Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Geoff Horsman

Advisor Role

Supervisor

Second Advisor

Masoud Jelokhani-Niaraki

Advisor Role

Committee Member

Third Advisor

Stephanie DeWitte-Orr

Advisor Role

Committee Member

Abstract

New strategies are urgently needed to combat infectious diseases in an era of rising antibiotic resistance. Furthermore, an emerging appreciation for the human microbiome’s role in maintaining health motivates discovery of species-specific antibiotics that minimally disrupt our native bacterial communities. Small molecule modifications to bacterial cell surfaces represent a potentially rich source of new targets for next generation antibiotics, as these molecules mediate virulence and evasion of the host immune response. Phosphocholine (PCho) is a rare cell surface modification that contributes to virulence, and modifications with phosphonates like 2-aminoethylphosphonate (AEP) are even more unusual and therefore provide opportunities for species- and pathway-specific targeting.

Cytidylyltransferase enzymes are required to activate these unique substrates. The cytidylyltransferase LicC was previously known to activate PCho through addition of a cytidine monophosphate (CMP) moiety, and here we demonstrate that the homologous protein that we have termed PntC activates AEP. PntC homologs and resulting cell-surface phosphonate modifications have been identified across diverse phyla of bacteria. Among these bacterial species are the known pathogens Atopobium rimae, Olsenella uli, Treponema denticola, and Streptococcus pneumoniae. NMR analysis and continuous spectrophotometric assays were performed to compare LicC from S. pneumoniae (Spn-LicC) to PntCs from the Gram positive A. rimae (Ari-PntC) and Gram negative T. denticola (Tde-PntC). The results demonstrated that: (i) PntC enzymes generate a previously unreported compound CMP-AEP; (ii) PntCs exhibit specificity towards CTP and Mg2+; (iii) PntCs possess >400-fold preference for AEP over PCho, while LicC exhibits a 200-fold preference for PCho over AEP; and (iv) LicC is capable of accepting a range of larger substrates. These findings have provided insight into the activity and versatility of these proteins to utilize uncommon molecular substrates, setting the stage for the development of molecular probes and effective protein-specific inhibitors designed to halt the overall phosphonate-tailoring pathway which could ultimately disrupt the ability of the pathogen to confer virulence in a host.

Convocation Year

2019

Convocation Season

Fall

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