CHARACTERIZATION OF Mycoplasma alligatoris IMMUNODOMINANT ANTIGENS By NIORA J. FABIAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010 1 © 2010 Niora J. Fabian 2 To N.N., E.N., S.N., A.N., and my entire family 3 ACKNOWLEDGMENTS I thank my main advisor, Dr. Daniel Brown, and my committee members for their support and education. I would also like to thank Kevin Kroll for his kind advice with Western blotting optimization and Mengmeng Zhu for her recommendations with IEF and 2DGE equipment. Finally, I would like to thank Diane Duke and Linda Green of the ICBR Hybridoma Lab, and Carolyn Diaz and Dr. Sixue Chen of the ICBR Proteomics Core. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 7 LIST OF FIGURES .............................................................................................................. 8 ABSTRACT.......................................................................................................................... 9 CHAPTER 1 INTRODUCTION ........................................................................................................ 11 Discovery and Characterization of M. alligatoris ....................................................... 11 Mycoplasmosis ........................................................................................................... 12 Reptile Immunology .................................................................................................... 14 Immunodominance ..................................................................................................... 17 Cellular Host Responses Affecting Immunodominance ............................................ 18 Immunogenic Proteins and Epitopes ......................................................................... 19 Antigenic Variation and Immunodominance .............................................................. 22 Previous Studies on the Temporal Antibody Response of Crocodilians to M. alligatoris .................................................................................................................. 23 Objectives and Experimental Approaches ................................................................. 25 Long Term Goals of Research on M. alligatoris ................................................. 25 Objectives ............................................................................................................. 25 Hypotheses .......................................................................................................... 26 Specific Aims ........................................................................................................ 26 2 MATERIALS AND METHODS ................................................................................... 29 Affinity Chromatography Antigen Purification ............................................................ 30 First Column Configuration ......................................................................................... 31 Second Column Configuration ................................................................................... 32 Third Column Configuration ....................................................................................... 32 SDS-PAGE Separation of Affinity Chromatography Elutions.................................... 33 Second Affinity Column Monoclonal Antibody Coupling Validation .......................... 33 2-Dimensional PAGE Antigen Separation ................................................................. 34 Active Rehydration ............................................................................................... 36 Isoelectric Focusing (IEF) .................................................................................... 37 Second Dimensional SDS-PAGE ........................................................................ 37 Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) .................................. 38 Western Blotting ......................................................................................................... 38 Phosphopentomutase Knockout Mutant Evaluation ................................................. 40 Peptide Sequencing and Protein Identification .......................................................... 40 Protein Reduction, Alkylation and Enzymatic Digestion ..................................... 40 5 LC-MS/MS ............................................................................................................ 42 Database Searching ............................................................................................ 42 De Novo Sequencing Protein Identifications....................................................... 45 PEAKS Criteria Settings ...................................................................................... 46 3 RESULTS AND DISCUSSION ................................................................................... 47 Affinity Columns .......................................................................................................... 47 Knockout Mutant Immunodominant Antigen Expression Evaluation ........................ 53 2DGE Protein Separation ........................................................................................... 54 2DGE Membrane Protein Separation ................................................................. 54 2DGE Total Protein Separation ........................................................................... 55 Blue Native PAGE ...................................................................................................... 57 4 CONCLUSIONS AND FUTURE DIRECTIONS ......................................................... 68 Conclusions ................................................................................................................ 68 Future Directions ........................................................................................................ 68 Technical Improvements ............................................................................................ 70 Alternative Immunodominant Antigen Candidate Screening Techniques ................ 72 Vaccine Development ................................................................................................. 73 Algorithm-Based Evaluations of Diagnostic Value .................................................... 74 Alternatives to Hypotheses ......................................................................................... 75 Responses to Future Potential M. alligatoris Outbreaks ........................................... 75 APPENDIX: DE NOVO SEQUENCES AND FASTA ZP_06610777 FRAGMENT PEPTIDE SEQUENCE ............................................................................................... 78 LIST OF REFERENCES ................................................................................................... 81 BIOGRAPHICAL SKETCH................................................................................................ 90 6 LIST OF TABLES Table page 3-1 M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold (peptide sources: in-gel trypsin or AspN digestion of third column configuration LAMP antigen elutions separated by SDS-PAGE). ........................ 65 3-2 M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold (peptide sources: in-gel trypsin digestion of M. alligatoris membrane protein separated by 2DGE). ............................................................................................. 65 3-3 M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold (peptide sources: in-gel trypsin digestion of M. alligatoris total protein separated by 2DGE). ............................................................................................. 66 3-4 M. alligatoris proteins identified by protein-protein BLAST searches of de novo sequence data reported in Appendix Tables A-1, A-2 and A-3 ................... 67 A-1 De novo sequence data obtained from LC-MS/MS spectra using PEAKS Studio software (peptide source: in-gel trypsin digestion of third column configuration LAMP antigen elutions separated by SDS-PAGE). ........................ 78 A-2 De novo sequence data obtained from LC-MS/MS spectra using PEAKS Studio software (peptide source: in-gel Asp-N digestion of third column configuration LAMP antigen elutions separated by SDS-PAGE). ........................ 79 A-3 De novo sequencing data obtained from LC-MS/MS spectra using PEAKS Studio software (peptide source: in-gel trypsin digestion of total protein separated by 2DGE). ............................................................................................. 80 7 LIST OF FIGURES Figure page 1-1 Representative Western blots of M. alligatoris antigens probed with plasma from crocodilians exposed to M. alligatoris. .......................................................... 28 3-1 Coupling validation of affinity column (second configuration). ............................. 58 3-2 M. alligatoris LAMP antigen eluted from affinity chromatography column (third configuration).. .......................................................................................................