Year of Graduation

2020

Level of Access

Restricted Access Thesis

Embargo Period

5-12-2020

Department or Program

Biochemistry

First Advisor

Danielle Dube

Abstract

Overuse and misuse of antibiotics have contributed to the rise of antibiotic-resistant bacteria and alterations in the gut microbiome of treated individuals. Thus, an urgent need exists for alternative approaches to target bacterial pathogens. Immunotherapies that target distinctive cell surface glycans on bacterial pathogens offer a potential alternative. Previous work in the Dube Laboratory has described a metabolic oligosaccharide engineering-based approach to first label cellular glycans with azide-containing sugars and then covalently deliver immune stimulants to azide-coated cells via bioorthogonal chemistry. This platform has enabled the targeting of azide-coated bacterial cells with a phosphine-based 2,4-dinitrophenol (DNP) conjugate to trigger immune-mediated killing of Helicobacter pylori, but these preliminary results require follow-up. The present work seeks to determine the viability of the bioorthogonal reaction partner dibenzoazacyclooctyne (DIBAC), a cyclooctyne-based azide-reactive component that undergoes strain promoted azide-alkyne cycloaddition (SPAAC). I explored whether azide-tagged Helicobacter pylori or Bacteroides fragilis cells would experience immune-mediated cell death after exposure to DIBAC dissolved in either phosphine-buffered saline (PBS) or dimethyl sulfoxide (DMSO). Metabolically labeled bacteria treated with DIBAC-PBS showed some evidence of immune-mediated cell death that decreased over time from experiment to experiment, likely due to low frequency of reactions of SPAAC between insoluble DIBAC and the azide tag. Metabolically labeled bacteria treated with DIBAC-DMSO also showed some evidence of immune-mediate cell death, but the extent of cell death is far lower compared to other immunotherapies. These results suggest that SPAAC with DIBAC may not act as a viable bioorthogonal reaction partner.

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