Genetic Variability and its Relationship to Acanthamoeba Pathogenesis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Monica Jane Crary ********** Graduate Program in Molecular Genetics The Ohio State University 2012 Dissertation Committee: Dr. Amanda Bird Dr. Gregory C. Booton, Advisor Dr. Susan Cole Dr. David Denlinger Dr. Paul A. Fuerst, Advisor Copyright by Monica Jane Crary 2012 ABSTRACT Acanthamoeba is a pathogenic protist that causes a sight-threatening eye infection, Acanthamoeba keratitis (AK). The central focus of this research is to analyze the genetic relationships between Acanthamoeba isolates and how that contributes to pathogenesis. Acanthamoeba is a diverse genus with more than 18 genotypes based on the 18S ribosomal RNA gene. DNA barcoding has suggested that the mitochondrial cytochrome oxidase c subunit 1 (COI) gene can be accurately used to identify a eukaryotic organism. As part of understanding more about the phylogenetic structure of the genus of Acanthamoeba, representative isolates of most of the genotypes were analyzed using their COI genes. The phylogenetic relationships based on the COI gene were nearly identical to those produced using the 18S rRNA gene. This allows COI to be used as a reasonable substitute to the 18S rRNA gene. This project found an alternative method of classifying Acanthamoeba and allowed Acanthamoeba phylogenetics to be examined at more than one locus. A majority of this research focused on the ongoing Acanthamoeba keratitis outbreak in Chicago, Illinois since 2002. The dramatic increase of AK cases has occurred following EPA mandated water treatment changes which have been hypothesized to have increased microorganisms in the water system. To understand the phylogenetic structure of Acanthamoeba within this outbreak, a multilocus sequence typing (MLST) protocol ii was created. This project involved identifying and developing sequencing methods for five highly conserved housekeeping genes in Acanthamoeba. Sequences from these genes were used to determine the degree of variability amongst Acanthamoeba as well as to further our understanding of sub-lineages within the most common genotype, T4. In addition to the Chicago AK review, three surveys were conducted to elucidate the diversity of environmental Acanthamoeba from Chicago, Sonora, Mexico and on an Ohio farm. These surveys combined with the MLST study demonstrate how diverse the genus of Acanthamoeba is, regardless of source. Acanthamoeba are also known to contain potentially pathogenic endosymbionts including Legionella and Pseudomonas. The presence of endosymbionts has been shown to increase the pathogenicity of Acanthamoeba. To determine if endosymbionts in Acanthamoeba could contribute to the Chicago outbreak, Acanthamoeba was analyzed for the presence of Legionella and Pseudomonas. More than 50% of Acanthamoeba from Chicago contained one or both bacterial genera and these bacteria were located intracellularly in Acanthamoeba. We hypothesize that increases in microorganisms in the Chicago water systems increased the opportunity for Acanthamoeba to associate with pathogenic bacteria. Future research will examine the mechanism that these bacteria use to increase Acanthamoeba’s virulence as well as monitoring if diseases associated with these bacteria are also increasing. Together, these projects are meant to further our understanding on the diversity of the genus Acanthamoeba and the underlying factors of Acanthamoeba’s virulence. iii This dissertation is dedicated to my parents, Roger and Leota. iv Acknowledgements I would like to thank both of my advisors, Dr. Greg Booton and Dr. Paul Fuerst, for their guidance and patience. Thank you to my dissertation committee and fellow lab members, Daryl and Mike for their technical assistance. To my parents, thank you for your unwavering encouragement and confidence. To Nil, thank you for showing me all the How's and Why's in the lab. I would like to also thank my friends, Ashley, Kaylan, Mindy and Bibi for their ears. Finally, thanks to my family, friends and fellow graduate students for their support. v Vita 2003 Madison Central School 2007 B.S. Biology, Ursinus College 2007 to present Graduate Teaching Associate, Department of Molecular Genetics, The Ohio State University Publications Crary, M.; Narayanan, S.; Hopkins, A.; Booton, G.; and Fuerst, P. Phylogenetic analysis and Taxon Barcoding of the Opportunistically Pathogenic Protistan genera Acanthamoeba and Balamuthia. In preparation. Crary, MJ.; Lares-Villa, F.; Booton, GC.; Pearlman, E.; Shoff, M.; Joslin, C.; Tu E.; Fuerst, PA. Acanthamoeba keratitis outbreak in Chicago, Illinois are Associated with the Presence of the Pathogenic Bacteria Legionella pneumophila and Pseudomonas aeruginosa. In preparation. Shoff, M. Crary, MJ.; Joslin, C.; Tu, E.; Booton, GC.; Fuerst, PA. Analysis of Acanthamoeba isolates from the Greater Chicago Area Tap Water. In preparation Cherukuri, NC.; Zhu, Y.; Wolf, JN.; Wu, Z.; Crary, M.; Buckley, K.; Bisaro, K.; and Parris, DS.; 2012 Characterization of the RNA Silencing Suppression Activity of the Ebola Virus VP35 Protein in Plants and Mammalian Cells. Journal of Virology. 86:(6)30-38. Visvesvara, G.; Shoff, M.; Sriram, R.; Booton, G.; Crary, M.; Fuerst, P.; Hanley, C.; and Garner, M. 2010 Isolation, morphologic, serologic and molecular identification of Acanthamoeba T4 genotype from the liver of a Temminck's tragopan (Tragopan temminckii). Veterinary Parsitology. 170(3)194-200. Fields of Study Major Field: Molecular Genetics vi Table of Contents Page Abstract..............................................................................................................................ii Dedication..........................................................................................................................iv Acknowledgements............................................................................................................v Vita.....................................................................................................................................vi List of Tables.....................................................................................................................ix List of Figures.....................................................................................................................x Chapters: 1: Introduction..............................................................................................................1 2: Cytochrome Oxidase I Taxon Barcoding and Phylogenetic Analysis of the Pathogenic Protistan Genera Acanthamoeba, Protacanthamoeba, and Balamuthia.............................................................................................................22 Methods..................................................................................................................27 Results....................................................................................................................29 Discussion..............................................................................................................40 3: Environmental Acanthamoeba Isolates Exhibit High Levels of Genetic Diversity and Indicate Possible Means of Acanthamoeba-Human Interaction……….........44 Methods..................................................................................................................47 Results....................................................................................................................49 Discussion..............................................................................................................65 4: Acanthamoeba in Chicago Illinois are Associated with the Presence of the Pathogenic Bacteria Legionella pneumophila and Pseudomonas aeruginosa..............................................................................................................71 Methods………………..........................................................................................72 vii Results....................................................................................................................79 Discussion..............................................................................................................87 5: Multilocus Sequence Typing of Acanthamoeba Keratitis-Associated Clinical Isolates from a Chicago Outbreak..........................................................................93 Methods..................................................................................................................96 Results..................................................................................................................100 Discussion............................................................................................................123 6: Discussion............................................................................................................126 Bibliography...................................................................................................................134 Appendix.........................................................................................................................144 viii List of Tables Table 1. Acanthamoeba species names with associated genotypes based on the 18S rDNA gene.........................................................................................................................12 Table 2. Protists