Histoplasma capsulatum: Drugs and Sugars Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kristie Goughenour Graduate Program in Microbiology The Ohio State University 2020 Dissertation Committee Dr. Chad A. Rappleye, Advisor Dr. Stephanie Seveau Dr. Jesse Kwiek Dr. Jason Slot 1 Copyrighted by Kristie Goughenour 2020 2 Abstract Histoplasma capsulatum is a thermally dimorphic fungal pathogen capable of causing clinical symptoms in immunocompetent individuals. It exists as hyphae in the environment but transitions to the yeast phase upon encountering the human host, with exposure to mammalian body temperature triggering this phase change. It is endemic to the Ohio and Mississippi River Valleys in the United States, Latin America (specifically Brazil, Venezuela, Argentina, and Columbia), and parts of Africa, with limited reports in China and India and demonstrates a clear medical relevance and healthcare burden. Antifungal options for Histoplasma are limited due to a lack of fungal-specific targets. Additionally, most studies do not take into account clinically relevant testing of fungal morphotypes and assume a one-size-fits-all approach to fungal drug testing, contributing to false leads and inaccurate frequencies of resistance. In this thesis, we develop and standardize an appropriate method for Histoplasma antifungal susceptibility testing. We show that current CLSI testing methodology is insufficient for testing of Histoplasma and that antifungal susceptibility is often phase-dependent in Histoplasma. In addition to a need for novel antifungals, there is a real need for novel, fungal-specific drug targets. As a result, target identification is an important stage in antifungal ii development, particularly for large-scale screens of compound libraries where the target is completely unknown. In this thesis, we combine a traditional genetics approach and a novel co-fractionation mass spectrometry approach to identify target genes for a novel antifungal compound, 41F5. We generated 41F5-resistant Cryptococcus neoformans strains and identified 11 potential target genes containing SNPs. In addition, we developed a biochemical approach using size co-fractionation of proteins with unmodified drug via size co-fractionation and compound detection by LC-MS/MS. Drug co-fractionating proteins were identified by proteomics, generating 34 candidate targets. We are currently validating candidates through overexpression of the candidate genes. This thesis also investigates virulence factors that could serve as novel fungal-specific drug targets. We work to characterize the O-linked mannosylated proteome in order to identify proteins that may contribute to growth at elevated temperatures (38°C). Using a combined bioinformatics approach and a novel lectin-free biotinylation-based O-linked mannosylated purification we are working to identify O-linked mannosylated proteins and determine which contribute to Histoplasma thermotolerance. We were additionally interested in other fungal-specific characteristics such as chitin as a main structural component of the fungal cell wall. As chitinases have not been comprehensively studied in fungi as a whole, this thesis generates a comprehensive phylogenetic survey of fungal chitinases in addition to characterization of enzymatic activities and gene expression of Histoplasma chitinases. We determined that the iii previous phylogenetically determined A clade of chitinases was not monophyletic and that the A and C clades need to be recategorized. In Histoplasma, we identify a hyphal phase-specific chitinase, Cts3. We also identify a recent gene duplication event in Histoplasma (Cts2 and Cts4) with differences in expression and enzymatic activity. Finally, we show that enzymatic activity of chitinases does not follow previous phylogenetic predictions, indicating that more chitinases need to be studied to identify their roles in individual fungi. iv Dedication This work is dedicated to my mom and dad for all their support over the years. I couldn’t have done it without them. It’s also dedicated to my friends both from grad school and before for all the good times and awesome science conversations over the years. v Acknowledgments I want to begin by thanking and acknowledging by advisor Dr. Chad Rappleye. Your support and willingness to let me try new projects and techniques have been really fundamental to my development as a scientist. I’ve learned so much on how to do research well from being in his lab. In addition, I’d like to thank Jason Slot, for all his invaluable assistance with the phylogenetic work for the chitinases project. I’ve learned so much from it and his mentorship was critical for the project and helping me to develop new skills. I’d also like to thank my committee for their support and advice throughout the years. I want to acknowledge my amazing colleagues (past and present). Andrew, Qian, Stephanie, Peter, Amita and Janice. You have provided great scientific advice and amazing companionship throughout the years. I’d also like to acknowledge the other OSU Microbiology graduate students and SAM. You all have provided a welcoming, helpful and collaborative environment. vi Vita 2014…………………………………………………………………….B.A. Microbiology Ohio Wesleyan University 2014-2015…………………………………………………………...University Fellowship Ohio State University 2015-2018………………………………………………… Graduate Teaching Associate Department of Microbiology, The Ohio State University 2018, 2019……………………………………………...NIH/NIAID T32 Graduate Fellow CMIB/IDI The Ohio State University 2020-Pesent………………………………………………… Graduate Research Associate Department of Microbiology, The Ohio State University vii Publications Tan CY, Wang F, Anaya-Eugenio G, Gallucci J, Goughenour KD, Rappleye CA, Spjut R, Carcache de Blanco E, Kinghorn AD, Rakotondraibe LH. 2019. α-Pyrone and Sterol Constituents of Penicillium aurantiacobrunneum, a Fungal Associate of the Lichen, Niebla homalea. Journal of Natural Products. 82 (9), 2529-2536 DOI: 10.1021/acs.jnatprod.9b00340 Garfoot AL, Goughenour KD, Wüthrich M, Rajaram MVS, Schlesinger LS, Klein BS, Rappleye CA. 2018. O-mannosylation of proteins enables Histoplasma yeast survival at mammalian body temperatures. mBio doi: 10.1128. Goughenour KD, Rappleye CA. 2017. Antifungal therapeutics for dimorphic fungal pathogens. Virulence. 8(2):211-221. doi:10.1080/21505594.2016.1235653. Goughenour KD, Balada-Llasat J-M, Rappleye CA. 2015. Quantitative microplate- based growth assay for determination of antifungal susceptibility of Histoplasma capsulatum yeasts. J Clin Microbiol 53:3286–3295. doi:10.1128/JCM.00795-15. viii Fields of Study Major Field: Microbiology ix Table of Contents Abstract ....................................................................................................................... ii Dedication ................................................................................................................... v Acknowledgments ...................................................................................................... vi Vita ............................................................................................................................ vii List of Tables ............................................................................................................. xiii List of Figures ............................................................................................................ xiv Chapter 1. Introduction ............................................................................................... 1 1.1 Histoplasma capsulatum infection .................................................................................. 1 1.2 Antifungal treatment for Histoplasma ............................................................................ 3 1.2.1 Current Antifungals avaibile for Clinical use .................................................................. 3 1.2.2 New and Alternative antifungal developments ............................................................. 7 1.3 O-Linked Mannosylation and Chitinases in Histoplasma ............................................... 10 1.4 Conclusions .................................................................................................................. 11 Chapter 2. Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts ...................................... 14 2.1 Introduction ................................................................................................................. 14 2.2 Materials and Methods ................................................................................................ 17 2.2.1 Fungal strains and culture ........................................................................................... 17 2.2.2 Preparation of Histoplasma inocula ............................................................................ 18 2.2.3 Microtiter plate growth ............................................................................................... 19 2.2.4 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide–based yeast vitality assay. .................................................................................................................................... 19 2.2.5 Resazurin-based yeast vitality assay ............................................................................ 20 2.2.6 Antifungal