Understanding glycosaminoglycans’ (GAG) interaction with proteins is of growing interest for therapeutic applications. For instance, heparin is a GAG exploited for its ability to inhibit proteases, therefore inducing anticoagulation. For this reason, heparin is extracted in mass quantities from porcine intestine in the pharmaceutical field. Following a contamination in 2008, alternative sources for heparin are desired. In response, much research has been invested in the extraction of the naturally occurring polysaccharide, heparosan, from Escherichia coli K5 strain. As heparosan contains the same structural backbone as heparin, modifications can be made to produce heparin or heparin-like molecules from this source. Furthermore, isotopically labeled batches of heparosan can be produced to aid in protein-GAG interaction studies. In this study, a comparative look between extraction and purification methods of heparosan was taken. Fed-batch fermentation of this E. coli strain followed by subsequent purification yielded a final 13C/15N labeled batch of 90mg/L of heparosan which was then N-sulfated. Furthermore, a labeled sulfated disaccharide from this batch was utilized in a protein interaction study with CCL5. With NMR analysis, it was found that this heparin-like molecule interacted with CCL5 when its glucosamine residue was in a β-conformation. This represents an interaction reliant on a specific anomericity of this GAG molecule.

As a major cause of nosocomial infections, biofilms such as those caused by Staphylococcus aureus and Staphylococcus epidermidis pose large concerns in the field of healthcare due to their extreme durability and resistance to treatment. While all biofilms grow similarly in a series of three stages: 1. Adhesion 2. Maturation 3. Dispersal, Staphylococcal species such as S. aureus and S. epidermidis make use of unique growth factors in order to form prolific and durable biofilms. Due to the prevalence and risks associated with bacteria, many antibacterial methods have been created to treat bacterial infections. Although many antibacterial methods exist, there is still a great need for additional and more effective methods to treat and prevent serious bacterial infections associated with biofilm growth, because incidences of bacterial infection and resistance, especially in medical settings, are on the rise. In recent research, the exotoxin tolaasin, produced by the bacterium Pseudomonas tolaasii has briefly been shown to exhibit antibacterial effects. Based on previous research and tolaasin's observed pore forming and detergent properties, it is hypothesized that tolaasin will disrupt and prevent staphylococcal biofilm growth either independently or synergistically with existing antibiotics. If this is confirmed, tolaasin may have major implications within the future of healthcare, particularly in the field of antibiotics. In order to optimally use tolaasin as an anti-biofilm agent, potential anti-biofilm applications would aim to prevent and treat biofilm infections at the most common sites of biofilm growth such as catheters, medical instruments, implanted medical devices, and surgical sites. In addition, under the assumption that tolaasin will be found effective in inhibiting biofilm growth and infection, this thesis proposes future anti-biofilm technologies that could use tolaasin as an anti-biofilm agent in order to prevent biofilms and associated infections. While there are many potential and promising ways that tolaasin could be used as an anti-biofilm agent in the future, there are still possible limitations that would need to be investigated through further research before these applications can come to fruition. Ultimately, if future research successfully determines that tolaasin can be used to make anti-biofilm technologies that are biocompatible, durable, and effective, then technologies using tolaasin as an anti-biofilm agent may more effectively ensure sterility of medical devices and prevent bacterial biofilms and infections, and may eventually save lives.