University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 3-22-2017 A Novel Abi-domain Protein Controls Virulence Determinant Production in Staphylococcus aureus Stephanie Michelle Marroquin University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Microbiology Commons, Molecular Biology Commons, and the Public Health Commons Scholar Commons Citation Marroquin, Stephanie Michelle, "A Novel Abi-domain Protein Controls Virulence Determinant Production in Staphylococcus aureus" (2017). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/6725 This Thesis is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. A Novel Abi-domain Protein Controls Virulence Determinant Production in Staphylococcus aureus by Stephanie M. Marroquin A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health with a concentration in Global Communicable Diseases Department of Global Health College of Public Health University of South Florida Major Professor: Lindsey N. Shaw, Ph.D. Thomas R. Unnasch, Ph.D. John H. Adams, Ph.D. Date of Approval: March 16, 2017 Keywords: Pathogenesis, Blood Survival, Mass Spectrometry, Macrophage Infection, Site-Directed Mutagenesis, agr Quorum Sensing Copyright © 2017, Stephanie M. Marroquin Acknowledgments Many people deserve acknowledgements for the guidance and support they have provided throughout my master’s degree program. Firstly, I would like to thank my major professor Dr. Lindsey Shaw, for providing encouragement and direction during this process. You are an exemplary role model, and have consistently challenged me in order to bring out the best in me as a graduate student. Thank you for believing in me and ultimately encouraging the continuation of my academic career as a Ph.D. student. I would also like to thank my co-major professor and academic advisor Dr. Thomas Unnasch, you have assisted me immensely throughout this journey. You were always readily available when I had a question, and went above and beyond in helping me understand the correct processes for obtaining my degree. I truly appreciate your guidance and I look forward to keeping in touch with such an incredible mentor. Additionally, my committee member Dr. John Adams, you have provided significant input throughout my meetings, and I am highly appreciative, as it has helped me greatly in my ability to present professionally. I would like to thank all members of the Shaw lab, you have all made this process truly enjoyable and I look forward to more years with you. I showed up as a rotating student in a research lab that isn’t in my program’s department, but everyone truly made me feel like I was part, and I slowly became an “adopted” member of the CMMB department. Brittney, you really took the time in helping me understand protocols and were always available when I had a question. We have become great friends and I look forward to embarking on the pursuit of our doctorate degrees together. Andy, you have been an amazing mentor throughout this and I am so incredibly proud of you; you are going to do amazing things and I can’t wait to hear about them. In a short time, we have become incredible friends, and you have provided so much insight on not only science but also on life; thank you for helping me grow as a scientist and for believing in me. Last but not least, I would like to thank my family, who have supported me through all these years of college, and continue to do so in my decision to pursue a Ph.D. degree. There has never been a moment that you have not believed in me, and have always been there for me during my hardships. My brother has always believed in me, and elicited interest in my work. He is constantly rooting me on to do the next thing on my mind; you are my rock. Mom and dad, you both came to this country to provide your family with a better future and endless possibilities. Your perseverance and work ethic have driven me to be the person I am today, and for that I am eternally grateful. Table of Contents List of Tables .......................................................................................................... iii List of Figures ......................................................................................................... iv Abstract .................................................................................................................. vi Chapter One: Introduction ....................................................................................... 1 Staphylococcus aureus ................................................................................... 1 Disease Manifestation in S. aureus .................................................................. 2 Antibiotic Resistance in S. aureus ................................................................... 3 Hospital Acquired MRSA and Community Acquired MRSA .................................. 5 Global Relevance of MRSA .............................................................................. 6 Virulence Factors in S. aureus ........................................................................ 7 agr Quorum-Sensing System and Virulence Regulation ................................... 12 Abi-Domain Protein Family ........................................................................... 15 Project Aims ................................................................................................ 17 Chapter Two: Materials and Methods ...................................................................... 19 Strains, Plasmids, and Primers ...................................................................... 19 Media and Growth Conditions ....................................................................... 19 Buffers ....................................................................................................... 23 Bacterial Construct ...................................................................................... 24 Native Complement ........................................................................... 24 Non-Native Catalytic Site Mutant Complement ..................................... 24 Native His-Tagged Complement .......................................................... 25 Transcriptional Assays ................................................................................. 26 Reporter Gene Fusions ....................................................................... 26 Quantitative Real Time-PCR ................................................................ 26 Protein Assays ............................................................................................. 27 Protein Extraction .............................................................................. 27 Protein Digestion ......................................................................................... 28 Desalt ............................................................................................... 28 Label-Free Mass Spectrometry ............................................................ 29 i Virulence Assays .......................................................................................... 30 Human Whole Blood Survival Assay .................................................... 30 THP-1 Macrophage Infection Assay ..................................................... 30 Chapter Three: Results .......................................................................................... 32 Exploration and Phenotypic Identification of 1984 as a Virulence Determinant in Staphylococcus aureus ..................................................... 32 Bioinformatic Analysis of 1984 as a Member of the Abi Protein Family ............. 34 Transcriptional Studies Given the Suggested Influence of 1984 on Virulence Determinant Production ............................................................ 38 Role of 1984 on Other Known Regulators and Effectors of Staphylococcus aureus ................................................................................................... 41 Proteomic Approach to Determine the Role of 1984 on Protein Production ....... 45 Influence of 1984 on Virulence in Staphylococcus aureus ............................... 48 Chapter Four: Discussion ....................................................................................... 53 Future Directions ................................................................................................... 62 References ............................................................................................................ 64 Appendix .............................................................................................................. 73 ii List of Tables Table 1. Strains and Plasmids ................................................................................ 20 Table 2. Primer Sequences .................................................................................... 21 Table 3. Notable Cytoplasmic Protein Fold Change in the 1984 mutant ..................... 48 Table 4. Notable Membrane Protein Fold Change in the 1984 Mutant ....................... 49 Table A-1. Proteins identified in Proteomic Analysis of Cytoplasmic Fraction .............