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

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

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

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

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

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. 2007, Vandeputte et al. 2011). There are only 4 classes of antifungal drugs available for the treatment of systemic invasive fungal infections. These 4 drug classes (polyenes, echinocandins, fluoropyrimidine analogs, and azoles) target only 3 fungal metabolic pathways. Polyenes disrupt the synthesis of ergosterol while echinocandins inhibit the synthesis of cell wall glucans and fluorpyrimidines serve as nucleotide analos (Vandeputte et al. 2011). There are over 4 available systemic antifungal drugs for Candida species while there are very few systemic antifungals for monomorphic filamentous fungi (Perlroth et al. 2007, Low and Rotstein 2011). For example, the first line antifungal agent in clinical settings for Aspergillus and Fusarium infections is voriconazole. If voriconazole is not effective alone, it can be combined

with a polyene (Perlroth et al. 2007, Low and Rotstein 2011). If the polyene and voriconazole combination therapy is not successful, posaconazole is used. Furthermore, there have been reports of antifungal resistance in some monomorphic filamentous fungal strains (Perea and Patterson 2002, White et al. 1998, Vandeputte et al. 2011). If an Aspergillus or Fusarium strain demonstrates resistance to either voriconazole or posaconazole, there are no other available drug therapies (Perlroth et al. 2007, Low and Rotstein 2011). For Mucor species, amphotericin B is the first line antifungal treatment. If amphotericin B is not effective by itself, it can be administered with a polyene, echinocandin, or posaconazole. For other monomorphic fungi, a polyene like amphotericin B is used as the first line antifungal and the only other alternative is to combine the polyene with voriconzole (Perlroth et al. 2007, Low and Rotstein 2011). In addition to the limited number of available antifungals, some antifungal medications cause unfavorable side effects. Voriconazole causes eye light sensitivity as well as other changes to the vision of patients while other antifungals such as amphotericin B are nephrotoxic (Stanzani et al. 2007, Vandeputte et al. 2011). Another problematic issue lies in the fact that animals, plants, and fungi are all eukaryotes. This creates a conundrum because it is difficult to synthesize drugs that will only target fungal cells without causing cytotoxicity in plant and/or animal cells (Rai and Mares 2003, Vandeputte et al. 2011). Due to these factors, as well as the rise in the number of immunocompromised individuals, there is a need to search for safe and effective novel antifungal compounds that specifically target fungal cells. Therefore, in this research we seek to identify safe and novel antifungal compounds that target fungi specific pathways and/or structures.

Fusarium solani, Fusarium oxysporum, Paecilomyces variotii Recent epidemiological data indicate a shift towards fungi that were not commonly considered human pathogens over 30 years ago (O’Donnell et al. 2008, Sahin and Akova 2005, Houbraken et al. 2010). Among such fungi include members of the ascomycete genera Fusarium and Paecilomyces. Members of Fusarium and Paecilomyces can be found worldwide and cause a wide array of opportunistic infections (O’Donnell et al. 2008, Sahin and Akova 2005, Ploetz 2006). Due to the epidemiological significance of these fungi, it is important to identify possible novel antimycotic drugs that act against them.

Fusarium species have become increasingly common etiologic agents in invasive disseminated fungal infections in immunocompromised individuals since the 1970s (Alastruey- Izquierdo et al. 2008, Boutati and Anaissie 1997, Nucci et al. 2004, O’Donnell et al. 2004, Sahin and Akova 2005). Invasive fusariosis is rare and occurs exclusively in immunocompromised individuals (Campo et al., 2010, Stanzani et al. 2007, Stempel et al. 2015). Members of the Fusarium solani species complex are the most implicated etiologic agents of fusarial infections in humans (Alastruey-Izquierdo et al. 2008). Fungal species complexes are fungi grouped together because they share similar morphology and evolutionary genetic profiles (O’Donnell et al. 2008). In addition to humans, members of the genus Fusarium also cause infections in plants. F. oxysporum causes vascular wilt in economically important crops such as bananas and tomatoes. In banana, vascular wilt caused by F. oxysporum is also commonly referred to as Panama disease. F. oxysporum has been credited with single-handedly causing the widespread loss of the predominant banana cultivar used worldwide before 1960, the Gros Michel cultivar, in South America between 1940 and 1960 (Ploetz 2006). Like F. solani, Paecilomyces variotii is also considered an emerging etiologic agent of opportunistic mycoses in immunocompromised individuals (Aguilar et al. 1998, Houbraken et al. 2010). Members of the genus Paecilomyces cause an array of opportunistic human infections (Aguilar et al. 1998, Houbraken et al. 2010). Of all Paecilomyces species, P. variotii is one of the most commonly encountered strains in human infections. In addition to causing human infections, P. variotii is an often encountered fungal contaminant of food and raw materials. It tends to contaminate foods such as cereals, nuts, cheeses, fruits, and seeds that contain oils (Aguilar et al. 1998).

General Anamorphic Filamentous Fungal Life Cycle Fungal food contamination and invasive infections are facilitated by the dispersal of conidia by wind, animals, or insects. If conidia are able to remain adhered onto substrate where they have been dispersed and conditions there are conducive, germination will ultimately occur. The life cycle of anamorphic filamentous fungi, fungi that undergo asexual reproduction, begins with the swelling of spores or conidia (sing. conidium). Conidia are asexual reproductive propagules produced by filamentous fungi such as F. oxysporum, F. solani, and P. variotii. Before germination, conidia exist in a state of dormancy with minimal metabolic activity. Activation of

germination does not occur until certain environmental requirements are met. Furthermore, environmental changes and/or conditions may also serve as germination activators or inhibitors. The presence of high levels of moisture in the form of liquid water or water vapor is one requirement for conidial germination. Conidia take up large quantities of water and swell to over a double fold increase in the conidial diameter in some species. Another factor that affects the activation of fungal germination is the proximity of other conidia. Generally, close proximity of other spores may play an inhibitory role against germination. In some species, such as Rhizopus stolonifer, close proximity between spores promotes the activation of germination (Isaac, 1998). Some species also require oxygen for germination. Germ tubes generally emerge a few hours after activation. At an initial concentration of 4.26 x 106 conidia/ml, it takes approximately 12 hours for at least 80% of F. oxysporum and F. solani conidia to germinate. For strains of P. variotii (Pv19, Pv06 and Pv23) with an initial concentration of 4.26 x 106 conidia/ml, it takes 18 hours for at least 80% of the conidia at to germinate. After the initiation of germination, growth continues in the form of extension at the hyphal apical tip. Such extension is achieved via the deposition of cell wall macromolecules that are carried by vesicles near the apical tip. Germination is required for the formation of fungal mycelia, masses of fungal hyphae that is often visible on contaminated food and raw materials and is indicative of invasive fungal growth. Once conidia have germinated, new hyphae (branches) ultimately develop, often perpendicular to the initial germ tube (Figure 1). Branching events occur exponentially to create mycelia (sing. mycelium). Over time, specialized hyphae referred to as conidiophores are formed to support conidia (Figure 1). As previously mentioned, these new conidia remain in a dormant state until favorable germination conditions are present to trigger germination in order to start a new cycle. In order to cause invasive infections, fungi must remain adhered to host substrate. To achieve adhesion, filamentous fungi rely on hydrophobins to help mediate interactions between fungal cell wall surfaces and host substrate. Hydrophobins are cysteine-rich proteins that are secreted onto fungal cell walls only by filamentous fungi. They form a hydrophobic water- repellent coating on the surface of objects and help fungi adhere to substrate. Several studies have linked decreases in adhesion to decreases in the presence of hydrophobins (Dubey et al. 2014, Sevim 2012, Talbot et al. 1996, Zhang et al. 2011). Decreases in adhesion are known to cause a decrease in the virulence of some filamentous fungi (Dubey et al. 2014). Zhang et al. (2011) found that the virulence of Beauvaria bassiana significantly decreased when its

hydrophobin genes were deleted. In addition to those from B. bassiana, hydrophobins in Metarhizium bruneum play a role in virulence (Dubey et al. 2014, Sevim 2012). Since hydrophobins play a significant role in fungal adhesion, it is a possible antifungal target.

Plants as Sources of Novel Antifungal Compounds There are several plants such as the tea tree Melaleuca alternifolia that secrete different compounds used in pharmaceutical and cosmetic preparations to ward off bacterial and fungal infections. Furthermore, within the last 6 years, antifungal naphthoquinone compounds produced by the carnivorous plant Nepenthes khasiana have been discovered. Production of these antifungal compounds in N. khasiana is induced by the presence of fungi or fungal derived chitin (Ellenberg et al. 2010). Ellenberg et al. (2010) noted that the synthesis of compounds as a means of defense against organisms that pose a threat to their survival is common in plants. Singh et al. (2007) found that alkaloid compounds from Argemone mexicana, the Mexican prickly poppy plant, inhibited conidial germination in plant pathogenic fungi including Fusarium udum. Similarly, Akiyama et al. (2005) found that exudate from Lotus japonicus plant promotes hyphal branching. Therefore, based on the fact that plants have evolved the ability to produce secondary defense metabolites, they appear to be a viable avenue for the discovery of novel antifungal drug therapies.

Aloe Taxonomy, History and Characteristics Plants of the genus Aloe have been used in folk medicine as far back as the oldest recorded civilizations until now (Park and Lee 2006, Rodriquez et al. 2005, Njoroge and Bussman 2007). Aloe spp. are a part of the perennial plant family Liliaceae. They are typically found in tropical climates of Southern and Central Africa. Park and Lee (2006) note that Aloe barbadensis (commonly referred to as aloe vera) was described as a folk medicine on Sumerian clay tablets and papyrus ebers that date back to approximately 1500 BCE. Furthermore, aloe plants were referred to as plants of immortality on Egyptian temple engravings that date back to 4000 BCE (Park and Lee 2006). The first detailed description of the pharmalogical effects of aloe was written in a book called The Greek Herbal which was written by Discorides during the first century CE. Discorides described Aloe plants as possessing panacea-like healing properties on wounds, burns, frostbites, constipation, insomnia, pain, stomach disease, as well as a wide

variety of other ailments. In recent studies, A. barbadensis has been reported to have antibacterial and antifungal properties effective in the treatment of skin ulcers (Ali et al. 1999, Park and Lee 2006). The majority of antifungal Aloe research mainly focuses on domesticated Aloe barbadensis (commonly known as aloe vera) (Ali et al. 1999). Shamim et al. (2004) found that Aloe barbadensis inner leaf gel extract inhibited the mycelial growth of 10 filamentous fungal species (Aspergillus flavus and Trichophyton rubrum) and 3 yeast species (Candida albicans, Candida tropicalis, and Candida glabrata) while Babaei et al. (2013) found that A. barbadensis inner leaf gel extracts caused a reduction in the growth of Aspergillus niger. Rosca-Cain et al. (2007) found that A. barbadensis also caused a decrease in fungal mycelial growth in 4 monomorphic fungal species including Botrytis gladiolorum. Antifungal Aloe research has predominately focused on Aloe barbadensis’ inhibition of fungal mycelial growth, but has failed to adequately evaluate how Aloe species affect fungal growth and ability to cause invasive infections. Since much of what we understand about the antifungal properties of Aloe species is based on fungal mycelial growth experiments with A. barbadensis, we cannot attribute antifungal properties to all Aloe species. It is important to characterize the effect of some of the less studied Aloe species such as A. striata, a less studied Aloe plant, to further understand whether the previously noted antifungal effects of A. barbadensis are also found in A. striata. Furthermore, there is a need to study how Aloe species affect early hyphal development because the formation and function of fungal hyphae are key in pathogenesis. Studying the antifungal effect of A. striata on the early stages of fungal development may lead to possible novel fungal specific targets. In filamentous fungi, the tip of fungal hyphae is used to thigmotrope, determine whether there are changes in the topography of the host surface such as wounds and redirect their apical tip of the hypha to orient itself in a manner that allows it to gain entry into openings such as host tissue (Bowen et al. 2007, Gow 2003). Furthermore, hyphae provide abundant surface area for fungi to form adhesion bonds with surfaces in order to remain adhered to the substrate and successfully colonize it.

Project Statement The goal of this project was to characterize the effects of A. striata on the development and morphology of F. oxysporum, F. solani, and P. variotii during the early stages of hyphal

development. We focused on F. oxysporum, F. solani, and P. variotii because of their status as emerging fungal pathogens of epidemiological significance due to the rise in the number of immunocompromised individuals that have remained alive as a result of advancements in medical care and technology. We initially examined the effect of A. striata on hyphal germination and branching frequency as well as the germ tube lengths of our fungi of interest. We also observed that treatment with A. striata causes the appearance of hyphal aberrations that are characterized by increased hyphal diameters at intervals along extending hyphae. We posit that these aberrations lead to a decrease in the ability of filamentous fungi to effectively adhere to substrate and ultimately colonize hosts/substrates due to the fact that hydrophobins in hyphal regions next to aberrated portions of hyphae do not have physical contact with substrate and subsequently are not be able to facilitate adhesion in the affected locale. Although A. striata causes a significant decrease in conidial germination and germ tube lengths as well as significant increases in hyphal branching frequency in F. oxysporum, F. solani, and P. variotii, the magnitudes of the decreases and increases are minimal. This work indicates that A. striata causes a significant increase in the frequency of aberrated hyphae. We found that aberrated hyphae correlated to a decrease in the adhesion frequency of 3 strains of P. variotii. We were also able to identify the portion of A. striata responsible for these aberrations. These findings indicate that A. striata is a source of compounds whose antimycotic properties may be used to prevent undesirable fungal colonization.

Figure 1. The fungal life cycle beginning with conidial germination, hyphal branching that leads to formation of mycelium. Diagram has been modified from Ingold and Hudson (1993).

Fungal conidia Germ tube = a single nascent hypha

Branching hyphae

Mycelium

Modified from Ingold and Hudson, 1993

CHAPTER 1 Inner leaf gel of Aloe striata induces adhesion-reducing morphological hyphal aberrations

Gloria A. Wada, Michael A. Vincent, Marcia R. Lee Submitted to Mycologia 2015 for review.

ABSTRACT Fungi, particularly molds that are cosmopolitan in soils, are frequent etiologic agents of opportunistic mycoses. Members of the Fusarium solani and Fusarium oxysporum species complexes are the most commonly implicated etiologic agents of 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., offer potential sources for novel antimycotic compounds. To screen for antifungal properties of a non-domesticated Aloe species, Aloe striata, we used germination assays with homogenized and filtered inner leaf gel against conidia of 3 strains each of F. solani, F. oxysporum, and P. variotii. Exposure to gel homogenates caused only minimal inhibition of conidial germination in F. solani, F. oxysporum and P. variotii strains. However, it significantly increased the frequency of hyphal aberrations characterized by increased hyphal diameters that resulted in intervals of non-parallel cell walls. Non-parallel cell walls ostensibly reduce the total hyphal surface area available for adhesion, an important fungal virulence factor. We also found a significant decrease in the ability of aberrated P. variotii hyphae to remain adhered to microscope slides after repeated washing with reverse osmosis water. Our results suggest that treatment with A. striata contributes to a decrease in the adhesion frequency of 3 strains of P. variotii.

INTRODUCTION

Due to a limited number of non-toxic and effective antimycotic drugs as well as the increasing number of new emerging human fungal pathogens, it is imperative that novel antifungal treatments be identified. Filamentous fungi, such as members of the genera Fusarium and Paecilomyces, that were not considered human pathogens over 30 years ago have been increasingly recognized as emerging pathogens (O’Donnell et al. 2008, Sahin and Akova 2005, Aguilar et al. 1998, Houbraken et al. 2010, Pfaller and Diekema 2004). Paecilomyces spp. were previously once only regarded as contaminants often encountered in food and raw materials such as cereals, nuts, cheeses, fruits, and seeds that contain oils (Aguilar et al. 1998). However, in recent years they have been implicated in an array of opportunistic human infections. (Aguilar et al. 1998, Grossman and Fowler 2005, Houbraken et al. 2010, Steiner et al. 2013, Tarkkanen et al. 2004). Paecilomyces variotii is one of the most commonly encountered Paecilomyces species in human infections, including onychomycosis, pneumonia, sinusitis, otitis media, osteomyelitis, and hyalohyphomycosis (Byrd et al. 1992, Dhindsa et al. 1995, Eloy et al. 1997, Otcenasek et al. 1984, Saddad et al. 2007, Vasudevan et al. 2013, Tarkkanen et al. 2004). Similarly, Fusarium species have become increasingly common etiologic agents in invasive disseminated fungal infections in immunocompromised individuals such as burn victims as well as HIV and neutropenic cancer patients (Alfonso et al. 2006, Campo et al., 2010, Eljaschewitsch et al. 1996, Latenser 2003, Nucci et al. 2003, O’Donnell et al. 2008, Okuda et al. 1987, Sahin and Akova 2005, Zhang et al. 2006). They are implicated in localized mycoses such as ocular infections, onychomycosis, and cutaneous mycoses (Alfonso et al. 2006, Gugnani et al. 1976, de Hoog et al. 2004, Ledbetter et al. 2007, Leslie and Summerell 2007, Narang et al. 2001, Romano et al. 2010). Members of the Fusarium solani species complex are the most frequently implicated etiologic agents in invasive fusariosis in humans and a diversity of other animals, including dogs and turtles (Kano et al. 2002, Phillot et al. 2001). In addition to animals, members of the genus Fusarium also cause infections in plants. Fusarium oxysporum causes vascular wilt in economically important crops such as bananas and tomatoes. It single-handedly caused the widespread loss of the Gros Michel banana cultivar, the predominant banana cultivar used worldwide before 1960, in South America between 1940 and 1960 (Ploetz 2006). In filamentous fungi such as F. solani, F. oxysporum and P. variotii, germination is an important virulence factor because it marks the beginning of a new cycle of fungal development. 12

The ability to germinate, form hyphae, and ultimately branch is important for the persistence of fungi in the vegetative stage of development, the stage when fungi can colonize hosts. It is also early in this stage that fungal hyphae can significantly contribute to virulence (Brand 2012, Isaac 1998). Branching increases the number of apices available to thigmotrope, the ability to contact sense and re-direct itself into openings associated with topographical changes in the environment. Since germination and other physiologically essential events of early fungal development are necessary for successful adherence to host substrate and subsequent invasion and colonization, it is important to find new antimycotic compounds that target the early stages of filamentous fungal development. Plants are potential sources of compounds that inhibit germination and/or early post- germination developmental processes. Singh et al. (2007) found that alkaloid compounds, dehydroxorydalmine and oxyberberine, extracted from the plant Argemone mexicana inhibited spore germination in 5 plant pathogenic fungi including Fusarium udum. In addition to germination, plant-derived compounds are implicated in the regulation of branching in fungi. In the absence of a plant host, spores from fungal genera Gigaspora and Glomus germinate, but fungal growth is retarded and branching frequency decreases (Harris 2008). Exudate from the plant Lotus japonicus promotes fungal branching in arbuscular mycorrhizal fungi (Akiyama et al. 2005). Previous experiments in our lab suggest that Aloe species possess antifungal compounds that target early hyphal development. Following treatment with A. barbadensis, A. cameronii and A. striata extracts, a significant decrease in germination frequency was observed in different strains of Candida albicans (Lee et al., unpublished data). Although Aloe inner leaf gel has been used in folk medicine since approximately 1500 BCE, most research has focused on Aloe species’ ability to reduce mycelial growth (Sung 1999). Exposure to inner leaf gel filtrates from Aloe barbadensis caused reduced fungal colony diameters in Botrytis gladiolorum, F. oxysporum f.sp. gladioli, Heterosporium pruneti and Penicillium gladioli, Aspergillus spp., Cladosporium herbarum and Fusarium moniliforme, Trichophyton spp., and Epidermophyton floccosum (Rosca-Casian et al. 2007, Ali et al. 1999, Shamim et al. 2004). Most investigations into the antifungal effects of Aloe involve A. barbadensis and exclude other Aloe spp. Furthermore; few investigations have studied the specific effects of Aloe species on early fungal development and function. Therefore, Aloe spp. appear to be promising sources of novel compounds to disrupt germination as well as other early

hyphal developmental events including hyphal branching as well as overall growth in filamentous fungi. To determine whether A. striata inner leaf gel possesses antifungal properties against early hyphal development, our study examines the effect of Aloe striata gel on the germination frequency of conidia from 3 strains each of F. solani, F. oxysporum, and P. variotii. We analyzed the effect that A. striata inner leaf gel had on other characteristics associated with early hyphal development, such as germ tube length and branching frequency. We also observed hyphal aberrations in germinated A. striata treated fungi and evaluated whether the aberrations affect adhesion.

MATERIALS AND METHODS

Aloe striata propagation, filtrate harvest, and filtrate lyophilization. Aloe striata plants, originally from South Africa, were deposited in Krohn Conservatory (Cincinnati, OH) where we obtained them. Plants were grown in a fungicide-free room in the Miami University greenhouse for at least 3 years before gel harvest. A voucher (M. Lee s.n., MU 258172) was deposited in the Willard Sherman Turrell Herbarium at Miami University (Oxford, OH). Inner gel, aseptically removed from harvested leaves, was homogenized and sequentially filtered through 300 µm nylon mesh, 30 µm nylon mesh, 5 µm durapore and 0.2 µm Media-Kap® pore-size filters to obtain sterile, low-viscosity (ca. 1.00 centipoise) filtrates. The harvested leaves were 32.90 + 7.54 x 7.07 + 1.26 cm (length x width) (mean + SD). The average yield of 0.2 µm filtrate per ml extracted inner leaf gel was 57.3% of total extracted gel. The A. striata filtrates were lyophilized and stored at 4°C for future use. The lyophilized filtrates were rehydrated immediately before use.

Fungal cultures and conidial harvest. We initially screened 3 strains each of P. variotii, F. oxysporum and F. solani, which are referred to as Pv19, Pv06, & Pv23 respectively (Table 1). Fusarial strains are referred to as Fo24, Fo57, Fo69, Fs20, Fs53, and Fs02 (Table 1). All Fusarium strains were obtained from the Agricultural Research Service (ARS). We chose these strains because they are easy to culture and they consistently generated characteristically conidiating mycelia for their respective species. Furthermore Pv19 and all of the Fusarium strains have portions of their genome already sequenced. This may become beneficial in the future if it becomes necessary to investigate Aloe inner leaf gel’s effect on fungi on the molecular level (Castelli et al. 2008, O’Donnell et al. 2008). Stock cultures of the aforementioned fungi from -80 C were inoculated onto 4 potato dextrose agar (PDA) slants. Cultures were grown at 25 C until they were visibly mature with copious amounts of conidia. For each strain, 2 or 3 of the 4 slants that had characteristically conidiating mycelia were used for conidial harvest. Conidia were harvested from slants by flooding the slants with 10 ml of sterile 0.85% saline. The mycelial surface of each slant was gently disturbed with glass Pasteur pipettes. The resultant mixture of conidia and hyphal fragments were transferred to sterile vortex tubes. After the transfer, the suspensions were left untouched for 15-20 minutes to allow hyphal particles to settle

to the bottom of the tube while the fungal conidia remained suspended in the upper layer of the solution. After 15-20 min, the upper homogenous top layer was transferred into Falcon conical tubes. The reclaimed conidial suspension was then centrifuged at 694 x g for 15 min. The resultant supernatant was discarded and the pellet was washed by adding approximately 7 ml of reverse osmosis water (ROH20), vortexed and centrifuged at 694 x g for another 15 min. The washing step was repeated once more. After the second washing, 2 ml of 0.85% saline solution was added to the resultant pellet. Conidial concentrations for each strain were determined using a hemocytometer and diluted accordingly to obtain a final concentration of 4.25  106 conidia/ml that was used for subsequent assays.

Germination assays. The germination assay protocol was based on NCCLS/CLSI standards with modifications (NCCLS/CLSI, 2002). For each fungal strain, there were 3 or 4 controls and A. striata treatments. In each microcentrifuge tube, we placed 75 µl RPMI, 785 µl A. striata filtrate (ca. 90% total assay mixture), and 30 µl of fungal-conidial inoculum. Samples were incubated at 25 C in the dark for 12-18 h depending on the strain (NCCLS/CLSI 2002, Leslie and Summerell 2007). Control assay tubes had the same contents, but ROH2O was substituted for A. striata filtrate. After incubation, assay tubes were centrifuged at 9300 x g for 10 min, the assay media was then removed and 0.5 ml of lactophenol-carbofuschin stain was added to tubes containing fungal pellets. The tube and its contents were mixed and centrifuged at 9300 x g for 10 min. After centrifugation, the resultant supernatant was removed leaving a 0.05 ml solution containing the fungal pellet that was then mixed and mounted onto microscope slides, coverslips were then added and sealed with clear nail polish and left to dry before fungi were enumerated under the microscope. For quality control of contamination in the A. striata extract used in each experiment, 1 µl of the A. striata filtrate was plated onto PDA plates. These plates were incubated at both 36 C and 25 C. Germination assays were then repeated 3 times on one strain with decreased branching frequency and germ tube length (Fo57), one strain with reduced germination frequency (Fo69) and one strain from each of the fungal species with significant hyphal aberrations (Fo69, Fs02, and Pv19).

Quantification of germination, branching, and aberration frequencies. From the prepared germination assay slides, the number of germinated, branched, and aberrated hyphae was

determined for the first 200 hyphae that were encountered on a given slide via raster pattern slide navigation (Lee et al. unpublished). Frequency values were calculated as the quotient of the total number of germinated, branched, and aberrated hyphae divided by 200, multiplied by 100 to yield a percentage value. Hyphae were counted as having germinated if their germ tubes are equal to or greater than the diameter of swollen conidia from which they emerge. Hyphae were counted as being branched if at least one branched hyphae emerged along the length of an original hypha. Hyphae were considered as being aberrated if there were any visible increases in their diameters at any given location on the hyphae that caused non-parallel cell walls. All quantification was performed via a light microscope (Zeiss Axiostar) at 400 total magnification.

Measurement of hyphal and conidial diameters of P. variotii. To quantitatively confirm increases in hyphal diameter, hyphal diameters at the sub-conidial and sub-apical regions of hyphae were measured via scanning electron microscopy (SEM). To understand whether increases in diameter are specific to hyphae or whether increases also occur in the parent cells (conidia), conidial diameters were also measured. Germination assays with Pv19, Pv06, Pv23 and A. striata were performed as described in the aforementioned germination assay protocol with the following modifications. After the first centrifugation post incubation, samples were sequentially washed with ROH2O and centrifuged each time to remove debris. Samples were then placed on 1% poly-L-lysine coated coverslips and vapor fixed with 1% OsO4 for 4 days and left to dry for another 4 days. After drying, the coverslips were mounted onto stubs and gold coated (90 nm of gold). Conidial diameters were measured at the widest area in the middle of each conidium. Due to field of view limitations at 5000x total magnification, we could not measure the midpoint region of hyphae. The measured sub-conidial region is the region extending less than ca. 1µm from the conidium and corresponds to the nascent hypha. The measured sub-apical region corresponds to the hyphal location ~ 1 µm from the apical tip.

Adhesion assay. The aforementioned germination assay protocol was followed using 3 strains of P. variotii (Pv19, Pv06, and Pv23) and A. striata. Instead of microcentrifuge tubes, Lab-Tek two chamber slides (EW-01838-23, Thermo Scientific Nunc, Cole-Parmer, and Vernon Hills, IL) were used. To aide fungal enumeration, 20 mm  20 mm adhesive grids (GRID-1000, Diversified Biotech, and Dedham, MA), stickers with grids printed on them, were affixed to the

bottom of each chamber slide. Only one chamber (the chamber furthest from the slide label area) was used. Once the fungal inoculum, RPMI, and A. striata or ROH2O (control) was added to the chamber slide, the mixture was gently swirled to mix the contents. Slides were incubated in the dark at 25 C (Leslie and Summerell 2007, NCCLS/CLSI 2002). Replicates for each treatment were staggered by 30 min to allow time between replicates to count and wash slides. Samples were removed from incubation after 18 h. The assay media including ROH2O or A. striata was carefully pipetted away and the incubation chamber was detached from the slide. The total number of fungi within each of the chosen 40 test quadrants on the grid was then counted. In addition to the total number of fungi present, we also recorded the total number of adhered fungi belonging to previously observed morphotype groupings (non-aberrated (NA), swollen with parallel cell walls (SP), swollen with non-parallel cell walls up to hyphal midpoint (SNPM), swollen with non-parallel cell walls throughout hyphal length (SNPL), and swollen with non- parallel cell walls in hyphal sub-conidial region (SNPSC), Figure 2A-E). We alternated between the 10x and 40x objectives to locate test quadrants and examine fungal morphology. After the pre-wash enumeration, slides were inserted into a modified slide holder (Lock Mailer TM Microscope Slide Mailer and Staining Jar without Capinserts TM, Ted Pella # 21096) and washed 3 times. To wash slides with uniform water flow, jars were modified by cutting 0.3 cm diameter holes in the center of the bottom of the jar and the lid (Doss et al. 1993, Doss et al. 1995). Slide holder jars holding one slide was placed into a 4000 ml beaker that contained 3000 ml of ROH2O while a gloved finger is held over the hole on the jar lid. After the tube was submerged in water with 5 mm of space left between the jar and beaker bottom, the finger covering the hole on the jar lid was removed to allow water to fill the jar. Once the jar was filled with water, the finger was replaced back over the jar lid hole. With a finger still over the hole on the lid, the jar was moved over to a separate waste container, the finger was then removed from the lid hole to allow the water to drain from the slide holder and staining jar into a waste beaker. This washing step was repeated 2 more times for each slide. After the washing step, slides were patted dry on the bottom and the side of the slide with samples was stained with 10 µl lactophenol blue stain. A 22 mm  22 mm coverslip was placed over the stained area and the same test quadrants that were counted in the pre-wash count were counted again alternating between the 10x and 40x objectives. Cells on the left and bottom borders of a grid square were included in the total count for any given square while ones on the right and top borders of a square were excluded (Doss et

al. 1993, Doss et al. 1995). Results from treatments and controls (the number of conidia or hyphlets that adhered to slide 18 h post incubation) were compared to determine whether fungi with A. striata induced hyphal aberrations adhere to slides well. Results are reported as the percent adhesion, and for each test quadrant, the percent adhesion is calculated by dividing the post-wash count by the pre-wash counts times 100.

Statistical analyses. All statistical analyses were carried out in R (R Core Team, 2015). Germination, aberration, and branching frequency data were analyzed using logistic regression. Germ tube length data were analyzed via ANOVA. Hyphal and conidial diameter data were analyzed using linear mixed-effects model via the lme4 package (Bates et al. 2015a, b). Adhesion assay results were analyzed using the Mann-Whitney Rank Sum test.

Table 1. List of all the strains used in this research with their corresponding source.

Strain Id In This Paper Organism Source Source Strain Id No.

Fs02 Fusarium solani ARS (NRRL) NRRL 22402

Fs20 Fusarium solani ARS (NRRL) NRRL 22820

Fs53 Fusarium solani ARS (NRRL) NRRL 22153

Pv19 Paecilomyces variotii ATCC ATCC 22319

Pv06 Paecilomyces variotii ATCC ATCC 28806

Pv23 Paecilomyces variotii ATCC ATCC 16023

Fo69 Fusarium oxysporum ARS (NRRL) NRRL 25369

Fo57 Fusarium oxysporum ARS (NRRL) NRRL 25357

Fo24 Fusarium oxysporum ARS (NRRL) NRRL 26924

ATCC = American Type Culture Collection, ARS = United States Department of Agriculture Agricultural Research Service (formerly known as NRRL) Culture Collection.

RESULTS Characterization of A. striata’s effect on early hyphal development. Preliminary assays indicated that A. striata inner leaf gel filtrate decreased germination by 1.7% to 24.0% when compared to controls. These decreases were significant in one strain of P. variotii (Pv19), one strain of F. solani (Fs02), and 2 strains of F. oxysporum (Fo69 and Fo24) (p < 0.05, Table 2). Of the 2 F. oxysporum strains, Fo69 demonstrated the greatest decrease of 19.0%. A. striata also significantly increased germination frequency in the Fs53 strain of F. solani and two strains of P. variotii (Pv06 and Pv23) (p < 0.001, Table 2). Preliminary data also indicated that 2 F. oxysporum strains (Fo57 and Fo24), 1 strain of P. variotii (Pv23) and all F. solani strains significantly increased branching frequency (p < 0.01). Branching frequency significantly increased in Fs53, Fo57, and Fo24 while a significant decrease in branching frequency was observed in Fs02 (p < 0.05, Table 2). Only Fo57 demonstrated a significant decrease in germ tube length (p < 0.05, Table 3). When the germination assay was repeated with Fo57, it demonstrated branching frequencies of 8.0% ± 6.0% in controls and 13.0% ± 6.0% in treatments (p < 0.00). When assays were repeated on Fo69 to verify results from preliminary experiments, Fo69 demonstrated a germination frequency of 92.0% ± 5.0% in controls and 83.0 % ± 16.0% in treatments (p < 0.001, data not shown). We also observed hyphal morphological aberrations characterized by increased hyphal diameters resulting in intervals of nonparallel regions of the hyphal cell wall as well as increased parent cell (conidium) diameter (Figure 2C-2E). Of all the strains, Pv19, Fo69, and Fs02 exhibited the most significant average aberration frequencies of 96.0 %, 37.0 %, and 26.0 % respectively when compared to controls (p < 0.05, Table 2). Repeated assays on Fo69, Fs02, and Pv19 had average aberration frequencies of 64.0% ± 21.0%, 62.0% ± 19.0%, and 90.0% ± 10.0%, respectively, in A. striata treated samples and 27.0%

±16.0%, 15.0% ± 11.0%, and 5.0% ± 0.3% respectively in ROH2O treated samples (p < 0.001, Figure 3). Jk6

Hyphal and conidial diameter increases. There was a significant increase in the parent cell diameter of A. striata treated Pv19 on all 3 days (p < 0.001, Table 4). Sub-conidial and sub- apical diameter measurements indicated an increase across all days (p < 0.001), except for the Day 1 sub-conidial measurements of Pv19 (p= 0.54, Table 4). Pv06 and Pv23 had a significant diameter increase in their sub-apical regions on all 3 days (p < 0.001, Table 4), and a significant

increase in sub-conidial and conidial diameters (p < 0.001, Table 4) except for the Day 1 conidial diameter of Pv06 (p = 0.33) and Day 1 sub-conidial diameters of Pv23 (p = 0.51, Table 4).

Effect of A. striata on fungal adhesion. In A. striata treatments, the average total percent adhesion of Pv19, Pv06, and Pv09 was 5.0% ± 12%, 31.0% ± 13%, and 36.0% ± 24.0% respectively while controls had 95.0 ± 5.0%, 81.0% ± 15.0%, 79.0% ± 10.0%, respectively, ( p < 0.001, Figure 4A, see Table 5 for the number of fungi adhered to slides before the washing step). Of the total adhered fungi, we found that 75.0 ± 0.0%, 86.0% ± 13%, and 52.0% ± 35.0% of NA from treatment slides remained adhered to slides after the washing step for Pv19, Pv06, and Pv23, respectively. Pv06 controls had 84.0% ± 12.0% adhesion and A. striata treatments had 86.0 %± 13.0% adhesion among NA morphotypes. Fungi with SP morphology demonstrated 100.0 ± 0.0%, 84.0% ± 12.0%, and 73.0% ± 35.0% in controls while treatments demonstrated 60.0% ±28.0%, 88.0% ± 33.0%, and 84.0% ± 23.0% for Pv19, Pv06, and Pv23 respectively (p < 0.001, Figure 4B, see Table 6 for the number of fungi with SP morphology adhered to slides before the washing step). The SNPM morphotype demonstrated 13.0% ± 23.0%, 0.0% ± 0.0%, and 0.0% ± 0.0% adhesion percentage in controls of Pv19, Pv06, and Pv23, respectively, while adhesion percentages in A. striata treatments were 16.0% ± 22.0%, 39.0 ± 31.0% and 9.0% ± 19.0% respectively. SNPL morphotypes demonstrated adhesion percentages of 0.0% ± 0.0% for both Pv19 and Pv06. There were no SNPL observed in the pre-wash count of Pv23. The SNPL adhesion percentages for A. striata treatments were 19.0% ± 37.0%, 0.0% ± 0.0%, and 17.0% ± 41.0% for Pv19, Pv06, and Pv23, respectively. In Pv06 and Pv23, the average number of adhered SNPSC in controls is 0.0% ± 0.0% and 1.0% ± 3.0 % for Pv19. SNPSC demonstrated 111.0 % ± 52.0%, 22.0 ± 20.0%, and 31.0 % ± 28.0% for Pv19, Pv06, and Pv23 respectively (Figure 4B, see Table 6 for number of fungi adhered to slides before the washing step).

Figure 2. Examples of the various fungal morphologies encountered in germination and adhesion assays. (A) Reverse osmosis water (ROH2O) (control) treated fungi. (B-E) Different morphologies observed in A. striata treated Pv19; (B) swollen with parallel cell walls (SP), (C) swollen with non-parallel cell walls in the sub-conidial hyphal region (SNPSC), (D) swollen with non-parallel cell walls up to the midpoint hyphal region (SNPM), (E) swollen with non-parallel cell walls throughout the hyphal length (SNPL). Arrows point to locations on hyphae where non- parallel cell walls are present.

B C D

B B

Table 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). ______

Strain Germination Frequency (%) Branching Frequency (%) Aberration Frequency (%)

______

Control Treatment ∆ Control Treatment ∆ Control Treatment ∆

Fo69 87.0±12.0 67.0±9.5* 20.0 45.0±12.4 36.0±5.6 -8.0 51.0±10.0 89.0±27* 38.0

Fo57 50.0±2.0 48.0±10.0 2.0 1.0±1.7 55.0±1.2* 54.0 84.0±6.4 85.0±20.8* 1.0

Fo24 100.0±2.0 93.0±5.0 * 7.0 12.0±5.0 57.0±7.0* 45.0 45.0±37.0 65.0±19* 20.0

Fs20 53.0±18.0 41.0±36.0 12.0 50.0 ±19.0 37.0± 31.0 -13.0 22.0±26.0 80.0±29.0* 58.0

Fs02 99.0±3.0 69.0±40.0* 30.0 22.0±2.0 3.0±6.0 * -19.0 36.0±22.0 61.0±37.0* 25.0

Fs53 54.0±25.0 67.0±24.0* 13.0 28.0±12.0 50.0±13.0* 22.0 28.0±26.0 60.0±29.0* 32.0

Pv19 100.0± 1.0 96.0±2.0 * -4.0 7.0±0.0 9.0±4.0 2.0 3.0±2.0 95.0±47.0* 92.0

Pv06 71.0 ±7.0 96.0±2.0* 25.0 0.0±0.0 9.0±2.0 9.0 2.0±1.0 40.0±51.0* 38.0

Pv23 71.0±15.0 81.0±11.0* 10.0 5.0±1.0 14.7±7.0 9.7 3.0±1.0 90.0±9.0* 87.0

N= 200, ∆ denotes difference between control and treatments where positive change implies an increase and negative change value implies a decrease when compared to controls. Statistical significance is denoted where * p < 0.05

Table 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).

Strain Germ Tube Lengths (µm) ∆

______

Control Treatment

Fo69 105.6±17.7 93.6±18.1 12.0

Fo57 155.6±26.4 102.8±1.6* 52.8

Fo24 137.0±54.1 129.6±26.1 7.4

Fs20 222.6±36.2 193.1±36.0 29.5

Fs02 119.4±14.5 110.5±11.9 8.9

Fs53 140.3±28.6 239.5±76.7 99.2

Pv19 67.7±31.4 183.4±266.9 115.7

Pv06 37.7±9.1 20.2±8.9 -17.5

Pv23 40.0±10.6 29.5±8.3 -10.5

*denotes statistically significant increase or decrease where P < 0.05 and N=200. ∆ denotes difference between control and treatments where positive change value implies an increase and negative change value implies a decrease in comparison to controls.

Figure 3. Effect of A. striata inner leaf gel on the aberration frequency of selected F. oxysporum (Fo69), F. solani (Fs02) and P. variotii (Pv19) strains in repeated germination assays. * indicates statistical significance, error bars represent standard error of the mean, n = 200, p-values: Fo69 = 0.0001, Fs02 = 0.0066, and Pv19 = 0.0001.

100 *

80 *

(%) 60

40 RO water As

Frequency 20 Average Aberration Average 0 Fo69 Fs02 Pv19 Fungal Strains

Table 4. Average conidial, sub-conidial, and sub-apical diameter increases in A. striata treated Pv19, Pv06, and Pv23 when compared to controls. Experiments were performed with 3 replicates on 3 separate days.

Strain Day Observed average diameter increase (µm)

______

Conidial Sub-conidial Sub-apical

Pv19

1 0.65±0.11* 0.05±0.09 0.82±0.12*

2 0.89±0.15* 0.49±0.12* 0.94±0.12*

3 0.62±0.17* 0.30±0.10* 0.43±0.09*

Pv06

1 0.14±0.14 0.37±0.08* 0.25±0.08*

2 1.12±0.11* 0.93±0.14* 1.24±0.15*

3 0.75±0.14* 0.86±0.13* 1.22±0.13*

Pv23

1 0.00±0.14 0.07±0.11 0.29±0.10*

2 0.40±0.15* 0.54±0.12* 0.38±0.08*

3 0.83±0.19* 0.49±0.11* 0.69±0.09* ______* denotes statistically significant increase, P < 0.001.

Table 5. Total number of P. variotii strains, Pv19, Pv06, and Pv23 adhered to control and treatment slides before slides were washed with RO water in adhesion assays where ∆ denotes difference between control and treatments where positive change value implies an increase and negative change value implies a decrease.

Total fungi on slides before adhesion assay washing step ∆ ______

Strain Control Treatment

Pv19 5696.0 1348.0 -4348.0

Pv06 1742.0 422.0 -1320.0

Pv23 7236.0 1161.0 -6075.0

______

Table 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, and Pv23. NA = non-aberrated, SP = swollen with parallel cell walls, SNPM = swollen with non-parallel cell walls up to the hyphal midpoint, SNPL = swollen with non-parallel cell walls throughout the hyphal length, SNPSC = swollen with non-parallel cell walls in the sub conidial hyphal region.

Strain Pre-wash total adhered A. striata treated fungi of each morphotype (N) ______NA SP SNPM SNPL SNPSC Pv19 8.0 301.0 281.0 48.0 530.0 Pv06 6.0 7.0 15.0 3.0 272.0 Pv23 88.0 75.0 74.0 17.0 844.0

Figure 4. Average percent adhered fungi in adhesion assays. (A) Average total percentage of adhered P. variotii strains (Pv19, Pv06, and Pv23) after treatment with A. striata (As) and reverse osmosis water (RO water) in adhesion assays that were repeated on 3 separate days with 3 replicates per day. (B) Average total percentage of the total adhered fungi per morphotypes (NA, SP, SNPM, SNPL, and SNPSC) per P. variotii strain. * Indicates p-value < 0.001.

* * *

DISCUSSION We initially identified strains of F. solani (Fs02, Fs20, Fs53), F. oxysporum (Fo69, Fo24, Fo57), and P. variotii (Pv19, Pv06, Pv23) via germination assays that exhibited sensitivity to treatment with A. striata in regards to early hyphal developmental events such as germination, branching, and germ tube length. Repeated germination assays on Fo69 indicated a significant decrease in germination frequency. Similarly, repeated germination assays with Fo57 found a significant increase in branching frequency as well as a significant decrease in germ tube length, differences in germ tube length and branching frequency were marginal. While A. striata inner leaf gel does cause differences in branching and germination frequencies as well as germ tube length when compared to the controls of certain strains, the observed differences are marginal in respect to the prevention and treatment of opportunistic fungal infections. Due to the nature of mycelial formation, if at least 1 conidium can germinate, it can grow exponentially to form mycelia and colonize hosts and/or surfaces. Although germination and branching frequency decreases were marginal, we were able to confirm that A. striata caused a significant increase in the aberration frequency of Fs02, and Pv19 in repeated germination assays. While Fo69 demonstrated a significant increase in aberration frequency, the error bars on the graph did overlap for treatment and controls. This may be due to a possible day effect. Pv19 demonstrated the highest increase in aberration frequency. The diameter increases associated with hyphal aberrations were confirmed via SEM measurements in 3 P. variotii strains. Significant increases in hyphal diameter were noted in the sub-apical regions on all 3 experiment days. Pv06 samples had a significant increase in the sub-conidial region on all 3 experiment days. However, Pv19 and Pv06 demonstrated sub-conidial hyphal increases on 2 of the experiment days. As discussed previously, it is difficult to get uniform conidial harvests, because, since fungal cultures are allowed to grow for 3 days before conidial harvest, the age of conidia present within any given culture may differ. Therefore, a day effect was observed when data was analyzed. In addition to the sub-apical and sub-conidial regions of hyphae, the conidia of A. striata treated Pv19 demonstrated a significant increase in diameter for all 3 experiment days. Pv06 and Pv23 demonstrated conidial increases on 2 experiment days. While conidia swell before germination, these conidial diameter increases were greater than those of the controls. Therefore, we can conclude that at least in Pv19, treatment with A. striata induces swelling throughout the fungi that is not exclusive to hyphae. Similar diameter increases were observed in Saprolegnia treated with Congo red (Nodet et al. 1986, Nodet 1987).

To understand whether hyphal aberrations cause a decrease in the ability of three P. variotii strains (Pv19, Pv06, Pv23) to adhere to substrate, we performed adhesion assays with Lab-Tek chamber slides. Adhesion assay results support our hypothesis that hyphal aberrations characterized by diameter increases leading to pronounced intervals of non-parallel cell walls along hyphae contribute to a decrease in the ability of fungi to adhere to substrate. The percent adhesion was higher in controls than in treatments for all 3 P. variotii strains with hyphal aberrations. Furthermore, when the total number of adhered fungi was separated into morphotypes, fungi, except for Pv19 SNPSC, with non-parallel cell walls consistently demonstrated lower adhesion percentages when compared to morphotypes with parallel cell walls irrespective of whether morphotypes were on control or treatment slides. In Pv19 controls, SNPSC morphotypes had a percent adhesion of over 100%, because such results indicated that fungi with the SNPSC morphotype that were not present within test quadrants were displaced into the test quadrants after slides were washed 3 times. Since the SNPSC morphotype has non- parallel cell walls in the sub-conidial location, it has a reduced amount of surface area available to adhere to slides compared to morphotypes with parallel cell walls, but more surface area available for adhesion when compared to the SNPM and SNPL non-parallel cell wall morphotypes. SNPSC morphotypes could be displaced. However, because of the force from the washing step was not enough to displace them completely off the slide, they instead dislocated into neighboring quadrants, at least in the case of Pv19. Therefore, considering that SNPSC are able to be displaced, but due to the fact that aberrations are only in the sub-conidial area, the majority of the length of the hyphal cell wall is parallel and affords displaced hyphae more surface area that is available for adhesion. Non-parallel cell walls correlate with a decrease in hyphal surface area available for adhesion to substrates and morphotypes with the most regions of non-parallel cell walls have decreased adhesion frequencies. This phenomenon is evident when the interactions among fungi on the slides are considered. We noted instances where SNPSC, SNPM, and SNPL morphotypes appeared to be pinned onto slides by SP and NA morphotypes. Since SP morphotypes have the most readily available surface areas for adhesion, they have higher adhesion percentages. Pv19 treatment slides had at least 4 times more SP morphotypes then Pv06 and Pv23, leading to an increase in the likelihood of such interactions between individual Pv19 fungi. Moreover, comparison of the total number of adhered fungi in the pre-wash counts across the 3 strains,

indicates that Pv19 adheres better to surfaces than Pv06, which has relatively poor adhesion overall. The Pv19 morphotypes with parallel cell walls, NA and SP, showed slightly higher displacement when treated with A. striata than controls. However, when compared to morphotypes with non-parallel cell walls, the displacement of NA and SP is significantly lower. Furthermore, the SP morphotype, due to the fact that it is swollen with parallel cell walls does have more surface area readily available to adhere to surfaces than all the observed morphotype. Therefore, it is not surprising that SP morphotypes would demonstrate higher adhesion percentages then NA morphotypes. While our results unequivocally suggest that hyphal morphology plays a significant role in decreasing adhesion of 3 P. variotii strains, we must acknowledge that there are other factors that contribute to fungal adhesion. Our studies indicate that there is a direct connection between A. striata inner leaf gel-induced non-parallel fungal cell walls and adhesion. Mechanically, the non-parallel cell wall regions cause portions of hyphae to not be able to touch substrate. Ostensibly, in order for hydrophobins to mediate adhesion, they have to be in contact with both fungal cell wall surfaces as well as substrate surfaces. Without contact to substrate, secreted hydrophobins are not able to mediate adhesion in those regions. Hyphal regions that are not in contact with substrate surfaces demonstrate similar adhesion capabilities as B. bassiana strains with deleted hydrophobin genes from Zhang et al. (2011). Zhang et al. (2011) linked a decrease in hydrophobins to a decrease in adhesion and a subsequent virulence. Since hyphal aberrations cause a decrease in substrate hydrophobin interactions, our experiments have demonstrated a significant decrease in adhesion and we also anticipate a decrease in the virulence of fungi with A. striata-induced non-parallel cell walls. However, we do not expect the decrease to be of the same magnitude of that reported by Zhang et al. (2011) since fungi with hyphal aberrations also have hyphal regions that are able to maintain contact with the surface of substrate. Experiments need to be performed to confirm that A. striata-induced hyphal aberrations also correlate to a decrease in P. variotii’s virulence. Our findings connecting A. striata -induced hyphal aberrations to adhesion have many real world applications. To prevent opportunistic fungal infections such as keratitis, A. striata gel may be formulated into prophylactic eye drops so that any fungal conidia that has found its way into the ocular regions of immunocompromised individuals and managed to germinate could be prevented from adhering to ocular surfaces if their hyphae become aberrated. Several studies

suggest that A. barbadensis and A. arborescens inner leaf gel extracts can be used in eye drop preparations to safely treat inflammatory disturbances and/or diseases of the cornea and other external ocular parts of the human eyes (Kodym et al. 2002, Kodym et al. 2003, Wozniak and Padnuch 2012). Furthermore, Wozniak and Padnuch (2012) found that A. barbendensis extract had no cytotoxic effects on human corneal cells. Since A. barbadensis and A. arborescens inner gel were not toxic to human cells, it is likely that A. striata will also be safe when used on human cells. To confirm that A. striata is not cytotoxic, cytotoxicity tests should be performed against human corneal cells as well as epithelial cells. Furthermore, since F. solani and F. oxysporum are also emerging human pathogens and they exhibit increased hyphal aberration frequencies when treated with A. striata in germination assays, future studies should include germination assays on the F. solani and F. oxysporum strains to determine whether hyphal aberrations affect their ability to adhere to surfaces. These findings may also be useful in settings where fungal biofilm formation is difficult to combat. It can be difficult to completely remove fungal and/or bacterial biofilm from water systems as well as medical devices and/or instruments such as catheters because only the top layers of biofilm are washed off during the sanitation process, while the first colonizing microbes at the bottom first layer of biofilms are protected from being washed off (Mack et al. 2006, Tautner and Darouiche 2004). To combat fungal biofilm formation on medical devices, fungi that are commonly encountered on such devices can be tested in germination assays to determine whether they A. striata causes aberrations on their hyphae. If such fungi do demonstrate increased hyphal aberration frequencies, pilot studies can be performed to determine whether it would be feasible to coat medical devices colonized by tested fungi with A. striata inner leaf gel and possibly gel from other Aloe spp. to reduce the ability of the initial layer of colonizing fungi from being able to adhere to the devices permanently after sterilization and sanitation steps . Similarly, feasibility tests can also be performed pipes within water systems to determine whether the routine flushing of pipes with A. striata inner leaf gel minimizes fungal biofilm formation. Minimizing fungal biofilm in the pipes of water systems is important for preventing pipe blockages (Siqueira et al. 2011). Furthermore, if other filamentous fungi, particularly those molds found on shower tiles, also exhibit such aberrations after treatment with A. striata, A. striata may also be employed as a household cleaner or shower tile coating.

AKNOWLEDGEMENTS We thank Mark House (Krohn Conservatory, USA), Harry Friedman and John Keegan (Belk Greenhouse, Miami University, OH), and Michael Hughes (Statistical Consulting Center, Miami University, OH). A grant from Miami University supported some of this work.

CHAPTER 2 Chemical Analysis, Isolation, and Identification of the A. striata compound(s) that cause(s) hyphal aberrations in Paecilomyces variotii and Fusarium oxysporum

Gloria A. Wada, Richard Bretz, Marcia Lee

ABSTRACT The prevention and treatment of opportunistic infections caused by emerging fungal pathogens, such as Fusarium oxysporum, Fusarium solani and Paecilomyces variotii, are problematic because available antimycotics are limited and often have toxic side effects. Therefore, it is neccessary to find novel antifungal compounds. Our previous research indicates that A. striata causes an increase in strains the hyphal aberration frequencies of F. oxysporum, F. solani and P. variotii. The hyphal aberrations contributed to a decrease in the adhesion frequency of 3 strains of P. variotii. With a combination of chromatographic techniques, we identified a fraction of A. striata inner leaf gel, AIa, that causes the most significant increase in hyphal aberration frequency.

INTRODUCTION Since our previous findings indicate that A. striata causes hyphal aberration frequency increases in Fusarium oxysporum, Fusarium solani, as well as Paecilomyces variotii and the hyphal aberrations in tested P. variotii strains correlate with a decrease in adhesion, an important fungal virulence factor, it is important to isolate A. striata compound(s), fraction, and/or physical and chemical characteristics that cause hyphal aberrations. Such information would lay a foundation for discovering the mechanism by which observed hyphal aberrations occur as well as identify a novel antifungal compound(s). While specific compounds may be responsible for the aberrations, compounds or fractions of A. striata gel may also possess characteristics such as pH and osmolality that cause aberrations. Therefore, in addition to identifying antifungal compounds and/or fractions, it is equally important to evaluate the effect of pH and osmolality on the appearance of hyphal aberrations and determine whether these characteristics of A. striata are responsible for the aberrations. Literature search on the relationship between pH and fungal hyphal morphology changes yielded no results from current research. However, based on available literature some inferences can be made about a possible relationship between pH and hyphal aberrations. Congo red caused hyphal aberrations in Saprolegnia that are similar to those observed in strains of F. oxysporum, F. solani, and P. variotii after treatment with A. striata inner leaf gel filtrate. A. striata has a pH of 4.5 at room temperature while Congo red has a pH of 6.7-7 at room temperature (Nodet et al. 1986, Nodet 1987). Therefore, since A. striata is acidic and Congo red has a neutral pH, we hypothesize that pH is not responsible for hyphal aberrations. Other chemical and/or physical properties of compounds or fractions from A. striata must be responsible for the hyphal aberrations. Literature search on the relationship between osmolality and hyphal growth and morphology rendered contrasting findings. Allaway and Jennings (1970) found that the marine filamentous fungus, Dendryphiella salina, demonstrated an increase in growth in the presence of 200 mM NaCl and KCl. Whereas, Wucherpfennig et al.(2011) found that 2 strains of Aspergillus niger demonstrated increased mycelial growth with increased osmolality. Furthermore, researchers found that fungal pellets were more elongated with rougher surfaces as osmolality increased (Wucherpfennig et al. 2011). Since the aforementioned studies were only able to make macroscopic observations, they do not report hyphal aberrations similar to those

that we have observed when filamentous fungi were treated with the inner leaf gel of A. striata. At the later stage of development, it is hard to differentiate the different hyphae that compose mycelia from each other in order to make such microscopic observations. Our observations of hyphal aberrations were made at the pre-mycelial stage of development whereas those of the aforementioned studies were made at the mycelial stage. Since osmotic pressure has varying effects on filamentous fungi, it is difficult to arrive at a conclusion as to whether the osmotic pressure introduced by A. striata inner leaf gel causes the observed hyphal aberrations that are characterized by hyphal diameter increases that lead to intervals of non-parallel cell walls. If increased osmotic pressure correlates to increased aberration frequencies, we predict that osmotic pressure is not solely responsible for hyphal aberrations because there may be another property of A. striata inner leaf gel that contributes to the appearance of hyphal aberrations in conjunction with osmotic pressure. Since the inner leaf gel of Aloe spp. is composed of a plethora of compounds, it is possible for there to be many other chemical reactions that occur among the components of the inner leaf gel of A. striata. Thus far, research of Aloe spp. inner leaf gels has identified anthraquinones, anthranols, anthrones, pyrones, chromones, and an assortment of other compounds such as chlorinated amides and isoflavone glycosides (Park and Lee 2006). Most of the separation and chemical analyses of Aloe gels has involved high performance liquid chromatography (HPLC). Using HPLC to separate macromolecule rich solutions offers many advantages over other chromatography techniques (Cock 2008, Park and Lee 2006, Light 2012, Chitarra 2003). The use of higher applied pressure (50-350 bar) is one factor that distinguishes HPLC from traditional liquid chromatography. Another difference between HPLC and traditional liquid chromatography involves the column dimensions and components. HPLC columns, stationary phases, are made with smaller solvent particles with internal column diameters of 2.1 - 4.6 mm and 30 - 250 mm in length (Gerber et al. 2004). To further improve resolution, reverse phase HPLC (RP-HPLC) can be used. RP-HPLC refers to HPLC that employs columns that are composed of hydrophobic material. Hydrophobic stationary phases allow investigators to take advantage of polarity differences. With hydrophobic stationary phases, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase and the hydrophilic molecules in the mobile phase pass through the column and are eluted first while hydrophobic molecules are retained on the column longer

(Gerber et al. 2004). To increase eluent retention times, the affinity of hydrophobic analytes for hydrophobic stationary phases can be increased relative to the mobile phase by adding more water to the mobile phase to make it more hydrophilic. Conversely, retention time can be decreased by adding more organic solvents, such as methanol, to the mobile phase. As a result of these distinguishing features and capabilities, RP-HPLC provides a better separation resolution than traditional liquid chromatography that make it ideal for the separation of Aloe inner leaf gel into its fractions and/or compounds. Since there is quite a large amount of compounds in Aloe inner leaf gel and very small quantities of samples can be loaded onto RP-HPLC injectors during a run, flash chromatography can be used to crudely separate gel components and injecting a portion of the fractions as samples to streamline the process. Flash chromatography, also referred to as medium pressure chromatography, is a form of chromatography that differs from traditional chromatography because it uses smaller silica gel particles (250- 400 mesh) as stationary phase material (Still et al. 1978). As a result of the smaller silica gel particles, the flow of the mobile phase is restricted and pressurized gas is used to drive the mobile phase through the stationary phase to make the overall flow rapid (Still et al. 1978). However, while the resolution is better than that of traditional chromatography, it is poor compared to the resolution achieved via RP-HPLC because the silica particles in flash chromatography stationary phases are larger than those of HPLC. The larger size of column particles in flash chromatography allows it to accommodate the large quantity of compounds in Aloe gels. Therefore, flash chromatography can be coupled with RP-HPLC to streamline the overall separation process in order to achieve the most superior separation of mixtures. Since RP-HPLC columns have much smaller silica particles, it would take a long time to run 1 chromatography cycle or even cause systemic blockage if whole A. striata were injected directly into the RP-HPLC system. Therefore, in respect to the separation of Aloe gel filtrate, it is best to first separate Aloe filtrate via flash chromatography and then select flash chromatography fractions of interest to be further separated via RP-HPLC. While studies have identified Aloe derived compounds believed to have some antimicrobial effects in bacteria and fungi, none of them have sought to further characterize their antifungal effects on filamentous fungi outside of observing mycelial growth patterns (Rodriquez et al. 2005, Rosca-Casian 2007). Furthermore, these studies have not addressed the other properties of Aloe inner leaf gel such as pH (4.5) and osmotic pressure that may play a role in the

inhibitory effect of Aloe inner leaf gels. Thirdly, the majority of the chromatography work and antifungal testing of Aloe inner leaf gel has primarily involved A. barbadensis, the most commercially available Aloe gel (Cock 2008, Rodriquez et al. 2005, Rosca-Casian et al. 2007). Therefore, the objective of this study is to investigate the chemical and physical properties of A. striata filtrate that cause the increases in hyphal aberration frequencies as well as isolate and identify the compounds that cause increases in hyphal aberrations. The effect of osmotic stress and pH of 4.5 (the pH of A. striata at room temperature) on the hyphal aberration frequency of a P. variotii strain, Pv19, was investigated. We also isolated a fraction of A. striata gel that causes hyphal aberrations in our fungal strains of interest via the use of combined chromatography techniques. Flash chromatography was first used to separate whole A. striata gel into 3 groups (groups 1-3). These groups were screened against fungi to determine whether they had aberration causing properties. The flash chromatography group that caused the greatest increase in the percentage of aberrated hyphae was further separated via RP-HPLC. Products from these separations were analyzed for aberration causing properties via modified germination assays. With the aforementioned combination of chromatography techniques, we identified a fraction from A. striata inner leaf gel that causes observed increases in aberration frequency. Our research also indicates that neither the pH nor the osmolality of A. striata inner leaf gel are responsible for increases in hyphal aberration frequency in 1 strain of P. variotii.

MATERIALS AND METHODS

Aloe striata propagation, harvest and lyophilization. Aloe striata plants were obtained from Krohn Conservatory in Cincinnati, Ohio. To ensure that the plants were not contaminated with fungicides, they were propagated for 3 years at Miami University’s greenhouse in a fungicide free room. After the 3 year period, we began to harvest A. striata inner leaf gel. A. striata leaves were cut from the main plant leaving the base of each leaf in order to allow for future growth. The gel within the inner portions of the leaves were harvested by cutting the sides of the leaves with a sterile knife which allowed the outer layer of a leaf to be peeled back to expose the inner gel of the leaf. The gel was then scooped into a sterile beaker, homogenized, and then sequentially filtered through 300, 30, 5, and 0.2 µm filters in order to obtain a microbe-free filtrate and to ensure that the resultant filtrate will have a viscosity of 1.0 centipoise. A. striata that had previously been harvested and passed through 0.2 µm filtration were frozen at -80 C and lyophilized, and stored at 4 C.

Fungal cultures and conidial harvest. P. variotii strain Pv19 and F. oxysporum strains Fo69 and Fo57 were used in the experiments outlined in this chapter (Table 7). Fo69 was obtained from Dr. Kerry O’Donnell (Agricultural Research Service, Illinois) while Pv19 was from our lab’s collection. Stock cultures of the aforementioned fungi from -80 C were then inoculated onto potato dextrose agar (PDA). Cultures were allowed to grow at 25 ±1 C until they became visibly mature with copious amounts of conidia. For each strain, 2-3 out of the 4 slants that have characteristically conidiating mycelia were used for conidial harvest. Conidia were harvested by flooding the slants with 10 ml of sterile 0.85% saline. The mycelial surface of each slant was gently disturbed with glass Pasteur pipettes. The resultant mixture of conidia and hyphal fragments were then transferred to sterile vortex tubes. After the transfer, the suspensions were left untouched for approximately 20-45 minutes to allow hyphal particles to settle to the bottom of the tube while the fungal conidia remained suspended in the upper layer of the solution. After 20-45 minutes, only the upper homogenous top layer was then transferred into Falcon conical tubes. The reclaimed conidial suspension was then centrifuged at 694 x g for 15 minutes. The resultant supernatant was discarded and the pellet was washed by adding approximately 7 ml of sterile saline, vortexed and then centrifuged at 694 x g for another 15 minutes. The washing step

was repeated once more. After the second washing, 2 ml of saline solution was added to the resultant pellet. Conidial concentrations for each strain were determined via the use of a hemocytometer and diluted accordingly to obtain a final concentration of 4.25 x 106 conidia/ml that was used in germination assays.

General germination assay protocol. Germination assays were performed via the use of P. variotii strain Pv19 (except where noted), 1 of the strains that demonstrated the most statistically significant increases in aberration frequency after treatment with whole A. striata inner leaf gel filtrate from Chapter 1. For each experiment there were 3 control and 3 treatment replicates except where noted otherwise. Each treatment tube contained 75 µl RPMI, 482 µl A. striata (or respective treatment or fraction), and 30 µl of fungal-conidial inoculum. Samples were incubated for 12-18 h (12 h for Fo69 and 18 h for Pv19). After incubation, assay tubes were centrifuged at 9300 x g for 10 minutes, the assay media was then removed and 0.5 ml of lactophenol- carbofuschin stain was added to tubes containing fungal pellets. The tube and its contents were mixed and centrifuged at 9300 x g for 10 minutes. After centrifugation, the stain was removed leaving a 0.05 ml solution containing the fungal pellet that was then mixed and mounted onto microscope slides, coverslips were then added and sealed with clear nail polish and left to dry before fungi were enumerated under the microscope. For quality control of contamination in the A. striata extract used in each experiment, 1 µl of the A. striata filtrate was plated onto PDA plates. These plates were incubated at both 36 C and 25 C.

Effect of pH on hyphal aberration frequency. Based on pH readings of A. striata filtrate, A. striata inner leaf gel filtrate has a pH of 4.5. To ascertain whether the aberration causing property of A. striata in some strains of F. oxysporum, F. solani, and P. variotii is due, in part, to pH, we set up germination assays as previously described with the following modifications. Instead of using A. striata as the treatment, a solution of HCl with pH 4.5 was used against Pv19 conidia. These experiments were repeated 3 separate times.

Effect of osmolality on hyphal aberration frequency. To determine whether any osmotic imbalance caused by A. striata’s presence leads to an increase in hyphal aberrations, osmolality control experiments were carried out with a 0.21 g/ml solution of dextran. An osmometer (model

3320, Advanced Instruments, Norwood, MA, USA) was used to obtain osmolality readings. Germination assays were performed as previously described with 3 replicates where the dextran solution was used in place of the whole A. striata filtrate in germination assays where the aberration frequencies were calculated. These experiments were repeated 3 separate times.

A. striata separation via flash chromatography. All of our chromatography analyses began with separation via flash chromatography irrespective of subsequent chromatography techniques that were used to further separate samples (Figure 5). This step helped reduce the amount of A. striata compounds that were injected into RP-HPLC columns since loading too many compounds onto a column at once may result in sub-standard separation. Based on earlier assays to determine the appropriate starting volume of A. striata needed to cause aberrations after separation in 1 replicate, we decided to use reconstituted A. striata filtrate powder that corresponds to approximately 72.0 ml of original filtrate for all experiments unless otherwise noted. The 72.0 ml allowed 3 replicates of each tested fraction. To prepare sample cartridges for separation via flash chromatography, lyophilized A. striata inner leaf gel filtrate was reconstituted drop wise with a solution of methanol, water, and acetic acid in a ratio of 45:55:1

(MeOH: H2O: HOAc). This ratio of methanol, water, and acetic acid was also used as the mobile phase for the actual flash chromatography separation. To ensure even sample distribution throughout the cartridge, the reconstituted solution of A. striata was loaded onto a C18 cartridge over the course of 2 days. Once the C18 column was cleaned with methanol, filled with mobile phase, and the collector was set to collect 80 total fractions with 2 waste strokes, the loaded sample cartridge was placed into the cartridge holder. UV/Vis analysis of the fractions from flash chromatography was performed to group the separated fractions, according to wavelengths, into 3 separate groups (F1, F2, and F3). F1 contains flash fractions 1-5, F2 contains flash fractions 6- 60, and F3 contains fractions 61-80. To prepare samples for use in germination assays towards aberration frequency enumeration, methanol and water were removed from each fraction via rotary evaporation then held under vacuum pressure to remove acetic acid from the mobile phase. Germination assays were performed as previously described, except F1, F2, and F3 were used instead of whole A. striata inner leaf gel filtrate and a total of 1476 µl of ROH2O (492 µl for each replicate) was added to each sample vile to produce 3 replicates of each test group.

General protocol for reverse phase high performance liquid chromatography (RP-HPLC). Based on the results of the germination assay screenings for hyphal aberrations with flash chromatography fractions (F1, F2, & F3), we decided to use flash chromatography group 2 (F2) for all further analyses since it caused the most hyphal aberrations when screened against Pv19. Methanol and water were removed from the F2 solution via rotary evaporation before further separation began with reverse phase HPLC on a C18 column with a total cycle run time of 45 minutes. Each cycle consists of a column cleaning run with 100% MeOH, a run to fill the column with mobile phase, and the separation run where samples (20 µl) were injected into the column for separation. Each run lasted approximately 15 minutes. The mobile phase was composed of MeOH, ROH2O, and HOAc. The ratio of mobile phase components varied depending on the goals of each separation strategy listed under the respective experiment descriptions below.

Separation of F2 into fractions A-D via RP-HPLC and aberration frequency increase via germination assays. Reconstituted filtrate was loaded onto cartridges and underwent flash chromatography as described earlier. RP-HPLC separations were then performed as previously described. For these RP-HPLC separations, flash chromatography group 2, F2, was separated into fractions A-D with a 45:55:1(MeOH: H2O: HOAc) mobile phase (Figure 6). Components of fraction A had retention times between 2.0 and 4.12 minutes. Components of fraction B had retention times between 4.12 and 7.0 minutes while fractions C had retention times between 7.0 and 10.0 minutes. Fraction D components had retention times between 10.0 and 15.0 minutes (Figure 7). These separations were repeated 3 separate times. Once the different fractions were separated, they were prepared for germination assays as previously described to quantify aberration frequencies.

Separation of Fraction A into 2 Fractions (AI & AII) via RP- HPLC. To isolate the compounds that cause aberrations, A. striata fraction A was further separated into 2 fractions

(Fractions AI & AII) (Figure 8 and Figure 9) with a 45:55:1(MeOH: H2O: HOAc) mobile phase via HPLC as described earlier with the retention times indicated in Figure 3. The original starting volume of A. striata used for this analysis was 86 ml.

HPLC fractions AI & AII sample preparation and aberration screening via germination assays. Germination assays as previously described were performed with AI and AII RP-HPLC fractions against Fo69, a strain that demonstrated significant increases in aberration frequency in preliminary assays, to determine which of the fractions contain the aberration causing compounds by determining the percentage of aberrated hyphae observed after exposure to fractions AI & AII.

Identification of suitable mobile phase for resolution of fraction AI Peaks. In order to better separate the peaks/compounds in fraction AI (Figure 10) to obtain pure samples that can undergo NMR analysis, different ratios of water to methanol with a constant ratio of acetic acid (35:65:1, 25:75:1, 15: 85:1, 10:90:1 and 5:95:1) were analyzed (Figures 11-15). For this analysis, flash chromatography and UV/Vis techniques outlined above were employed. Fraction AI was first isolated via RP-HPLC with the 45:55:1 mobile phase and then underwent separation using mobile phases of different water concentrations as indicated above. These evaluations were performed at least twice on separate days for the 15:85:1, 10:90:1 and 5:95:1 mobile phases to ensure reproducibility due to their low methanol ratio.

RP-HPLC separation of AI into AIa and AIb. Fraction AI was collected as earlier described with the 45:55:1(MeOH: H2O: HOAc). Based on the previous analysis of mobile phases, the

15:85:1(MeOH: H2O: HOAc) mobile phase was used to further separate AI into 2 fractions (AIa & AIb) via RP-HPLC as previously described with the exception of the use of a different mobile phase constituents ratio.

Screening of fraction AIa for aberration causing compounds. To determine whether fraction AIa contains aberration causing compounds, germination assays were performed with the fraction collected during the same RP-HPLC runs to separate AIa and AIb for the identification of the resolved peak in fraction AIb as previously mentioned with the original A. striata starting material volume of 511 ml. The RP-HPLC separated sample used for these assays was re- suspended in enough ROH2O in order to yield 3 replicates. Although we could have added enough water to have 9 total replicates, we decided to only have 3 replicates in case adding more water would dilute the concentration of our compounds of interest.

Statistical Analyses. All statistical analyses were carried out in R. Aberration frequency data were analyzed using logistic regression (R Core Team, 2015).

Table 7. List of all the strains used in this chapter and their corresponding source.

Strain id Organism Source Source Strain Id No. Pv19 Paecilomyces variotii ATCC ATCC 22319 Fo69 Fusarium oxysporum ARS (NRRL) NRRL 25369

ATCC =American Type Culture Collection, ARS = United States Department of Agriculture Agricultural Research Service (formerly known as NRRL) Culture Collection.

Figure 5. Schematic diagram of flash chromatography separation of A. striata inner leaf gel into

F1, F2, & F3. A. striata was separated via flash chromatography with a MeOH: H2O: HOAc (45:55:1, v/v/v) mobile phase. Flash chromatography fractions were combined into 3 groups (F1, F2, and F3) based on UV/Vis readings at 293 nm.

Aloe striata Inner Leaf Gel Extract

Flash Chromatography

F1 F2 F3

Figure 6. Schematic diagram of flash chromatography and RP-HPLC separations of A. striata inner leaf gel into fractions A, B, C, and D. A. striata was first crudely separated via flash chromatography with a MeOH: H2O: HOAc (45:55:1, v/v/v) mobile phase. Flash chromatography fractions were combined into 3 groups (F1, F2, and F3) based on UV/Vis readings. F2 was further separated into fractions A, B, C, D with RP- HPLC with a MeOH: H2O: HOAc (45:55:1) mobile phase.

Aloe striata Inner Leaf Gel Extract

Flash Chromatography

F1 F2 F3

RP-HPLC

A B C D

Figure 7. Representative RP-HPLC chromatogram of A. striata flash chromatography fraction 2, F2, separated into 4 HPLC fractions A-D. The 4 HPLC fractions (A-D) were collected at the retention times indicated on the chromatogram via a C18 RP- HPLC column with a mobile phase composed of methanol, water, and acetic acid at a ratio of 45:55:1.

Figure 8. Schematic diagram of flash chromatography and RP-HPLC separations of A. striata inner leaf gel into AI and AII. A. striata was first crudely separated via flash chromatography with a MeOH: H2O: HOAc (45:55:1, v/v/v) mobile phase. Flash chromatography fractions were combined into 3 groups (F1, F2, and F3) based on UV/Vis readings. F2 was further separated into fractions A, B, C, D with HPLC with a MeOH: H2O: HOAc (45:55:1) mobile phase.

Fraction A was then separated via RP-HPLC with a MeOH: H2O: HOAc (45:55:1) mobile phase into 2 fractions, AI and AII.

Aloe striata Inner Leaf Gel Extract

Flash Chromatography

F1 F2 F3

RP-HPLC

A B C D

AI AII

Figure 9. Representative RP-HPLC chromatogram for the separation of A. striata fraction A into 2 fractions AI and AII. The fractions were collected on a C18 reverse phase HPLC column with a mobile phase composed of methanol, water, and acetic acid at a ratio of 45:55:1.

Figure 10. Schematic diagram of flash chromatography and RP-HPLC separations of A. striata inner leaf gel into AIa and AIb. A. striata was first crudely separated via flash chromatography with a MeOH: H2O: HOAc (45:55:1, v/v/v) mobile phase. Flash chromatography fractions were combined into 3 groups (F1, F2, and F3) based on UV/Vis readings. F2 was further separated into fractions A, B, C, D with HPLC with a MeOH: H2O: HOAc (45:55:1) mobile phase.

Fraction A was then separated via RP-HPLC with a MeOH: H2O: HOAc (45:55:1) mobile phase into 2 fractions, AI and AII. AI was further separated into 2 fractions, AIa and AIb.

Aloe striata Inner Leaf Gel Extract

Flash Chromatography

F1 F2 F3

RP-HPLC

A B C D

AI AII

AIa AIb

Figure 11. Representative chromatogram from RP-HPLC separation of A. striata fraction AI via a 35:65:1 (MeOH: H2O: HOAc) mobile phase.

Figure 12. Representative RP-HPLC chromatogram from the separation of A. striata fraction AI via a 25:75:1 (MeOH: H2O: HOAc) mobile phase.

Figure 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. Fraction AIb contains the lone resolved peak.

Figure 14. Representative RP-HPLC chromatogram from the separation of A. striata fraction AI via a 10:90:1 (MeOH: H2O: HOAc) mobile phase.

Figure 15. Representative RP-HPLC chromatogram from the separation of A. striata fraction AI with a 5:95:1 (MeOH: H2O: HOAc) mobile phase.

RESULTS Analysis of Chemical and Physical Characteristics of A. striata. Pv19 treated with HCl solution with a pH of 4.5, had an average aberration frequency of 14% ± 11% while RO water treated samples had an average aberration frequency of 12% ± 8% (p = 0.93, Figure 16A). Treatment with a solution of 0.21 g/ml dextran that corresponds with the osmolality of whole A. striata extract caused an average aberration frequency of 6.67% ± 6.20%. Control samples yielded an average aberration frequency of 1.3% ± 2.06% (p = 0.31, Figure 16B).

Isolation and identification of hyphal aberration causing A. striata fractions. Pv19 treated with flash chromatography groups F2 (p = 0.0001), and F3 (p = 0.002) caused aberration frequency increases of 98% ± 0.00 and 43 ± 0.04 respectively (Figure 17A). Group F1 caused an average aberration frequency of 12.5% ± 11.7% (p = 0.3407, Figure 17A). All of the fractions (A-D) caused average aberration frequencies of 73% ± 19%, 27.0% ± 11%, 9.2% ± 8.0%, 13.5% ± 12.2% respectively (p < 0.001, Figure 17B). Of all the fractions, Fraction A caused the most significant increase in hyphal aberration frequencies. Fraction AI caused an average aberration frequency of 76.8% ± 15.3 while fraction AII caused an average aberration frequency of 23% ±

20.3% (p < 0.001, Figure 17C). We determined that the 15:85:1(MeOH: H2O: HOAc) mobile phase composition ratio was the lowest ratio that we could us to further separate fraction AI into AIa and AIb because the 10:90:1 and 5:95:1 ratios did not yield reproducible chromatograms during each of the 3 performed runs. Pv19 treated with fraction AIa had an average aberration frequency of 97 % ± 4.9 % (p < 0.001, Figure 17D).

Figure 16. Chemical and physical properties (pH and osmolality) of A. striata and their individual effect on aberration frequency. Average aberration frequency of Pv19 after treatment with HCl solution with pH of 4.5 (A) and solution of dextran at 96 ± 2 mOsm/kg osmolality (B) where p= 0.93 and 0.31 respectively, N= 200.

B DISCUSSION

Figure 17. Average aberration frequency of Pv19 when treated with flash chromatography groups F1-F3 (A), RP-HPLC fractions A-D (B), fractions AI-AII (C), and fraction AIa from A. striata. * indicates p < 0.001 when compared to RO water controls and N=200.

B *

C *

D *

DISCUSSION Experiments with HCl (pH 4.5) indicate that pH 4.5 does not contribute to the increase in the aberration frequency of Pv19 and by extension the aberration frequencies of all tested strains. Moreover, we also noted that the osmolality of A. striata is not responsible for causing hyphal aberrations since treatment with a solution of dextran at the relative osmolality of A. striata did not cause the same type of hyphal aberrations caused by A. striata inner leaf gel. Since the pH and osmolality of A. striata are not responsible for the increases in aberration frequency, there must be a compound(s) within the inner leaf gel of A. striata that leads to the aberration frequency increases. From chromatography separations and corresponding germination assays, we have been able to conclude that the aberration causing compounds in A. striata are primarily present in the first half of fraction A, AI. When Cock (2008) fractionated the inner leaf gel of A. barbadensis into 14 fractions, they found that the first fraction, a fraction that has a similar chromatogram profile as fraction A from our research, caused a zone of mycelial inhibition in a nystatin resistant strain of Aspergillus niger. When RP-HPLC fraction A was further separated into 6 fractions via HPLC (Lee et al. unpublished data), no significant increases in aberration frequency were observed. Such a phenomenon suggests that hyphal aberrations are created via a synergistic mechanism involving more than 1 compound where the compounds within fraction AI could be a mixture of compounds needed in the synthesis of a bioactive compound(s) responsible for the aberration frequency increases observed when fungi are treated with the inner leaf gel of A. striata. After AI was further separated into 2 fractions ( AIa and AIb) via RP-HPLC with a mobile phase of MeOH: H2O: HOAc (15:85:1), AIa caused significant increases in the aberration frequency of Pv19. The 15:85:1 (MeOH: H2O: HOAc) mobile phase was used because it contains the lowest amount of methanol that can be used to generate reproducible chromatograms.

AKNOWLEDGEMENTS We thank Mark House (Krohn Conservatory, USA), Harry Friedman and John Keegan (Belk GreenhouseMiami University, OH), and Michael Hughes (Statistical Consulting Center, Miami University, OH). A grant from Miami University supported some of this work. We would also like to thank the Bretz lab (Department of Chemistry and Biochemistry Miami University, OH) for help with the chromatography analyses.

SUMMARY Based on the ability of A. striata’s inner leaf gel to cause a significant decrease in the germination frequency of C. albicans, we initially screened 3 strains each of F. oxysporum (Fo69, Fo24, & Fo57), F. solani (Fs02, Fs20, & Fs53), and P. variotii (Pv19, Pv06, & Pv23) for decreases in germination frequency after treatment with whole A. striata inner leaf gel. We did not find any significant decreases in germination frequency except in Fo69. Therefore, we went on to analyze whether germ tube lengths and branching frequencies were affected by treatment with whole A. striata in all strains from these preliminary screenings. We also did not find any significant increases or decreases in all the branching frequencies in A. striata treated fungal strains except for 2 F. oxysporum strains, Fo57 and Fo24. Repeated assays on Fo69 indicated a significant decrease in germination frequency. Similarly, repeated germination assays with Fo57 demonstrated a significant increase in branching frequency as well as a significant decrease in germ tube length. Although A. striata caused significant decreases in germination and germ tube lengths or increases in branching frequencies in only a few fungal strains, it did cause significant increases in hyphal aberrations in all of the screened strains. These aberrations are characterized by increased hyphal diameters that result in non-parallel cell walls at intervals along hyphae. When germination assays were repeated 3 different times with A. striata inner leaf gel against the strains that were the most significantly aberrated in the initial screenings (Fo69, Fs02, & Pv19), all strains demonstrated statistically significant aberration frequency increases. In order to understand the extent of the increases in hyphal diameters as well as understand whether diameter increases also occur in conidia, randomly selected hyphae and associated conidia of 3 different P. variotii strains (Pv19, Pv23, and Pv06) were measured at 3 different locations via SEM. Conidial diameters were measured at the widest portion of conidia. Hyphal diameter measurements were acquired at 2 different locations, the sub-conidial and sub- apical hyphal regions. There was a significant increase in the parent cell diameter of A. striata treated Pv19 on all 3 days. Sub-conidial and sub-apical diameter measurements indicated an increase across all days, except for the day 1 sub-conidial measurements of Pv19. Pv06 and Pv23 had a significant diameter increase in their sub-apical regions on all 3 days, and a significant increase in sub-conidial and conidial diameters except for the day 1 conidial diameter of Pv06 and day 1 sub-conidial diameters of Pv23.

To understand whether A. striata-induced fungal hyphal aberrations have any effect on fungal adhesion, we performed adhesion assays with chamber slides. Adhesion assay results suggest that hyphal aberrations characterized by diameter increases that result in intervals of non-parallel cell walls along hyphae significantly contribute to a decrease in the ability of fungi to adhere to substrate. The percent adhesion was higher in controls than in treatments for all 3 P. variotii strains. When the total number of adhered fungi was separated into morphotypes, fungi, except for Pv19 SNPSC, with non-parallel cell walls consistently demonstrated lower adhesion percentages when compared to morphotypes with parallel cell walls irrespective of whether morphotypes were on control or treatment slides. To examine whether A. striata’s pH of 4.5 is responsible for the noted aberrations, the germination assay protocol was employed. However, instead of whole A. striata filtrate, a hydrochloric acid (HCl) solution with pH of 4.5 was used. We decided to use HCl instead of other acids such as lactic acid because HCl does not introduce additional carbon sources into the incubation media. When treated with HCl solution at pH 4.5, Pv19 did not demonstrate a significant increase in aberration frequency. This data suggests that A. striata’s pH of 4.5 does not cause the observed increase in hyphal aberration frequency. We also observed that the osmolality of A. striata is not responsible for causing hyphal aberrations since treatment with a solution of dextran at the relative osmolality of A. striata did not cause the same type of hyphal aberrations caused by A. striata inner leaf gel. Based on our earlier findings that A. striata causes hyphal aberrations in our fungi of interest and that these hyphal aberrations plays a role in causing a reduction in adhesion frequencies of P. variotii strains, identifying the compounds in A. striata that cause an increase in aberration frequency is important. Identification of the compounds helps establish a foundation for engineering antifungal treatments that will reduce and/or prevent invasive fungal infections caused by filamentous fungi. To isolate the compound(s) responsible for increasing aberration frequencies in the fungal strains that we tested in Chapter 1, we first employed flash chromatography to separate whole A. striata filtrate into eighty fractions with a mobile phase of methanol (MeOH), reverse osmosis water (ROH2O), and acetic acid (HOAc) in a ratio of 45:55:1. These eighty fractions were pooled into 3 groups (F1, F2, & F3). When each of the 3 groups was screened against Pv19 for aberration causing activity, F2 and F3 caused significant increases in aberration frequencies.

F2 caused the most significant aberration frequencies. Therefore, another set of A. striata separations were performed to isolate F2 via flash chromatography with a mobile phase of

MeOH: H2O: HOAc (45:55:1). F2 was subsequently further separated via RP-HPLC with a mobile phase of MeOH: H2O: HOAc (45:55:1) into 4 fractions (fractions A-D). Pv19 and Fo57 were treated in germination assays with each of the fractions, all 4 fractions demonstrated significant increases in aberration frequency in both Pv19 and Fo57, but fraction A demonstrated the most significant increases in both tested strains. Therefore, one may infer that the aberration causing compound(s) may be present in all 4 fractions, but fraction A must contain a concentration of the compound(s) that causes aberrations. To confirm whether the aberrations caused by fraction A demonstrate the same hyphal diameter increases noted in chapter 1 when we measured the parent cell and hyphal diameters of fraction A treated Pv19. Similar to results from chapter 1, fraction A treated Pv19 demonstrated diameter increases in the midpoint and sub-apical hyphal regions. Fraction A was further separated into 2 fractions, AI and AII via RP-HPLC with a mobile phase of MeOH: H2O: HOAc (45:55:1). When AI and AII were screened for aberration causing properties in Pv19, AI caused significant increases in aberration frequency. Another set of separations were performed to isolate AI via HPLC with a mobile phase of MeOH: H2O: HOAc (45:55:1). The isolated AI was further separated into 2 fractions, fractions AIa and AIb via RP-

HPLC with a mobile phase of MeOH: H2O: HOAc (15:85:1). When screened against Pv19, AIa caused significant increases in aberration frequency. Our research has identified fraction AIa as the aberration-causing portion of A. striata. To date, this is the most exhaustive separation of early A. striata RP-HPLC eluents with retention times between 2 and 3 minutes.

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