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Transferrin Binding Protein B Structure, Function, and Export in Neisseria Gonorrhoeae

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Transferrin Binding Protein B Structure, Function, and Export in Neisseria Gonorrhoeae Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2012 Transferrin binding protein B structure, function, and export in Neisseria gonorrhoeae Meredith L. Weck Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medicine and Health Sciences Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/382 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Meredith L. Weck 2012 All Rights Reserved TRANSFERRIN BINDING PROTEIN B STRUCTURE, FUNCTION, AND EXPORT IN NEISSERIA GONORRHOEAE A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University by MEREDITH L. WECK Bachelor of Science, University of Richmond, 2010 Director: CYNTHIA NAU CORNELISSEN, PH.D. PROFESSOR OF MICROBIOLOGY AND IMMUNOLOGY Virginia Commonwealth University Richmond, VA July 2012 ii Acknowledgements First and foremost, I would like greatly thank my advisor, Dr. Cynthia Cornelissen. She is a wonderful mentor who has always been there to provide support and advice. I am truly grateful for the knowledge she has provided me. She is an exceptional scientist and teacher, whom I deeply respect and admire. Thanks to my committee members, Dr. Suzanne Barbour and Dr. Todd Kitten, who have provided indispensable suggestions and advice. I am grateful for the knowledge and support they have given me throughout my degree. I would also like to thank Dr. Gail Christie, the MBG Program Director. She has always been there to help and support me with anything and everything. I would like to thank previous lab member Dr. Amanda DeRocco whose work has led to the development of this project. I would also like to acknowledge summer intern Shannon Pettitt who contributed to the transposon mutagenesis project and rotation student Spencer Harris who contributed to the C-lobe deletion project. Thanks to previous lab members Dr. Aimee Hollander, Heather Strange, and Jacqueline Thomas who were always there to help me and answer questions when I first started. I would like to thank previous lab member Dr. Rosuany Vélez Acevedo and current lab members Sophonie Jean, Shreni Mistry, Aminat Oki, Dr. Karen Ostberg, and Abena Waston-Siriboe. They helped keep me going when I was having a rough time and cheered me on for all of my accomplishments. A special thanks goes to my rotation advisor, Dr. Karen Ostberg, for her contribution to this work as well as always being there to support me, providing me with the skills needed to complete this work, and teaching me pretty much everything I know. My labmates are not only people I worked with but are also my close friends so I thank them for all of the support they have provided me, both in and out of lab, throughout this experience. I am grateful to all of my wonderful friends from VCU and the University of Richmond who have supported me throughout this process. They listened to me complain, had fun with me when I needed a break, and kept me grounded. I would also like to thank my family who are there for me through anything. My sister and brother-in-law, Allyson and Stephen Rinker, gave me an escape and made me laugh when I needed it. I especially want to thank my parents, Candy and Phil Weck, who have always been supportive and loving no matter what. I greatly appreciate everything they have done for me and cannot begin to express my gratitude for the amazing parents they are to me. I am truly blessed to have them in my life. iii Table of Contents Page Acknowledgements...……………………………………………………………………………...ii List of Tables…………………………………………………………………………………….vii List of Figures…………………………………………………………………………………...viii List of Abbreviations……………………………………………………………………………...x Abstract………………………………………………………………………………………….xiv CHAPTERS 1. Introduction……………………………………………………………………………...1 I. The Genus Neisseria……………………………………………………………………1 II. Meningococcal Disease………………………………………………………………..2 A. Epidemiology…………………………………………………………………2 B. Infection……………………………………………………………………….3 C. Treatment and Prevention……………………………………………………..4 III. Gonococcal Diseases…………………………………………………………………5 A. Epidemiology…………………………………………………………………5 B. Infection……………………………………………………………………….5 C. Treatment……………………………………………………………………...7 IV. Gonococcal Virulence Factors………………………………………………………..8 A. Pilus…………………………………………………………………………...8 B. Opacity proteins……………………………………………………………….9 iv C. Porin………………………………………………………………………….10 D. Lipooligosaccharide………………………………………………………….11 E. Reduction-modifiable protein………………………………………………..12 F. IgA1 protease………………………………………………………………...13 V. Iron Sources and Transport in the Human Host……………………………………...13 A. Transferrin……………………………………………………………………14 B. Lactoferrin……………………………………………………………………14 C. Ferritin………………………………………………………………………..15 D. Heme binding proteins…………………………………………………….....15 VI. Iron Acquisition Systems in Bacteria…………………………………………….....17 VII. Iron Acquisition by Pathogenic Neisseria………………………………………….18 VIII. Transferrin Iron Acquisition System of Neisseria………………...………………20 IX. Vaccine Development……………………………………………………………….24 X. Objectives………………………………………………………………………….....25 2. Materials and Methods…………………………………………………………………26 I. Bacterial Growth Conditions………………………………………………………….26 II. Generation of Mutants using a Transposon Library………………………………….27 III. Screening Transposon Library Transformants………………………………………27 IV. Ligation-mediated PCR and DNA sequencing……………………………………...30 V. Generation of the TbpB C-lobe ……………………………………………………...34 A. Construction of the gonococcal TbpB C-lobe deletion gene………………...34 B. Gonococcal transformation…………………………………………………..37 C. Construction of plasmid to express C-lobe deletion in E. coli……………….38 v D. Recombinant protein expression……………………………………………..39 VI. Immunoblotting and Detection……………………………………………………...40 A. Whole cell lysate preparation, SDS-PAGE, and protein transfer……………40 B. TbpA detection……………………………………………………………….40 C. TbpB detection……………………………………………………………….41 D. TonB detection……………………………………………………………….41 VII. Protease Accessibility Assay………………………………………………….........41 VIII. Solid-phase Binding Assays……………………………………………………….42 IX. Transferrin Utilization Assays………………………………………………………42 X. Saturation of Human Transferrin…………………………………………………….43 3. Identification of the TbpB Transport Mechanism through Transposon Mutagenesis……………………………………….…………………………………….44 I. Introduction………………………………………………………………….………..44 II. Results………………………………………………………………………………..50 A. Screening of transposon library revealed multiple potential TbpB export mutants………………………………………………………….………………..50 B. Identification of the transposon insertion sites……………….………………53 C. Characterization of the surface exposure of TbpB in Mutant 4912………….58 D. Mutant 4912 is a mixed population…………………………………………..58 III. Discussion…………………………………………………………………………...61 4. Characterization of C-lobe Deletion of TbpB…………..………………….…………67 I. Introduction…………………………………………………………………………...67 II. Results……………………………………………………………………………..…71 A. Deletion of the C-lobe causes degradation of TbpB…………………………71 vi B. TbpB N-lobe is unable to bind transferrin in vitro and in vivo………………76 C. Utilization of transferrin is impaired in the TbpB C-lobe deletion…………..88 III. Discussion …………………………………………………………………………..93 5. Summary…………..…………………………………………………………………...102 Literature cited………………………………………………………………………………….107 vii List of Tables Page Table 1. Strains used in this study………………………………………………………………28 Table2. Plasmids used in this study……………………………………………………………..29 Table3. Oligonucleotides used in this study…………………………………………………….33 viii List of Figures Page Figure 1. Schematic of the Neisseria transferrin iron acquisition system……………................22 Figure 2. Schematic of Ligation-mediated PCR………………………………………………...31 Figure3. Schematic of DNA splicing by directed ligation………………………………………35 Figure 4. Schematic of the Lol system in E. coli………………………………………………..46 Figure 5. Growth phenotype of potential TbpB export deficient mutants on CDM plates……..51 Figure 6. Protein expression of Tbps and TonB in mutant 4912………………………………..54 Figure 7. Protein expression of Tbps and TonB in mutant 13614………………………………56 Figure 8. Protease accessibility of TbpB in mutant 4912……………………………………….59 Figure 9. Growth phenotype of mutant 4912 is inconsistent………………………………........62 Figure 10. Degradation of TbpB in gonococcal C-lobe deletion strains grown in liquid cultures...........................................................................................................................................72 Figure 11. Degradation of TbpB in gonococcal C-lobe deletion strains grown on plate….……74 Figure 12. Degradation of induced TbpB N-lobe construct in E. coli…………………………..78 Figure 13. Detection of TbpB C-lobe deletion over time……………………………………….79 Figure 14. TbpB C-lobe deletion is unable to bind transferrin in gonococcal strains…..............82 Figure 15. Detection of transferrin binding by gonococcal TbpB C-lobe deletion over time…..84 Figure 16. TbpB C-lobe deletion is unable to bind transferrin in E. coli strains………………..86 Figure 17. Solid-phase binding assays of TbpB C-lobe deletion strains………………………..90 Figure 18. Solid-phase binding assays of TbpB C-lobe deletion strains over time……………..91 ix Figure 19. TbpB C-lobe deletion is able to support limited
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