Date

2017

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Chemistry

First Adviser

Pires, Marcos M.

Other advisers/committee members

Thevenin, Damien; Vezenov, Dmitri; Berger, Bryan

Abstract

Although many experts projected that the discovery of penicillin signaled an end to infectious diseases, the emergence of antibiotic resistant bacteria has unfortunately rendered many antimicrobial therapeutics ineffective. The arsenal of drugs that physicians once had to combat infections has been reduced to last resort antibiotics with high patient toxicity. In conjunction with the emergence of antibiotic resistant bacteria, we have witnessed a languid pursuit in the discovery of novel antimicrobials as the last clinically useful antibiotic was identified thirty years ago. As the number of efficacious antibiotics continues to rapidly dwindle without replenishment, the possibility of entering a post-antibiotic era threatens to quickly become an ominous reality. To reverse this disconcerting trend, it is essential that novel antimicrobial therapeutics that target resistant bacteria be discovered. The use of therapeutics which alter the host immune response have become attractive strategies for the treatment of cancer and virus affiliated diseases. As discussed in Chapter 1, these strategies act to stimulate or attenuate the host immune system in a manner that is advantageous to clearance of infection. Although these strategies have demonstrated great potential, especially in cancer treatment, the use of immunomodulation to target pathogenic bacteria has been lagging. We hypothesized that clearance of bacterial infections could be achieved by tethering hapten molecules recognized by the immune system to the bacterial cell surface. Hapten recognition was theorized to recruit endogenous antibodies to stimulate the innate immune system and ultimately achieve eradication of the pathogen. In developing this method, it was recognized that facile modulation of the bacterial surface would be necessary. Chapter 2 discusses different methods in which both Gram-positive and Gram-negative bacteria surfaces can be modified. Through careful examination of these cell surface remodeling techniques, the use of exogenous D-amino acid cell wall analogues, which remodel the oligopeptide of peptidoglycan, were identified as an appropriate approach for incorporating hapten molecules onto the Gram-positive bacterial cell surfaces. The work discussed in Chapter 3 describes the initial efforts towards the development of D-amino acid Antibody Recruitment Therapy (DART). In this novel immunomodulation strategy, dinitrophenol (DNP) haptens, which are readily recognized by antibodies circulating in the human blood stream, were conjugated to the side chain of diverse D-amino acids. Incubation of bacteria in the presence of these DNP-D-amino acids led to extensive peptidoglycan remodeling and demonstrated enhancement of antibody recruitment to the bacterial surface. Furthermore, we demonstrated that incorporation of DNP-D-amino acids into bacterial peptidoglycan stimulates macrophage antibody-dependent cellular phagocytosis of bacteria. These studies indicate the potential of DART as an immunomodulation strategy to combating pathogenic bacterial infections and demonstrate DNP-D-amino acids could serve as a new antimicrobial agent. From the initial development of DART, several questions were raised about D-amino acid substrate side chain promiscuity exhibited by penicillin-binding protein (PBP) transpeptidase. In developing a highly efficacious DART agent, a D-amino acid substrate that is readily incorporated into the peptidoglycan would prove advantageous as more hapten molecules would be attached to the bacterial cell surface. To determine the extent of PBP transpeptidase side chain promiscuity, a series of fluorescent D-amino acids were synthesized and analyzed across several bacterial strains for peptidoglycan incorporation. Additionally, an assay to rapidly assess if non-fluorescent D-amino acids are incorporated into the peptidoglycan was developed. From these studies, several trends were observed which could direct future generations of DART and are discussed in Chapter 4. Additionally, we hypothesized that the accessibility of DNP epitopes could drastically effect antibody recruitment to the bacteria surface and therefore reduce immune response. In Chapter 5, the development of a method to accentuate the protrusion of antibodies from the bacterial peptidoglycan for enhanced antibody recruitment is discussed. In this two-step method, a dipeptide cell wall analogue containing a reactive chemical handle was incorporated into the bacterial peptidoglycan. Following dipeptide incorporation, subsequent chemical reaction facilitated covalent attachment of peptide-based DNP linkers varying in length. Through analysis of this two-step modulation system, it was determined that larger linkers, although less readily modulate bacterial peptidoglycan, are more adept to recruit antibodies to the bacterial surface. Lastly, in the pursuit of DART agents that could generate enhanced immune response, we decided to explore the ability of PBP transpeptidases to incorporate D-amino acids possessing diverse C-terminus modifications. Through this study, it was observed that exogenous D-amino carboxamide variants are more readily incorporated into the peptidoglycan than the natural carboxylic acid containing D-amino acid substrate. With this knowledge in hand, it was hypothesized that D-amino carboxamides displaying DNP haptens could engender a greater immune response. In Chapter 6, the enhanced antibody recruitment facilitated by D-amino carboxamides modified with DNP is discussed. Additionally, in this study, the recruitment of antibodies from pooled human serum is shown, thus validating the biological relevance of this immunomodulation strategy. The work described in this thesis represents the initial developments of DART. Although future in vivo experiments must be completed to ascertain if this strategy will be viable in live organisms, the work described herein demonstrates the potential of targeting pathogenic bacteria through host immune system stimulation.

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