AN ABSTRACT OF THE THESIS OF Daniel D. Rockey for the degree of Doctor ofPhilosophy in the Department of Microbiology presented on November 10.1989. Title:Virulence Factors of Aeromonas salmonicida and Their Interaction with the Salmonid Host. Redacted for Privacy Abstract approved: S. Rohovec Selected secreted and cellular virulence factors ofAeromonas salmonicida were examined. A protocol wasdeveloped for the separation of two secreted proteases (P1 and P2protease), and a trout erythrocyte specifichemolysin (T-lysin) from supernatants of cultures of thebacterium.Distinctions between the proteases were demonstrated using molecularweight determinations, substrate specificities,sensitivity to chemical protease inhibitor sensitivities, andpolyacrylamide gel electrophoresis using gels containing protease substrates(G- PAGE).P1, but not P2, protease was detected inG-PAGE analyses of protease from lesions of cohosalmon (Oncorhynchus kisutch) infected by injection.Other proteases of apparent host origin were also detected in these assays.Analysis of the T- lysin demonstrated that although thebacterium produced high titers of the enzyme in vitro, no hemolyticactivity was detected in vivo nor in cultures grown in salmonid sera.Subsequent experiments demonstrated that salmonid sera possess an inhibitor of hemolysis capable of protecting erythrocytesfrom enzymatic or chemical lysis.The inhibitor was partially purified using molecular sieve chromatography andpreparative isoelectric focusing.Analysis of P1 protease, P2 protease, and T-lysin production was continued by examiningtheir production in the presence of salmonid sera and in the presence of high concentrations of selected salts added tobrain heart infusion broth (BHI).The spectrum of proteases produced in serum was similar to the spectrumproduced in BHI.However, a larger phenylmethylsufonylfluoride sensitive fraction was detected in supernatants from bacterial cells grownin serum. Analysis of supernatants from the cultures grown inhigh salts indicated that P1 protease and T-lysin production were inhibited by these salts but P2 protease production was not. Growth in high concentrations of magnesiumsalts also affected the cellular morphology of the bacteriumand this effect was associated with the presence of an outer membraneprotein layer, the A layer. Four monoclonal antibodies (Mabs) were producedwith specificity towards A. salmonicida lipopolysaccharide(LPS). These Mabs were used to identify two distinctepitopes on LPS and to show that the presence of eachepitope varied among different strains.The antibodies were also used to demonstrate the difference in the host responseof rabbits and rainbow trout (Oncorhynchus mykiss) to A. salmonicida. Virulence Factors of Aeromonas salmonicida and Their Interaction with the Salmonid Host by Daniel D. Rockey . A THESIS submittedto Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed November 10, 1989 Commencement June, 1990 APPROVED: Redacted for Privacy Assoc JeProfessor of Microbiology in charge of Major Redacted for Privacy ChCinan,Departmeof Microbiology Redacted for Privacy Dean ofGrate School Date thesis is presented November 10. 1989 ACKNOWLEDGEMENTS I dedicate this thesis to my family- my father Dean, my mother Frances, my brothers Bill and Brian, my wife Martha and my son Tyler.Without their love and guidance I would not have been able to accomplish this longtime goal.I am especially indebted to Martha and Tyler who, especially at the end, have helped me understand that there are other things in life. I am also indebted to the following people: My graduate student friends and co-workers-Scott "Woody Marie" Lapatra, Jerri "Canyon Way" Bartholomew, Rich "Workaholic" Holt, Susan "Drano queen" Gutenberger, Steve "Up for the stuff' Piacentini, Yen-Ling "Singa" Song, "Kung foo" Carla Mason, Scott "A, spe" Manning, Bob "Hum baby" Gilmore, Mark "Big Red" Engelking, Greg "Skunked again" Wiens, Mary Arkoosh (the gum junkie), Prasad "Where's the beef' Turaga, Ralph "Missed a big one" Tripp, Jeff "Daguto" Miner, Glacier "Mac attack" Tajwall, and the list goes on and on.... Rich, Tony, Craig, John, and Leslie of the Oregon Department of Fish and Wildlife for their advice and assistance.I also thank the ODFW for fish used throughout the course of this work. Undergraduate research assistants Stan van de Wetering, Mary Beer, and especially Leah A. Shook for significant contributions to this work. Carlene Pelroy, Teresa Curry, Rachel le Nelson, Joy Asbury, Bonnie Casper, and Barbara Overholser for administrative and clerical assistance. Dr. S. L. Kaattari for allowing me to finish my thesis while employed in his laboratory. Oh yes, and don't forget Harriet. Finally, Dr. John Fryer and Dr. John Rohovec for their assistance and guidance during the progress of my thesis work. Dr. Rohovec in particular was both sadistic and masochistic during the course of our five year interaction.Sadistic because of the abuse heaped upon the author during the course of this work, and masochistic because he decided to teach me how to "write good".A sincere thanks for the diligent guidance. The principal funding for this research was received from USDA Science and Education grant no. 85-CRSR-2-2578 andfrom the N. L. Tartar Fellowship Fund. CONTRIBUTION OF AUTHORS Dr. J. S. Rohovec and Dr. J. L. Fryer were advisors onall manuscripts included in this work.Ms. L. A. Shook was responsible for protease assays, hemolysin asays and proteingel electrophoresis described in Chapters 4 and 5.Dr. T. Lunder assisted in the comparison of strains reported inChapter 7.Mr. C. Dungan was responsible for the care and propagationof the monoclonal antibodies described in Chapter 7. TABLE OF CONTENTS Chapter Page 1. Introduction 1 2. Literature Review 4 The Etiology of Furunculosis in Fish and the Classification of the Causative Agent 4 The Pathology of Furunculosis and Virulence Factors Produced by Aeromonas salmonicida 6 Aeromonas salmonicida and the Immune Response in Fish 20 Literature Cited 26 3. Separation and in vivo Analysis of Two Extracellular Proteases and the T-hemolysin from Aeromonas salmonicida 41 Abstract 42 Introduction 43 Materials and Methods 45 Results 51 Discussion 67 Literature Cited 71 4. Salmonid Sera Inhibit the Hemolytic Activity of the Secreted Hemolysin of Aeromonas salmonicida 74 Abstract 75 Introduction 76 Materials and Methods 77 Results 80 Discussion 91 Literature Cited 98 5. Protease and Hemolysin Activity of Aeromonas salmonicida Cultures Grown in Modified Media and Trout Serum 100 Abstract 101 Introduction 102 TABLE OF CONTENTS Chapter Page 5. (Continued) Materials and Methods 104 Results 109 Discussion 124 Literature Cited 129 6. Cellular Pleomorphism Induced in Virulent Aeromonas salmonicida by Growth in Media Containing High Concentrations of Magnesium Salts 133 Abstract 134 Introduction 135 Materials and Methods 136 Results 138 Discussion 144 Literature Cited 147 7. Monoclonal Antibodies against Aeromonas salmonicida Lipopolysaccharide Identify Differences among Strains 149 Abstract 150 Introduction 151 Materials and Methods 152 Results 157 Discussion 170 Literature Cited 174 CONCLUSIONS 177 BIBLIOGRAPHY 180 LIST OF FIGURES Page CHAPTER 3. 3.1. Flow diagram of protocol used to isolate secreted virulence factors from Aeromonas salmonicida culture supernatants. 52 3.2.Results of SDS-PAGE of Aeromonas salmonicida P1 and P2 proteases.1. Molecular weight standards, values in kilodaltons.Standards apply to lanes 2 and 3.2. Material eluted from DEAE at 0.15 M NaCl.3. P1 from hydroxylapetite (HAP).4 and 5.P2 from HAP (4) electrophoresed adjacent to a P1 preparation (5). 54 3.3. Results of SDS-PAGE of T-lysin and P1 protease of Aeromonas salmonicida.1. Molecular weight standards, values in kilodaltons.2. T-lysin preparation.3. P1 protease preparation. 54 3.4. Enzyme activities of samples from the 0.15 M and 0.35 M NaC1 elutions from a DEAE column using the material from the 0-45% NH4SO4 concentration. 57 3.5.Enzyme activities of samples of hydroxylapetite- separated proteases in the presence of PMSF or EDTA. Dark bars; P1, light bars; P2. 5 7 LIST OF FIGURES Page 3.6. Substrate PAGE of Aeromonas salmonicida culture supernatant and purified proteases.1. Gelatin- PAGE of culture supernatant.2. Casein-PAGE of culture supernatant.3 and 4. Gelatin-PAGE of isolated P1 protease (3) and P2 protease (4). 59 3.7. Gelatin-PAGE of Aeromonas salmonicida proteases from culture supernatant (1) andof cell free exudate from infected fish (2) in the presence and absence of the proteaseinhibitors PMSF and EDTA. 62 3.8.Rainbow trout red blood cell (RBC) preparations subjected to treatments for selective lysis of outer membranes.Normal RBC (1), treated with T-lysin (2), 35% PBS (3), and 0.1% NP40 (4). 64 CHAPTER 4. 4.1. Hemolysis by Aeromonas salmonicida culture supernatants of washed ( 0) orunwashed ( ) rainbow trout erythrocytes. 82 4.2. A. Hemolysis inhibition by rainbow trout serum in assays with Aeromonas salmonicida supernatants (a) added to serumbefore rainbow trout erythrocytes or (b) with supernatantadded after the erythrocytes.B.Hemolysis and hemolysis inhibition assays using A. salmonicida (dark bars) or A. hydrophila(light bars) culture supernatants as source of hemolysinand rainbow trout serum as the sourceof hemolytic inhibitor. 84 LIST OF FIGURES Page 4.3. A.Hemolysin inhibition assays using serafrom selected species as inhibitor of A.salmonicida hemolysin.B.Hemolysin assays with erythrocytes from