East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works 5-2012 An Investigation of Bacterial Ribonucleases as an Antibiotic Target Ashley Denise Frazier East Tennessee State University Follow this and additional works at: https://dc.etsu.edu/etd Part of the Bacteriology Commons Recommended Citation Frazier, Ashley Denise, "An Investigation of Bacterial Ribonucleases as an Antibiotic Target" (2012). Electronic Theses and Dissertations. Paper 1417. https://dc.etsu.edu/etd/1417 This Dissertation - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. An Investigation of Bacterial Ribonucleases as an Antibiotic Target A dissertation presented to the faculty of the Department of Biochemistry and Molecular Biology East Tennessee State University In partial fulfillment of the requirements for the degree Doctor of Philosophy of Biomedical Sciences by Ashley Denise Frazier May 2012 W. Scott Champney, Ph.D., Chair Mitchell E. Robinson, Ph.D. Douglas P. Thewke, Ph.D. J. Russell Hayman, Ph.D. Bert C. Lampson, Ph.D. Keywords: Ribonuclease Mutants, Ribosomal Subunit Assembly, Aminoglycosides, Escherichia coli, Staphylococcus aureus, Protein Synthesis, Vanadyl Ribonucleoside Complex ABSTRACT An Investigation of Bacterial Ribonucleases as an Antibiotic Target by Ashley Denise Frazier Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance. Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of an antibiotic. This makes ribonucleases a potential target for novel antibiotics. It was shown that Escherichia coli mutants that were deficient for RNase III, RNase E, RNase R, RNase G, or RNase PH had an increase in ribosomal subunit assembly defects. These mutant bacterial cells also displayed an increased sensitivity to neomycin and paromomycin antibiotics. My research has also shown that an inhibitor of RNases, vanadyl ribonucleoside complex, potentiated the effects of an aminoglycoside and a macrolide antibiotic in wild type Escherichia coli, methicillin sensitive Staphylococcus aureus, and methicillin resistant Staphylococcus aureus. 2 RNases are essential enzymes in both rRNA maturation and degradation. Based on this and previous work, the inhibition of specific RNases leads to an increased sensitivity to antibiotics. This work demonstrates that the inhibition of RNases might be a new target to combat antibiotic resistance. 3 DEDICATION This manuscript is dedicated to: My mom and dad: Thank you for your support and confidence. I couldn’t have done this without you. Both of you have always led by example and for that I am grateful. I wouldn’t be the person that I am today without you. I love you both! 4 ACKNOWLEDGEMENTS First and foremost, I would like to thank God for this achievement. Thank you to Dr. Champney for your incredible guidance and support. It has been a rewarding experience to work in your lab. I couldn’t have asked for a better mentor! I would like to thank my committee members: Dr. Robinson, Dr. Thewke, Dr. Hayman, and Dr. Lampson for their time and advice. Thank you to Beverly Sherwood, Angela Thompson, and Sandra Davis-Osterhaus. Each of you provided me with much needed support, advice, and encouragement through my educational experience. A special thank you to Beverly for everything that you have done for me in the program. To my friends and fellow graduate students, thank you for your advice, support, and the occasional shoulder. This work was supported by the National Institutes of Health AREA grant awarded to Dr. W. Scott Champney and the ETSU Graduate School research grant awarded to Ashley D. Frazier. 5 CONTENTS Page ABSTRACT..................................................................................................................... 2 DEDICATION................................................................................................................. 4 ACKNOWLEDGEMENTS............................................................................................. 5 LIST OF TABLES........................................................................................................... 10 LIST OF FIGURES......................................................................................................... 11 Chapter 1. INTRODUCTION..................................................................................................... 13 Antibiotic Overview............................................................................................. 13 Antibiotic Targets................................................................................................. 15 Bacterial Ribonucleases........................................................................................ 23 RNase I....................................................................................................... 26 RNase III.................................................................................................... 26 RNase E...................................................................................................... 28 RNase G...................................................................................................... 30 RNase R...................................................................................................... 31 RNase II...................................................................................................... 32 PNPase........................................................................................................ 34 RNase PH.................................................................................................... 35 Vanadyl Ribonucleoside Complex....................................................................... 37 Research Hypothesis............................................................................................ 38 6 Specific Research Aims........................................................................................ 39 2. INHIBITION OF RIBOSOMAL SUBUNIT SYNTHESIS IN AMINOGLYCOSIDE TREATED RIBONUCLEASE MUTANTS OF ESCHERICHIA COLI................................................................................................ 40 Abstract............................................................................................................... 41 Introduction......................................................................................................... 42 Material and methods.......................................................................................... 43 Analysis of cellular growth and viability.................................................... 43 Analysis of ribosomal subunit assembly..................................................... 44 Analysis of total cellular RNA with the Agilent Bioanalyzer..................... 44 Analysis of 16S rRNA and 23S rRNA by Northern blot hybridization......................................................................... 45 Statistical analysis....................................................................................... 46 Results................................................................................................................. 47 Discussion............................................................................................................ 56 Acknowledgements.............................................................................................. 59 References............................................................................................................. 60 3. INHIBITION OF RIBOSOMAL SUBUNITS SYNTHESIS IN ESCHERICHIA COLI BY THE VANADYL RIBONUCLEOSIDE COMPLEX.................................................................................................................. 62 Abstract................................................................................................................ 63 Introduction.......................................................................................................... 64 Material and Methods........................................................................................... 65 7 Cell growth and viability............................................................................. 65 Rate of protein synthesis............................................................................. 66 Ribosomal subunit assembly....................................................................... 66 Uridine pulse and chase labeling................................................................. 66 Agilent Bioanalysis of RNA....................................................................... 67 Northern blot hybridization........................................................................