Date of Award

6-2020

Document Type

Thesis

Publisher

Santa Clara : Santa Clara University, 2020.

Department

Bioengineering

First Advisor

Zhiwen Zhang

Abstract

The rapid escalation of the antibiotic resistance crisis has brought attention to the decline in efficiency of antibiotics which have long been a cornerstone of modern medicine. This project aims to provide novel drug targets for the creation of anti-infective immunotherapies that can treat drug-resistant infections. The identification of said drug target(s) (Molecule X) will allow for the development of an antibody based drug that will neutralize bacterial virulence rather than killing the bacteria. Molecule X will be bound using the protein Sortase A (SrtA). SrtA is a surface protein that controls the virulence of gram positive bacteria by anchoring virulence factors to the cell surface. Molecule X is the signal from mammalian cells that initiates this process. Blocking this signal prevents the activation of bacterial virulence.

SrtA is obtained from E. coli cells using affinity chromatography and Molecule X is obtained by collecting the entire mammalian proteome of Chinese Hamster Ovary (CHO) cells. The SrtA and mammalian proteome are mixed together with a crosslinker to covalently bind Molecule X to SrtA. The cross-linked products are purified and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS-MS) and the proteins are identified using a proteomic database. The resulting data is analyzed for Molecule X candidates by grouping the proteins into clusters based on function and subcellular location. The identified clusters are calcium ion dependent, membrane associated, G protein-coupled receptors (GPCR) associated, and adenosine triphosphate (ATP) or guanosine triphosphate (GTP) binding proteins.

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