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An Abstract of the Dissertation of Melanie J. Harriff

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An Abstract of the Dissertation of Melanie J. Harriff AN ABSTRACT OF THE DISSERTATION OF MELANIE J. HARRIFF for the degree of Doctor of Philosophy in Molecular and Cellular Biology presented on June 8, 2007. Title: Mechanisms for the Interaction of Environmental Mycobacteria with Host Cells Abstract approved: Luiz E. Bermudez Michael L. Kent Environmental mycobacteria are important opportunistic pathogens for many hosts, including humans, cattle, and fish. Two well-studied species are Mycobacterium avium subsp. avium, a significant cause of disseminated bacterial disease in patients with AIDS, and Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s disease in cattle. Many other species that are considerable sources of infections in fish, such as Mycobacterium chelonae and Mycobacterium marinum, also have zoonotic potential. To gain knowledge about the invasion of epithelial cells by environmental mycobacteria, selected genes and proteins involved in the uptake of M. avium by HEp-2 cells were analyzed by a variety of methods. Two transcriptional regulators (MAV_3679 and MAV_5138) were identified as being involved in invasion. A mycobacterial protein (CipA) with an amino acid sequence suggestive of an ability to be a part of the scaffolding complex that forms during cell signaling leading to actin polymerization was found to putatively interact with host cell Cdc42. Fusion of CipA to GFP, expressed in Mycobacterium smegmatis, revealed that CipA localizes to a structure on the surface of bacteria approaching HEp-2 cells. To establish whether species of environmental mycobacteria isolated from different hosts use similar mechanisms to M. avium for interaction with the mucosa, and for survival in macrophages, assays to determine invasion and replication were performed in different cell types, and a custom DNA microarray containing probes for known mycobacterial virulence determinants was developed. Bacteria cultured from macrophages indicated differences between the ability of M. avium and other environmental species to replicate in human, mouse, and carp cell lines. Hybridization of genomic DNA isolated from EM species against the sequenced MAC104 strain of M. avium showed that there are genes and regions of the chromosome absent from a subset of those species that may be important in determining host specificity and the in differences in virulence observed in vitro and in vivo. Finally, the intestinal epithelium was determined to be the primary route of infection for mycobacteria in zebrafish, similar to M. avium infection in humans. M. avium does not infect zebrafish, but the comparative virulence and genomics of some species isolated from fish suggest that they could be used as a surrogate model to continue progress in understanding the interaction between environmental mycobacteria and epithelial cells. ©Copyright by Melanie J. Harriff June 8, 2007 All Rights Reserved Mechanisms for the Interaction of Environmental Mycobacteria with Host Cells by Melanie J. Harriff A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented June 8, 2007 Commencement June 2008 Doctor of Philosophy dissertation of Melanie J. Harriff presented on June 8, 2007. APPROVED: Co-Major Professor, representing Molecular and Cellular Biology Co-Major Professor, representing Molecular and Cellular Biology Director of the Molecular and Cellular Biology Program Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Melanie J. Harriff, Author ACKNOWLEDGEMENTS Thank you to Mike Kent and Luiz Bermudez for providing me the opportunity to complete my degree at Oregon State University, and for their many contributions to my professional and personal development. Thank you also to the members of my committee, Chris Bayne, John Fowler, and Doug Barofsky, who have freely offered their time and advice. A special thanks to those with whom I have worked in the laboratory. Without their friendship, professional collaboration, and contribution to my projects, I wouldn’t be here today. To name a few: Julie Early, Lia Danelishvili, Martin Wu, Marta Alonso-Hearn, Heather Duchow, Sanjai Tripathi, and Mike McNamara from the Bermudez lab; and Chris Whipps, Virginia Watral, Janell Bishop-Stewart, and Jen Ramsey from the Kent lab. For Tracey, Duke, and Satchel, whose endless love, friendship, and loyalty have been a light in each day, and who have sustained me throughout, I have immeasurable gratitude, appreciation, and love. It has been a joy to go through this experience with you at my side. I would like to acknowledge the encouragement of friends who have passed through my life during this chapter, and to thank my family for their willingness to examine ideas beyond their own experience. Finally, thanks to the Molecular and Cellular Biology Program and the Department of Biomedical Sciences for funding and support over the past few years. I appreciate being given the opportunity to represent the faculty, staff, and students of these programs, and also am grateful for the contribution that they have made in my graduate career. I am indebted to Denny Weber for her assistance and support while I have been here, and much appreciate the efforts of Gail, Rosa, and Sheri in the MCB office. CONTRIBUTION OF AUTHORS Chapter 2: Martin Wu collected the RNA and performed the original microarray hybridization comparing MAC109 and MAC109:∆fadD2; Cara Wilder, as a rotation student under my supervision, made the strains over-expressing MAV_3679 and MAV_5138 and performed invasion assays with those strains; and Michael McNamara made the MAV_4671:GFP construct. Chapter 3: Martin Wu performed the macrophage invasion assays for the six strains of M. salmoniphilum. Drs. Luiz Bermudez and Mike Kent were advisors on all manuscripts. TABLE OF CONTENTS Page Chapter 1: Introduction 1 The Genus Mycobacterium ……………………….. 1 Mycobacterial infection in humans ……………….. 2 Mycobacterial infection in fish ……………………. 4 Invasion of intestinal epithelial cells by prokaryotic pathogens …….……………………….. 7 M. avium invasion of intestinal epithelial cells …….………………………………. 10 Gene regulation and expression by intracellular pathogens upon epithelial cell invasion …..……….. 13 Zebrafish as a host model for mycobacteriosis …….. 14 Concluding remarks ……………………………….. 15 References ……………………………………….. 17 Chapter 2: MAV_3673 and MAV_5138 are transcriptional 22 regulators that play a role in Mycobacterium avium invasion of epithelial cells, in part by the regulation of CipA, a putative surface protein interacting with host cell signaling pathways Abstract ……………………………………….. 23 Introduction ……………………………………….. 24 Materials and Methods ……………………….. 26 Results ……………………………………….. 37 Discussion ……………………………………….. 51 Acknowledgements ……………………………….. 60 References ……………………………………….. 60 TABLE OF CONTENTS (Continued) Page Chapter 3: Species of environmental mycobacteria vary in 65 their ability to grow in human, mouse, and carp macrophages, and differ regarding the presence of mycobacterial virulence genes observed by DNA microarray hybridization Abstract ………………………………………… 66 Introduction ………………………………………… 67 Materials and Methods ………………………… 70 Results ………………………………………… 78 Discussion ………………………………………… 87 Acknowledgements ………………………………… 98 References ………………………………………… 98 Chapter 4: Experimental exposure of zebrafish (Danio 113 rerio Hamilton) to Mycobacterium marinum and Mycobacterium peregrinum reveals the gastrointestinal tract as the primary route of infection: a potential model for environmental mycobacterial infection Abstract ………………………………………… 114 Introduction ………………………………………… 115 Materials and Methods ………………………… 117 Results ………………………………………… 122 Discussion ………………………………………… 132 Acknowledgements ………………………………… 138 References ………………………………………… 138 Chapter 5: Discussion 143 Aim 1. Interaction of M. avium with intestinal epithelial cells 143 Aim 2. Comparative analysis of the presence of virulence 148 genes in species of EM isolated from fish Aim 3. Identification of the route of infection in fish by 152 mycobacteria. TABLE OF CONTENTS (Continued) Page Bibliography …………………………………………………… 156 Appendix I: Molecular characterization of a Mycobacterium 170 species in non-native poeciliids in Hawaii using DNA sequences Appendix II: Environmental amoeba and 182 mycobacterial pathogenesis Appendix III: Abstracts for additional publications 201 LIST OF FIGURES Figure Page 1.1 A phylogenetic tree showing the relationship between a 2 subset of mycobacterial species, including many of those used in this study, based on molecular methods. 1.2 A model from Sangari et al. (1999) showing the interaction 4 of M. avium with the intestinal epithelium. 1.3 A lesion on the skin of a striped bass (www.vims.org), 5 and granulomas in the visceral organs of a European tench (www.was.org) infected with mycobacteria. 1.4 Mycobacteria in zebrafish tissues. 6 1.5 A drawing showing the two mechanisms by which intracellular 8 pathogens trigger their entry into host epithelial cells. 1.6 Electron micrograph of Mycobacterium avium invading 11 epithelial cells of the mouse intestine. 1.7 Proposed model of regulation of the Salmonella pathogenicity 14 island 1 (SPI-1) by hilA and other upstream regulators. 2.1 Real-time PCR results comparing the fold-change in gene 41 expression upon 15 minute exposure to HEp-2 cells between the MAC109 and MAC109:∆fadD2 strains. 2.2 Percent
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