Author ORCID Identifier

https://orcid.org/0000-0002-8750-6279

Date of Award

8-24-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Cynthia Nau Cornelissan, PhD

Second Advisor

Timothy L. Denning, PhD

Third Advisor

Zehava Eichenbaum, PhD

Abstract

Neisseria gonorrhoeae causes the eponymous sexually transmitted infection gonorrhea, a global disease that afflicts millions of individuals worldwide per year. To date, no effective vaccine to prevent gonococcal infection has been produced, and the pathogen rapidly acquires and maintains mechanisms for antimicrobial drug resistance. Further, natural infection by N. gonorrhoeae elicits no protective immune response, making reinfection a common outcome. During infection, the gonococcus employs an arsenal of TonB-dependent outer-membrane transporters (TdTs) which facilitate the acquisition of essential nutrients such as iron and zinc from the human host, which itself goes to great lengths to restrict these metals’ availability. The TdTs are well conserved, surface accessible, and play critical roles in gonococcal pathogenesis, making them promising targets for therapeutic and/or vaccination efforts. One TdT, called TdfJ, allows the gonococcus to acquire zinc from S100A7, an innate immunity protein that typically suppresses bacterial growth via zinc sequestration. TdfJ contains an α-helix motif in its extracellular loop 3 (L3H), a conserved structure which has been shown to participate in the binding of and subsequent iron extraction from transferrin by another TdT, TbpA. In this report, we generated a series of mutations in the TdfJ L3H and assessed their impacts on S100A7 binding and utilization. We found that proline insertions at residues K261 and R262 fully abrogated the interaction between the two proteins, highlighting the importance of this motif in metal acquisition. We went on to explore the functionality of TdfJ as a vaccine antigen via two approaches. In one approach, we grafted extracellular loop sections of TdfJ onto a soluble lipoprotein scaffold and immunized mice, whose post-immune sera and vaginal secretions were screened for TdfJ-reactive antibodies. In another, we formulated recombinant TdfJ with multiple adjuvants to assess the duration of gonococcal colonization in mice and whether adjuvant type affected efficacy. These two approaches demonstrated that TdfJ as a vaccine antigen is protective in a mouse model of infection, and that antibodies generated during upon vaccination may inhibit gonococcal ability to utilize S100A7. Taken together, these data may direct the path towards future prevention of gonococcal infections.

DOI

https://doi.org/10.57709/31312707

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