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View of Fatal Burn Wound Cases, 33% of Deaths from Invasive Infections Were Caused by Fungi (Murray Et Al MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Gloria Achibi Wada Candidate for the Degree: Doctor of Philosophy ________________________________ Dr. D.J. Ferguson, Director ________________________________ Dr. Marcia Lee, Reader ________________________________ Dr. Xiao-Wen Cheng, Reader ________________________________ Dr. Rachael Morgan-Kiss ________________________________ Dr. Richard Edelmann Graduate School Representative ABSTRACT EFFECT OF ALOE STRIATA INNER LEAF GEL ON EARLY HYPHAL DEVELOPMENT AND ADHESION IN PAECILOMYCES VARIOTII, FUSARIUM OXYSPORUM, AND FUSARIUM SOLANI by Gloria Achibi Wada Members of the Fusarium solani and Fusarium oxysporum species complexes are the most implicated etiologic agents in opportunistic fusarial infections in mammals while Paecilomyces variotii is one of the most frequently encountered Paecilomyces species in human infections. Prevention and treatment of these mycoses are problematic because available antimycotics are limited and often have toxic side effects. Popular folk medicines, such as the inner leaf gel from Aloe spp., are potential sources for non-toxic novel antimycotic compounds. To screen for antifungal properties of a non-domesticated Aloe species, Aloe striata, germination assays with homogenized 0.2 µm filtered A. striata inner leaf gel were performed against conidia of 3 strains each of P. variotii, F. solani and F. oxysporum. Although exposure to A. striata inner leaf gel caused only minimal inhibition of conidial germination for all strains, it caused visible hyphal aberrations characterized by increased hyphal diameters that lead to intervals of non- parallel hyphal cell walls as well as increased parental cell diameters. Adhesion assay results indicated that A. striata inner leaf gel induced hyphal aberrations significantly contribute to a decrease in the ability of 3 P. variotii strains to successfully remain adhered to microscope slides. To isolate and identify the fractions of A. striata inner leaf gel responsible for hyphal aberrations, a combination of chromatographic techniques was used. A reverse phase high performance liquid chromatography (RP-HPLC) generated fraction, fraction A, demonstrated the most significant induction of hyphal aberrations. When fraction A was further separated, we identified fraction AIa as the portion of fraction A that caused the most significant hyphal aberration frequency increase in P. variotii ATCC 22319. Our findings implicate A. striata inner leaf gel fraction AIa as the source of hyphal aberration frequency increases in P. variotii, F. oxysporum, and F. solani. Since hyphal aberrations contribute to a decrease in adhesion frequency, an important fungal virulence factor, we have identified A. striata inner leaf gel fraction AIa as a mixture of compounds with novel antimycotic properties that could potentially be used to combat adhesion and help reduce and/or prevent fungal colonization of hosts and/or substrates. EFFECT OF ALOE STRIATA INNER LEAF GEL ON EARLY HYPHAL DEVELOPMENT AND ADHESION IN PAECILOMYCES VARIOTII, FUSARIUM OXYSPORUM, AND FUSARIUM SOLANI A Dissertation Submitted to the Faculty of Miami University in partial fulfillment Of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Gloria Achibi Wada Miami University Oxford, Ohio 2016 Dissertation Director: D.J. Ferguson TABLE OF CONTENTS List of Tables iii List of Figures iv Dedication vi Acknowledgements vii Introduction 1 Chapter 1. Inner leaf gel of Aloe striata induces adhesion-reducing morphological hyphal aberrations 10 Chapter 2. Chemical Analysis, Isolation, and Identification of A. striata compound(s) that cause(s) hyphal aberrations in Paecilomyces variotii and Fusarium oxysporum 39 Summary 81 References 84 ii LIST OF TABLES Table Page 1 List of all the strains used in this research with their corresponding source 20 2 Summary of results from the preliminary screening of the effect of A. striata filtrate on the average germination, branching, and aberration frequencies of 3 strains each of F. oxysporum (Fo69, Fo57, & Fo24), F. solani (Fs20, Fs02, & Fs53), P. variotii (Pv19, Pv06, & Pv23) 25 3 Results from preliminary screening of the effect of A. striata filtrate on the average germ tube lengths of 3 strains each of F. oxysporum (Fo69, Fo57, & Fo24), F. solani (Fs20, Fs02, & Fs53), P. variotii (Pv19, Pv06, & Pv23) 26 4 Average conidial, sub-conidial, and sub-apical diameter increases in A. striata treated Pv19, Pv06, &Pv23 when compared to controls on 3 separate days 29 5 Total number of Pv19, Pv06, and Pv23 adhered to control and treatment slides before slides were washed with RO water in adhesion assays 30 6 Total number of adhered fungi for each morphotype after exposure to A. striata before slides were washed with RO water in adhesion assays for 3 strains of P. variotii, Pv19, Pv06, & Pv23 31 7 List of all the strains used in Chapter 2 and their corresponding source 51 iii LIST OF FIGURES Figure Page 1 Diagram showing fungal life cycle beginning with conidial germination, hyphal branching that leads to formation of mycelium 8 2 Examples of the various fungal morphologies encountered in germination and adhesion assays 23 3 Effect of A. striata on the aberration frequency of selected F. oxysporum (Fo69), F. solani (Fs02) and P. variotii (Pv19) strains in repeated germination assays 27 4 Average percent adhered fungi in adhesion assays 32 5 Schematic diagram of flash chromatography separation of A. striata inner leaf gel into F1, F2, & F3 52 6 Schematic diagram of flash chromatography and RP-HPLC separations of A. striata inner leaf gel into fractions A, B, C, and D 54 7 Representative HPLC chromatogram of A. striata flash chromatography fraction 2, F2, separated into 4 HPLC fractions A-D 56 8 Schematic diagram of flash chromatography and RP-HPLC separations of A. striata inner leaf gel into AI and AII 58 9 Representative RP-HPLC chromatogram of further separation of A. striata fraction A into 2 fractions AI and AII 60 10 Schematic diagram of flash chromatography and RP-HPLC separations of A. striata inner leaf gel into AIa and AIb 62 11 Representative chromatogram from RP-HPLC separation of A. striata fraction AI via a 35:65:1 (MeOH: H2O: HOAc) mobile phase 64 12 Representative RP-HPLC chromatogram from the separation of A. striata fraction AI via a 25:75:1(MeOH: H2O: HOAc) mobile phase 66 13 Representative RP-HPLC chromatogram from the separation of A. striata fraction AI into 2 fractions, AIa and AIb via a 15:85:1 (MeOH: H2O: HOAc) mobile phase 68 14 Representative RP- HPLC chromatogram from the separation of A. striata fraction AI via a 10:90:1 (MeOH: H2O: HOAc) mobile phase 70 iv 15 Representative RP-HPLC chromatogram from the separation of A. striata fraction AI into peaks via a 5:95:1 (MeOH: H2O: HOAc) mobile phase 72 16 Chemical and physical properties (pH and osmolality) of A. striata and their individual effect on aberration frequency 75 17 Average aberration frequency of Pv19 when treated with flash chromatography groups F1-F3, RP-HPLC fractions A-D, fractions AI-AII, and fraction AIa from A. striata 77 v To my great-grandfather Mr. Okolo Ukpuchanawo, my grandparents, Mr. Wada Okolo, Mrs. Diana Omẹko Wada, Mr. Ikani Salifu, and Mrs. Achana Ikani Ochai, my parents, Dr. Emmanuel Tijani Wada and Mrs. Ikede Ikani Wada, my sister Eunice Wada and brother Emmanuel Wada Jr. vii ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor, Dr. Marcia Lee, for the mentorship she provided during my doctoral studies. I will forever be indebted to her for fostering my enthusiasm for mycology. I would also like to thank everyone that served on my committee, Drs. Gary Janssen, D.J. Ferguson, Richard Edelmann, Rachael Morgan-Kiss, and Xiao-Wen Cheng for guidance and thought provoking suggestions that each of them offered me over the years. In the same vein, I would also like to thank Drs. Eileen Bridge, Jenna Dolhi, and Anand Prakesh for help throughout the course of my time at Miami as well as Dr. Richard Bretz and his lab for help with the chromatography portion of this work. Finally, I’d be remiss if I didn’t acknowledge the immeasurable support provided by my immediate and extended family throughout the course of my studies. vii INTRODUCTION There has been a dramatic increase in opportunistic mycoses in recent years due to the increased prevalence of susceptible hosts. According to the United States Department of Health and Human Services Organ Procurement Transplantation Network (OPTN) data as of January 16, 2016, there was a nearly 10% increase in the number of individuals that underwent organ transplants between 2005 and 2015. Furthermore, as a result of advances in medical care and technology, the life span of immunocompromised individuals such as AIDS, organ transplant, and burn wound patients has increased (Centers for Disease Control and Prevention (CDC) MMWR 2014, U.S. Department of Health and Human Services Health Resources and Services Administration (HRSA) 2014, Behr et al. 2008, Kowalske 2011). Although their lifespan has increased, these patients are still left in immunocompromised states that make them more vulnerable to opportunistic infections (Kowalske 2011, McNeil et al. 2001). In their study of burn wound patients, Pruitt et al. (1998) found that while cases of infections from bacteria and yeast-like organisms have decreased between 1986 and 1996, monomorphic filamentous fungal infections have not decreased. In a 12 year review of fatal burn wound cases, 33% of deaths from invasive infections were caused by fungi (Murray et al. 2008). Furthermore, invasive fungal infections have also been a major cause of mortality in immunocompromised patients with hematopoietic stem cell transplantation as well as organ transplant patients (De Pauw et al. 1999, Nucci et al. 2003, Stanzani et al. 2007). The prevention and treatment of opportunistic mycoses are problematic for a number of reasons. The number of safe and effective systemic antifungal medications for humans, approximately 20, is small compared to the hundreds of available antibacterial medications (Perlroth et al.
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