistant bacteria forming as only the target bacteria will be infected while the others remain untouched. This is very different from antibiotics that cause drug-resistant strains of numerous species. If, and when, a bacteria does become resistant to a phage, it is much easier and faster to find a new bacteriophage than it is to develop a new antibiotic. (1) Finally, while a phage’s ability to mutate could be seen as a safety threat, it also provides a built-in mechanism to continue to infect bacteria that have adapted a resistance to the original strain. (5)
While phage therapy is not a new technique, it has only recently started a comeback as a viable antibacterial treatment. Bacteriophages are viruses that can be selected to target and lyse bacteria of choice. Phage therapy has overcome many of the obstacles that cleared the way for antibiotics to take over, and is now in many ways superior to antibiotics. The new surge of research being done on phage therapy continues to reveal exciting new options for the technique.
 "What Is Phage Therapy?" Phage Therapy. Phage Therapy Center, 2013. Web. 20 Aug. 2015. .
 Potera, Carol. "Phage Renaissance: New Hope against Antibiotic Resistance." Environmental Health Perspectives. 1 Feb. 2013. Web. 20 Aug. 2015. .
 "Lytic Cycle." New World Encyclopedia. 22 Dec. 2008. Web. 20 Aug. 2015. .
 Sulakvelidze, Alexander, Zemphira Alavidze, and Glenn J. Morris. "Bacteriophage Therapy." Antimicrobial Agents Chemotherapy. National Center for Biotechnology Information, 1 Mar. 2001. Web. 20 Aug. 2015. .
 Carlton, Richard M. "Phage Therapy: Past History and Future Prospects." Archivum Immunologiae Et Therapiae Experimentalis, 1999. Web. 20 Aug. 2015. .