AN ABSTRACT OF THE DISSERTATION OF Igor H. Wierzbicki for the degree of Doctor of Philosophy in Pharmaceutical Sciences presented on September 28, 2016. Title: Characterization and Validation of Neisseria gonorrhoeae Proteins GmhAGC and NGO1985 as Molecular Targets for Development of Novel Anti-gonorrhea Therapeutic Interventions. Abstract approved: ______________________________________________________ Aleksandra E. Sikora 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, GmhAGC and NGO1985, as targets for development of new antibiotics and a vaccine antigen, respectively. The sedoheptulose-7-phosphate isomerase, GmhAGC, is the first enzyme in the biosynthesis of nucleotide-activated-glycero-manno-heptoses. We demonstrate that N. gonorrhoeae GmhAGC is essential for lipooligosaccharide (LOS) synthesis and pivotal for bacterial viability. Our crystallization studies have shown that GmhAGC 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 GmhAGC 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 GmhAGC and NGO1985 on gonococcal physiology, we conclude that these proteins are promising molecular targets for development of new anti-gonorrhea interventions. ©Copyright by Igor H. Wierzbicki September 28, 2016 All Rights Reserved Characterization and Validation of Neisseria gonorrhoeae Proteins GmhAGC and NGO1985 as Molecular Targets for Development of Novel Anti-gonorrhea Therapeutic Interventions by Igor H. Wierzbicki A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented September 28, 2016 Commencement June 2017 Doctor of Philosophy dissertation of Igor H. Wierzbicki presented on September 28, 2016 APPROVED: Major Professor, representing Pharmaceutical Sciences Dean of the College of Pharmacy Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Igor H. Wierzbicki, Author ACKNOWLEDGEMENTS I would like to thank Dr. Aleksandra Sikora for giving me the opportunity to do my Doctor of Philosophy education under her guidance. Thanks to her dedication in my training, as well as her never-ending patience with my actions and behavior, I was able to learn all of the aspects of being a successful scientist. All of my achievements, recognitions, and awards throughout the last four years would not be possible without her great input. I would also like to thank all members, past and present, of the Sikora laboratory for creating a welcoming atmosphere and always being there for me. I would like give special thanks to Rachael Ryner for being a wonderful undergraduate trainee. I enjoyed every moment of being her teacher and mentor. Watching her rapid progress made me more than proud. I want to thank everybody in the College of Pharmacy for helping me make it through my graduate education with special thanks to Debra Peters and Patricia Beaumont for brightening days with their smiles and help with all of the overwhelming paper work; Dr. McPhail for being part of my graduate committee, for the training, and help with all of my uncertainties related to the graduate program; and Dr. Adam Alani for being a wonderful and understanding mentor. I want to thank all of my friends that I have made here in Oregon for all of the hikes, camping trips, barbecues, potluck dinners, concerts, parties, movie nights, and all other spontaneous occasions. I am happy that I had the chance to meet and know you. I give thanks to my mother and grandparents for believing in me and supporting my life decisions. Lastly, but definitely not least, I want to thank my beloved wife, Dorota, for encouraging and convincing me to pursue my doctorate education. Without her patience, understanding, support, caring, and love I would not succeed. TABLE OF CONTENTS Page Chapter 1. General introduction…………………………………………………..…...1 Neisseria genus overview…....………………………………………………...2 Neisseria gonorrhoeae and gonorrhea...…………………………………........3 Treatment and prevention of gonorrhea………………………………………..6 Proteomic mining of cell envelopes and naturally released membrane vesicles for identification of novel targets for development of anti-gonorrhea interventions……………………………………………………….…10 NGO1985…...………………………………………………………………..12 β-barrel Assembly Machinery (BAM) complex……………………………...13 Nucleotide-activated-glycero-manno-heptoses and GmhA………………….15 Scope of dissertation…………………………………………………………16 Chapter 2. Functional and structural studies on the Neisseria gonorrhoeae GmhA, the first enzyme in the glycero-manno-heptose biosynthesis pathways, demonstrate a critical role in lipooligosacharide synthesis and gonococcal viability………20 Abstract...…………………………………………………………………….21 Introduction…………………………………………………………………..22 TABLE OF CONTENTS (Continued) Page Materials and Methods……………………………………………………….25 Results………………………………………………………………………..32 Discussion…………………………………………………………………....40 Acknowledgements…………………………………………………………..44 Chapter 3. NGO1985 is a surface-exposed lipoprotein interacting with BAM complex and a gonorrhea vaccine candidate…………………………………………...61 Abstract..……………………………………………………………………..62 Introduction…………………………………………………………………..63 Materials and Methods……………………………………………………….65 Results………………………………………………………………………..77 Discussion……………………………………………………………………85 Chapter 4. Dissecting the signal of lipoprotein sorting and function of BON domains in Neisseria gonorrhoeae lipoprotein NGO1985……………...…………….116 Abstract……………………………………………………………………..117 Introduction…………………………………………………………………118 TABLE OF CONTENTS (Continued) Page Materials and Methods……………………………………………………...120 Results………………………………………………………………………123 Discussion…………………………………………………………………..128 Chapter 5. Discussion and Conclusion………...……………………………………142 Overview……………………………………………………………………143 GmhAGC involvement in physiology of Neisseria species…………………..144 NGO1985 is a surface-exposed lipoprotein of N. gonorrhoeae…………..…147 NGO1985 lipobox +2 residue implication in protein localization and function……………………………………………………………..150 NGO1985 functions in maintaining cell envelope homeostasis…………….151 NGO1985 association with β-barrel Assembly Machinery (BAM) Complex…………………………………………………………….154 BON domains of NGO1985………………………………………………...156 Conclusions and future directions…………………………………….…….158 TABLE OF CONTENTS (Continued) Page Bibliography ……………………………………………….……………………….160 Appendices: Abstracts of Additional Manuscripts………………………………….189 LIST OF FIGURES Figure Page 1.1. Cell envelope of N. gonorrhoeae. ………...……..………………………………18 2.1. GmhAGC is pivotal for N. gonorrhoeae growth and LOS synthesis……………...45 2.2. Assessment of GmhAGC subcellular localization and expression patterns……….47 2.3. GmhAGC expression in a panel of N. gonorrhoeae isolates………………………49 2.4. Hindering GmhAGC isomerase activity does not influence N. gonorrhoeae growth………………………………………………………………………..50 2.5. Homologues of GmhA from N. gonorrhoeae and N. meningitidis function interchangeably………………………………………………………………51 2.6. The structure of N. gonorrhoeae GmhAGC……………………………………….52 S2.1. Purification of rGmhAGC……………………………………………………….56 S2.2. Loading controls for immunoblotting experiments…………………………….57 S2.3. Analysis of single nucleotide