UNIVERSITY OF CINCINNATI Date: February 22, 2007 I, _ Samuel Lee Hayes________________________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Biological Sciences It is entitled: Response of Mammalian Models to Exposure of Bacteria from the Genus Aeromonas Evaluated using Transcriptional Analysis and Conjectures on Disease Mechanisms This work and its defense approved by: Chair: _Brian K. Kinkle _Dennis W. Grogan _Richard D. Karp _Mario Medvedovic _Stephen J. Vesper Response of Mammalian Models to Exposure of Bacteria from the Genus Aeromonas Evaluated using Transcriptional Analysis and Conjectures on Disease Mechanisms A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of Biological Sciences of the College of Arts and Sciences 2007 by Samuel Lee Hayes B.S. Ohio University, 1978 M.S. University of Cincinnati, 1986 Committee Chair: Dr. Brian K. Kinkle Abstract The genus Aeromonas contains virulent bacteria implicated in waterborne disease, as well as avirulent strains. One of my research objectives was to identify and characterize host- pathogen relationships specific to Aeromonas spp. Aeromonas virulence was assessed using changes in host mRNA expression after infecting cell cultures and live animals. Messenger RNA extracts were hybridized to murine genomic microarrays. Initially, these model systems were infected with two virulent A. hydrophila strains, causing up-regulation of over 200 and 50 genes in animal and cell culture tissues, respectively. Twenty-six genes were common between the two model systems. The live animal model was used to define virulence for many Aeromonas spp. Strains that demonstrated mortality and produced an average up-regulation of > 3-fold, at challenged doses of 107-108 CFU, were considered virulent. Mortality results correlated well with dose and transcript up-regulation. Cell cultures were infected with representative virulent and avirulent Aeromonas strains. Transcriptional response from live animal and cell culture models were compared to find common transcripts unique to virulent infections. Two genes with potential for predicting virulence (Jun and Fos) were identified. Confirmation testing (qRT-PCR) indicated that the Jun oncogene is potentially predictive of Aeromonas virulence using cell culture. Aeromonas caviae is associated with gastrointestinal disease but lacks obvious virulence factors (VFs). Microarray profiling of mouse intestinal extracts after A. caviae infection demonstrated a Th1 immune response characterized by gamma-interferon induced genes. This suggests A. caviae causes a dysregulatory cytokine response leading to an irritable bowel-like syndrome. To evaluate loss of single VFs, isogenic mutants were produced using a transposable element. Mutations in VFs associated with lateral flagella, O-antigen and secretion systems were created. Swarming motility, associated with intestinal colonization, was eliminated in a lateral flagella mutant, but subsequent colonization testing was inconclusive. Murine monolayers demonstrated no difference in gene expression after infection with lateral flagella mutant and wild type organisms. As a result of this research, I suggest that Aeromonas has at least two pathways to virulence. The first functions by activation of multiple VFs to pathway(s) thru Jun. The second, as demonstrated by some A. caviae strains, is activated through gamma interferon, producing a chronic infection. ACKNOWLEDGMENTS Many individuals were instrumental in helping me complete this dissertation and associated research. I owe the following people an enormous amount of gratitude. First, many thanks to my advisor, Dr. Brian Kinkle for his advice and guidance, especially for keeping me focused on the science. My EPA contact, Dr. Steve Vesper, a very passionate and driven scientist, infused his enthusiasm for this project into me and was very inspirational. I want to also thank the other members of my committee, Dr. Dennis Grogan, Dr. Richard Karp and Dr. Mario Medvedovic for sticking with me over the past four years and for their input and expertise. I have also had the support and advice of a group of scientists (our Aeromonas research group), with whom I have been meeting with almost weekly over this same time period. Dr. Dennis Lye, Dr. Maura Donohue, Dr. Mark Rodgers and Dr. Gerard Stelma have been incredibly supportive and have offered invaluable advice to me regarding research direction. I am very grateful to this group and look forward to a continuing collaboration on similar research efforts. The following people have provided technical assistance to me during various phases of the research, Ms. Bethany Lye, Ms. Shannon Murphy, Mr. Brent Bertke, Ms. Karen White and Ms. Cathy Kelty. Many of these marvelous professionals have moved on to bigger and better endeavors, though I have the pleasure of still having some as colleagues. Thanks for their assistance over the years. As she always has, the most important support I received was from my wife, Kim. The biggest lesson I have learned from her is that with the right support, anything is possible. She has been, and always will be, my rock. Table of Contents Table of Contents ...................................................................................................................... i List of Tables ........................................................................................................................... iii List of Figures...........................................................................................................................iv LITERATURE REVIEW .........................................................................................................1 1.0 Aeromonas, Taxonomy and General Information ...................................................1 1.1 Aeromonas Occurrence/Epidemiology/Significance ................................................4 1.2 Known Aeromonas Virulence Factors (VFs)...........................................................12 1.2.1 Enterotoxins .....................................................................................................13 1.2.1.1 Aerolysin........................................................................................................13 1.2.1.2 Act .................................................................................................................14 1.2.1.3 Ast and Alt .....................................................................................................15 1.2.2 Hemolysins .......................................................................................................16 1.2.3 Lipase ...............................................................................................................17 1.2.4 Adhesion Factors..............................................................................................17 1.2.5 Metallo- and serine proteases ..........................................................................20 1.2.6 Type III Secretion Systems ..............................................................................21 1.2.7 S-Layer .............................................................................................................21 1.2.8 Plasmid associated virulence factor.................................................................22 1.3 Additional Research of VFs and Aeromonas Virulence .........................................22 1.4 DNA chip technology (Affymetrix) .........................................................................25 1.4.1 Gene Chip Preparation and Technology Description.....................................26 1.4.2 Gene Chip Data Analysis .................................................................................28 1.5 Bacterial-host interaction research using microarray technology.........................31 1.5.1 Research of host bacterial interactions, epithelial cell models .......................33 1.6 Animal models for assessing Aeromonas virulence.................................................36 1.7 References ................................................................................................................41 Chapter 1, Identification by microarray of a common pattern of gene expression in intact intestine and cultured intestinal cells exposed to virulent Aeromonas hydrophila isolates...60 Abstract ...............................................................................................................................61 INTRODUCTION...............................................................................................................62 MATERIALS and METODS..............................................................................................63 RESULTS and DISCUSSION ............................................................................................67 CONCLUSIONS .................................................................................................................70 REFERENCES....................................................................................................................72 Chapter 2, Evaluating virulence of waterborne and clinical Aeromonas isolates using gene expression and mortality in neonatal mice followed by assessing cell culture's ability to predict virulence based on transcriptional response .............................................................81