Characterization of Pathogens for Potential Diagnostic Tests A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) in the Department of Molecular Genetics, Biochemistry, & Microbiology of the College of Medicine by KAREN M. GALLEGOS B.S., Universidad Nacional Agraria La Molina, Perú 2013 Committee Chair: Alison A. Weiss, Ph.D. ABSTRACT Traditional methods for detection of microbial pathogens, which are based on antibody recognition, often are limited by microbial antigenic changes. Characterization of pathogens and identification of stand-alone features with diagnostic value has the potential to replace the typical approaches of current diagnostic test. The approach taken here is to use invariant features of pathogens to develop new diagnostics and potential treatment. In this thesis, we characterized intrinsic features of two very common human pathogens: influenza and the main virulence factor of E. coli O157:H7, Shiga toxin. In the first part of this study, we characterize influenza neuraminidase (NA) activity as a marker for influenza detection. Since influenza NA activity is an invariant characteristic of this virus, our objective was to develop an assay to detect and differentiate influenza NA activity from other respiratory pathogens such as parainfluenza and Streptococcus pneumoniae. We analyzed the influence of pH, calcium and NA inhibitors (oseltamivir and zanamivir) on NA using intact, viable pathogens. We found that these three pathogens display different pH optimum and the calcium requirement was different in each pathogen NA activity. We also found that influenza NA with H274Y mutation, which confers oseltamivir-resistance, displayed up to ten fold- difference in levels of drug resistance. Our results show that measuring NA activity in different conditions can be used to detect and differentiate NA activity of influenza virus, parainfluenza and S. pneumoniae. In the second study, we analyzed the ability of host factors to bind influenza surface proteins as an alternative to replace antibodies as capturing agents. Three types of host factors were considered here: (1) natural (e.g. fetuin) and synthetic receptors containing sialic acid, which mimic the traditional influenza receptor; (2) host proteins binding to the invariable glycosylation pattern of influenza glycoproteins, high mannose core, (e.g. mannose binding ii | P a g e lectin) and (3) factors playing a role in influenza-host interaction (e.g. galectin). Binding of influenza to host factors was assessed by a capturing assay. Our results show that influenza virus can be captured using all three approaches, and all of these factors show promise for replacing classical antibodies in diagnostic applications. In the third study, we characterized the binding of two immunologically distinct forms of Shiga toxin (Stx1 and Stx2) to understand their differences in cell toxicity and different affinity to the reported receptor, the glycolipid globotriaosylceramide (Gb3). Binding of Stx1 and Stx2 to Gb3, various glycans, glycosphingolipids and their mixtures was studied in the presence or absence of membrane components, phosphatidylcholine and cholesterol. Our results show that in addition to the glycan, Stx2 binding is also influenced by the Gb3 lipid and the Gb3 lipid environment; while Stx1 binds primarily to the Gb3 glycan. These results show that Stx1 and Stx2 recognize different residues in the Gb3 molecule and that Gb3 lipid environment modulates Stx binding; this is especially true for Stx2. Our results contribute to the potential development of new therapeutics and diagnostic tests. Overall, our results demonstrate that studying invariant characteristics of pathogens show promise to improve current diagnostic assays. iii | P a g e iv | P a g e Dedicado a mis padres, mis hermanos y a mi amor Rafael por ellos, por siempre y para siempre KMGV Cada día sabemos más y entendemos menos Albert Einstein No des sólo lo superfluo, da vuestro corazón Madre Teresa de Calcuta v | P a g e ACKNOWLEDGMENTS En primer lugar, quiero agradecer a mi familia, mis padres Zulema y Alberto, a mis hermanos Miguel, Giancarlo, Francisco y Susan por siempre brindarme su constante apoyo, cariño, tranquilidad y por hacerme recordar que no hay cosas más importantes en la vida que la de tener una familia unida, llena de amor, risa y franqueza. Así también quiero agradecer a Rafael Masitas por su soporte, su amor, su apoyo, su comprensión, su ayuda incondicional y por convencerme varias veces de no desistir y alcanzar mis metas. I am grateful to Dr. Alison Weiss for giving me the opportunity to work my Ph. D. in the exciting field of influenza diagnostics and Shiga toxin. I benefited a lot from her enormous experience and scientific guidance, which allow my scientific mind to sharpen. I am sincerely thankful to her. I also would like to acknowledge the members of my dissertation committee — Dr. A. Herr, Dr. R. Thompson, Dr. T. Thompson, and Dr. M. Staat— who provided helpful insight and feedback regarding this work. My thesis could not have been completed without the help my friends in the program, who listen patiently while I tried to understand the wonderful world of influenza. Special thanks to my lab mate, Sayali Karve for giving valuable support to completed this thesis. Finally, warm and heartfelt thanks to my family and my love Rafael, for making it all worthwhile and reminding me that the life outside science holds me many warm sunny days at Peru’s best beaches with awesome food and the best company ever. vi | P a g e TABLE OF CONTENTS ABSTRACT ...................................................................................................................................... 2 ACKNOWLEDGMENTS ...................................................................................................................... 6 TABLE OF CONTENTS ...................................................................................................................... 7 FIGURES AND TABLES ................................................................................................................... 12 ABBREVIATIONS ............................................................................................................................ 15 CHAPTER I: BACKGROUND .............................................................................................................. 1 1 Introduction ............................................................................................................................ 1 2 Biology of influenza virus ....................................................................................................... 2 2.1 History ..................................................................................................................... 2 2.2 Classification ........................................................................................................... 2 2.3 Characteristics of influenza virus ............................................................................. 3 2.4 Influenza life cycle ................................................................................................... 8 2.4.1 Binding to receptor ........................................................................................... 9 2.4.2 Internalization into endosome. ......................................................................... 9 2.4.3 Fusion ............................................................................................................ 10 2.4.4 Uncoating. ...................................................................................................... 12 2.4.5 Import of RNP to the nucleus ......................................................................... 13 2.4.6 Replication of genome ................................................................................... 13 2.4.7 Nuclear export and assembly ......................................................................... 14 vii | P a g e 2.4.8 Virus budding and packaging ......................................................................... 15 2.4.9 Viral release ................................................................................................... 16 2.5 Immune response to influenza infection ................................................................ 17 2.6 Epidemic and pandemic influenza ......................................................................... 19 2.6.1 Antigenic Drift ................................................................................................. 19 2.6.2 Antigenic Shift ................................................................................................ 20 3 Developing diagnostic test ................................................................................................... 21 3.1 Virus cell culture .................................................................................................... 22 3.2 Serology ................................................................................................................ 22 3.2.1 Hemagglutination inhibition (HAI) ................................................................... 22 3.3 Reverse-transcription polymerase chain reaction (RT-PCR) ................................ 23 3.4 Neuraminidase activity .......................................................................................... 24 3.5 Immunofluorescence ............................................................................................