Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2015 The Characterization of a Putative Protease Expressed by Sneathia amnii Rana Mehr 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/3931 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]. CHARACTERIZATION OF A PUTATIVE PROTEASE EXPRESSED BY SNEATHIA AMNII A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University by RANA MEHR B.S., Virginia Commonwealth University 2011 Director: Kimberly Jefferson, Ph.D. Associate Professor, Department of Microbiology and Immunology Virginia Commonwealth University Richmond, Virginia Virginia Commonwealth University Richmond, Virginia July, 2015 Acknowledgements I would first like to express my deepest gratitude to my mentor Dr. Kimberly Jefferson. Her continuous mentorship, trust, and support in academic, scientific, and personal experiences have empowered me to successfully complete my graduate career both academically and scientifically. She has aided my development as an independent scientist which would have not been possible without guidance. I would also like to thank the members of my graduate advisory committee: Dr. Dennis Ohman and Dr. Darrell Peterson. Their advice and direction have allowed me to better understand my project and their invaluable knowledge has made me a better scientist. Furthermore, I would like to thank Dr. Gail Christie and Dr. Cynthia Cornelissen for allowing me access to their laboratory resources and equipment. Finally, I would like to thank all of my lab-mates, both past and present: Dr. Shreni Mistry, Dr. Zhumei Cronk, Dr. Michael Harwich, Melissa Prestosa, Abdallah Abdelmaksoud, and Matthew Allen-Daniels. They have provided invaluable input, guidance, and patience throughout my graduate career. I would like to specially thank my two lab-mates Dr. Jamie Brooks and Amy Sanford for their never ending laughter, friendship, advice, and support that has abetted in my successful conclusion to my graduate career. My closing acknowledgement is extended Dr. Kristin Lane and Laura Klenow, who both always have advice whenever I have needed it. ii Dedication I would like to dedicate this work to my parents for their unyielding love, support, dedication, and guidance which have led me to my successes. I would also like to dedicate this to my brother who has taught me to be a strong independent person. Finally, I must thank my boyfriend, Eric Hall, who has supported me and encouraged me throughout my scientific career. My successes are due to the endless love and support my loved ones have given me. iii Table of Contents Page Acknowledgments ..................................................................................................................................... ii Dedication ................................................................................................................................................. iii List of Figures ........................................................................................................................................... vi List of Tables ............................................................................................................................................ vii Abbreviations ............................................................................................................................................ viii Chapter 1. Introduction ............................................................................................................... 1 Clinical significance of Preterm Birth ........................................................................... 1 Intrauterine and Amniotic Cavity Invasion by Vaginal Bacteria .................................. 2 Bacterial Vaginosis ........................................................................................................ 5 Sneathia amnii strain Sn35 ............................................................................................ 8 Research Objectives ..................................................................................................... 10 2. Materials and Methods ............................................................................................ 12 3. Analyses of Pathogenicity Potential of Sneathia amnii isolate Sn35 ..................... 32 Introduction .................................................................................................................. 32 Invasion of human fetal membranes by Sneathia amnii .............................................. 34 Sn35 adhesion capability with VK2 cell line ............................................................... 36 Selection of virulence determinant expressed by Sn35 ............................................... 36 iv Expression and purification of recombinant Peg1 ....................................................... 41 Edman Degradation ..................................................................................................... 41 Extracellular Matrix Component Degrading Activity ................................................ 48 Coagulase potential of Peg1 ........................................................................................ 49 Analysis of cytotoxicity of Peg1 to ME-180s .............................................................. 52 Analysis of biofilm formation capability of Peg1 ........................................................ 52 Bacteriolytic Activity ................................................................................................... 54 Collagen degradation ................................................................................................... 54 Analysis of Peg1 antisera production .......................................................................... 56 Discussion, Conclusion and Future Perspectives ......................................................... 56 Literature Cited ...................................................................................................................................... 63 Vita ......................................................................................................................................................... 69 v List of Figures Page Figure 1.1: Schematic representation of bacterial ascension into the amniotic cavity............................... 4 Figure 3.1: Fetal membranes incubated with or without Sn35 .................................................................. 35 Figure 3.2: Gene Map of Putative Zinc Metalloprotease .......................................................................... 37 Figure 3.3:DNA gel of Peg1 PCR amplification ....................................................................................... 40 Figure 3.4: DNA agarose gel analysis of restriction digest of BL21 DE3 RIL E.coli cells containing pET32xT+Peg1. ......................................................................................................................................... 42 Figure 3.5: Confirmation of induced Peg1 with 1 mM IPTG. ................................................................... 43 Figure 3.6: Purified protein by cobalt resin column confirmed with SDS PAGE. .................................... 44 Figure 3.7: Thrombin cleavage of Thio-His tag with CaCl2 ..................................................................... 45 Figure 3.8: Thrombin cleavage without CaCl2 allows for site specific degradation. ................................. 46 Figure 3.9: Schematic representation of site specific degradation. ............................................................ 48 Figure 3.10: SDS PAGE fibronectin degradation assay ........................................................................... 50 Figure 3.11: SDS PAGE laminin degradation assay. ................................................................................. 51 Figure 3.12: Cytotoxic effect of Peg1on ME-180s. ................................................................................... 53 Figure 3.13: Bacteriolytic effect of Peg1 on Micrococcus luteus. ............................................................. 55 Figure 3.14: Role of Peg1 in collagen degradation. ................................................................................... 57 Figure 3.15: Western analysis of Peg1 expression location. ...................................................................... 58 Figure 3.16: Temporal expression of Peg1. ............................................................................................... 59 vi List of Tables Page Table 1: Bacterial strains ........................................................................................................................... 13 Table 2: Primer list ..................................................................................................................................... 15 vii List of Abbreviations °C ....................................................................................................................................