Date of Award

2020

Embargo Period

8-12-2025

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Christopher Davies

Second Advisor

Shaun Olsen

Third Advisor

Ozlem Yilmaz

Fourth Advisor

Yi-Te Hsu

Fifth Advisor

Eric Meissner

Abstract

According to the World Health Organization, 78 million cases of gonorrhea occur worldwide each year, comprising 22% of new sexually transmitted infections. If untreated, such infections can have lasting results, including pelvic inflammatory disease or infertility. Gonorrhea is caused by Neisseria gonorrhoeae, a Gram-negative diplococcus. Over time, N. gonorrhoeae has developed resistance against most antibiotics used to treat gonorrhea, leaving extended spectrum cephalosporins (ESCs) and azithromycin as the current treatment. Recently, new strains of Neisseria gonorrhoeae have emerged that exhibit resistance to ESCs, including strains H041 (isolated in Japan), 35/02 (isolated in Sweden), F89 (isolated in France), and FC428 (isolated in Japan). A major factor in cephalosporin resistance is mutations in the penA gene, encoding penicillin-binding protein 2 (PBP2). Strains F89 and FC428 exhibit a similarly high level of ESC resistance as H041. However, PBP2 from F89 appears more closely related to that from intermediate-resistant strain 35/02 with only one amino acid change, an A501P mutation, while the PBP2 from FC428 has nine mutations compared to H041. This suggests the molecular mechanism of cephalosporin resistance varies between F89, FC428, and H041. To investigate this, we have solved the structures of PBP2 derived from FC428 at 2.5 Å and with cefoperazone at 2.8 Å, and the PBP2 from F89 at 1.3 Å and with ceftriaxone at 1.7 Å. There appear to be minimal structural changes between these PBP2s. This observation raises the question of the role internal protein dynamics play in cephalosporin resistance. To understand this, we have collected a series of 3D nuclear magnetic resonance (NMR) spectra and have assigned approximately 52% of PBP2-H041. When we compare the NMR relaxation experiments of PBP2-H041 to the crystal structure of PBP2-H041 there is consistency between their dynamic regions. Overall, our structural and protein dynamic information provide insight into the resistance mechanisms conferred by PBP2 variants, with the potential of designing new therapeutics against resistant N. gonorrhoeae.

Rights

All rights reserved. Copyright is held by the author.

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