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INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 COMPARATIVE ANALYSIS OF PSEUDOMONAS AERUGINOSA LASA AND LASD DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sukjoon Park, B. S. ***** The Ohio State University 1996 Dissertation Committee: Approved by Dr. Darrell R. Galloway Dr. Charles J. Daniels Dr. Donald H. Dean Advisor Graduate Program in Molecular, Dr. Gary E. Means Cellular and Developmental Biology ÜMX Number: 9710640 UMI Microform 9710640 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT Pseudomonas aeruginosa is widely known as an opportunistic pathogen. The organism produces many virulence factors including both proteolytic and non- proteolytic enzymes. Among the proteolytic virulence factors, pseudolysin (elastase) has long been considered to be of major significance due to its ability to digest elastin tissues. However, LasA has been the focus of research for the past several years particularly due to its ability to enhance the elastolytic activity of pseudolysin. In addition, LasA was characterized as a staphylolytic enzyme; LasA causes lysis of Gram-positive organisms such as Staphylococcus aureus. In this study, another protease, LasD, was purified and characterized from various strains of P. aeruginosa. LasD is a 23 kDa protease which shares many of the enzymatic properties of LasA, including the ability to lyse heat-killed staphylococci and hydrolyzing synthetic peptides such as pentaglycine. However, LasD is distinct from the 22 kDa LasA for the following reasons: (i) the N-terminal sequence of LasD shares no homology with LasA or the LasA precursor sequences; (ii) P. aeruginosa LasA negative mutant strains AD 1825 and FRD2I28 do not produce LasA yet produce LasD, and (iii) specific antibodies to each protease do not exhibit any cross-reactivity. Furthermore, the purified LasD shows strong u staphylolytic activity only under alkaline pH conditions, while LasA exhibits staphylolytic activity over a broad pH range. The staphylolytic activity of both LasA and LasD may be related to hydrolysis of the pentaglycine bridge in the Gram- positive cell wall. To verify this possibility, various synthetic peptides containing glycine residues were analyzed and the results show that both LasA and LasD cleave peptides with internal diglycine, and the minimum size of the peptide is a tetramer such as tetraglycine. In addition to the study on the substrate specificity of LasA and LasD, kinetic analysis of both proteases was also performed using acetylated pentaglycine as a substrate. The results indicate that the acetylated pentaglycine is better substrate for LasA than LasD, thereby indicating another difference between LasA and LasD. LasD cuts the acetylated pentaglycine more efficiently at higher pH consistent with the observation made using heat-killed S. aureus cells as a substrate. Finally, the processing events of LasA from an inactive 41 kDa precursor form to an active 22 kDa form was studied primarily to elicit possible roles of other proteases, including LasD, in the processing event. Surprisingly, LasD was found to be involved in the processing of LasA in vitro, indicating LasA and LasD work together for greater efficiency. However, the fact that LasA negative strains still produce active LasD suggests that the possible processing event for activation of LasD is independent of LasA. m Dedicated to my parents IV ACKNOWLEDGMENTS I express my sincere appreciation to Dr. Darrell R. Galloway for his advice and guidance throughout these years. Without his support, it would not have been possible to come this far. I also thank Mrs. Galloway for inviting me to every holiday celebration for the last six years; I appreciate her willingness to make me a part of the Galloway family. Additional thanks is given to the other members of my committee: Drs. Charles Daniels, Donald Dean, and Gary Means for their encouragement. I would also like to thank Dr. John Lowbridge at the Ohio State University Biochemical Instrumentation Center for his advise on HPLC. My sincere thanks to the former and present members of the Galloway Lab: Dr. John Peters, Dr. Christine Sauer, Dr. Leena Hiremath, Dr. Scotty Walker, Dr. Christine Besson, Dr. Xiang-Yang Han, Dr. Pil-Jung Kang, Monte Kendricks, Todd Kelly, Madhura Pradhan, Kimberly Denis-Mize, and Susan Meyer. I especially thank Kimberly Denis-Mize for patiently proofreading my draft over and over again. Most of all, I would like to thank my parents and my wife without whom I could not finish my long journey as a graduate student. Dad, you will never know how grateful I am that you encouraged and supported me for so long. VTTA February 27,1964 ................................. Bom - Seoul, Korea 1990...................................................... B.S. Botany, Ohio University, Athens, Ohio 1990-96..................................................Graduate Teaching and Research Associate, Ohio State University, Columbus, Ohio PUBLICATIONS 1. Sulqoon Park and D. R. Galloway (1995). “Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa.'' Molecular Microbiology 16: 263-270. 2. Peters, J. E., S. J. Park, A. Darzins, L. C. Freck, J. M. Saulnier, J.M. Wallach and D. R. Galloway (1992). “Further studies on Pseudomonas aeruginosa LasA: analysis of specificity.” Molecular Microbiologv 6: 1155-1162. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology Minor Field: Biochemistry VI TABLE OF CONTENTS Page Abstract.................................................................................................................. ü Dedication ............................................................................................................. iv Acknowledgments .................................................................................................... v Vita.......................................................................................................................... vi List of Tables ........................................................................................................... xi List of Figures ........................................................................................................xiii List of Abbreviations ........................................................................................... xvi Chapters; 1. Literature Review ........................................................................................ 1 A. Classification of proteases ................................................................. 1 1. Serine proteases ....................................................................... 4 2. Cysteine proteases ....................................................................8 3. Aspartic proteases .................................................................. 11 4. Metallopro teases ....................................................................12 B. Pseudomonas aeruginosa as an opportunistic pathogen ....................19 C. Non-proteolytic Pseudomonas aeruginosa virulence factors ............. 22 1. Exotoxin A ............................................................................ 22 2. Exoenzyme S ....................................................................... 24 3. Phospholipase C .................................................................. 24 D. Pseudomonas aeruginosa proteases .................................................. 25 1. Pseudolysin ............................................................................25 2. LasA...................................................................................... 31 vii 3. Alkaline protease ....................................................................34 4. Lysine-specific protease ......................................................... 35 E. Regulation of Pseudomonas aeruginosa proteases