Characterization of the Gonococcal Calprotectin Zn-Acquisition System, TdfH, and the Efficacy of Metal Starvation for Treating Gonococcal Disease
Doctor of Philosophy
Microbiology & Immunology
Cynthia Nau Cornelissen
Characterization of the Gonococcal Calprotectin Zn-Acquisition System, TdfH, and the Efficacy of Metal Starvation for Treating Gonococcal Disease.
By Michael T. Kammerman, B.S.
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University
Virginia Commonwealth University, 2020
Major Director: Cynthia Nau Cornelissen, Ph.D.
Professor, Department of Microbiology and Immunology
Neisseria gonorrhoeae, the bacterial agent that is responsible for the human disease gonorrhea, has had a steady increase in the number of infections per year. In 2018 the WHO estimated over 87 million infections occurred world-wide, and the CDC estimated that over 800,000 infections happened in the United States. Accompanying the increase in gonococcal infections per year is the increase in the number of antibiotic resistant isolates being recovered. The recent recovery of a gonococcal isolate that was resistant to the current CDC recommended dual-treatment, coupled with the lack of a preventative vaccine, marks the beginning of an era where there may be no effective treatments for gonococcal disease. The gonococcus possesses a conserved set of proteins that enables it to pirate metal ions from host proteins, and is essential in order for N. gonorrhoeae to establish an infection in the human urogenital tract. The importance of TdTs for gonococcal survival and their sequence conservation make them ideal candidates to be included in a gonococcal vaccine, or as targets for potential new therapeutics that are capable of disrupting the interaction with their ligands. In this study we developed a competition assay and probed gonococcal cells with either human (hCP) or mouse calprotectin (mCP) to determine if the gonococcus is species restricted for its ligand interaction. We also performed Isothermal Titration Calorimetry experiments to characterize the binding affinity between the gonococcal calprotectin (CP) binding protein TdfH and calprotectin. In this study we also continued a mutational and small molecule analysis of TbpA aimed at disrupting the interaction with its ligand, human transferrin (hTf). Finally, we investigated if the gonococcal efflux pump, MacA, was responsible for heme export via heme-dependent growth assays of a MacA deficient gonococcal strain. We report that the interaction between TdfH and hCP is high affinity and that the Zn piracy of TdfH occurs optimally at the non-canonical metal binding site of hCP known as site 1. We determined that multiple mutations, both in the loop 3 helix and in loop 2 of TbpA, minimally reduce the total binding of hTf, similar to what Cash et al. has previously described. A screening of a database of small molecules found that several first-generation small molecules were able to significantly reduce the ability of TbpA to interact with hTf. Finally, we found that the efflux pump MacA, does not meaningfully contribute to gonococcal heme export. However, more in-depth studies interrogating MacA substrates are still needed. These studies determined the species specificity of the gonococcal calprotectin binding protein and found that Zn- piracy occurs optimally from one specific site on hCP. This study also shows the promise in investing in new therapeutics that disrupt TdT function.
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