University of Ghana http://ugspace.ug.edu.gh ANALYSIS OF ANTIMICROBIAL RESISTANCE IN CANDIDA ALBICANS USING MODULATORS OF MDR/CDR GENE EXPRESSION A THESIS PRESENTED TO THE WEST AFRICAN CENTER FOR CELL BIOLOY OF INFECTIOUS DISEASES, DEPT. OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY BY REBECCA YEBOAH M.PHIL MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES (10343581) JULY 2018 University of Ghana http://ugspace.ug.edu.gh i University of Ghana http://ugspace.ug.edu.gh ANALYSIS OF ANTIMICROBIAL RESISTANCE IN CANDIDA ALBICANS USING MODULATORS OF MDR/CDR GENE EXPRESSION A THESIS PRESENTED TO THE WEST AFRICAN CENTER FOR CELL BIOLOY OF INFECTIOUS DISEASES, DEPT. OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY BY REBECCA YEBOAH M.PHIL MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES (10343581) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF M.PHIL MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE JULY 2018 ii University of Ghana http://ugspace.ug.edu.gh DECLARATION I Rebecca Yeboah (West African Center for Cell Biology of Infectious Pathogens, Dept. of Biochemistry, Cell and Molecular Biology, University of Ghana) hereby declare that this thesis is the outcome of my own research under the supervision of Dr. Patrick Kobina Arthur and Dr. Winfred-Peck Dorleku (West African Center for Cell Biology of Infectious Pathogens, Dept. of Biochemistry, Cell and Molecular Biology, University of Ghana). To the best of my knowledge, this thesis contains neither materials which has been accepted for the award of any degree or any material previously published by another author, except where due reference is made in the text of the thesis. …………………………………… Rebecca Yeboah (Student) ………………………………………….. Dr. Patrick Kobina Arthur (Principal Supervisor) ………………………………………… Dr. Winfred-Peck Dorleku (Co-Supervisor) iii University of Ghana http://ugspace.ug.edu.gh DEDICATION I dedicate this thesis to the past and present members of the Laboratory for Chemical Systems Biology of Infectious Pathogens, especially to Ethel Juliet Serwaa Blessie. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I wish to express my gratitude to my Supervisor, Dr. Patrick Kobina Arthur for giving me the opportunity to work on this project and also for his mentorship. I would also like to gratefully thank WACCBIP for offering me full scholarship all throughout my Masters studies, for funding this research and also for sponsoring my attendance to the Gordon Research Conference on Drug Resistance in the USA. My appreciation to the technical staff of Dept. of Biochemistry, Cell and Molecular Biology, University of Ghana for their support throughout my project. Lastly, I would like to thank the past and present members of the Laboratory for Chemical Systems Biology of Infectious Pathogens, especially to Ethel Juliet Serwaa Blessie, Isaac Carilo, Leonard Asare, Benaiah Abbey, Bismarck Kyei-Amaniampong and Dr. Vincent Amarh for their support. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ............................................................................................................................ iii ACKNOWLEDGEMENT ............................................................................................................... v TABLE OF CONTENTS ............................................................................................................... vi LIST OF FIGURES .........................................................................................................................ix LIST OF ABBREVIATIONS ............................................................................................................ x ABSTRACT .....................................................................................................................................xi CHAPTER ONE .............................................................................................................................. 1 1.1 INTRODUCTION ............................................................................................................ 1 List of Selected Stress Response Genes for RT-PCR Analysis .................................................... 9 1.3 Aim: .................................................................................................................................... 9 1.4 Specific Objectives: ............................................................................................................. 9 1.5 Rationale............................................................................................................................ 10 CHAPTER TWO........................................................................................................................... 11 2.0 LITERATURE REVIEW .................................................................................................... 11 2.1 C. albicans Infections......................................................................................................... 11 2.2 Current Antifungals in clinical use, cellular targets, toxicities and the Development ........... 13 of Resistance ........................................................................................................................... 13 2.3 Fungi as Sources of Novel Antimicrobials and Antimicrobial Chemosensitizers ................. 16 2.4 Utilizing small molecules to probe antimicrobial phenotypic resistance .............................. 17 2.5 Selecting molecular drug targets based on analysis of stress response. ................................ 19 2.6 Use data to guide selection and isolation of new bioactive compounds ............................... 21 2.7 Stress Responses to Antifungals ......................................................................................... 23 2.8 Antifungal Chemosensitization To Overcome Efflux Pump-mediated Resistance ............... 24 CHAPTER THREE....................................................................................................................... 28 3.0 MATERIALS AND METHODS ......................................................................................... 28 3.1 Chemicals and Reagents ..................................................................................................... 28 3.2 Fungal strains used ............................................................................................................. 30 3.3 Inoculum preparation ......................................................................................................... 30 3.4 Determination of Minimum Inhibitory Concentrations (MICs) of compounds and Phenotypic Array of Organisms in The Presence of Compounds ................................................................ 31 3.4.1 Determination of breakpoint Concentration of Unique Interactions .................................. 31 3.4.2 Alamar Blue Assay ......................................................................................................... 32 3.5 Measurement of Efflux Activity in the presence of Efflux Modulating and Phenotype Modifying Compounds ............................................................................................................ 32 vi University of Ghana http://ugspace.ug.edu.gh 3.6 Chemosensitization of C. albicans to fluconazole by SBF and TEF Fungal Extracts ........... 34 RESULTS .................................................................................................................................. 36 4.1 Determination of minimum inhibitory concentrations of Efflux Pump Modulators and other phenotype modifying compounds............................................................................................. 36 4.2 Determination of antimicrobial phenotypes in the presence of efflux pump modulators and other phenotype modifying compounds .................................................................................... 38 4.4 Determination of breakpoint Concentration of Unique Interactions ..................................... 49 4.5.1 Confirmation of resistant breaking compound-antifungal interactions in C. albicans ........ 52 4.6.2 S. cerevisiae efflux activity in the presence of efflux modulators and phenotypic modifiers without and with deferasirox .................................................................................................... 63 4.7 Initial screening of the SBF and TEF extracts against C. albicans and S. cerevisiae ............ 68 4.6 Fluconazole chemosensitization ......................................................................................... 71 4.7 Analysis of Efflux Activity of Chemosensitizing TEF and SBF extracts ............................. 73 5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ......................................... 75 5.1 DISCUSSION ....................................................................................................................... 75 5.1.1 Chemical compounds modify the antimicrobial phenotypes of Candida albicans and Saccharomyces cerevisiae ........................................................................................................ 76 5.1.2 Efflux activity of C. albicans and S. cerevisiae are affected by Phenotype Modulators .... 79 5.1.3 Soil Borne and Terrestrial Endophytic Fungal Extracts Chemosensitize C. albicans and S. cerevisiae To Fluconazole........................................................................................................ 80 5.2 CONCLUSION .................................................................................................................... 82 5.3 RECOMMENDATIONS AND FUTURE OULOOK ......................................................... 83 APPENDIX .................................................................................................................................... 84 REFERENCES .............................................................................................................................. 89 vii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 2.1: List of compounds used in this study and information from literature……….…….26 Table 2.2: List of compounds used in this study and information from literature……………..27 Table 3.1: List of compounds used in this study…………………………..……………...…...26 Table 3.1: List of antifungals and concentrations………………………………….…………29 Table 4.1: Minimum inhibitory concentrations of compounds……..…………………..…….37 Table 4.2: Ranking of phenotypic compounds in C. albicans……………….…………….…….46 Table 4.3: Raking of phenotypic compounds in S. cerevisiae……………………..…………….49 Table 4.4: Break point concentration determination to confirm unique interactions in C. albicans……...………………………………………………………………………………………….52 Table 4.5: List of Selected fungi and zones of inhibition………………………………..........71 APPENDIX Table 1.1: Antifungal resistance or susceptibility patterns of C. albicans With Modulators of Efflux Pumps…………………………………………………………………………...…….85 Table 1.2: Antifungal resistance or susceptibility patterns of S. cerevisiae With Phenotype modifiers……………………………………………………………………………………...85 Table 1.3: Antifungal resistance or susceptibility patterns of C. albicans With Phenotype Modifiers……………………………………………………………………………………..86 Table 1.4: Antifungal resistance or susceptibility patterns of S. cerevisiae With Phenotype Modifiers……………………………………………………………………………………..86 Table 1.5: Antifungal resistance or susceptibility patterns of C. albicans With Phenotype Modifiers……………………………………………………………………………………..87 Table 1.7: Antifungal resistance or susceptibility patterns of S. cerevisiae With Phenotype Modifiers……………………………………………………………………………………..87 viii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figures 1: Antimicrobial Phenotype Modification in C. albicans and S. cerevisiae.……………………………………………………………………………………………….43 Figures 2: Sample assay plates from antimicrobial phenotype modification in S. cerevisiae………………………………………………………………………………………………..44 Figure 3: Confirmation of resistant breaking compound-antifungal interactions in C. albicans………………………………………………………………………………………54 Figure 4: Confirmation of resistant inducing compound-antifungal interactions in C. albicans………………………………………………………………………………………………….56 Figure 5: Confirmation of resistant breaking compound-antifungal interactions in S. cerevisiae………………………………………………………………………………………………..58 Figure 6: Confirmation of resistant inducing compound-antifungal interactions in S. cerevisiae………………………………………………………………………………………………..60 Figure 7: Analysis of the C. albicans efflux activity in the presence of efflux modulators and phenotypic modifiers without and with deferasirox…………………...……………………..63 Figure 8 A: Analysis of the S. cerevisiae efflux activity in the presence of efflux modulators and phenotypic modifiers without (A) and with (B) deferasirox……………..……………….66 Figure 9: Analysis of the C. albicans (A) and S. cerevisiae (B) efflux activity in the presence of efflux modulators and phenotypic modifiers………………………………………………68 Figure 10: Initial screening of the SBF and TEF extracts against C. albicans and S. cerevisiae……………………………………………………………………………………..70 Figure 11: Screening for chemosensitizing agents from SBF and TEF extracts against C. albicans…………………………………………………………………….…………….......73 Figure 12: Analysis of the C. albicans and S. cerevisiae efflux activity in the presence of fungal extracts………………………………………………………………...……...……75 APPENDIX Figure 1: Analysis of the C. albicans and S. cerevisiae efflux activity in the presence of efflux modulators and phenotypic modifiers………………………………………...………………88 Figure 2 A and B: Analysis of the C. albicans (A) and S. cerevisiae (B) efflux activity in the presence of fungal extracts ……………………………………………………………..……89 ix University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS YPDA Yeast Peptone Dextrose Agar NB Nutrient Broth TEF Terrestrial Endophytic Fungi SBF Soil Borne Fungi 5-FU 5-Fluorouracil Amp B Amphotericin B CyH Cycloheximide Para Paromomycin Ben Benzoic acid Flu Fluconazole Gri Griseofulvin Set Sertraline x University of Ghana http://ugspace.ug.edu.gh ABSTRACT Majority of life-threatening fungal infections in clinics are caused by Candida albicans. The emergence of azole resistance in fungi complicates patient management. In response to chemical stress, C. albicans make transient changes in the gene expression for survival. Notable among these is the upregulation of efflux pump which is known to be the main mechanism of antifungal resistance. Potent therapeutic agents targeting this resistance mechanism are urgently needed. Chemo-sensitization is postulated as one way to overcome antifungal resistance. Endophytic fungi produce bioactive metabolites which are used as chemotherapeutic agents. The aim of this study is to use modulators of CDR and MDRs genes as probes to study chemo-sensitization and resistance phenotypes. Also, fungal metabolites (alone and in combination with chemosensitizers) will be used to reverse antifungal resistance. On analysis of phenotypic switching of the fungal cells in the presence of efflux modulators and phenotypic modifiers, S. cerevisiae was frequently observed to switch phenotypes as compared to C. albicans. Chemical compounds, including, compounds PC04-10, PC04-11, PC04-16 and PC04-23, significantly modified the antimicrobial phenotypes of Candida albicans and Saccharomyces cerevisiae and could be considered for use as synergistic partners of antifungal drugs to overcome resistance. Also, it was realized that some compounds including rifampicin, estradiol, PC04-09 and PC04-14 caused resistance. A total of 40 out of 507 bioactive and 90 chemosensitizing extracts were identified from SBF and TEF fungal extracts. In the Rhodamine efflux assay, six compounds were found to inhibit S. cerevisiae efflux, these well trifluoperazine, trifluoprozerazine, thioridazine, chlorpromazine, deferasirox and ibuprofen, whereas in C. albicans only the last four out of the six compounds were active. Also, 13 out of 20 chemosensitizing extracts significantly inhibited efflux activity of C. albicans and S. cerevisiae. Thus, fungi are good sources of novel and potent antifungal and chemosensitizing compounds. xi University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.1 INTRODUCTION Unicellular organisms have developed adaptations to respond appropriately to changes in their environment. Generally, microorganisms respond to stress in the environment by transient changes in expression of genes that regulate stress response to enhance their survival (Enjalbert, Nantel et al. 2003). The presence of antifungal compounds is recognized as stress in Candida albicans, therefore, in response to the stress, various changes are made in the genome, eventually conferring antifungal resistance. The main mechanism through which C. albicans develop resistance has been found to be the overexpression of the ATP binding cassette (ABC) transporters, which are found in the cell membrane as well as the major facilitator superfamily (MFS)- membrane efflux proteins (Łącka, Konieczny et al. 2015). Some efflux proteins identified in C. albicans, include the CaMDR1, Mdr1p, Cdr1p, Cdr2p and Flu1p. These pumps reduce the accumulation of antifungals as a self-defense mechanism (Prasad and Goffeau 2012). Over the years, several screening as well as chemical synthesis programs and strategies have been devised and has led to the discovery of numerous bioactive fungal secondary metabolites. Despite these efforts, new bioactive products significant to human therapy are rare and urgently needed, mainly due to large rise in the numbers of immunocompromised patients such as recipients of various organ transplants and AIDS patients. And more notably, the high incidence of drug resistance among clinically relevant fungal species against a significant proportion of the already limited number of antifungals currently in clinical use. Also, development of novel compounds especially from natural sources is necessary due to the considerable toxic side effects of some of the existing drugs. It is reported that high mortality rate resulting from C. albicans infections are mainly attributed to limited collection of 1 University of Ghana http://ugspace.ug.edu.gh antifungal agents ((Ła̧cka et. al, 2015) as well as the development of resistance against the few available ones. New antifungal agents are therefore urgently needed. Current research findings in drug resistance and discovery fields has revealed chemosensitization as one way to avert fungal resistance, especially to the azole group of antifungals including fluconazole which is the first line of antifungal used in clinics for treatment of most fungal infections. It is known that, a number of milbemycins chemosensitize Candida species to fluconazole (Niimi, Harding et al. 2004). In an investigation conducted by Maesaki et al., 2000 it was revealed that, C. albicans clinical isolates which were resistant to fluconazole and mutant Saccharomyces cerevisiae strains with the overexpression of C. albicans ABC or MFS efflux pumps were made more susceptible to fluconazole in the presence of FK50 and cyclosporine, which are originally the immunosuppressors (Maesaki, Hossain et al. 2000). In a study conducted by (Del Poeta, Cruz et al. 2000) demonstrated that combining FK506 and fluconazole increased the sensitivity of C. neoformans and C. albicans to the triazoles, making them fungicidal. Thus, research geared toward discovering and developing novel antifungal chemosensitizers that increase potency against C. albicans is a promising alternative to overcoming fungal infections and to prolong the clinical life of antifungals. In this study, modulators of C. albicans ABC or MFS efflux proteins and their combinations will be used as probes to study chemosensitisation. Also, fungal metabolites obtained from terrestrial endophytic fungi (TEF) and soil borne fungi (SBF), either alone or in combination with chemosensitising agents will be tested against C. albicans for their ability to reverse antifungal resistance via downregulation or total inhibition of efflux pumps. It is estimated that 30% of the most frequently given clinical drugs are of fungal origin, this suggests that fungi are a great source for the search and discovery new antimicrobial agents (Dame, Silima et al. 2016). 2 University of Ghana http://ugspace.ug.edu.gh 1.2 Project Concepts In this current study, a panel of small molecules that modulate the activity of efflux pumps as well as other phenotype modifying compounds are used to probe and study resistant phenotypes in C. albicans and S. cerevisiae. Various resistant genes induced and expressed in the presence of each compound would be analyzed so as to have an idea of resistance pathways and associated genes and genes products. This observation and knowledge will subsequently be used to identify potent antifungal agents to overcome any possible mode of resistance to our identified compound. Based on the above-mentioned scenario being used in our study, four different concepts (figures 1.1 to 1.5) below has been drawn to depict the interactions with various molecules, possible outcomes and deductions from each interaction. In figure1.1, inhibitors of efflux pump and bioactive extracts will be used in an interaction study with standard antifungals. Expected outcomes includes increase, decrease or maintenance of antifungal activity. An increase in antifungal activity could inform a strategy for studying reversal of resistance. A decrease in the activity of the antifungal could be adopted as strategy for inducing cellular resistance. A decrease in the activity of the extract on interaction with efflux pump inhibitors could indicate the new antifungal is affected by accumulated antagonist or affected by cell remodeling due to the inhibitor or affected by newly induced class of efflux pumps. Maintenance of the activity of the extract on interaction with efflux pump inhibitors could indicate possible discovery of a new antifungal unaffected by efflux pump. However, it could be said that, the activity of the extract is affected by efflux pump on increase of the activity of the bioactive extract. and potent activities during these screens will further be taken through product isolation. 3 University of Ghana http://ugspace.ug.edu.gh Figure 1.1: Shows interaction between inhibitors of efflux pumps with standard antifungals and bioactive extracts and possible outcomes In figure 1.2, inhibitors of efflux pump and bioactive extracts will also be used in an interaction studies with standard antifungals. A decrease in the activity of the extracts could inform a strategy for screening for new sources of resistant breaking antifungals, maintenance of the activity could mean the new antifungal is unaffected by efflux pump and an increase in activity could indicate a new resistant breaking antifungal. The bioactive extracts will also be used in an interaction study to analyze the effects of efflux pump inducers and standard antifungals on them (figures 1.1 and 1.2). Here, an increase in the activity of the antifungals in the presence of efflux pump inhibitors could serve as a strategy for studying reversal of resistance while a decrease in antifungal activity could serve as a strategy for inducing cellular resistance. On interaction of the bioactive extracts with the efflux pump inhibitors, a decrease in bioactivity could indicate the antifungal that is affected by efflux pump overexpression, an increase in bioactivity could indicate new antifungal increased by 4 University of Ghana http://ugspace.ug.edu.gh efflux pump overexpression the bioactivity is increased by efflux pump overexpression via the extrusion of an antagonist from the cell or cell remodeling increases the antifungal activity and a maintenance of bioactivity cold indicate a new antifungal unaffected by efflux pump. On interaction of bioactive extracts with antifungals a decrease in bioactivity would serve as a strategy for screening for new antifungals to overcome efflux pump based resistance, an increase in bioactivity could indicate a new antifungal whose activity is increased by efflux pump overexpression and a maintenance of activity indicates that, the new antifungal is unaffected by efflux pump due to antagonist being extruded from the cell or a cell remodeling occurs, increases the antifungal activity. Figure 1.2: Shows interaction between inducers of efflux pumps with standard antifungals and bioactive extracts and possible outcomes. Also, the inhibitors of efflux pump will be used in an interaction study with standard antifungals and extracts that are initially inactive but active on combination with various MICs 5 University of Ghana http://ugspace.ug.edu.gh of fluconazole (figure 1.3). These extracts will be referred to as chemosensitizers (ChemoS). An increase or decrease in the activity of standard antifungals could serve as a strategy for studying reversal of resistance or strategy for inducing cellular resistance respectively. On interaction of ChemoS with efflux pump inhibitors, a decrease, increase or maintenance of activity of the ChemoS could indicate the ChemoS require efflux pump for activity, it is activated by efflux pump or is unaffected by efflux pump respectively. On the other hand, a decrease, increase or maintenance of activity of the ChemoS could indicate the ChemoS is affected by accumulated antagonist or affected by cell remodeling due to the inhibitor or affected by newly induced class of efflux pumps; it accumulates and activity increases with efflux pump inhibition; or is unaffected by efflux pump respectively. Figure 1.3: Shows interaction between inhibitors of efflux pumps with standard antifungals and inactive extracts but active on sub-MIC of fluconazole and possible outcomes 6 University of Ghana http://ugspace.ug.edu.gh In figure 1.4, on interaction of ChemoS with efflux pump inducers, a decrease in activity could inform a strategy for screening for new ChemoS to overcome efflux pump-based resistance. An increase in the activity of the ChemoS indicates its activity could be due to antagonist is extruded from the cell or cell remodelling increases its activity. Maintenance of activity of indicates the new ChemoS is unaffected by efflux pump overexpression. Figure 1.4: Shows interaction between inhibitors of efflux pumps with standard antifungals and inactive extracts but active on sub-MIC of fluconazole and possible outcomes Finally, in figure 1.5, a summarized and general project concept. To begin, extracts obtained from soil borne and terrestrial endophytic fungi, (SBF and TEF) will be tested against C. albicans and S. cerevisiae using the disc diffusion method. Extracts that turn out to be active will be tested in the presence of modulators of efflux pumps, thus both inducers and inhibitors of efflux. Extracts that still maintains their activities under these conditions will be termed as special active extracts. Here, they are suspected to contain novel and potent antifungal 7 University of Ghana http://ugspace.ug.edu.gh compounds, since they able to maintain antifungal properties under such stringent conditions. However, extracts that do not show any bioactivity would be tested against C. albicans and S. cerevisiae in the presence of sub-inhibitory concentrations of fluconazole. Active extracts here will then be termed as chemosensitizers (ChemoS). Chemosensitizing extracts will also be screened in the presence of efflux modulating compounds and other phenotype modifiers to identify new compounds that modulate efflux to cause fluconazole sensitivity. Also, molecular analysis of phenotypic patterns of organisms will be done to identify genes that are involved in phenotypic changes in the presence of compounds. SBF and TEF extracts that show interesting activities would be taken through natural product isolation. Figure 1.5: Shows a summarized general experimental design. 8 University of Ghana http://ugspace.ug.edu.gh List of Selected Stress Response Genes for RT-PCR Analysis STRESS GENES SELECTED Hyperosmotic Stress gpd1, ena1 Oxidative Stress trx1, cta1 Heat inducible hsp70, hsp78 Carbohydrate reserve metabolism gph1, glc3 Antifungal stress response mdr, cdr1, cdr2 and CaMDR1 Sporulation Dit1, Spr3 Hyphae development Cla4 ,Flo11 1.3 Aim: To determine resistance phenotypes in C. albicans using efflux pump modulators and to apply the lessons to the isolation of novel fungicidal and chemosensitizing agents from TEF and SBF extracts. 1.4 Specific Objectives: 1. To determine resistance phenotypes in the presence efflux pump modulators in C. albicans and S. cerevisiae using disc diffusion. 2. To analyze the expression pattern of mdr/cdr genes and stress response genes using RT- PCR and the rhodamine 123 (Rh123) transport assays. 3. To screen and select a priority set of bioactive SBF and TEF extract for product isolation based on the interaction with the mdr/cdr modulators. 4. To screen for chemosensitizers from non-active extracts using MIC-infused fluconazole agar and select a priority set for product isolation based on the interaction with the mdr/cdr modulators 9 University of Ghana http://ugspace.ug.edu.gh 1.5 Rationale Fungi in the genus Candida mostly cause opportunistic infections in humans including mucosal, cutaneous, and systemic infections. They are also known to causes serious diseases in immunocompromised patients such as those with cancer, transplants or human immunodeficiency virus (HIV) infection. An estimate of 40% of candedemia results in deaths and Candida albicans are known to contribute nearly half of these cases (Pfaller and Diekema 2007). The limited number of antifungals in clinical use and the misuse of available ones have led to increased antifungal resistance which is a global menace (Pfaller 2012). High mortality rate resulting from C. albicans infections are mainly attributed to limited collection of antifungal agents ((Ła̧cka et. al, 2015). New and potent antifungal agents are therefore urgently needed. The development of novel antifungals especially from natural sources is necessary due to the considerable reduction in toxic side effects of natural products. Studies have shown that endophytic fungi produce secondary metabolites that are potent anticancer and antibacterial agents. This coupled with their abundance in nature makes them a good source of novel and potent antifungal compounds. Evidence of increase in sensitivity of Candida sp. to a the combination fluconazole and FK506 (Reedy, Filler et al. 2010) suggests that, development of novel antifungal chemosensitizers that increase potency against C. albicans may offer alternative ways to overcome fungal infections and to prolong the commercial life of antifungals. Extracts obtained at the end of the study will be potent antifungals that work by overcoming expression of efflux pumps, which is the main route of antifungal resistance. 10 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 C. albicans Infections Several fungal species are known to cause serious adverse health conditions in humans, which when left untreated, could be fatal. Some of these diseases include aspergillosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetomas, mucormycosis, and candidosis (Miceli, Díaz et al. 2011). The dermatophytic and keratinophilic fungi causes infections of the eyes, nails, hair, and especially skin. They are also known to cause mostly localized infections including athlete’s foot and ringworm (Badiee and Hashemizadeh 2014). Fungal spores also cause allergic reactions, with different taxonomic classes of fungi causing different allergies (Ruhnke 2014). Nearly 40% of fungal infections results in deaths and about half of these deaths are caused by Candida species (Picazo, González-Romo et al. 2008). Candida albicans infection constitutes the most common fungal pathogenic disease in humans. Candida albicans is mostly opportunistic human fungus that causes cutaneous, mucosal as well as systemic infections. In immunocompromised patients such as those with cancer, transplants or human immunodeficiency virus (HIV) infection C. albicans causes serious diseases. Naturally, C. albicans is part of the normal microflora in human body, existing as commensal organism. Within the body, it is found in diverse niches, including the urogenital tracts, gastrointestinal surfaces, skin and oral cavity of healthy individuals. However, in situations such as when the immune system is compromised, the fungus can survive in the bloodstream, resulting in its infection of certain vital internal organs including the spleen, liver, kidney and brain (Romani 2012). 11 University of Ghana http://ugspace.ug.edu.gh Candida infections is also known to be the fourth most common nosocomial infections, more than 50% of which could be fatal in some groups of patients. C. albicans are known to successfully adapt to a wide range of surfaces including patient catheters and artificially implanted heart valves (Nobile and Johnson 2015). Candida albicans belong to the phylum ascomycete, which also consist of Saccharomyces cerevisiae. S. cerevisiae has often been used as model for Candida albicans infections. S. cerevisiae is non-pathogenic and share high genetic similarity with C. albicans, although the organisms are known to be distantly related to S. cerevisiae (circa 150 million years) (Galagan, Henn et al. 2005). C. albicans, unlike other ascomycetes, do not undergo meiosis to generate spores. C. albicans exist in two main forms; the yeast forms are known for spreading of infection while the filamentous facilitates tissue penetration. A number of factors contribute to virulence and establishment of infection by C. albicans. These include hyphal formation and penetration of tissues. During establishment of an infection via hyphal penetration, the cells express various cell surface adhesins to help facilitate adherence to the host tissues (Brown, Budge et al. 2014). The adhesin, Als3 is specifically known to play a major role during brain infection by facilitating invasion of the brain endothelial cells (Sheppard and Filler 2014). Also, during renal infections, there is the induction of the expression of certain substances needed during assimilation of zinc and iron and this ultimately plays a role in virulence. To enhance pathogenicity during disease progression, certain groups of proteases including phospholipases and lipases are secreted, which modulates the host immune responses and provide nutrients for fungal growth (Pietrella, Rachini et al. 2010). 12 University of Ghana http://ugspace.ug.edu.gh 2.2 Current Antifungals in clinical use, cellular targets, toxicities and the Development of Resistance Research efforts have extensively dedicated time and resources in an attempt to discover new antifungals. Despite this, there are only a few therapeutic agents available to treat fungal infections. Currently there are only four main classes of agents accessible for clinical use during the treatment of invasive fungal diseases. Although other classes including the morpholines and allylamines are available, these are only used topically owing to their unwanted side effects and low to negligible effectiveness when used to treat systemic infections (Davis 2005). The four main molecular classes of antifungals target three separate metabolic pathways in fungal cells. Fluoropyrimidines, polyenes, azoles and echinocandins constitute the four main classes. 2.2.1 The fluoropyrimidines The fluoropyrimidines are synthetic structural analogues of cytosine, a DNA nucleotide. An example of a fluoropyrimidine is 5-fluorocytosine (5-FC) which is known to have a broad- spectrum antifungal activity. It is most known to be effective against Cryptococcus spp. and Candida spp. Using specified transporter proteins including cytosine permeases or the pyrimidine transporter, 5-FC enters fungal cells, where it is metabolised into 5-fluorouracil, using the enzyme cytosine deaminase which in turn is metabolised to 5- fluorouracil monophosphate (5-FUMP) using uridine phosphoribosyl transferase (UPRT). 5-FUMP is further converted to 5-fluorouracil triphosphate, to be inserted into the strand of RNA, instead of UTP during RNA synthesis. This then results in protein synthesis inhibition. Alternatively, 5-FC can be converted to 5 fluorodeoxyuridine monophosphate, an inhibitor of thymidylate synthase. Thymidylate synthase is a crucial enzyme involved in DNA metabolism, thus 13 University of Ghana http://ugspace.ug.edu.gh inhibiting the fungal cell replication (Vandeputte, Ferrari et al. 2011). In spite of several known pharmacological advantages such as small molecular size and high-water solubility, there has been decreased clinical use of 5-FC due to regular occurrence of acquired or innate resistance. 2.2.2 The polyenes The bacteria belonging to Streptomyces spp. are known to produce a wide range of chemical compounds with antifungal activities belonging to the polyenes class of chemicals. Three of these compounds, however have been confirmed to be cytotoxic to fungal cells and are currently employed in clinical management of fungal infections; amphotericin B (AmpB), nystatin and natamycin. Targeting of ergosterol, which is the major sterol component making up the cell membrane of fungi is the mode of action polyenes. The polyenes are amphipathic in nature which facilitates their binding to the lipid bilayer, forming pores in the membrane. For over four decades, AmpB was the gold standard for systemic fungal infections due to its wide spectrum of activity, and low incidence of recorded resistance (Dodgson, Dodgson et al. 2004). AmpB is employed to treat infections due to Aspergillus spp., Rhizopus spp., Candida spp., Mucor and Fusarium spp. thus, AmpB treats infections caused by both yeast and filamentous fungi. 2.2.3 The azoles Azoles represent the most frequently used antifungal. Azoles inhibit lanosterol 14-alpha demethylase which is encoded by the ERG11 gene. This is a vital enzyme during the biosynthesis of ergosterol. Inhibition of his enzyme leads to the build-up and metabolism of 14-alpha methylated sterols resulting in synthesis of toxic compounds, consequently depleting ergosterol amounts in the cell membrane, leading to high cellular permeability and eventually, cell death (Martel, Parker et al. 2010). Fluconazole has been the gold standard for treating 14 University of Ghana http://ugspace.ug.edu.gh fungal infections since its introduction in the 1990s as it is perceived to provide several advantages over the previously used clotrimazole, econazole, and miconazole whose use was limited to topical applications owing to their high toxicity upon oral administration (Delye, Laigret et al. 1997). Fluconazole is used to treat a broad range of infections, including disseminated, superficial and cutaneous candidiasis, cryptococcal meningitis and coccidioidomycosis. Its highwater solubility allows its intravenous administration, complete absorption through the gut and diffusion to various parts of the human body, including cerebrospinal fluid (Martel, Parker et al. 2010). However, over prescription of this drug for prophylaxis or treatment has resulted in an increased resistance to azole drugs. 2.2.4 The echinocandins Within the past one and half decade, echinocandins have remained the most recently discovered antifungals in used in clinics for treatment of fungal infections that are invasive. Caspofungin, micafungin and anidulafungin are the three echinocandins in current use. They function by non-competitive inhibition of β(1-3)- glucan synthase, therefore, preventing the synthesis of β(1-3) glucan, consequently affecting cell wall rigidity and integrity of fungi (Pfaller, Boyken et al. 2008). Poor absorption of echinocandins in the gut due their high molecular weight restricts their use to intravenous only. 2.2.5 Resistance mechanisms Various fungal species have however developed various mechanisms of resistance against these groups of antifungals. These mechanisms include: Drug efflux via upregulation of protein pumps. In Candida albicans, the ATP Binding cassette, ABC transporters including CDR1 and CDR2 are known to be the main efflux proteins. These proteins mainly provide resistance 15 University of Ghana http://ugspace.ug.edu.gh against the azoles. These protein pumps reduce the concentration or prevent the accumulatio of the drugs in the cell, rendering them ineffective. Mutation of drug target is also another mechanism of drug resistance. This leads to decrease in affinity of the drug for the target. This happens in the case of azoles where a point mutation in ERG 11, coding for lanosterol 14α-demethylase, results in decreased affinity (Miceli, Díaz et al. 2011). Another mechanism is via the deregulation of the drug target. C. albicans on exposure to azoles transiently upregulates the ERG family of gene, this leads to decreased drug susceptibility. 2.3 Fungi as Sources of Novel Antimicrobials and Antimicrobial Chemosensitizers In the search for novel bioactive compounds to be developed into new drugs by industrial pharmaceuticals, natural products have proven superior to combinational chemistry (Schulz, Boyle et al. 2002). Organisms that inhabit a particular biotope produce natural compounds that are adapted to and perform specified roles in its environment. Schulz et al, 2002 confirms that, there exist a correlation between biological activity and biotope. The quest for new bioactive secondary metabolites which concentrates on species inhabiting particular biotopes is therefore most likely to be successful and yield very potent compounds. Soil borne fungi (SBF) and terrestrial endophytic fungi (TEF) inhabit such a biotope. Endophytic fungi for the production of various bioactive metabolites of different chemical classes including the cytochalasines, steroids, chinones, phenols, isocoumarins, terpenoids and xanthones with diverse anticancer, antimicrobial and antiviral activities (Li et al., 2005). Recently, it has been estimated that, fungal species is 1.5 million globally (Hawksworth 2001). A reported percentage of only 10 out of the 1.5 million, are known and revealed and described, while scarcely 1% have been studied for their secondary metabolites production capacities (Hawksworth 2004). 16 University of Ghana http://ugspace.ug.edu.gh 2.4 Utilizing small molecules to probe antimicrobial phenotypic resistance Exposure to small molecules imposes stress on cells and are recognized as stress in Candida albicans, therefore, in response to the stress, various changes are made in the genome, eventually conferring antifungal resistance (Cowen and Steinbach 2008). Thus, certain small molecules induce antifungal resistant phenotypes in Candida albicans. On the other hand, another group of small molecules, sometimes referred to as resistant breakers or chemosensitizers induce sensitivity of fungal pathogens to the action of antimicrobials. These two groups of chemicals include metabolites, other antimicrobials and non-antimicrobial drugs. It is therefore important to consider the influence of these molecules on fungal cells in an attempt to discover new and potent antifungal agents that can stand the test of time. Traditionally, antifungal resistance and general antimicrobial resistance are studied mostly based on genetic alterations in pathogens and seldom via phenotypic factors. Phenotypic modifications have been found to contribute immensely to antifungal resistance in fungal pathogens, specifically, Candida albicans. Historically, small molecules have been known to alter the genotype as well as phenotype of yeast cells (Shareck and Belhumeur 2011). In Saccharomyces cerevisiae, genome-wide studies done after exposure to drugs have been known to induce the expression of genes that are implicated in drug resistance (Karababa, Coste et al. 2004). Similarly, exposure to specific small molecules revealed clusters of genes, collectively regulated under a common regulatory element recognized by the same transcription factors (Bamford, d'Mello et al. 2009). Phenotypic switch from yeast to hyphal forms is a well-known phenomenon which is essential in Candida albicans biology. This particular characteristic has been implicated in C. albicans virulence via various mechanisms. The yeast to hyphae conversion has been linked to 17 University of Ghana http://ugspace.ug.edu.gh adherence, tissue invasion, biofilm formation, phagocyte escape, and pathogenesis (Shareck and Belhumeur 2011). Farnesol, a quorum sensing molecule has been known to be secreted by a number of Candida laboratory strains as well as clinical isolates. In Candida albicans, farnesol is known to inhibit yeast to hyphal transition (Hornby and Nickerson 2004). N-acetylglucosamine is known to induce farnesol secretion, which then blocks hyphal growth (Hornby, Jensen et al. 2001). Another small molecule that is known to affect morphology of C. albicans is propranolol, an inhibitor of calmodulin (Shareck and Belhumeur 2011). This is shown to inhibit formation of hyphae via reduction of EFG1 expression levels in the presence of serum (Toenjes, Stark et al. 2009). Similarly, nisin Z, an antimicrobial peptide reduces germ tube formation as well as adhesion of C. albicans cells to gingival monolayer cultures in the presence of serum (Akerey, Le‐ Lay et al. 2009). Thus, in C. albicans, these molecules could increase drug susceptibility as hyphal formation is a known virulence factor in C. albicans. In a study conducted by Bachewich and colleagues in 2005, hydroxyurea a compound that causes cell cycle arrest by inhibiting ribonucleotide reductase leading to the depletion of ribonucleotides and therefore inhibiting DNA synthesis was also noticed to trigger hyperpolarized growth of C. albicans cells. Upon exposure to hydroxyurea, the candida cells developed pseudohyphal morphological features (Bachewich, Nantel et al. 2005). Thus, based on this observation, development of virulent features which could led to drug tolerance or resistance could result in the presence of this small molecule. Thus, profiling the resistance or susceptibility phenotypes of C. albicans in the presence of different phenotype modifying compounds during the search of new antifungals could lead to the discovery potent drugs. 18 University of Ghana http://ugspace.ug.edu.gh In this current study, a panel of small molecules that modulate the activity of efflux pumps as well as other phenotype modifying compounds are used to probe and study resistant phenotypes in C. albicans. Various resistant genes induced and expressed in the presence of each compound would be analyzed so as to have an idea of resistance pathways and associated genes and genes products. This observation and knowledge will subsequently be used to identify potent antifungal agents to overcome any possible mode of resistance to our identified compound. 2.5 Selecting molecular drug targets based on analysis of stress response. It is well-known that most antifungal drugs in clinical use fail because either they are toxic or are no longer effective due to resistance. In view of this, target identification and selection constitute a significant step in the drug discovery process. Targets broadly refers to proteins, DNA and RNA of the pathogens which are directly involved in the pathology of the disease. One of the desirable qualities of a good target is its ability to induce biological response both in vivo and in vitro. A good target identification and validation process therefore gives an indication of how well aiming at a specific target would lead to elimination of a disease condition. Various methods that have been used in target selection include; data mining which involves the use of bioinformatics in detecting, choosing and prioritizing potential targets of disease conditions from gene expression data, proteomics data, transgenic phenotyping and compound profiling data (Yang, Adelstein et al. 2012). Alternatively, target based phenotypic screens are done to find molecular targets that play major roles the disease condition of interest (Kurosawa, Akahori et al. 2008). Small molecules are significant to the survival of organisms. Some of the roles small molecules play include providing nutrients to support growth, facilitate cell-cell communication and exert toxicity towards other organisms in the same niche that may be competing for survival. 19 University of Ghana http://ugspace.ug.edu.gh However, in pathogenic organisms, the presence of certain small molecules is seen as stress (Toenjes, Munsee et al. 2005). In response to this stress, transient changes are made in the genome in order to cope with the stress. Some of these genetic changes in response to stress have also been implicated in the development of drug resistance. Based on the fact that genetic changes in response to small molecule stress are able to induce drug resistance, we propose a new method of target identification which involves inducing small molecules stress, then analyzing and identifying unique genes whose expression are induced or repressed during response to the stress, enabling survival of the organism. Blocking small molecule stress by targeting such genes is an indication of good therapeutic target Analysis of these stress responses are done so as to give an indication of an ideal or most appropriate druggable target in the pathogen. This method, thus subjecting pathogens to various stress conditions, analyzing stress responses and subsequently selecting an appropriate target is advantageous as it has the potential of being a novel target and also on that would be able to withstand resistance. Efflux pump genes are known to be expressed in the presence of small molecule stress (Rogers and Barker 2002). Upregulation of the expression of these pumps allows extrusion of these molecules, alleviating the stress. In the presence of fluconazole, the transcription factor, Tac1p which regulates cdr1 and cdr2 are known to be highly expressed. Also, Mrr, Upc2 and Cap1 which are MDR1 regulators, are found to be highly expressed. Upc2p, the main gene that regulates ergosterol biosynthesis is known to upregulate the biosynthesis of ergosterol in response to azoles (Enjalbert, Nantel et al. 2003). Candida albicans are known to induce certain unrelated stress responses in order to accommodate chemical stress. Stress response genes including heat-shock response such as hsp12, hsp70, hsp78, and hsp104 as well as hsf1p a transcription factor, which is an important regulator during heat-shock have been found to be upregulated in chemical stress (Enjalbert, 20 University of Ghana http://ugspace.ug.edu.gh Nantel et al. 2003). The hyperosmotic stress genes ena1, ena2, gpd2 and gpp1 in C. albicans are also known to have an increasing level of expression in response to hyperosmotic shock. Enjalbert and colleagues also found out that several transcriptional regulators and signaling molecules are required for acquired oxidative resistance after hyperosmotic stress although these stresses are relatively unrelated. These include the stress-activated transcription factor msn2 and the a number of HOG signaling components (including hog1, pbs2, ssk2, ssk1, ste50, and cdc42). Similar to the above stated stress responses, several other stress response genes are also known to be expresses including that of oxidative stress, sporulation and hyphae development. The fact that small molecules are recognized as stress, are able to induce remarkable changes in cellular signaling and are everywhere in an organism’s surroundings, makes it important to probe into their role in cell function. Analyzing the types and levels of stress response genes induced by organisms during target selection is imperative. This reason is based on the fact that, a target which may be perceived as a good one could also be rendered ineffective by stress coping mechanisms induced by the organism via expression of alternative proteins (Shapiro, Robbins et al. 2011). Here, performing molecular analysis of stress response genes prior to selecting a target would help avoid resistance via mechanisms that permit organisms to survive drug-induced stress. These mechanisms include altering the target to block drug binding or increasing the production of multidrug transporter proteins to extrude the drug compounds outside the cell. 2.6 Use data to guide selection and isolation of new bioactive compounds On selecting and validating a target, hit compounds are identified by use of various screening assays. During these assays, compounds with the desired characteristics and whose activity 21 University of Ghana http://ugspace.ug.edu.gh could be confirmed after retesting are selected as leads. High throughput screening against the selected drug target is used to identify potential hits, which is usually followed by further secondary screens for confirmation (Fox, Farr-Jones et al. 2006). In the discovery of new bioactive compounds from fungi, extracts are usually subjected at the same time to chemical screening and to various biological or pharmacological targets. Traditionally, activity guided fractionation has been used in the isolation of bioactive compounds. Here all fractions are biologically tested and those that continuously show activity are taken through various fractionation and isolation methods. However, during this process there could be re-isolating existing compounds. Here, we propose that patterns of expression of stress response genes in the presence of different compounds can be used in selecting a potent antifungal. This is done on the basis that, compounds that do not induce or prevent the expression of many virulent stress response genes but are yet fungicidal could potentially be very good antifungals as the development of resistant against such compounds via alternative genetic pathways and protein expressions could be avoided. Also, compounds that maintain their activity in the presence of expression of various virulent genes could be considered as potent as their fungicidal activity is maintained and unaffected by expression of stress response genes. Based on the target selection technique discussed earlier, certain classes of compounds could be found to target a specific group of stress response genes. This allows focused or knowledge- based detection and selection of molecules which are likely to yield potent bioactive compounds. Also, selection and isolation of bioactive compounds after molecular stress analysis allows profiling of each extract based on the molecular stress analysis profile they elicit. This would provide a means of dereplication of existing compounds. 22 University of Ghana http://ugspace.ug.edu.gh 2.7 Stress Responses to Antifungals Changes in the environment of fungi in the human host caused by intake of antifungal compounds poses physiological stress to which the pathogen has evolved responses to cope with the stress. Various fungal species respond differently to the antifungal stress, depending on the target of the drug. Well known antifungal resistant mechanisms include genetic mutations, mostly, point mutation in drug targets, metabolic pathway enzymes or transcription factors which results in overexpression of the gene (Cowen, Sanglard et al. 2014). Such genetic alterations occur over a long period of time and are usually stable. Yet, antifungals can initiate abrupt transient phenotypic stress responses. These temporary changes usually do not involve mutations or rearrangements in the genome and are thought to be reversible. However, there is a tendency for these short-term changes to become genetically stable over time, conferring fitness (Sanglard, Coste et al. 2009). Osmotic or membrane stress is produced in the presence of cell wall or membrane sterol biosynthesis antifungals. An example of such antifungal is amphotericin B. Such membrane stress induced by azoles is via the protein kinase C (Mkc1p) component of the MAPK pathway. There is also evidence of induction of oxidative stress in C. albicans on exposure to certain groups of antifungals. This signal is induced through the transcription factor CaCap1p. CaCap1p has been implicated in Candida multidrug resistance (Cannon, Lamping et al. 2009). The major facilitator superfamily (MFS) transporter gene CaMDR is upregulated upon activation of CaCap1p by a C-terminal truncation of the protein. Another pathway that is known to be involved in antifungal stress response in C. albicans is the cyclic AMP-protein kinase A signal transduction pathway. Here, adenylate cyclase mutants with overexpressed CaCDR1 are found not to respond to azoles (Cannon, Lamping et al. 2009). 23 University of Ghana http://ugspace.ug.edu.gh Calcineurin has been reported to mediate responses to a number of stress conditions in C. albicans. These include high salt concentrations and high pH. In addition to this, during membrane stress caused by the azole group of antifungals, calcineurin has been found to play an essential role in averting the stress, allowing survival of the organism. Inhibition of calcineurin in C. albicans results in fungistatic azoles becoming fungicidal, thus, this provides a means of abolishment of drug tolerance. In accordance to this, the immunosuppressant, cyclosporin A, which inhibit calcineurin has be found to synergize with fluconazole (Bader, Bodendorfer et al. 2003). 2.8 Antifungal Chemosensitization To Overcome Efflux Pump-mediated Resistance An expected characteristic of a good antifungal is its ability to avoid efflux-mediated resistance. Four main approaches have been proposed to overcome antifungal resistance caused by efflux. First of these is the use of antifungals that are not efflux pump substrates, an example of which are the echinocandins and polyenes. The next is the use of small molecules or other agents that prevent efflux by inhibiting the pumps, thereby protecting the efficacy of the drug. The third is to inhibit H+ ATPase in the cell membrane, depleting the energy required for drug efflux out of the cell. The last approach involves the use of a compound whose uptake exceeds the rate of its efflux out of the cell, allowing accumulation of the antifungal at high therapeutic doses. Chemosensitization has however been proposed as a potent way of overcoming antifungal resistance. Here, new compounds are combined with existing and failing antifungals in order to improve the efficacy of such antifungals. Screening compound libraries to identify transporter inhibitors that chemosensitize fungal pathogen to antifungals that are substrates of efflux proteins have yielded a number of candidate molecules. These include FK506, milbemycins, enniatin, beauvericin and 24 University of Ghana http://ugspace.ug.edu.gh unnarmicins, as specific ABC pump inhibitors (Holmes, Cardno et al. 2016). However, despite the high number of promising compound screens for chemosensitizers, there has not been any success in providing one for clinical use. This situation therefore calls for more investigations. 25 University of Ghana http://ugspace.ug.edu.gh Table 2.1. List of compounds used in this study and information from literature 26 University of Ghana http://ugspace.ug.edu.gh Table 2.2. List of compounds used in this study and information from literature 27 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Chemicals and Reagents Antifungal agents and chemical compounds were purchased from Sigma Aldrich and Carl Roth, unless otherwise indicated. The compounds included 7 efflux pump modulating compounds. In addition, 17 other compounds, some of which are known efflux modulators in bacterial cells were also included. The purpose of these other compounds was to serve as further potential sources of phenotype modifiers in yeast as they have different functional groups, have different cellular targets and also are able to induce cellular stress and morphological changes in biological cells. Since the effects of the 17 other compounds on C. albicans and S. cerevisiae are not yet known, they are identified throughout this work by their codes listed in table 3.1. 28 University of Ghana http://ugspace.ug.edu.gh Table 3.1: List of compounds used in this study 29 University of Ghana http://ugspace.ug.edu.gh Table 3.2: List of antifungals and concentrations 3.2 Fungal strains used Two different types of yeast were used: Candida albicans and Saccharomyces cerevisiae. The cells were stored and maintained on YPDA (Yeast extract Peptone, Dextrose and granulated Agar) slants at 4 °C and were sub-cultured on YPDA plates before the bioassay. 3.3 Inoculum preparation Starter cultures were prepared by inoculating a single loop of organism into a conical flask containing 50 ml of nutrient broth. The culture was incubated in an incubator shaker at room temperature (30 °C) for 24 hours. The 24-hour culture was then inoculated into the seed culture 50 ml nutrient broth at an O.D600 of 0.01 and also incubated in an incubator shaker at room temperature for 24 hours. 30 University of Ghana http://ugspace.ug.edu.gh 3.4 Determination of Minimum Inhibitory Concentrations (MICs) of compounds and Phenotypic Array of Organisms in The Presence of Compounds The disc diffusion assay was used to determine the MICs of all compounds as well as and determination of phenotypic patterns of C. albicans and S. cerevisiae. The O.D600 of the 24 hour cultures of C. albicans and S. cerevisiae from the inoculum preparation were adjusted to 0.7. Cells were spread on YPDA plates using a sterile swab stick. Whatman filter papers were cut into 6 mm disc and autoclaved. The discs were then infused with compounds (listed in table 1) to achieve a concentration of 40, 20, 10, 5, 1 and 0.5 ul per disc. The infused discs were placed on the YPDA plates with the organisms at room temperature (30 °C) for 24 hours. Zones of inhibition around each disc in mm were then measure using a ruler. To determine changes in resistance or susceptibility phenotypes of C. albicans and S. cerevisiae in the presence of various efflux modulating and phenotype modifying compounds, YPDA plates were modified with one-eighth or one-fourth of the minimum inhibitory concentration of each of the compounds determined earlier. Antifungals, each at its effective concentration (listed in table 2) were impregnated onto sterile 6 mm Whatman filter paper disc and were then placed on the modified YPDA plates. Plates were incubated for 24 hours at room temperature and zones of inhibition measured. These served as controls. 3.4.1 Determination of breakpoint Concentration of Unique Interactions Compound-antifungal pairs that showed unique resistant breaking or resistant inducing pairs were assayed in a decreasing or increasing concentration manner respectively using the disc diffusion methods as described. Compound-antifungal pairs analysed were PC04-11 and 5- 31 University of Ghana http://ugspace.ug.edu.gh fluorouracil (5-FU), PC04-18 and 5-5-FU and PC04-15 and 5-FU for resistant breaking while PC04-0 and Fluconazole (FLU), PC04-16 and FLU and rifampicin and Sertraline (SET). Amounts of compounds PC04-11, PC04-18 and PC04-15 were 0.25, 0.125, 0.0625, 0.03, 0.015, 0.007 ug per disc while that of 5-FU was 0.25 ug/uL. Amounts of compounds PC04-09 and PC04-16 used were 40, 20, 10, 5, 2.5 and 1.25 ug per disc. Amount of rifampicin used were 800, 400, 200 and 100 ug per disc, while that of fluconazole was 1.25ug/uL. Briefly the antifungals were spread on YPDA plates, C. albicans cells with O.D600 of 0.7 were the spread on the plates and 6 mm sterile filter paper discs impregnated with the indicated concentrations of compounds were placed on the plates. Standard antifungals were included as controls. Plates were incubated overnight at room temperature and zones of inhibition was taken. 3.4.2 Alamar Blue Assay Compound-antifungal pairs interaction was also confirmed in liquid medium using the alamar blue assay. Briefly, 100 uL C. albicans and S. cerevisiae cells at O.D600 of 0.7 were pipetted into 96-well plates, each to a final concentration of 5 ug/uL. Antifungals at indicated concentrations were added to each well. Plates were incubated for 4 hours. Alamar was added and florescence measured at times 0, 30, 45 and 60 minutes at excitation and emission wavelengths 545 nm and 590 nm respectively. 3.5 Measurement of Efflux Activity in the presence of Efflux Modulating and Phenotype Modifying Compounds The efflux activity of all the 24 phenotypic compounds were analysed using the Rhodamine 6G efflux assay, adopted from (Sun, Cheng et al. 2010) with modification. 32 University of Ghana http://ugspace.ug.edu.gh Starter cultures of C. albicans and S. cerevisiae cells were prepared by inoculating a single loop of organism into a conical flask containing 50 ml of nutrient broth. The culture was incubated in an incubator shaker at room temperature (30 °C) for 24 hours. The 24-hour culture was then inoculated into the seed culture 50 ml nutrient broth at an O.D600 of 0.01 and also incubated in an incubator shaker at room temperature for 24 hours. 3.5.1 Measurement of Rhodamine 6G Uptake To measure uptake of Rhodamine 6G dye, C. albicans and S. cerevisiae cells grown overnight were again inoculated into a 100 ml nutrient broth at od of 0.5 and incubated at room temperature for 4 hours to an OD of 0.7. Cells were centrifuged at 4000 g for 5 min in 50 ml falcon tube. Pellets were washed twice with physiological saline, then resuspended in saline. An aliquot of 960uL cells was made into 24 test tubes. A volume of 40uL of 2.5Ug/ul of each compound was added to the cells except the control tube and Rh 6G was added at a final concentration of 10 µmol/L at the same time. The reaction was incubated for 4hours. Each tube was centrifuged at 15000 g for 5 min and briefly washed two times with saline. After external Rh6G was removed, cells were resuspended in 1.5ml saline and 100ul of each reaction mixture was Pipette into 96-well plate in triplicates. Fluorescence was measured at excitation and emission wavelengths of 518 and 543 nm respectively. Further experiments were done by adding deferasirox during rhodamine 6G uptake. This was done to increase uptake of the dye. Here the same procedure described above was used. 33 University of Ghana http://ugspace.ug.edu.gh 3.5.2 Measurement of glucose-induced efflux To measure ATP-driven efflux, cells were centrifuged and washed twice to remove any external rhodamine, 1% glucose was then added to each tube and cells are incubated at room temperature for 40mins. Each tube was then centrifuged at 15000 g for 5 min and pellets were briefly wash two times with saline. Fluorescence was measured at excitation and emission wavelengths of 518 and 543 nm respectively. In a separate experiment, the above procedures were repeated, however, the same amount of compounds were added during the ATP-driven efflux step in order to effectively measure efflux. 3.6 Chemosensitization of C. albicans to fluconazole by SBF and TEF Fungal Extracts A library of 507 TEF and SBF fungal extracts were initially screened for antifungal activity against C. albicans and S. cerevisiae using the disc diffusion assay as previously described. The ability of inactive TEF and SBF fungal extracts to sensitize C. albicans to the antifungal activity of sub-inhibitory concentration of fluconazole was analyzed by the disc diffusion assay. Inoculum preparation: Starter culture of C. albicans was prepared by inoculating a single loop of organism into a conical flask containing 50 ml of nutrient broth. The culture was incubated in an incubator shaker at room temperature (30 °C) for 24 hours. The 24-hour culture was then inoculated into the seed culture 50 ml nutrient broth at an O.D600 of 0.01 and also incubated in an incubator shaker at room temperature for 24 hours. The O.D600 of the 24 hour culture was adjusted to 0.7. 34 University of Ghana http://ugspace.ug.edu.gh Whatman filter papers were cut into 6 mm disc and autoclaved. The discs were then infused with 40 ul SBF and TEF extracts. To determine sensitization of C. albicans to fluconazole, YPDA plates were modified with 2.5ug fluconazole. Cells with O.D600 0.7 were spread on YPDA plates using a sterile swab stick. The infused SBF and TEF extracts discs were placed on the YPDA plates with the organisms at room temperature for 24 hours. Zones of inhibition around each disc in mm were then measured using a ruler. Antifungals, each at its effective concentration (listed in table 2) were impregnated onto sterile 6 mm Whatman filter paper disc and were then placed unmodified YPDA plates. Plates were incubated for 24 hours at room temperature and zones of inhibition measured. These served as controls. The influence of chemosensitizing extracts on the efflux activity of C. albicans and S. cerevisiae were analyzed by the Rhodamine 6G efflux assay as previously described, using 10ul of extracts. 35 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1 Determination of minimum inhibitory concentrations of Efflux Pump Modulators and other phenotype modifying compounds The minimum inhibitory concentrations (MICs) and the phenotypes of S. cerevisiae and C. albicans in the presence of all 7 efflux pump modulating compounds were determined. In addition, the MICs of 17 other compounds, some of which are known efflux modulators in bacteria cells was also determined. The purpose of these other compounds was to serve as further potential sources of phenotype modifiers in yeast as they have different functional groups, have different cellular targets and also are able to induce cellular stress and morphological changes in cells. Here most of the MICs obtained for the efflux modulating compounds were all above 40ug/ul for both C. albicans and S. cerevisiae, with the exception of 4-notroquinoline oxide and 1,10-phenanathroline which were 1 ug/ul and 20 ug/ul respectively in both organisms. Thus, most of the compounds were not fungicidal on their own even at high concentrations with the exception of 4-notroquinoline oxide. Similar observations were made with the other group of phenotype modifying compounds. Also, compounds PC04- 10, PC04-11, PC04-12 and PC04-14 were found to have MICs of 20 ug/ul for both C. albicans and S. cerevisiae. PC04-14 had an MIC of 10 ug/ul for S. cerevisiae. 36 University of Ghana http://ugspace.ug.edu.gh Table 4.1. Minimum inhibitory concentrations (MIC in ug/uL) of compounds The minimum inhibitory concentrations of all compounds were determined using the disc diffusion method. 37 University of Ghana http://ugspace.ug.edu.gh 4.2 Determination of antimicrobial phenotypes in the presence of efflux pump modulators and other phenotype modifying compounds In this study, eight antifungal compounds were used. These include amphotericin B (AmpB), cycloheximide (CyH), paramomycin (Para), benzoic acid (Ben), fluconazole (Flu), 5- fluorouracil (5-FU), griseofulvin (Gri) and sertraline (Set). There was no observed antifungal activity in the presence of CyH, para, Ben and Gri in both the control and modified plates. However, the antifungal activities of AmpB, 5-FU and Set were modified to different extents in the presence of efflux modulators and phenotype modifiers as compared to the control. On analysis of phenotypic switch of the fungal cells in the presence of efflux modulators and various phenotypic modifiers, it was realized that, generally, the compounds had higher effects on both the susceptibility and resistant patterns of S. cerevisiae as compared to C. albicans. Analyzing phenotypic changes in the two organisms separately, both C. albicans and S. cerevisiae cells were found to be more resistant to the activity of antifungals in the presence of the chemical compounds than were susceptible. However, cumulatively, S. cerevisiae was found to be more resistant (Tables 4A and 4B). The term ‘partial zone of clearance’ as used here is defined as where thin layer of organisms is observed within the zone of clearance around an antifungal disc. 4. 2.1 Antifungal resistance or susceptibility patterns of C. albicans with 7 Modulators of Efflux Pumps The antifungal activity of amphotericin B was increased from 7 mm to 10 mm in the presence of rifampicin. Also, rifampicin boosted the activity of fluconazole, giving a clear zone of 13 mm instead of the observed 17 mm partial zone (Appendix table 1.1). However, rifampicin also rendered sertraline resistant to C. albicans. Following compound PC04-20, 1,10- phenanathroline monohydrate had the second highest effect on increasing the gave clear zones 38 University of Ghana http://ugspace.ug.edu.gh of 19 mm. From figure 1A, the activity of fluconazole was decreased while 4-nitroquinoline oxide increased the activity in C. albicans. Estradiol was also observed to cause a decrease in C. albicans susceptibility to fluconazole. This observation was more pronounced in S. cerevisiae, where there was induction of total resistance to fluconazole in the presence of estradiol. Contrary to the observation made in estradiol, sulfometuron methyl was found to increase C. albicans sensitivity to fluconazole. 4.2.2 Antifungal resistance or susceptibility patterns of S. cerevisiae with 7 Modulators of Efflux Pumps In S. cerevisiae, all zones of clearance observed for the control plates were partial, thus a thin layer of organisms were observed in the zone of clearance around the antifungal disc. In figure 1B, estradiol induced resistance to amphotericin B, fluconazole and sertraline. Here there were no zones of clearance observed for amphotericin B, fluconazole and sertraline while there were partial zones of 11, 27 and 12 mm observed in the no treatment control. On the other hand, estradiol induced susceptibility to 5-fluoruracil (figure 1B), creating a zone of 11 mm whereas there was no clearance of the organisms in the control table 3a. In the presence of 1,10-phenanthroline monohydrate, resistance to amphotericin B and fluconazole was realized, producing no zone of inhibition in both cases while the control had partial zones of 11 and 25 mm respectively. Benomyl also induced susceptibility of S. cerevisiae to cycloheximide and 5- fluorouracil from no zone of clearance to zones of 7 and 9 mm respectively (Appendix table 1.2). Similarly, the activity of fluconazole was enhanced by 10 mm in the presence of benomyl. Benomyl however induced resistance to amphotericin B, reducing the zone of clearance from 11 mm to 0. Methotrexate induced the activity of 5-fluorouracil against S. cerevisiae, giving a clearance zone of 12 mm as compared to the control where there was no clearance of the organisms. Methotrexate however induced resistance to amphotericin B, inducing 0 zone of clearance as opposed to the 11 mm partial zone in the untreated plate (Figure 1B). 39 University of Ghana http://ugspace.ug.edu.gh 4.2.3 Antifungal resistance or susceptibility patterns of C. albicans with 17 Phenotype Modifiers In figure 1A, PC04-09 induced resistance to fluconazole from a partial zone of 17 mm to 0 mm. On the other hand, compound PC04-11 increased the susceptibility of fluconazole from a partial zone of 17 mm to a clear zone of 18 mm while that of sertraline was increased from 7 mm to 10 mm (Appendix table 1.3). Compounds PC04-10 and PC04-12 with fluconazole totally cleared the organisms, giving zones of 14 mm in both cases Compounds PC04-10 also induced susceptibility of C. albicans to 5-fluorouracil, creating a partial zone of 10 mm while there was zero clearance in the case of the control. Thus, in the presence of compound PC04- 09 there was induction of fluconazole resistance in C. albicans. Compound PC04-08 also induced amphotericin B resistant phenotype in C. albicans. In C. albicans, compound PC04-23 was found to have one of the most remarkable effect (figure 1A). In the presence of PC04-23, the susceptibility of C. albicans to fluconazole was increased by almost 58% while the was induction of the activity of 5-fluorouracil from zero clearance to a zone of 7 mm. Similarly, compound PC04-24 increased the zone of fluconazole from 17 mm to 24 mm as well as inducing the activity of 5-fluorouracil from zero clearance to give a 9 mm zone of inhibition (Appendix figure 1E). Also in figure 1A, compound PC04-16 and PC04-22 induced resistance of amphotericin B and fluconazole against C. albicans from zones of 7 mm to 0 mm and 17 mm to 0 mm respectively. However, compounds PC04-18 and PC04-20 increased the susceptibility C. albicans to the activity of fluconazole, creating a zone of clear 17 ad 26 mm respectively instead of the partial 17 mm zone observed in the control plate. Of all the compounds put together, compound PC04- 40 University of Ghana http://ugspace.ug.edu.gh 20 with fluconazole gave the highest effect on phenotype switching to a more susceptible phenotype. Reduction in the activity of some antifungals, thus induction of C. albicans resistance in the presence of the compounds were also observed. Compound PC04-15 induced total resistance to fluconazole, giving zero zone of clearance as compared to the control where a partial zone of 17 mm was observed. Same observation was also made in the presence of PC04-15. Thus, these two compounds caused C. albicans to switch from fluconazole susceptible phenotype to resistant phenotype (Figure 1A). 4.2.4 Antifungal resistance or susceptibility patterns of S. cerevisiae with 17 Phenotype Modifiers Compound PC04-11 had one of the highest influences on the susceptibility and resistance phenotypic pattern of S. cerevisiae (Figure 1B). PC04-11 induced activity of 5-fluorouracil against S. cerevisiae, creating a partial zone of 20 mm while there was no zone of inhibition in the control. In the presence of PC04-11 there was induction of activity of 5-fluorouracil from no zone of clearance in the control to partial zone of 20 mm in the treated plate. There was also the observance of a clear zone of 12 mm instead of the partial 11 mm zone observed in the control for amphotericin B (Appendix table 1.4). A clear inhibition zone was observed for fluconazole and sertraline with the influence of PC04-11 as compared to the 27 and 12 mm zones of inhibition observed in the controls respectively. Similarly, the antifungal. In table 4b, the compound PC04-08 increased the susceptibility of S. cerevisiae to 5-fluorouracil, creating a zone of 10 mm while no zone of inhibition was observed in the case of the control plates. The susceptibility of S. cerevisiae to 5-fluorouracil was induced in the presence of compound PC04- 13 and PC04-14 from no zone of clearance to a zone of 13 and 12 mm respectively. However, 41 University of Ghana http://ugspace.ug.edu.gh compound PC04-14 induced resistance to sertraline, creating no zone of clearance while a partial zone of 11 mm was observed in the control. Activity of sertraline was increased such that the 12 mm partial zone observed in the control became a clear zone in the presence of PC04-14 (Appendix table 1.4). In figure 1B, resistance to amphotericin B and fluconazole was induced in the presence of compound PC04-18. Nevertheless, compound PC04-24 induced susceptibility of S. cerevisiae to the activity of 5-fluorouracil from 0 mm to 11mm. 42 University of Ghana http://ugspace.ug.edu.gh Figures 1. A and B show antimicrobial Phenotype Modification in C. albicans and S. cerevisiae respectively. The various chemical compounds altered the antimicrobial resistant or susceptibility pattern of the organisms as shown. 43 University of Ghana http://ugspace.ug.edu.gh Figures 2. Shows sample assay plates from antimicrobial phenotype modification in S. cerevisiae (A) and C. albicans (B) and respectively. Phenotypic analysis was done using the disc diffusion method. 44 University of Ghana http://ugspace.ug.edu.gh 4.3.1 Ranking of phenotypic compounds in C. albicans Based on their resistance inducing or susceptibility inducing activities, the 24 compounds tested in this study were ranked according to their cumulative zones of inhibition as compared to controls. This revealed the most resistance or susceptibility inducing compounds. As shown in table 4.2, compound PC04-23 had the most resistance breaking activity in C. albicans, inducing susceptibility to fluconazole and 5-fluorouracil. Here a cumulative zone of inhibition f 17 mm was observed. This was followed by compound PC04-15, a known compound for the treatment of psychotic conditions including schizophrenia. This compound induced susceptibility to 5-fluorouracil, resulting in higher clearance of organism as compared to the control plate. Compound PC04-10 ranked third in the induction of susceptibility to C. albicans. Again PC04-10 also induced 5-fluorouracil susceptibility in C. albicans. Considering table 4.2, amphotericin was the least likely antifungal whose activity could be manipulated in the presence of other chemical compounds as it does not appear in the ranks at all. Meanwhile the antifungal activity of 5-fluorouracil was the most likely one to change in the presence of other compounds as seen. Compound PC04-04 was found to be the least in induction of fluconazole sensitivity to C. albicans. On analyzing resistance inducing activities in the presence of compounds, fluconazole was found to be the antifungal whose activity was most frequently repressed in C. albicans. Here compounds PC04-09, PC04-14, PC04-15 and PC04-16 were found to induce resistance most in C. albicans. Each of these compounds had a cumulative zone of inhibition of 17 mm, relative to the control plates where there was no modification with any compound. Here, compounds PC04-21 and PC04-02 were found to be the least in induction of resistance to C. albicans. Each had a cumulative zone of inhibition of 6 mm and 7 mm respectively. 45 University of Ghana http://ugspace.ug.edu.gh Table 4.2: Ranking of phenotypic compounds in C. albicans The table represent a cumulative score of the zones of inhibition in mm for resistance inducing and resistance breaking activities of various compounds in C. albicans 46 University of Ghana http://ugspace.ug.edu.gh 4.3.2. Ranking of phenotypic compounds in S. cerevisiae The resistance inducing or susceptibility inducing activities of the 24 compounds tested in S. cerevisiae were also ranked according to their cumulative zones of inhibition, relative to the controls. This revealed the most resistance or susceptibility inducing compounds. As shown in table 4.3, compound PC04-13 had the most resistance breaking activity in S. cerevisiae, inducing susceptibility three different antifungals. These included fluconazole, amphotericin B and sertraline. Here a cumulative zone of inhibition of 31 mm was observed. This was followed by benomyl, which also induced susceptibility to three antifungals including 5-fluorouracil, fluconazole and cycloheximide. Here, there were larger zones of clearance of organism as compared to the control plate. Compound PC04-08 ranked third in the induction of susceptibility to S. cerevisiae. Again PC04-08 also induced 5-fluorouracil and amphotericin B susceptibility in S. cerevisiae. Considering table 4.3, sertraline was the least likely antifungal whose activity could be manipulated in the presence of other chemical compounds. Meanwhile the antifungal activity of 5-fluorouracil was the most likely one to change in the presence of other compounds as seen. Compound PC04-21 was found to be the least in induction of fluconazole sensitivity to S. cerevisiae. On analyzing resistance inducing activities in the presence of compounds, fluconazole was again found to be the antifungal whose activity was most frequently repressed in S. cerevisiae. Here the efflux modulating compounds, namely estradiol, 1,10 phenanthroline monohydrate and sulfometuron methyl were found to induce resistance most in S. cerevisiae. Each of these compounds had a cumulative zone of inhibition of 50 mm, 38 mm and 27 mm respectively, relative to the control plates where there was no modification with any compound. Here, compounds PC04-21 and PC04-17 were found to be the least in induction of resistance to S. cerevisiae. Each had a cumulative zone of inhibition of 7 mm. 47 University of Ghana http://ugspace.ug.edu.gh Table 4.3: Raking of phenotypic compounds in S. cerevisiae The table represent a cumulative score of the zones of inhibition in mm for resistance inducing and resistance breaking activities of various compounds in S. cerevisiae. 48 University of Ghana http://ugspace.ug.edu.gh 4.4 Determination of breakpoint Concentration of Unique Interactions Compound-antifungal pairs that showed unique resistant breaking or resistant inducing pairs were assayed in a decreasing or increasing concentration manner respectively using the disc diffusion assay. Resistant breaking interactions are those that increased zones of inhibition, therefore concentrations were reduced to determine the lowest concentration at which this activity is sustained. This was done to find the breakpoint concentrations where gene expression patterns in response to this can be analyzed. Thus, making titrations of concentrations that enabled determination of sub-lethal concentrations that also allowed optimum expression of genes in the presence of the compound in order to ascertain the genetic factors contributing to the phenotype changes in response to each compound-antifungal pair. From table 5A, a two-fold decrease in concentration of compounds PC04-11 and PC04-18 were tested and it was realized that, there was no bioactivity up to the tenth-fold decrease. Thus, the concentration at which organisms are able to survive in the presence of the compound- antifungal pair and at which optimum gene expression changes could be determined was found to be 0.25 ug/ul for both compounds PC04-11 and PC04-18. Compound PC04-15 however did not give any clearance of organisms even at the initial concentration of 0.25 ug/ul. Thus, the compound PC04-15 – 5-FU pair inhibited growth of C. albicans even at that low concentration. Therefore, the critical concentration for effective determination of gene expression changes in the presence of this condition is expected to be lower than the concentrations tested. Resistant inducing interactions are those that decreased zones of inhibition, therefore concentrations were increased to determine the highest concentration at which this activity is still seen. Similarly, this was also done to find the breakpoint concentrations where gene expression patterns can be analyzed. On analyzing the selected resistant inducing pairs by increasing concentrations, compounds PC04-09 and PC04-16 had breakpoint at the sixteenth- 49 University of Ghana http://ugspace.ug.edu.gh fold dilution, thus this concentration is where the lowest zone of inhibition was measured. At this concentration, gene expression analysis can then be done to identify critical genes expressed or repressed in response to this compound-antifungal pair, resulting in induction of resistance observed in the phenotypic analysis. The rifampicin-sertraline however continued to have activity even at the sixteenth-fold dilution with zone of clearance as high as 14 mm. Thus, the breakpoint concentration at which optimal gene expression could be examined is expected to be much lower than analyzed. 50 University of Ghana http://ugspace.ug.edu.gh Table 4.4: Break point concentration determination to confirm unique interactions in C. albicans 51 University of Ghana http://ugspace.ug.edu.gh 4.5.1 Confirmation of resistant breaking compound-antifungal interactions in C. albicans Selected resistant breaking interactions in C. albicans were further tested in a liquid culture format to determine changes in interactions in different assay systems. This was done using the alamar blue assay. Here, cells with or with antifungals and compounds and in combination were incubated for four hours and fluorescence was read at various time points. From figure 3A, there was no significant difference in fluorescence, between treated and untreated C. albicans at time 0 minutes. At time 30 minutes, there was an indication of proliferation activity in the presence of compound PC04-23 only, fluconazole and the combination of the two. However, at time 45 minutes, there was a reduction proliferation in the presence of fluconazole and even more inhibitory activity with combination of compound Pc04-23 and fluconazole. However, as compared to the control where there was no treatment, there was reduced proliferation. Thus, here, there was observation of induction of resistance with respect to controls. Contrary to observations made in solid culture, compound PC04-23 – Fluconazole interaction pair decreased proliferation, thus showing resistant breaking activity. Although compound PC04-10 only was seen to increase proliferation at 45 and 60 minutes, combination with 5-FU was seen to reduce proliferation, indicating resistance breaking activity (figure 3B). Compering this activity with the control however gave an indication of resistance inducing activity as the proliferation rate of the compound PC04-10-fluconazle combination was slightly higher than the control. In figure 3C, resistance breaking activity of the interaction between PC04-15 and 5-FU was seen at times 30 minutes, 45 minutes and 60 minutes. This confirms the observation made previously in the solid culture format. In general, there was lower proliferation rate in all conditions. 52 University of Ghana http://ugspace.ug.edu.gh Figure 3 A-C. Confirmation of resistance breaking compound-antifungal interactions in C. albicans. Alamar blue cell viability assay was performed to confirm selected interaction pairs. 53 University of Ghana http://ugspace.ug.edu.gh 4.5.2 Confirmation of resistant inducing compound-antifungal interactions in C. albicans. Three resistant inducing interactions were in C. albicans were also selected and further tested using the alamar blue assay. Similarly, cells with or with antifungals and compounds and in combination were incubated for four hours and fluorescence was read at various time points. On analysis of resistance inducing activity, rifampicin and sertraline was observed to induce proliferation on their own, interacting the two however reduced proliferation, indicating synergistic activity. Compared to the control set up, there was higher proliferation. This thus confirms the resistance inducing activity observed in the solid culture format (figure 4A). From figure 4B, there was no significant difference in fluorescence, between treated and untreated C. albicans at time 0 minutes. At time 30, 45 and 60 minutes, there was a significant reduction proliferation in the presence of compound PC04-08 only, as compared to amphotericin B only and combination of the two. At times 30, 45 and 60 minutes, amphotericin B only, as well was combination of amphotericin B and compound PC04-08 showed a significant reduction enhancement of proliferation of the C. albicans cells (figure 4B). Thus, here, similar to observations made in solid culture, compound PC04-08 – amphotericin B interaction pair decreased proliferation, thus showing resistant breaking activity. In figure 4C, there was slight reduction in proliferation in the presence of compound PC04-14 at times 30, 45 and 60 minutes. Higher proliferation was observed in the presence of fluconazole only at times 30, 45 and 60 minutes as compared to the control. However, there was a reduction in proliferation at these times with combination of PC04-14 and fluconazole although this reduction was not significantly lower the control set up. Here also, the resistance inducing activity of this interaction pair was confirmed. 54 University of Ghana http://ugspace.ug.edu.gh Figure 4 A-C. Confirmation of resistance inducing compound-antifungal interactions in C. albicans. Alamar blue cell viability assay was performed to confirm selected interaction pairs 55 University of Ghana http://ugspace.ug.edu.gh 4.5.3 Confirmation of resistant breaking compound-antifungal interactions in S. cerevisiae. Further tests were done in S. cerevisiae in a liquid culture format to confirm previous observations made with the disc diffusion assay or to determine changes in interactions in different assay systems. This was done using the alamar blue assay. Cells with or with antifungals and compounds ad in combination were incubated for four hours and fluorescence was read at various time points. From figure 5A, there was a slightly higher proliferation in the presence of PC04-23 only and fluconazole only as compared to the control. Also, at times 30,45 and 60, there was a significantly higher S. cerevisiae proliferation for PC04-23 only and fluconazole only. At these times however, proliferation was reduced significantly with compound PC04-23–Fluconazole interaction, indicting the resistance breaking activity of this combination. In figure 4B, there was a general low S. cerevisiae proliferation across the different times for all conditions. There was surprisingly high proliferation of cells in the presence of 5- fluorouracil and compound PC04-10. Although the two compounds alone were seen to increase proliferation at 30, 45 and 60 minutes, combination of the two was seen to reduce proliferation, indicating resistance breaking activity. Thus, interaction of the pair however led to a significant decrease in proliferation, confirming the resistance breaking activity previously observed in the solid culture format. In figure 5C, at 30 minutes, compound PC04-15 only and 5-FU only was seen to have reduced proliferation as compared to the control, at the same time also, there was an even higher reduction in proliferation in the presence of the compound antifungal pair. The resistant breaking activity of the interaction pair was maintained at times 45 and 60 minutes as there was higher inhibition of proliferation as compared to the control. 56 University of Ghana http://ugspace.ug.edu.gh Figure 5 A-C. Confirmation of resistance breaking compound-antifungal interactions in S. cerevisiae. Alamar blue cell viability assay was performed to confirm selected interaction pairs. 57 University of Ghana http://ugspace.ug.edu.gh 4.5.4 Confirmation of resistant inducing compound-antifungal interactions in S. cerevisiae. The resistant inducing interactions were in S. cerevisiae was also further tested using the alamar blue assay with three interaction pairs. Similarly, cells with or with antifungals and compounds and in combination were incubated for four hours and fluorescence was read at various time points. On analysis of resistance inducing activity in figure 6A, rifampicin and sertraline was observed to induce proliferation alone at time 0, 30 45 and 60 minutes. At times 45 and 60 minutes, there was observed reduction in proliferation in the presence of sertraline. Interacting rifampicin and sertraline led to a significant decrease in proliferation as compared to the no treatment control. This gives an indication of resistant breaking activity, contrary to the resistance inducing activity realized in the previous solid culture assay system. From figure 6B, tat time 0, there was an increase in proliferation for all treated cells as compared to the control. At 45 minutes, there was no observed difference in fluorescence, across all treated cells and control cells. At 60 minutes, there was a significant reduction proliferation in the presence of compound PC04-08 only, amphotericin B only as well as the combination. There was no observed difference in the florescence of the three treatment conditions at 60 minutes. Thus, here, contrary to observations made in solid culture, compound PC04-08 – amphotericin B interaction pair showed resistant breaking activity. In figure 6C, at time 0, there was higher proliferation for compound PC04-14 only, fluconazole only as well as combination of the pair as compared to the control. At times 30, 45 and 60 minutes, the fluconazole only treatment increased proliferation than the control and other conditions. At 60 minutes, there was a reduction in cell growth compared to the control, indicating resistance breaking activity. 58 University of Ghana http://ugspace.ug.edu.gh Figure 6 A-C. Confirmation of resistance inducing compound-antifungal interactions in S. cerevisiae. Alamar blue cell viability assay was performed to confirm selected interaction pairs. 59 University of Ghana http://ugspace.ug.edu.gh 4.6.1 C. albicans efflux activity in the presence of efflux modulators and phenotypic modifiers without and with deferasirox The activity of C. albicans efflux pumps were determined using the Rhodamine 6G assay as described by Sun et al., 2010 with few modifications. Cells were incubated with rhodamine 6G for 4hours, external dye was washed and dye uptake was read as fluorescence. Cells were then incubated for 4 hours with compounds and glucose, washed and florescence was read. Here, there was low intake of rhodamine 6G as compared to the control with the exception of 4-Nitroquinoline. Rifampicin, estradiol and benomyl are known inducers of efflux pump gene expression, however, they retained rhodamine 6G dye up to 99%, 79% and 80% as compared to the control where 42% of the dye was retained in C. albicans (Appendix figure 1A). Same observations were made for methotrexate, 1,10-phenanthroline and sulfometuron methyl, which are known suppressors of efflux activity, where there was two times increase in efflux activity as compared to the control. 4-nitroquinoline however had the same efflux activity as the control. In a different set of experiments where there was addition of compounds during the efflux phase, efflux activity of C. albicans increased significantly in the control as well as in the presence of efflux modifying compounds. Here, fluorescence retained in cells was 34% for the control while that for the efflux modifying compounds ranged between 47% to 62% (Figure 7A). On addition of deferasirox, uptake of rhodamine 6G was considerably increased. This allowed effective measurement of cellular efflux. On analysis of efflux rifampicin, estradiol, benomyl, methotrexate, 1,10-phenanthroline, sulfometuron methyl, and 4-nitroquinoline oxide were all found to activate efflux activity as compared to the control (Figure 7B). The efflux inhibition activity however observed correlated with changes in antimicrobial phenotypic patterns 60 University of Ghana http://ugspace.ug.edu.gh observed. For instance, estradiol in the phenotypic analysis was found to induce resistance to both amphotericin B and fluconazole was also seen to activate efflux. This correlation of efflux activity and phenotypic change was also observed in 4-nitroquinoline oxide, where induction of resistance to amphotericin B was observed. In addition to efflux modulating compounds, the efflux activity in the presence of 17 other compounds previously described were tested. The purpose of these other compounds was to serve as further potential sources of phenotype modifiers in yeast as they have different functional groups, have different cellular targets and also are able to induce cellular stress and morphological changes in different bacteria cells. Generally, in the presence of the other phenotype modulating compounds, there was higher uptake as compared to the efflux modulators. Compared to the control, most of these compounds significantly inhibited efflux. These includes compounds PC04-10, PC04-15, PC04-18, PC04-19 and PC04-23. However, compounds PC04-9, PC04-11 and PC04-12 was seen to significantly activate efflux in C. albicans. Compound PC04-16 however the highest dye uptake and was found to inhibit efflux (Appendix figure 1A). On addition of equal amounts of compounds during the efflux phase in a separate experimental setup, observations previously made were maintained however, compound PC04-16 and PC04- 21 was seen to have increased uptake up to 8 times higher than the control. However, contrary to observations made earlier, compound PC04-16 was seen to activate efflux. Compound PC04-21 however maintained its efflux inhibiting ability as seen previously (Figure 7A). On improvement of rhodamine 6G uptake by addition of deferasirox, compounds PC04-09, PC04-10, PC04-11, PC04-12, PC04-13, PC04-16 and PC04-21 was found to significantly inhibit efflux activity of C. albicans as compared to the control. Compounds PC04-08, PC04- 14, PC04-15, PC04-17, PC04-18, PC04-19, PC04-20, PC04-22, PC04-23 and PC04-24 were all found to activate efflux n C. albicans (Figure 7B). 61 University of Ghana http://ugspace.ug.edu.gh Figure 7. Analysis of the C. albicans efflux activity in the presence of efflux modulators and phenotypic modifiers without (A) and with (B) deferasirox using the rhodamine 6G efflux assay. 62 University of Ghana http://ugspace.ug.edu.gh 4.6.2 S. cerevisiae efflux activity in the presence of efflux modulators and phenotypic modifiers without and with deferasirox The efflux pump activity of S. cerevisiae was also determined using the Rhodamine 6G assay as described by Sun et al., 2010 with few modifications. Cells were incubated with rhodamine 6G for 4hours, external dye was washed and dye uptake was read as fluorescence. Cells were then incubated for 4 hours with compounds and glucose, washed and florescence was read. In S. cerevisiae, similar observations were made as previously described in C albicans. In the initial assays where compounds were only added at the uptake phase and without deferasirox, all efflux modulating compounds had very low uptake fluorescence, therefore, measured efflux activities of compounds and controls may not be a true reflection of efflux as uptake was very low. Here, as compared to the control, high efflux activities were observed in rifampicin, benomyl, 4-nitroquinoline oxide as well as sulfometuron methyl (supplementary figure 1B). Upon addition of compounds in different set of experiments during the efflux phase, although there was low rhodamine 6G uptake, significant efflux activation in majority of the compounds as compared to C. albicans as realised (figure 8A). Compounds PC04-09, PC04-10, PC04-11, PC04-12, PC04-16 and PC04-21 however had higher dye uptake and also significantly activated efflux, compared with the control. Addition of equal amounts of compounds in the efflux phase in the second experimental set up did not change observations made previously, except in compounds PC04-16, PC04-20 and PC04-21 where uptake was increased vastly but efflux activation was maintained (Figure 8A). Addition of deferasirox across all conditions increased dye uptake, measured efflux activity is considered more efficient and a true reflection of cellular efflux in the presence of the various compounds. Here, among the efflux modulating compounds, benomyl was seen to have the highest efflux activating activity in S. cerevisiae. Methotrexate and 4-nitroquinoline oxide also highly activated efflux. Compounds PC04-09, PC04-10, PC04-12, PC04-16 and PC04-21 63 University of Ghana http://ugspace.ug.edu.gh inhibited efflux while all other compounds activated efflux (Figure 7B). Compound PC04-11 has the highest efflux inhibiting activity, retaining 85% of dye in S. cerevisiae. Compound PC04-20 was observed to have a high efflux activating activity, where about 80% of dye was pump out of the cell. 64 University of Ghana http://ugspace.ug.edu.gh Figure 8. Analysis of the S. cerevisiae efflux activity in the presence of efflux modulators and phenotypic modifiers without (A) and with (B) deferasirox using the rhodamine 6G efflux assay. 65 University of Ghana http://ugspace.ug.edu.gh 4.6.3 C. albicans and S. cerevisiae efflux activity in the presence of efflux modulators and phenotypic modifiers Figure 9 shows the efflux activity of C. albicans and S. cerevisiae using a modified rhodamine 6G efflux assay. Here, deferasirox and rhodamine dye was added during the uptake phase of the experiment. After measurement of uptake, equal amounts of compounds were added to each reaction during the efflux phase and fluorescence measured. This analysis in C. albicans showed that, all the efflux modulating compounds significantly inhibited efflux activity, compared to the control, which contained deferasirox only. A number of the other 17 phenotype modulating compounds however showed efflux inhibition. These included compounds PC04-09, PC04-10, PC04-11, PC04-12, PC04-16 and PC04-21. Here, PC04-21 showed the highest efflux inhibition activity, followed by compounds PC04-16, PC04-10 and PC04-09. These observations confirmed previous results shown in figure 7B. The phenotype modulating compounds, PC04-15, PC04-17, PC04-18 and PC04-19 significantly induced efflux. In S. cerevisiae, as shown in figure 9B, observations made in C. albicans were replicated and even more pronounced. Again, all the efflux modulating compounds were seen to induce efflux, while compounds PC04-09 PC04-10, PC04-11, PC04-12, PC04-16 and PC04-21 were found to inhibit efflux. Here however, compounds PC04-11 and PC04-16 showed the high efflux inhibiting activity, followed PC04-16, which was the highest in C. albicans. Similarly, PC04-13. PC04-14, PC04-15, PC04-12, PC04-16 PC04-17, PC04-18, PC04-20 and PC04-24 significantly activated S. cerevisiae efflux activity. The highest efflux inducer here was PC04- 13 followed by PC04-20. 66 University of Ghana http://ugspace.ug.edu.gh Figure 9. Analysis of C. albicans (A) and S. cerevisiae (B) efflux activity in the presence of efflux modulators and phenotypic modifiers. Rhodamine 6G efflux assay was employed to analyze the effect of various chemical compounds on efflux. 67 University of Ghana http://ugspace.ug.edu.gh 4.7 Initial screening of the SBF and TEF extracts against C. albicans and S. cerevisiae Initial screening of 201 SBF and 306 TEF extracts was done using the disc diffusion method. This yielded 50 bioactive extracts against C. albicans and S. cerevisiae. The initial screening of the extracts yielded zones of inhibition between 6 mm and 14 mm. In terms of numbers, more active extracts were from TEFs as compared to SBFs. There were 20 active SBF extracts while 30 was active extracts were obtained from TEF extracts. However, in terms of percentages, 9.95% of SBF extracts were active while 9.8% of TEF extracts were found active. Also, the active SBF extracts however gave higher zones of inhibition as compared to the TEF extracts. While the highest zone of inhibition was observed to be 12.5 mm in TEF extracts, that of SBF extracts was found to be 17 mm (Figures 10 A-D). Thus, in this assay it could be said that, SBF extracts are more potent in terms of antifungal activity against C. albicans. Fungal isolates whose extracts yielded high bioactivity against C. albicans and S. cerevisiae were selected (table 4.5). These were mostly selected based on their consistency of bioactivity across the two organisms. SBF 188 and SBF 197 were selected due to their high bioactivities in S. cerevisiae even though there was no bioactivity against C. albicans. These selected fungi have been re-cultured for a period of three weeks to obtain pure isolates. Pure isolates of each fungi have then been grown in 2L yeast peptone malt dextrose (YPMD) both. Fungal cultures are expected to mature in three months to be taken through product isolation. Some of these are ready to be isolated and the fungal cultures has been stopped using ethyl acetate. 68 University of Ghana http://ugspace.ug.edu.gh Figure 10. Initial screening of the SBF and TEF extracts against C. albicans (A and B) and S. cerevisiae (C and D). The disc diffusion assay was employed in analyzing the bioactivity of 507 fungal extracts. 69 University of Ghana http://ugspace.ug.edu.gh Table 4.5: List of Selected fungi and zones of inhibition The table shows the list of selected fungi for test of chemosensitization activity and their zones of inhibition in mm. 70 University of Ghana http://ugspace.ug.edu.gh 4.6 Fluconazole chemosensitization In order to detect SBF and TEF extracts that sensitize C. albicans to fluconazole, YPDA plates were modified with sub-MIC of fluconazole, organisms were spread on the plate and extract disc were placed on it. Plates were incubated for 24 hours and zones of inhibition was measured. The remaining 457 inactive extracts were tested for their chemosensitizing ability against C. albicans. A total of 58 TEF inactive extracts was seen to sensitize C. albicans to the activity of fluconazole while 32 inactive SBFs had activity in the presence of fluconazole. Here, 15.9% SBF extracts were found to be active while 18.9% TEFs were active. As compared to initial screening, higher zones of inhibition were observed for chemosensitizing extracts as compared to the initial active extracts. In general, the SBF extracts were more bioactive, thus gave higher zones of clearance of organisms as compared to TEF extracts. The highest zone of clearance during the screen was 46 mm, observed for SBF 168. A zone of 40 mm was also observed for TEFs 090 and 119, being the highest observed for the TEF extracts. In all 90 extracts were found to act as chemosensitizers to fluconazole against C. albicans (Figures 11 A and B). Thus here, the most potent chemosensitizing extracts were observed among SBF extracts. Efflux-mediated resistance is an important resistant mechanism of resistance in C. albicans and has been found to be the main resistant mechanisms against fluconazole, the first line antifungal. Some of these sensitizing extracts from soil borne and terrestrial endophytic fungi identified are expected are suspected to act via inhibition of C. albicans efflux pumps, which allows high concentrations of fluconazole to be maintained in the cell in order to inhibit cellular growth. 71 University of Ghana http://ugspace.ug.edu.gh Figure 11. Screening for chemosensitizing agents from SBF (A) and TEF (B) extracts against C. albicans. The disc diffusion assay was employed in the screen using sub-inhibitory concentration of fluconazole. 72 University of Ghana http://ugspace.ug.edu.gh 4.7 Analysis of Efflux Activity of Chemosensitizing TEF and SBF extracts Efflux activity of C. albicans and S. cerevisiae was analyzed in the presence of extracts using the rhodamine 6G efflux assay. On analysis of efflux in the presence of chemosensitizing extracts, 13 out of 20 extracts tested were found to significantly inhibit efflux. This observation was replicated in both C. albicans and S. cerevisiae. The efflux inhibiting activity was however slightly higher in C. albicans as compared to S. cerevisiae. Most significant of this difference is that of SBF 094, TEF 119 and TEF 209 where the activity in C. albicans was over 60% higher than that in S. cerevisiae (Figures 12 A and B). The rhodamine efflux assay is reportedly more specific to and mostly used in analysis of the activity of CDR1p and CDR2p, which are mostly found in C. albicans as compared to S. cerevisiae. It was therefore not surprising to different levels of dyes in C. albicans as compared to S. cerevisiae although the efflux activity was replicated in the two organisms. In S. cerevisiae, the strongest inducers of efflux were SBF 080 followed by TEF 008, while that observed in C. albicans were SBF 187 and SBF 168. SBF 168 which was however observed to have the highest chemosensitizing activity. This suggest that the chemosensitizing activity of this extract might be through other mechanisms rather than efflux inhibition. Similar observation was made for SBF 044 which had an inhibitory zone as high as 40 mm but was seen to also significantly induce efflux in C. albicans. Some extracts which had high chemosensitizing activity were however observed to inhibit efflux in C. albicans. Such extracts include TEFs 044, 049 and 089 which had inhibitory zones as high as 36 mm, 37mm and 36 mm respectively. It could be said that inhibition of efflux could partly account for their mechanism of action. 73 University of Ghana http://ugspace.ug.edu.gh Figure 12. Analysis of the C. albicans (A) and S. cerevisiae (B) efflux activity in the presence of fungal extracts. Rhodamine 6G efflux assay was employed to analyze the effect of the extracts on efflux. 74 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS 5.1 DISCUSSION Candida albicans are mostly opportunistic fungal pathogens that causes infection in immunocompromised patients including cancer, transplant and HIV patients. The eukaryotic nature of this organism makes therapeutic development complicated as drug target in the pathogen are also mostly found in the host. The presence of antifungals in C. albicans is recognized as stress and in response to this stress, transient changes are made in the genome which eventually confers antifungal resistance. There has been reports of resistance to fluconazole, a first line antifungal. The main mechanism of antifungal resistance is reportedly the upregulation of efflux genes which reduce the accumulation of antifungals, rendering the drugs inactive. Chemosensitization has however been postulated as a way of overcoming antifungal resistance. This current study investigated the effects of chemical compounds that modulate efflux gene as well as other small molecules on the antimicrobial susceptibility or resistance phenotypes of Candida albicans and Saccharomyces cerevisiae. Although several studies have been done on the effect of various chemical compounds and other stress conditions on gene expression patterns of Candida albicans and Saccharomyces cerevisiae, little has been done on the phenotypic patterns. The effects of these compounds on the efflux activity of Candida albicans and Saccharomyces cerevisiae were investigated. Lastly, the ability of Soil Borne Fungi (SBF) and Terrestrial Endophytic Fungi (TEF) to sensitize C. albicans to the antifungal activity of fluconazole as well as the influence of the chemosensitizing extracts was studied. 75 University of Ghana http://ugspace.ug.edu.gh 5.1.1 Chemical compounds modify the antimicrobial phenotypes of Candida albicans and Saccharomyces cerevisiae Several studies have been conducted recently on the effects of non-antimicrobial compounds on the susceptibility or resistance phenotypes of different pathogenic microorganisms. The purpose of these studies is mostly to find compounds that can be repurposed for use as antimicrobials either alone or in combination with failing antimicrobial drugs in clinical use. In this study also, the same idea to was applied identify compounds that causes antimicrobial phenotypic changes in C. albicans and S. cerevisiae and to apply results obtained to inform strategies to reverse antifungal resistance, by using hit compounds identified as synergistic partners of various antifungals tested. The results obtained showed that generally, the compounds had higher effects on both the susceptibility and resistant patterns of S. cerevisiae as compared to C. albicans. This could be due to the pathogenic nature of C. albicans cells, making it mount various stress response defense mechanism against the compounds. C. albicans in host cells are faced with various chemical stresses and are therefore required to adapt to the environment in order to survive. This could lead to induction of various stress response mechanisms, accounting for the observation made in this study. S. cerevisiae, however being non-pathogenic are naturally not frequently exposed to stressful host and are more likely to be affected phenotypically in a modified environment as observed. Previous studies have shown that on exposure to heat shock, oxidative and osmotic response, S. cerevisiae exhibited strong response and made more genetic changes in general stress response genes as compared to C. albicans (Enjalbert, Nantel et al. 2003). Thus, genetically, S. cerevisiae is more affected by environmental changes as compared to C. albicans. This evidence therefore supports observations made in this study. Also, on analysis of phenotypic changes in the two organisms independently, although the two cells were more resistant than susceptible, S. cerevisiae cells were found to more resistant to the activity of antifungals in the presence of the various compounds than C. albicans. This is 76 University of Ghana http://ugspace.ug.edu.gh also supported by the report by Enjalbert et al., 2003. Thus, the several genetic changes made by S. cerevisiae allows it to adapt and survive exposure to the compounds, eventually making the cells more resistant to antifungal agents as compared to C. albicans. Estradiol induced resistance to amphotericin B, fluconazole and sertraline. Estradiol has been demonstrated to induce the overexpression of the efflux genes, CDR 1 and CDR 2 (Candida drug resistance 1 and 2). These genes produce proteins on the cell membrane that pumps drugs out of the cell, making antifungals ineffective (Micheli, Bille et al. 2002). These findings therefore confirm the results obtained in this study. Contrary to the above stated, estradiol in this current study was also found to induce susceptibility to 5-fluoruracil in C. albicans. This suggest that, the mechanism of these observed interaction of estradiol may be complex and therefore further molecular investigation might be needed to provide further understanding. Rifampicin, an antibiotic has also been known to induce MDR1 (multidrug drug resistance 1) gene expression in C. albicans, and is therefore known to cause antifungal resistance (Vogel, Hartmann et al. 2008). In this study however, rifampicin was seen to increase the activity of fluconazole, while it rendered sertraline resistant to C. albicans. Previous studies on antifungal resistance causing activity of rifampicin has focused on genetic analysis and not extended to analysis of actual phenotypes its presence. Results seen here could therefore suggest that although there is induction of multi drug resistance gene, under certain conditions, this characteristic is not translated into a phenotypic change. In a drug screen to identify potential inhibitors of Staphylococcus aureus biofilm inhibitors, PC04-11 was found as a potential source of S. aureus anti-biofilm compound (Van den Driessche, Brackman et al. 2017). This indicates the potential usefulness of this compound in treating various in antimicrobial infections. Here also, compound PC04-11 increased the susceptibility of fluconazole and caused a phenotype switch of C. albicans to sertraline 77 University of Ghana http://ugspace.ug.edu.gh susceptible phenotype. This indicates the potential usefulness of PC04-11 in treatment of fungal infections as well. Compound PC04-09, a phenothiazine, usually prescribed as an antidepressant induced resistance to fluconazole, making not useful to be repurposed as synergistic partner to fluconazole, as found in this current experiment. PC04-09 has however been found to render methicillin resistant strains of Staphylococcus aureus (MRSA) more susceptible to oxacillin in the presence of sub-inhibitory concentrations of PC04-09 (Kristiansen, Leandro et al. 2006). In C. albicans, compound PC04-23 was found to have a remarkable effect by acting synergistically with fluconazole and inducing 5-fluorouracil susceptible phenotype. Compound PC04-23 has so far not been recorded to have any antibacterial or antifungal activity either alone or in combination with another antibacterial or antifungal drug. It has however been found to synergize with the anti-helminthic drug, albendazole in treating human neurocysticercosis (Lima, Ferreira et al. 2011). This shows the diverse applications of this compound in eukaryotic infections, representing a promising compound of choice for drug repurposing. C. albicans causes mucosal, cutaneous and systemic infections. It was therefore necessary to repeat selected interactions in liquid media to verify changes in cellular behavior in different culture environments. Repeating rifampicin-sertraline interaction in liquid media, using the alamar blue assay however resulted in a resistance breaking interaction contrary to the resistance inducing interaction previously observed in solid culture format with the disc diffusion method. This confirms the observation that cells behave differently in different culture medium and adjust to stressful conditions in diverse ways, depending on their environment (Ernst 2000). 78 University of Ghana http://ugspace.ug.edu.gh 5.1.2 Efflux activity of C. albicans and S. cerevisiae are affected by Phenotype Modulators The efflux activity of all 24 compounds used in the phenotypic assays were analyzed. Deferasirox is an iron chelating compound that is recently being investigated for antibacterial activity (Chatterjee, Anju et al. 2016), anticancer activity (Kim, Na et al. 2016) among others. In this study however, deferasirox was found to increase intake of rhodamine 6G up to four times higher than control. This allowed effective measurement of rhodamine uptake and therefore efficient measurement of significant efflux activities of the various compounds Previous studies indicate that rifampicin upregulates C. albicans MDR1 gene, causing resistance to antifungal drugs (Vogel, Hartmann et al. 2008). Here, rifampicin was observed to significantly activate the efflux activity of C. albicans CDRI and CDR2 transporters, a different group of efflux pumps also found on the surface of the cell, performing the same function as MDRp. This result suggests that rifampicin could cause antifungal resistant via upregulating the activity of efflux transporters. This was certainly observed in the phenotypic analysis where rifampicin was observed to induce sertraline resistance phenotype in C. albicans. Estradiol has been reported to cause upregulation of several clinical isolates of drug resistant C. albicans (Karababa, Coste et al. 2004). This observed was also realized in this study where estradiol was observed to activate efflux in both C. albicans and S. cerevisiae. Efflux activation usually causes drug resistance, as drugs are rapidly pumped out of the cells, leaving sub- inhibitory cellular concentrations, making the drugs inactive. Interestingly, a switch to resistant phenotype was observed in S. cerevisiae in the presence of estradiol. S. cerevisiae was found resistant to fluconazole, amphotericin B and sertraline. 79 University of Ghana http://ugspace.ug.edu.gh Compound PC04-16 is a known iron chelator and has been found to synergize with imatinib to cause cell death in imatinib-resistant chronic myeloid leukemia cancer cells (Kim, Na et al. 2016). This reported occurred via the induction of apoptosis and cell cycle arrest. Here, compound PC04-16 was observed to significantly induce efflux in both C. albicans and S. cerevisiae. As observed before, PC04-16 was expected to cause resistant phenotypes in organisms, however, this was no observed. There was induction of fluconazole susceptible phenotype in C. albicans and amphotericin B susceptible phenotype in S. cerevisiae. Compound PC04-23 was also found to significantly activate efflux in both C. albicans and S. cerevisiae. However, it increased the activity of fluconazole and induce susceptibility to 5- fluorouracil in C. albicans. These observations the possibility of complex cellular mechanisms involved in phenotypic changes and efflux activities observed which needs to be investigated. 5.1.3 Soil Borne and Terrestrial Endophytic Fungal Extracts Chemosensitize C. albicans and S. cerevisiae To Fluconazole There has been growing concern over antifungal resistance and this is a major challenge to clinicians especially in the treatment of invasive and systemic fungal infections (Wiederhold 2017). There is limited number of antifungal agents and these are challenged by toxicities and overuse, causing resistance. Fluconazole resistance in both Candida albicans has become very common due to the high incidence of C. albicans infection in different geographical areas, leading to wide-spread resistance. There is therefore the need for novel and potent antifungals. However, the fact that it takes decades to discover and develop new drugs for clinical use has left a huge antifungal discovery void (Brown 2015). Chemosensitization has been postulated as a way to overcome antifungal resistance and prolong the market life of existing and failing antifungals. 80 University of Ghana http://ugspace.ug.edu.gh Here, initial screening of 507 SBF and TEF extracts yielded 50 bioactive extracts against C. albicans, which could be developed into therapeutic agents. Also, we found a total of 90 SBF and TEF extracts that sensitized C. albicans to the activity of sub-inhibitory concentrations of fluconazole. Efflux-mediated resistance is an important mechanism of resistance in C. albicans and has been found to be the main resistant mechanisms against fluconazole, the first line antifungal. Finding compounds that combat resistance via this mechanism is urgently needed. On testing the chemosensitizing extracts for efflux inhibition activity, 13 out of 20 extracts tested showed significant efflux inhibition in both C. albicans and S. cerevisiae. 81 University of Ghana http://ugspace.ug.edu.gh 5.2 CONCLUSION This study investigated the influence of chemical compounds on antimicrobial susceptibility or resistance phenotypes in Candida albicans and Saccharomyces cerevisiae and to subsequently apply result obtained to inform strategies to curb efflux-mediated antifungal resistance. Also, extracts from secondary metabolites of soil borne and terrestrial endophytic fungi (SBF and TEF) were analyzed for chemosensitizing efflux activities. The results indicated that, chemical compounds used in this study, including, compounds PC04-10, PC04-11, PC04-16 and PC04-23, significantly modified the antimicrobial phenotypes of Candida albicans and Saccharomyces cerevisiae and could be considered for repurposing as antifungals or synergistic partners of antifungal drugs to overcome resistance. Also, it was realized that some compounds including rifampicin, estradiol, PC04-09 and PC04- 14 caused resistance and should possibly not be co-administered with antifungals. Initial screening of 507 SBF and TEF extracts yielded 50 bioactive extracts against C. albicans, which has the potential to be developed into therapeutic agents. A total of 90 chemosensitizing extracts were identified from 457 inctive SBF and TEF fungal extracts. Also, 13 out of 20 chemosensitizing extracts significantly inhibited efflux activity of C. albicans and S. cerevisiae. Thus, fungi are good sources of novel and potent antifungal and chemosensitizing compounds. 82 University of Ghana http://ugspace.ug.edu.gh 5.3 RECOMMENDATIONS AND FUTURE OULOOK Further molecular analyses of differential gene expression of C. albicans and S. cerevisiae should be done to know the genetic factors involved in the phenotypic changes in the presence of compounds. This would help identify potent drug targets in these organisms. Also, SBF and TEFF extracts that were bioactive against C. albicans or sensitized C. albicans to fluconazole should be purified to identify active compounds. Toxicities should also be analyzed and compounds taken forward for antifungal drug development. In this study, it was realized that different chemical compounds affected the antimicrobial susceptibility and resistance phenotypes of C. albicans. Drug compound interactions that caused switch to more susceptible phenotypes could further be investigated and potentially considered for applications as antifungals or synergistic partners of different antifungals for clinical treatment of resistant C. albicans infections. Also, further investigations into drug- compound pairs that induced resistance should be made. This would provide more information that would inform clinicians to possibly avoid co-administration of such compounds during treatment of fungal infections. 83 University of Ghana http://ugspace.ug.edu.gh APPENDIX Table 1.1. Antifungal resistance or susceptibility patterns of C. albicans With Modulators of Efflux Pumps Table 1.2. Antifungal resistance or susceptibility patterns of S. cerevisiae With Phenotype modifiers 84 University of Ghana http://ugspace.ug.edu.gh Table 1.3. Antifungal resistance or susceptibility patterns of C. albicans With Phenotype Modifiers Table 1.4. Antifungal resistance or susceptibility patterns of S. cerevisiae With Phenotype Modifiers 85 University of Ghana http://ugspace.ug.edu.gh Table 1.5. Antifungal resistance or susceptibility patterns of C. albicans With Phenotype Modifiers Table 1.6. Antifungal resistance or susceptibility patterns of S. cerevisiae With Phenotype Modifiers 86 University of Ghana http://ugspace.ug.edu.gh Figure 1. Analysis of the C. albicans (A) and S. cerevisiae (B) efflux activity in the presence of efflux modulators and phenotypic modifiers. Rhodamine 6G efflux assay was employed to analyze the effect of various chemical compounds on efflux. 87 University of Ghana http://ugspace.ug.edu.gh Figure 2. Analysis of the C. albicans (A) and S. cerevisiae (B) efflux activity in the presence of fungal extracts. Rhodamine 6G efflux assay was employed to analyze the effect of the extracts on efflux. 88 University of Ghana http://ugspace.ug.edu.gh REFERENCES Akerey, B., C. Le‐ Lay, I. Fliss, M. Subirade and M. 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