Graduate Thesis Or Dissertation
 

Characterization and Validation of Neisseria gonorrhoeae Proteins GmhAGC and NGO1985 as Molecular Targets for Development of Novel Anti-gonorrhea Therapeutic Interventions

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  • The sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium and obligate human pathogen Neisseria gonorrhoeae, remains a significant health and economic burden worldwide. In the absence of a protective vaccine, antimicrobial agents are the only pharmacological intervention for patients with gonorrhea. However, due to the remarkable ability of gonococcus to develop antibiotic resistance, infections caused by N. gonorrhoeae are believed to become untreatable in the near future. Identification and elucidation of the physiological function of novel N. gonorrhoeae proteins is critical for the formulation of new therapeutic interventions. This work focuses on characterization and validation of two gonococcal proteins, GmhA[subscript GC] and NGO1985, as targets for development of new antibiotics and a vaccine antigen, respectively. The sedoheptulose-7-phosphate isomerase, GmhA[subscript GC], is the first enzyme in the biosynthesis of nucleotide-activated-glycero-manno-heptoses. We demonstrate that N. gonorrhoeae GmhA[subscript GC] is essential for lipooligosaccharide (LOS) synthesis and pivotal for bacterial viability. Our crystallization studies have shown that GmhA[subscript GC] forms a homo-tetramer in the closed conformation with four zinc ions in the active site. Site directed mutagenesis studies showed that active site residues E65 and H183 are important for LOS synthesis but not bacterial viability, suggesting that abolition of LOS synthesis is disconnected from the GmhA[subscript GC] involvement in N. gonorrhoeae viability. NGO1985 was initially described as a hypothetical lipoprotein containing two BON (Bacterial OsmY and Nodulation) domains, hypothesized to be involved in maintaining bacterial cell envelope integrity. In our studies we demonstrate that NGO1985 is a surface exposed lipoprotein, conserved among diverse gonococcal isolates. Deletion of ngo1985 results in bacterial cell envelope defects leading to a pleiotropic phenotype including increased susceptibility to antimicrobial agents, decreased survival during in vitro growth conditions mimicking the human host environment, and high attenuation in the murine model of infection. NGO1985 interactome studies indicated a broad network of interactions including potential association with β-Barrel Assembly Machinery (Bam) complex, antibiotic efflux pump(s), and several lipoproteins. Furthermore, we demonstrate that NGO1985 does not undergo lipoprotein sorting according to the +2 residue of the lipobox motif as characterized for Escherichia coli. We determined that both BON domains, in their native orientation, are essential for NGO1985 functionality and stability. Finally, for the first time we have investigated the importance of the BON domains’ conserved glycine residues and showed that these amino acids play a critical role in protein stability. Based on the importance of both GmhA[subscript GC] and NGO1985 on gonococcal physiology, we conclude that these proteins are promising molecular targets for development of new anti-gonorrhea interventions.
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file details WierzbickiIgorH2016.pdf 2017-08-22 Pubblico Scaricare