Abstract
Bacterial resistance has posed an immense threat to healthcare worldwide. Within the last year, nearly 2 million people were identified as having a drug resistant bacterial infection, and approximately 23,000 of these infections had a fatal outcome. The Centers for Disease Control and Prevention attributes this exponential increase in drug resistance to overuse of antibiotics resulting from over administration and improper prescribing. In the presence of antibiotics, a bacterium can undergo a mutation that induces drug resistance. Continued dosing of the antibiotic effectively eliminates the susceptible bacteria while the resistant phenotype rapidly divides, resulting in increased pathogenicity. Therefore, the development of new methods to treat this increasing number of resistant infections is crucial. Tailoring an immune response as a therapeutic modality has seen much success within the past several years, especially for the treatment of cancer. Interesting, oncological therapeutics such as preventative vaccines, monoclonal antibodies, and chimeric antigen receptor T cell therapy (CAR-T) are all FDA approved cancer treatments despite cancerous cells and healthy cells exhibiting high chemical and physical similarities. To this end, the bacterial cell surface is composed of structural modalities that are inherently unique from that of the host cell, ultimately increasing therapeutic specificity while limiting off target cytotoxicity. With this in mind, the focus of this thesis will be on applying immunotherapeutic applications to aid and enhance the immune system in response to bacterial infections.
Understanding the mechanisms of bacterial resistance is essential for developing new therapeutic measures to combat such pathogens. Chapter 1 will describe the history of antibiotics, their targets, and specific methods of resistance. The details of the bacterial cell envelope, the peptidoglycan (PG) layer in particular, will be described, as it is a primary target of antibiotics of which structural changes to the PG are a major contributing factor to antibiotic resistance. Additionally, the bacterial cell wall is the principal stimulating factor of both innate and adaptive immune responses against infection. As such, Chapter 2 will describe the host recognition and defensive mechanisms directed toward invading bacterial pathogens, along with a comprehensive review of the advances to date in immunotherapies targeted at bacteria pathogens.
Chapters 3 describes the use of a novel synthetic PG stem peptide mimic tagged with a hapten to re-direct an immune response against drug resistant bacteria. To date, immune modulation of bacterial cells utilizing metabolic processes has been limited to mimics of natural substrates that require millimolar concentrations for effective labeling, such as single D-amino acids. The synthetic nature of the PG stem peptide mimics enabled a large degree of bacterial cell labeling due to high substrate recognition and specificity by PG crosslinking enzymes. As a result, relatively low concentrations of the hapten modified PG stem peptide mimic were needed to induce antibody recognition and enhanced phagocytotic uptake of drug resistant pathogens.
The innate immune system employs many mechanisms to detect bacterial pathogens. Taking inspiration from natural bacterial binding proteins, Chapter 4 describes the tagging of bacterial PG with a peptide fragment derived from the immune system of ticks. Modification of this peptide with a hapten enabled specific binding of bacterial cell surfaces, and effectively directed an immune response against pathogenic Enterococci species. Additionally, it was demonstrated that both exogenous and endogenous haptens can be installed on the PG binding peptide to effectively target bacterial cells for clearance by the immune system.
Bacteria have evolved several means of immune evasion and pressure from antibiotics. One of which involves transitioning from existing extracellularly to being able to survive within the intracellular host environment. Chapter 5 aims to examine intracellular Staphylococcus aureus in regard to avoiding antibiotic pressure. S. aureus is a pathogenic bacterium that has evolved to evade immune detection by proliferating intracellularly within host and immune cells. A challenge with treating such infections lies within the ability to uncover if decreased antibiotic efficacy is a result of poor antibiotic permeation into the host cytosolic compartments or phenotypic changes to the bacterium in response to immune defense. We described the use of a PG tag to covalently anchor a biorthogonal click handle to the surface of S. aureus PG and assess the ability of azide modified antibiotics to react with the available click handle. We envisioned that comparing the reactions of the antibiotic between extracellular and intracellular S. aureus will give insight towards the permeability of antibiotics versus antibiotic efficacy within host cells.
