Author ORCID Identifier

https://orcid.org/0009-0004-5550-4529

Date of Award

12-11-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Cynthia Nau Cornelissen

Second Advisor

Andrew Ted Gewirtz

Third Advisor

Zehava Eichenbaum

Abstract

Gonorrhea, a prevalent sexually transmitted infection affecting millions annually worldwide, is caused by Neisseria gonorrhoeae (Ngo), a "superbug" resistant to all antibiotic classes. Compounding the challenge, the absence of protective immunity upon infection allows for reinfection, and a viable vaccine against gonococcal infection remains elusive.

In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).

Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.

Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.

As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine.

DOI

https://doi.org/10.57709/36337409

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