University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 2011 Characterization of Aminopeptidase PepZ in Staphylococcus aureus Virulence Tiffany Marie Robison University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the American Studies Commons, and the Microbiology Commons Scholar Commons Citation Robison, Tiffany Marie, "Characterization of Aminopeptidase PepZ in Staphylococcus aureus Virulence" (2011). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/3314 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]. Characterization of Aminopeptidase PepZ in Staphylococcus aureus Virulence by Tiffany M. Robison A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Cell Biology, Microbiology, Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Lindsey N. Shaw, Ph.D. James T. Riordan, Ph.D. Stanley M. Stevens, Ph.D. Date of Approval: October 31, 2011 Keywords: CA-MRSA, Exopeptidase, Nutrition, Mass Spectrometry, 2D-DIGE Copyright © 2011, Tiffany M. Robison Acknowledgements I owe my deepest gratitude to my mentor and advisor, Dr. Lindsey N. Shaw, for his encouragement, guidance and advice throughout my graduate studies and thesis writing. I would also like to thank my committee members, Dr. James T. Riordan and Dr. Stanley M. Stevens for their support and assistance throughout this project. Additionally, I am extremely grateful for all of the members of the Shaw, Riordan and Stevens labs for all of their assistance needed for the completion of this project as well. I am also very appreciative for all of the members and staff of the CMMB department for all of your encouragement and support while as a graduate student at USF. Table of Contents List of Figures .................................................................................................................... iv List of Tables ..................................................................................................................... vi Abstract ............................................................................................................................. vii Introduction ..........................................................................................................................1 Staphylococcus aureus is an Opportunistic Pathogen ............................................1 Manifestation of S. aureus Infection ........................................................................1 Antibiotic Resistance in S. aureus ...........................................................................2 Community-Acquired S. aureus ..............................................................................4 Toxin Production in S. aureus .................................................................................5 Proteolytic Enzymes ................................................................................................6 Proteases of S. aureus ..............................................................................................8 Aminopeptidases ......................................................................................................9 Metalloaminopeptidases ........................................................................................10 Leucine Specific Aminopeptidases ........................................................................11 Aminopeptidases of S. aureus ................................................................................11 Aminopeptidases in Bacterial Pathogenesis ..........................................................13 Materials and Methods .......................................................................................................15 Culture Media ........................................................................................................15 Buffers and Reagents .............................................................................................20 Bacterial Strains, Plasmids and Primers ................................................................24 Transformations .....................................................................................................24 Electroporation of S. aureus ..................................................................................25 Phage Transductions of S. aureus ..........................................................................25 Construction of a pepZ mutant ...............................................................................25 Construction of pepZ-lacZ Reporter Gene Fusions ...............................................26 Transcriptional Analysis ........................................................................................26 Transcriptional Analysis of pepZ Expression ............................................26 Transcriptional Disk Diffusion Assays ......................................................27 Growth and Nutrition Profiling .............................................................................27 Growth Analysis in Peptide Based Media .................................................27 Long-Term Starvation Analysis .................................................................28 Competitive Growth Analysis....................................................................28 Proteomic Analysis ................................................................................................29 i Cytoplasmic Protein Extraction .................................................................29 Secreted Protein Extraction ........................................................................29 Western Blot Detection ..............................................................................30 2D Difference Gel Electrophoresis (DIGE) ...............................................30 Trypsin Digestion.......................................................................................31 Desalt .........................................................................................................31 Virulence Assays ...................................................................................................32 Murine Model of Septic Arthritis ..............................................................32 Biofilm Formation Analysis ......................................................................32 Human Macrophage Survival and Clearance ............................................33 Murine Model of Wound Formation..........................................................33 Murine Model of Bacterial Sepsis .............................................................33 Phenotypic Characterization ..................................................................................34 Growth Profiling using Chemically Defined Media ..................................34 Evaluating Carbon Utilization ...................................................................34 Lysis Kinetics.............................................................................................34 Heat Shock .................................................................................................35 Heat Stress .................................................................................................35 Oxidative Stress .........................................................................................35 Disk Diffusions ..........................................................................................36 Results ................................................................................................................................37 Analysis of the Virulence of a S. aureus pepZ mutant using a Murine Model of Septic Arthritis .......................................................................................37 Profiling pepZ Expression using a lacZ Reporter Fusion ......................................39 Evaluating the Effects of Environmental Stimuli of pepZ Transcription ..............41 Investigation of the Role of PepZ during S. aureus Growth in Peptide Rich Media .....................................................................................................................43 Investigating the Role of PepZ during Long-Term Starvation ..............................45 Competitive Growth Analysis Reveals pepZ mutants are Impaired in their Ability to Compete with Wild-Type Strains ..........................................................50 Anaerobic Stress Profiling of PepZ using Chemically Defined Media .................55 Evaluating the Role of PepZ in S. aureus Carbon Utilization using Chemically Defined Media ....................................................................................57 Characterization of the Role of PepZ in Membrane Integrity and Autolysis ........58 Evaluating the Role of PepZ in Cellular Survival Following Exposure to Elevated Temperature ............................................................................................59 Assessing the Role of PepZ in Response to Oxidative Stresses ............................61 General Stress Profiling of pepZ mutant Strains using a Modified Kirby- Bauer Assay ...........................................................................................................62