Development of small interfering to treat fungal disease

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2018

Mireille Henriëtte van der Torre

School of Biological Sciences

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List of Contents List of Contents ...... 2

List of Figures ...... 5

List of Tables ...... 7

List of Abbreviations...... 8

Abstract ...... 11

Declaration ...... 12

Copyright statement ...... 12

Acknowledgement ...... 13

1. Introduction ...... 14

1.1 Fungi in the environment: from harmless useful fungi to fungi that cause serious disease ...... 15 1.2 Aspergillus fumigatus ...... 16 1.3 Antifungal drugs ...... 18 1.4 silencing mechanisms ...... 20 1.5 The RNA interference pathway in the N. crassa ...... 22 1.6 RNAi conservation in ...... 25 1.7 Different small RNAs (sRNAs) in fungal gene silencing pathways ...... 27 1.8 Uptake of siRNA by fungi ...... 29 1.9 Challenges with the use of siRNA as therapeutic agents...... 30 1.9.1 Stability of siRNA...... 30 1.9.2 Delivery of siRNA...... 32 1.10 Aims ...... 34 2. Materials and Methods ...... 35

2.1 Strains and conditions ...... 36 2.2 Polymerase Chain Reaction (PCR) ...... 37 2.3 RNAi mutant strains ...... 39 2.3.1 Generation of RNAi mutant strains by two step fusion PCR ...... 39 2.3.2 Verification of RNAi mutant strains ...... 39 2.3.3 Phenotypic screening of RNAi mutant strains ...... 39 2.4 Generation of siRNAs ...... 40 2.4.1 Unmodified synthetic siRNAs ...... 40 2.4.2 Modified synthetic siRNAs ...... 41 2.4.3 Preparation of diced-siRNAs pool ...... 42 2.5 ...... 42 2.5.1 Agarose gels ...... 42 2.5.2 Polyacrylamide gels ...... 43

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2.6 Amplification of al-1 DNA ...... 43 2.7 Analysis of siRNA treated cultures...... 43 2.7.1 Growth assay ...... 43 2.7.2 assay ...... 43 2.7.3 Confocal microscopy ...... 44 2.7.4 Live imaging ...... 44 2.7.5 RNA extraction and qRT-PCR...... 45 2.8 Minimal inhibitory concentration ...... 45 2.8.1 Hygromycin ...... 45 2.8.2 Amphotericin B ...... 46 2.9 Statistical analysis ...... 46 2.10 Protoplast transfection ...... 46 2.11 Labelling cornea with siRNA ...... 47 2.12 Mammalian cell cultures ...... 47 2.12.1 Transfection of A549 cell line ...... 47 2.12.2 Generation of exosomes ...... 48 2.13 Lipid fungal transfections ...... 49 2.13.1 Transfection reagents ...... 49 2.13.2 ...... 49 2.14 Cell penetrating peptides (CPP) PAF ...... 49 2.14.1 Synthesis of CPP-siRNA ...... 49 2.14.2 Co-cultures of siRNA-SMCC-PAF26 and B-siRNAs with peptide8 ...... 51 2.15 Small RNA library construction ...... 51 2.16 Small RNA sequencing and bioinformatics analysis ...... 52 3. Defining the effect of siRNA treatment on N. crassa and A. fumigatus 53

3.1 Introduction ...... 54 3.2 The generation of siRNAs used in this study ...... 56 3.3 The effect of synthetic al-1 siRNA treatment on N. crassa ...... 57 3.3.1 N. crassa after siRNA treatment ...... 57 3.3.2 No significant changes in mRNA levels after al-1 siRNA treatment in N. crassa 58 3.4 The effect of synthetic odcA siRNA treatment on A. fumigatus ...... 59 3.4.1 A. fumigatus phenotypes after siRNA treatment ...... 59 3.4.2 Growth of A. fumigatus is not affected in the presence of siRNA ...... 61 3.4.3 No significant changes in odcA mRNA levels after siRNA treatment in A. fumigatus ...... 62 3.5 The effect of a pool of siRNAs (d-siRNAs) against one target ...... 63 3.5.1 The effect of a pool of diced al-1 siRNAs (d-siRNAs) on N. crassa ..... 64 3.5.2 The effect of pptA d-siRNAs on A. fumigatus ...... 66

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3.5.3 The effect of hph d-siRNAs on N. crassa and A. fumigatus ...... 68 3.6 Discussion ...... 71 4. The effect of modified siRNAs on fungi ...... 75

4.1 Introduction ...... 76 4.2 Modified siRNA treatment does not affect N. crassa ...... 78 4.3 Modified siRNA treatment does not affect A. fumigatus ...... 81 4.4 B-siRNAs are concentrated at the cell wall and septa of N. crassa ...... 83 4.5 B-siRNAs are concentrated at the cell wall of A. fumigatus ...... 86 4.6 A variety of Aspergillus spp are labelled by B-siRNAs ...... 90 4.7 B-siRNAs can be used as a diagnostic tool for fungal keratitis...... 93 4.8 Discussion ...... 95 5. Development of siRNA delivery systems in A. fumigatus ...... 98

5.1 Introduction ...... 99 5.2 Optimising the concentration of siRNA treatment ...... 101 5.3 Disrupting the using the antifungal drug amphotericin B 105 5.4 Testing extracellular vesicles of mammalian cells for siRNA transfection 106 5.5 Testing lipid-based formulations for siRNA transfection...... 110 5.6 Testing cell penetrating peptides for siRNA transfection ...... 113 5.7 Discussion ...... 117 6. Characterisation of the small RNA transcriptome of A. fumigatus ..... 120

6.1 Introduction ...... 121 6.2 Conservation of RNAi in fungi ...... 123 6.3 Dicer and QDE-2 mediated siRNA synthesis are dispensable for viability in A. fumigatus...... 125 6.4 RNAi mutants are dispensable for growth and stress adaptation ...... 129 6.5 Variable sizes of sRNAs are detected in sRNA libraries ...... 132 6.6 Sequencing analysis of small RNA library ...... 133 6.7 Discussion ...... 142 7. Conclusions and future work ...... 146

References ...... 150

Total word count: 45,526

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List of Figures Figure 1.1. Schematic overview of RNA interference (RNAi) mechanism ...... 21 Figure 1.2. Overview of the RNAi pathway in N. crassa ...... 24 Figure 1.3. Overview of fungal small RNAs (sRNAs) involved in gene silencing ..... 27 Figure 1.4. Selection of chemical modifications ...... 31 Figure 2.1. Schematic overview of linking siRNA to PAF26-C...... 50 Figure 3.1. Polyacrylamide gel confirming primer annealing into siRNA ...... 57 Figure 3.2. No observable effects were seen in N. crassa phenotypes on siRNA coated agar medium ...... 58 Figure 3.3. al-1 mRNA levels did not change significantly after siRNA treatment in N. crassa ...... 59 Figure 3.4. Phenotype of A. fumigatus in presence of odcA siRNA is not affected . 60 Figure 3.5. Phenotype assay of A. fumigatus on siRNA coated agar medium ...... 61 Figure 3.6. odcA siRNA treatment in A. fumigatus did not affect growth ...... 62 Figure 3.7. odcA mRNA levels did not change after siRNA treatment in A. fumigatus ...... 63 Figure 3.8. Generation of d-siRNAs confirmed on 20% polyacrylamide gel ...... 64 Figure 3.9. d-siRNA coated agar medium does not change the phenotype of N. crassa ...... 65 Figure 3.10. d-siRNA treatment did not affect al-1 mRNA levels in N. crassa ...... 66 Figure 3.11. pptA d-siRNAs do not cause a change in phenotype ...... 67 Figure 3.12. No significant differences in pptA mRNA levels after d-siRNAs treatment ...... 68 Figure 3.13. The effect of hph d-siRNAs on N. crassa ...... 69 Figure 3.14. The effect of hph d-siRNAs on A. fumigatus ...... 70 Figure 4.1. Phenotype of N. crassa on modified siRNA coated agar medium was not affected ...... 79 Figure 4.2. al-1 mRNA levels in N. crassa after modified siRNA treatment ...... 80 Figure 4.3. qde2 mRNA levels in N. crassa after modified siRNA treatment...... 81 Figure 4.4. Phenotypes of A. fumigatus in presence of modified siRNAs did not show observable differences ...... 82 Figure 4.5. pptA mRNA levels were not affected in A. fumigatus after modified siRNA treatment ...... 83 Figure 4.6. Labelled siRNAs accumulated at N. crassa septa ...... 84 Figure 4.7. B-siRNAs concentrate on the outside of the septa in N. crassa ...... 85 Figure 4.8. Live-cell imaging of N. crassa reveal accumulation of B-siRNAs to septa and cell wall ...... 86 Figure 4.9. B-siRNA accumulation in A. fumigatus ...... 87

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Figure 4.10. A. fumigatus co-cultured with peptide8 did not improve B-siRNA uptake ...... 89 Figure 4.11. B-siRNAs are excluded from protoplasts ...... 90 Figure 4.12. Time lapse of A. nidulans B-siRNAs treatment ...... 91 Figure 4.13. Aspergillus lentulus and Aspergillus niger are labelled with B-siRNAs 92 Figure 4.14. Labelling of A. fumigatus infection in pig cornea ...... 93 Figure 5.1. Concentrations of pptA siRNA up to 250 nM did not affect A. fumigatus phenotypes ...... 102 Figure 5.2. Treatment of A. fumigatus with high concentrations of labelled pptA siRNA cause morphological differences ...... 103 Figure 5.3. Morphological changes in A. fumigatus cultures treated with 2500 nM siRNA ...... 104 Figure 5.4. Amphotericin B MIC did not change in presence of pptA siRNA ...... 105 Figure 5.5. Transfection of mammalian cells with labelled siRNA and A. fumigatus ...... 107 Figure 5.6. A. fumigatus treated with B-siRNA loaded mammalian exosomes ..... 109 Figure 5.7. B-siRNAs captured in lipofectamine lost their ability to accumulate to the cell wall of A. fumigatus ...... 110 Figure 5.8. Treatment of A. fumigatus with B-siRNA encapsulated in Viromer Blue or multi-lamellar liposomes ...... 112 Figure 5.9. Binding assay of radio-labelled siRNA to PAF peptides ...... 113 Figure 5.10. Treatment of A. fumigatus with B-siRNA-PAF26 complex did not change uptake of B-siRNA ...... 114 Figure 5.11. Polyacrylamide gel confirming the generation of siRNA-SMCC-PAF26 complex ...... 115 Figure 5.12. Treatment of A. fumigatus with siRNA covalently linked to PAF26 in the presence and absence of peptide8 ...... 116 Figure 6.1. Cladogram showing the conservation of RNAi components in several eukaryotes ...... 124 Figure 6.2. Generation of gene KO cassettes for qde-2, dicer-1 and dicer-2 ...... 126 Figure 6.3. Validation of RNAi mutant strains ...... 128 Figure 6.4. Phenotypic analysis of RNAi mutant strains ...... 129 Figure 6.5. Relative fitness of RNAi mutant strains ...... 130 Figure 6.6. DNA damage assay reveals similar responses in WT and RNAi mutants ...... 131 Figure 6.7. sRNA libraries of RNAi mutants reveal the variability in cDNA sizes ... 132 Figure 6.8. Identification of siRNA-like transcripts in A. fumigatus ...... 139 Figure 6.9. Overview of sRNAs annotated to the target gene pptA ...... 141

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List of Tables Table 1.1. Classes of antifungal drugs used to treat A. fumigatus ...... 20 Table 2.1. Fungal strains used in this study ...... 36 Table 2.2. PCR, RT-PCR and sequencing primers used in this study ...... 37 Table 2.3. Synthetic unmodified siRNAs synthesised by Eurofins Genomics ...... 41 Table 2.4. Modifications of antisense synthetic RNA oligos ...... 42 Table 2.5. Primers and indices used to generate the small RNA library ...... 52 Table 3.1. Summary of methods of previous siRNA studies on Aspergillus spp. .... 72 Table 6.1. Characterisation of conserved N. crassa RNAi proteins in A. fumigatus123 Table 6.2. Total sRNA sequencing reads per replicate ...... 133 Table 6.3. Classification of high-abundance annotated sRNAs ...... 135 Table 6.4. Properties of 19 – 25 bp sRNAs mapped to pptA ...... 140

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List of Abbreviations 2’F-ANA 2’deoxy-2’-fluoro-β-D-arabino sugar modification ABPA Allergic bronchopulmonary aspergillosis ACM Aspergillus complete medium AGO AIDS Acquired immune deficiency syndrome al Albino AMM Aspergillus minimal medium ANOVA Analysis of variance ApoB Apoliprotein B ASO Anti-sense ATP Adenosine triphosphate BLAST Basic local alignment search tool bp Basepairs BSA Bovine serum albumin B-siRNA BLOCK-iTTM labelled (Alexa FluorTM 555 or ) siRNA C3PO Component 3 promoter of RISC CFW CalcoFluor White chs Chalcone synthase CMV Cytomegalovirus retinitis CPA Chronic pulmonary aspergillosis CPP Cell-penetrating peptide CTAB Cetyl trimethyl ammonium bromide DIC Differential interference contrast disiRNA Dicer-independent small interfering RNA DMEM Dulbecco’s modified eagle medium DMEM++ DMEM supplemented with 10% fetal bovine serum and 1 µg/mL of the antibiotics streptomycin and ampicillin DPBB Double-psi beta-barrel d-siRNAs Pool of dicer cleaved siRNAs EDTA Ethylenediamine tetra-acetic acid esRNA Endogenous short RNA EV Extracellular vesicle FBS Fetal bovine serum FDA Food and Drug Administration FGSC Fungal genetics stock centre FIJI Fiji is just imageJ Fluoro 2’-fluoro sugar modification GFP Green fluorescent GTCF Genomic Technologies Core Facility GUS β-glucuronidase HeLa Henrietta Lacks

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HIV Human immunodeficiency virus infection hph Hygromycin B phosphotransferase HSD Honestly significant difference IA Invasive aspergillosis ICU Intensive care units LDL-C Low-density lipoprotein cholesterol masiRNA MSUD-associated small interfering RNA MFIG Manchester fungal infection group MIC Minimal inhibitory concentration milRNA miRNA-like RNA MOE 2′-O-methoxyethyl mRNA Messenger RNA MSN Mesoporous silica nanoparticle MSUD Meiotic silencing by unpaired DNA MW Molecular weight NAC National Aspergillosis Centre nt odcA Ornithine decarboxylase OMe 2’O-Methyl ORF Open reading frame PAF Penicillium AntiFungal PAZ PIWI, Argonaute and Zwille domain PBS buffer saline PBS-T PBS with 0.1% Tween-80 PCA Principal component analysis PCP Pneumocystis pneumonia PCR Polymerase chain reaction PEG pHLIP pH-low-insertion-peptide piRNA Piwi-interacting RNA PNA Peptide pptA Phospho-pantetheinyl transferase A PS Phosphorothioate linkage PS OMe Phosphorothioate linkage with 2’O-Methyl sugar modification ptrA Pyrithiamine resistance gene pyrG Orotidine-5’-monophosphate decarboxylase QA Quinic acid QDE Quelling-deficient QIP QDE-2-interacting protein qiRNA QDE-2-interacting sRNA qRT-PCR Quantitative real-time polymerase chain reaction RDE-1 RNAi-deficient-1

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RdRP RNA-dependent RNA polymerase RIP Repeat induced-point mutation RISC RNA inducing silencing complex RNAi RNA interference RNase Ribonuclease RPA Replication protein A RPMI Roswell Park memorial institute 1640 medium SAB Sabouraud dextrose SAD Suppressor of ascus dominance SAFS Severe asthma with fungal sensitisation shRNA Short hairpin RNA siRNA Small interfering ribonucleic acid SIS Sex induced silencing SMCC Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate SPT Skin prick test sRNA Small RNA TAE Tris-acetate-EDTA TBE Tris-borate-EDTA TLR Toll-like receptor TMR Tetramethylrhodamine TNF-α Tumor necrosis factor α tRNA Transfer-RNA UCSC The University of California Santa Cruz VMM Vogel’s minimal medium VMM+ Vogel’s minimal medium supplemented with 1% glucose VVC Vulvovaginal candidiasis

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Abstract

Aspergillus fumigatus is an ubiquitous saprophytic fungus that causes life-threatening disease in immunocompromised individuals. The increasing resistance to current antifungals makes the development of new antifungal drugs an important goal. Small RNA (sRNA) mediated gene silencing, whereby activation of the RNA interference (RNAi) pathway by dsRNA results in degradation of target mRNA, has shown to be promising in its ability to control a range of diseases. This pathway can potentially be exploited to target essential fungal leading to their down-regulation and resulting in growth inhibition or death. Major barriers for the use of small interfering RNA (siRNA) as therapeutics are the stability of the siRNA and its delivery into the cell. If these obstacles can be overcome a new class of antifungals based on siRNA could be developed.

This thesis focuses on the development of siRNAs to treat fungal disease. We show that the RNAi pathway is conserved and functional in A. fumigatus. Sequencing the small RNA transcriptome of wildtype and RNAi pathway mutants revealed siRNA-like sequences that are candidate therapeutics. We assessed the uptake and efficacy of siRNA treatment in A. fumigatus and the model filamentous fungus Neurospora crassa. We show that siRNAs had no significant impact on growth or target mRNA levels. Chemical modification of siRNAs (PNA, 2’-fluoro, 2’-O-methyl and/or phosphorothioate linkages) did not improve efficacy. Attempts to improve uptake of siRNAs using transfection reagents, cell-penetrating peptides and extracellular vesicles (EVs) were unsuccessful. However, lipid-based formulations, more specifically liposomes, were demonstrated to be a promising delivery method that should be tested in more detail in future.

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Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

Copyright statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and she has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=2442 0), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses.

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Acknowledgement

Firstly I would like to express my appreciation to my supervisors Mike Bromley and Sue Crosthwaite. Their knowledge, kindness and patience has brought me to this point. I have been very lucky with such great supervisors.

My deepest thanks go to my family and friends. You are the reason this thesis is finished as you all believed in me, especially when I did not. My mum and dad, for their infinite support and always encouraging me. My dearest sister, who is always there and listens to me even if she does not know what I am talking about. She never judges and is my greatest support. My grandpa as he is the greatest and my grandma as I am sure she would have been very proud of me. Thank you Michael, for your encouragement and always trying to keep my stress levels as low as possible. To my Dutch friends that never complained about me leaving the country and always supporting me. To my lovely friends I met along the way in Manchester, especially Cristina, Maria, Jenni and Lauren. Your positive energy helped me more than you think and it makes me smile so often. Thank you all so much.

A special thanks goes to the funding bodies VSBfonds, Quintusfonds and the BBSRC to make this project possible.

I would also like to thank everyone that helped me with my project along the way: All MFIG members with special thanks to Paul Bowyer and Can Zhao. Thanks to Peter March and Darren Thomson for their help with imaging. Thanks to Andy Hayes, Bharat Rash and Ping Wang for their help with the RNAseq library. Thanks to Cristian Heintzen and Steven Woods for all my questions in the first years of my PhD. I really enjoyed working with all of you and I appreciate the effort you have put in to help me. The MFIG lab was a great environment for learning as well as building friendships.

This road has not been easy, but because of all these lovely people I managed to get through it with a smile. I am forever grateful to them.

Being positive and loving is the key for success.

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Chapter 1

Introduction

1. Introduction

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1.1 Fungi in the environment: from harmless useful fungi to fungi that cause serious disease

Fungi are found everywhere in the environment. Ranging from pathogenic fungi found on plants and mammals to harmless useful fungi found in food. Organisms in the kingdom Fungi are eukaryotes and the kingdom includes microorganisms such as yeasts, moulds and mushrooms. Fungi are interesting in many ways, for instance they can be used to make useful products like cheese, beer or bread. Penicillium roqueforti, Penicillium camemberti and Penicillium glaucum are used to make the blue cheeses Roquefort, Camembert, Brie and Gorgonzola, respectively [1]. The same genus includes Penicillium chrysogenum, which is responsible for the production of the antibiotic penicillin [2]. In addition to the harmless fungi described above, a wide range of pathogenic fungi are found in our environment. A recent study on airborne samples in Italy revealed the presence of plant (e.g. Botrytis, Bipolaris, Ramularia and Stemphylium) and mammalian pathogens (e.g. Aspergillus, Candida, Cladosporium, Cryptococcus) [3]. Fungal infections on crops such as rice, wheat, cereals, vegetables and fruits can have devastating effects for the world’s food production, economy and natural ecosystems. Magnaporthe oryzae, also known as the rice blast fungus, is possibly the most important followed by Botrytis cinerea and Puccinia spp [4]. M. oryzae can infect over 50 plant species, including rice, wheat, barley and millet and is responsible for a 10 to 30% loss of rice crop each year [5]. B. cinerea can infect over 230 species including many species of fruit and vegetables. It infects its hosts by releasing degrading enzymes and penetrating the and since B. cinerea is still active at a temperature as low as 0°C, infection can occur even during transportation of crops [6].

Besides being plant pathogens, B. cinerea, Bipolaris spicifera and Stemphylium botryosum can also cause allergic reactions in humans [7]. Other fungal species that cause more serious diseases in humans belong to the genera Aspergillus, Candida, Cryptococcus and Pneumocystis. In fact, these species are responsible for 90% of all fungal-related deaths [8]. The seriousness of fungal disease is underestimated as there is no mandatory surveillance programme to report fungal infections. One million people with AIDS are killed by fungal meningitis or pneumonia every year [9]. Underlying diseases such as or AIDS are often recognised to be the cause of deaths but despite this misdiagnosis, the occurrence of 1.5 million deaths by fungal disease was higher than deaths caused by either tuberculosis or malaria in 2012 [8]. In 2016, the estimated burden of invasive fungal disease in the United Kingdom was between 241,525 and 662,987 cases per year [10].

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The main fungal diseases are known under the names aspergillosis, candidiasis and pneumocystis pneumonia. Aspergillosis, caused by Aspergillus spp, mainly affects people with lung disease or a weakened immune system. Candidiasis, caused by Candida spp, can be congenital, oral or invasive and the most common causative species is C. albicans. Candida is a common member of the human microbiota, but causes major problems when the commensal relation with the host is misbalanced. Most women experience a vaginal thrush caused by Candida at least once in their life. Some women experience multiple infections (4 or more) a year which is defined as vulvovaginal candidiasis (VVC), but this can also be continuous which is defined as chronic VVC [11]. The invasive form of candidiasis when Candida is found in the bloodstream, called candidemia, mainly occurs in immunocompromised individuals. 53.3% of patients admitted to intensive care units (ICU) were identified with candidemia [12]. Mortality rates in patients identified with candidemia range from 5 to 71% [13].

Pneumocystis pneumonia (PCP), caused by Pneumocystis jirovecii, is a high risk for immunosuppressed patients. It used to occur mainly in AIDS patients, with PCP in more than 63% of the patients in 1986 [14]. Due to the effective HIV treatments, the most susceptible group shifted from AIDS patients to non-HIV immunosuppressed patients where CD4+ levels are significantly reduced. This occurs in transplant patients, patients receiving immunosuppressive therapy to reduce tumour growth or patients with inflammatory and rheumatic diseases [15].

Cryptococcus spp, mainly C. neoformans and C. gattii, are also one of the main causative agents responsible for fungal disease. C. neoformans mainly infects the membranes covering the spinal cord and brain in immunocompromised patients, also known as cryptococcal meningitis. Cryptococcal meningitis is responsible for over 600,000 deaths per year in HIV-positive patients worldwide [16]. Moreover, healthy individuals risk infection with C. gattii which is common in (sub-) tropical climates [17].

1.2 Aspergillus fumigatus

The general name of fungal infections caused by Aspergillus spp is aspergillosis. Of all fungal diseases, aspergillosis has the highest mortality rate, ranging from 30% to 95% [8]. Aspergillus fumigatus is responsible for approximately 90% of the aspergillosis cases and therefore this thesis is focussed on A. fumigatus. The other 10% of aspergilloses is caused by either A. niger, A. flavus, A. nidulans or A. terreus [18]. A. fumigatus grows optimally at a pH between 3.7 to 7.6 and at 37°C, but it can survive in a wide range of environmental conditions such as temperatures between 12°C and 65°C. The green conidia, with a size between 2 µm – 3 µm, are

16 airborne and therefore ubiquitous in the environment. The occurrence of spores in the air, the adaptability to the environment, and its small conidia that can reach human airways are important for its pathogenicity. Aspergillosis is initiated by inhalation of fungal spores resulting in airway infections and an entire lobe or lung can be destroyed if it remains untreated. It is estimated that each individual inhales hundreds of spores a day [19]. A recent study on air samples in Bejing hospital showed that Aspergillus spp. were the most common fungi and of these 34% to 50% were characterised as A. fumigatus [20]. These numbers are worrying, especially in hospital environments, as mainly immunocompromised individuals are susceptible to aspergillus infections. Even though aspergillosis is rarely a risk for healthy individuals, some aspergillosis cases have been reported in non-immunocompromised individuals [21, 22].

Aspergillosis can occur either in invasive or allergic forms. The allergic form called allergic bronchopulmonary aspergillosis (ABPA) is found in individuals suffering from asthma or cystic fibrosis and affects over 4 million people worldwide [23]. Furthermore, asthma patients can be sensitised to different fungal species, a disease characterised as severe asthma with fungal sensitization (SAFS), and this can result in hospital admission [24]. SAFS can be caused by either A. fumigatus, C. albicans, B. cinerea, Penicillium notatum, Cladosporium herbarum or Alternaria alternata and is identified by allergy skin prick tests (SPT) and specific IgE tests [25]. The invasive form of aspergillosis is common in immunocompromised individuals and mostly affects the lungs or sinuses [26]. One form of IA, chronic pulmonary aspergillosis (CPA), is common in individuals with underlying lung diseases. The estimated number of people in the world who have CPA is around 3 million and approximately 450,000 patients die per year in their first six months of infection [8]. Another type of aspergillosis, aspergilloma, involves the growth of a fungal ball in pre-existing pulmonary cavities. These cavities can be caused by a variety of conditions such as sarcoidosis, cysts, bronchiectasis, but most commonly tuberculosis. An aspergilloma consists of fungal mycelia, mucus, inflammatory cells and tissue debris and can be treated by surgical removal to prevent life-threatening haemoptysis (coughing up blood) [27].

In addition to respiratory infections, A. fumigatus and Candida spp are often associated with burns. Open wounds can be easily colonised and combined with the impaired immune system of burn patients and the use of broad-spectrum antibiotics on burn wounds present ideal sites for infection [28]. Almost 93% of the 97 patients autopsied at the US army institute of surgical research burn centre suffered from an A. fumigatus infection which contributed to their death [29].

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A. fumigatus is also one of the most common causative agents responsible for eye and ear infections. Ear mould infections or otomycosis are often mixed with bacterial infections of the external ear canal. These infections can result in perforation of the tympanic membrane and loss of hearing [30]. Fungal eye infections, keratomycosis, are most common in developing and (sub-) tropical countries. In developing countries, keratomycosis mainly occurs in men working in the agricultural sector suffering ocular trauma from vegetative or sand particles [31]. The main sources of infections in developed countries are contact lenses or contact lens fluid. The percentage of patients diagnosed with microbial keratitis, caused by either or fungi, that were wearing contact lenses was as high as 62.7% in the Netherlands and 33.7% in Australia [32, 33]. The diagnosis is time consuming as growing cultures from corneal scrape samples can take up to 2 weeks and when cultures are ready, PCR or microscopy needs to be performed [34]. Delay in diagnosis of 2 or more weeks in contact lens wearers significantly increases the chance of surgery, hence early diagnosis and treatment is important to prevent serious reduced visibility or even blindness [35].

1.3 Antifungal drugs

There are four major families of drugs available to treat A. fumigatus infections (Table 1.1). The first family includes azoles that disrupt the ergosterol biosynthetic pathway by inhibiting the action of the lanosterol 14 alpha-demethylase enzyme, encoded by cyp51A. Because ergosterol is a major component of cell membranes, inhibition of lanosterol 14-alpha demethylase enzyme has fatal consequences for the fungus. This class of agents represent the first line therapy for treatment of Aspergillosis, however resistance to the class is a significant problem. The second family of drugs are the polyenes, for example Amphotericin B. Polyenes also target ergosterol, but in a different way. Amphotericin B binds to ergosterol and creates pores at the cell membrane. In addition, this drug induces an oxidative burst which promotes its antifungal effect. Fungi resistant to this class of drugs exist but occur much less frequently [36]. The key problem with Amphotericin B is that it is poorly tolerated by the human host and leads to nephrotoxicity due to channel formation over kidney cell membranes. Another family of agents are classified as allylamines. These drugs target ergosterol by inhibiting an enzyme essential for ergosterol biosynthesis, squalene epoxidase, however these drugs are the least effective against Aspergillus spp. [30]. The last family, echinocandins, inhibit formation of the cell wall molecule (1,3)-β-D-glucan and one of the drugs in this class is caspofungin. However, antifungal resistance is also reported for this class [37]. Two additional agents not yet consistent to a class, flucytosine and griseofulvin, are used in antifungal treatments. [30]. Flucytosine is in most cases only used in combination with another

18 antifungal drug as flucytosine rapidly evolves to be antifungal resistant when used on its own [38].

The emergence of resistance, particularly to the azoles, is concerning. The median time in which the wildtype isolate developed azole resistance in patients treated with azoles was only 4 months [39]. In some centres in the Netherlands, around 10% of patients are infected with azole resistant isolates and the National Aspergillosis Centre (NAC) in the UK report as many as 20% of patients with resistant strains [40]. The prognosis for these patients is poor with mortality levels exceeding 88% [41]. Two routes to the development of resistance have been postulated. It is likely that resistance develops de novo after treatment with azoles as patients, particularly those with chronic infections, are on long term therapy. Some patients however are infected with azole resistant strains prior to treatment and these individuals have acquired resistant strains from the environment. The resistant isolates identified in the environment were particularly found in agricultural areas where azoles are used extensively as crop protection agents [37]. The most common mechanism of resistance involves point mutations in cyp51A. Sequencing the cyp51A gene in azole resistant strains revealed a replacement for Glycine in codon 54 [42]. In a more detailed study on A. fumigatus clinical isolates, 18 alterations were found in cyp51A, including four novel mutations in codons 147, 216, 431 and 434 [43]. Analysis of antifungal drug resistant clinical strains collected from 7 different countries revealed another dominant resistance mechanism, namely the combination of point mutations in cyp51A with tandem repeats (TR). Besides the substitution of leucine 98 for histidine (L98H), two copies of a 34 bp sequence were found in the cyp51A promoter. The TR/L98H substitution was found to be present in 69% of itraconazole resistant strains [44]. Another resistance mechanism requires increased expression of ATP-binding cassette transporters (ABCTs) which mediate drug efflux. Itroconazole resistance was associated with a 5-fold increase in the level of ABCT atrF mRNA [45].

The serious diseases A. fumigatus can cause, the increase of drug resistant strains and the toxicity in human cells, make research on this fungus a high priority.

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Table 1.1. Classes of antifungal drugs used to treat A. fumigatus infections

Resistant A. Efficacy Antifungals fumigatus Class Target (mg/L in examples strains mice) found? Ergosterol (cell Voriconazole: Itraconazole, membrane) by 0.064-0.094 Azoles Voriconazole, Yes disrupting its Posaconazole: Posaconazole production 0.047-0.064 Ergosterol (cell Amphotericin Amphotericin Polyenes membrane) by binding Yes B B: 0.125-0.19 and creates pores Caspofungin, (1,3)-β-D-glucan (cell Caspofungin: Echinocandins Yes Micafungin wall) 0.032 Ergosterol (cell Terbinafine: membrane) by Allylamines Terbinafine 5 mg/g Yes inhibiting squalene (topical only) epoxidase

1.4 Gene silencing mechanisms

The mechanism in fungi where copies of a specific gene are introduced and cause the post-transcriptional silencing of this specific gene in vivo is called quelling. Quelling is one of the three silencing mechanisms active in the filamentous fungus Neurospora crassa and is also known by the RNA interference (RNAi) mechanism that is active in many eukaryotic species [46]. The RNAi (RNAi) mechanism serves originally as a host defence mechanism against exogenous invading nucleic acids such as or transposons [47]. RNAi is activated by introduction of (external) double-stranded RNA and results in post-transcriptional gene silencing (Figure 1.1). Single-stranded RNA (ssRNA) has to be generated into double-stranded RNA (dsRNA) by RNA- dependent RNA polymerases (RdRPs) [48]. Dicer-like proteins (DCL) recognise dsRNA and cut them into smaller RNA fragments of 21-25 nucleotides that are called small interfering RNAs (siRNAs) [49]. siRNAs are loaded onto the RNA-induced silencing complex (RISC), mainly composed of Argonaute proteins, and cause reduction of related mRNA levels by binding to complementary mRNA and preventing its .

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Figure 1.1. Schematic overview of RNA interference (RNAi) mechanism RNAi, a mechanism resulting in post-transcriptional gene silencing, is activated by the recognition of long dsRNA by Dicer enzymes. These enzymes digest the dsRNA into small interfering RNAs (siRNAs) with a length of generally 21 – 25 nt. siRNAs are loaded into RNA-induced silencing complex (RISC), mainly consisting of Argonaute proteins, and the passenger strand of the siRNA gets cleaved. The antisense strand of the siRNA is guided to its complementary mRNA which causes the cleavage of mRNA and its degradation prevents translation.

Van der Krol et al. were the first that described an interference mechanism in plants. Their aim was to analyse the effect of increased expression of the pigmentation gene, chalcone synthase (chs) in petunia [50]. The result was surprising, because 25% of the transformants were lighter instead of darker. The same phenomenon was discovered in N. crassa two years later by Romano et al. This research was focussed on increasing the expression of the pigmentation gene albino-1 (al-1). 30% of the transformed N. crassa strains were white instead of the wildtype colour orange [51].

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In addition to quelling, N. crassa has two other gene silencing mechanisms. The second mechanism is meiotic silencing of unpaired DNA (MSUD) which is another post-transcriptional gene silencing mechanism only active in meiotic cells [52]. Homologous chromosomes normally pair during meiosis and are subsequently evenly distributed to each daughter cell. If pairing does not occur, the unpaired DNA is recognized and silenced by the MSUD mechanism. Similar to quelling, this mechanism requires DCL-1 and the QIP exonuclease for the production of small RNAs, but their production also depends on another protein, suppressor of ascus dominance (SAD) [53]. This suggests that there are different pathways for sexual and vegetative post-transcriptional gene silencing. The third silencing mechanism is repeat induced point-mutation (RIP) which also occurs during the sexual phase of the life cycle, but this is not a post-transcriptional gene silence mechanism. RIP occurs in the haploid nuclei of premeiotic cells before the fusion of the nuclei [54]. RIP targets duplicated DNA sequences above 155 bp that may have arisen due to mistakes in DNA replication, or be due to invading viral DNA or transposons [55]. The mechanism of RIP causes silencing of the duplicate sequences by mutating the nucleic acid resulting in G:C to A:T transitions [56].

1.5 The RNA interference pathway in the model organism N. crassa

N. crassa is a model organism for research on the RNAi mechanism. Neurospora spp. are filamentous ascomycetes, grow well in the laboratory, have a short and fast haploid life cycle and are more closely related to complex eukaryotes than the other well-known model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe [57]. Neurospora was discovered and named the red bread mould after contamination in French bakeries around mid-1800s. One decade later, Dodge classified the different Neurospora species and discovered their sexual structures [58]. The fungus has a vegetative growth cycle and a sexual growth phase [59]. The conidia of N. crassa are either A or a and two different mating types are required for reproduction during the sexual growth phase. One of the biggest advantages in using N. crassa as a model, is that the entire sequence of seven chromosomes (43 Mb) of N. crassa was completed in 2003 [60]. Also, an almost complete library of gene deletion strains, available from the Fungal Genetic Stock Center (FGSC), is an useful resource for the study of gene function. To date, N. crassa has been widely investigated and many useful strains and tools have been generated. Nowadays it is an important organism to study the circadian rhythms, gene silencing, light-signal transduction, morphogenesis and epigenetics [61]. Many studies have been performed revealing genes involved in quelling in N. crassa and this will be discussed in the following section.

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Qde-3 encodes for a RecQ DNA helicase which causes unwinding of double-stranded DNA and therefore allows the DNA to be transcribed into ssRNA [62]. Qde-1 encodes an RNA-dependent RNA polymerase which is important for the synthesis of dsRNA from the (external) ssRNA [63]. However, QDE-1 is also able to convert ssRNA from aberrant DNA [64]. Replication protein A (RPA), a single stranded DNA binding complex, is a necessary component in converting DNA into dsRNA since it interacts with QDE-1 [65]. QDE-1, RPA and QDE-3 are thought to work upstream of the RNAi pathway, because they are involved in converting introduced DNA into dsRNA. The dsRNA is cut into 21- 25 nt long siRNAs by DCL-1 and DCL-2 [66]. Strains harbouring mutations in both dcl-1 and dcl-2 showed no quelling, indicating the two Dicer proteins are necessary for an optimal RNAi mechanism pathway. However, mutation of either dcl-1 or dcl-2 showed a quelling activity of 20% which indicates both proteins can cause quelling activity individually [49]. Once the RNA is processed by Dicer-like proteins, the siRNAs are loaded into RISC. The Argonaute protein encoded by qde-2, which is the core of RISC, is essential for the RNAi mechanism [67]. Furthermore, Choudhary et al. examined RNAi responses after induction of dsRNA. They revealed that the production of dsRNA by QDE-1 and QDE-3 induces the levels of QDE-2 and DCL-2 and post-transcriptional regulation of qde-2 is dependent on dcl- 1 and dcl-2 [68].

Combining all these studies gives a clear view of the quelling mechanism in N. crassa and the specific genes involved (Figure 1.2). First of all, aberrant DNA is converted into dsRNA by the combination of QDE-1, QDE-3 and RPA proteins [62] [63] [64]. When dsRNA is present, the RNAi pathway in N. crassa starts with the recognition of the dsRNA by Dicer-like proteins (DCL-1, DCL-2). Once the dsRNA is cut into siRNAs, they are loaded onto the RISC and the passenger strand is nicked by the QDE-2 and removed by QIP, a QDE-2-interacting protein, with an exonuclease domain [69]. The remaining siRNA strand, which is complementary to the target mRNA, is used as a guide to find the target mRNA. RNA complementary to siRNAs are degraded by QDE-2 and therefore causes the gene silencing [70].

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Figure 1.2. Overview of the RNAi pathway in N. crassa Quelling is the RNAi pathway of N. crassa in the vegetative growth phase. This pathway starts with the recognition of aberrant DNA or RNA and this is converted into dsRNA by a collaboration of QDE-1, QDE-3 and RPA proteins (1). DCL proteins cut the dsRNA into smaller RNAs named small interfering RNAs (siRNAs) (2). siRNAs activate the RISC complex which separates the two strands and the passenger strand gets degraded by QIP (3). The activated RISC carries the remaining siRNA strand to its complementary mRNA and causes the cleavage and degradation of target mRNA which results in gene silencing (4).

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1.6 RNAi conservation in eukaryotes

RNA interference is described in many other organisms besides N. crassa [71]. RNAi was described in C. elegans for the first time in 1998 [72]. Studies on RNAi in C. elegans reveals an amplification step where it replicates siRNA to silence target sequences more effectively. It is assumed RdRP activity is responsible for amplification of the dsRNA, because it generates dsRNA from aberrant RNA or DNA. It is possible amplification of siRNA continues for some time [73]. N. crassa contains homologues of C. elegans RNAi components. For example, RNAi-Deficient-1 (RDE-1) is homologous to QDE-2 in N. crassa and EGO-1 is homologous to the RdRP QDE-1 in N. crassa [74, 75].

Another example of an RNAi mechanism in organisms of the Animal kingdom is found in the fruit fly Drosophila melanogaster. Also this RNAi mechanism is similar to that in N. crassa. For example Drosophila’s C3PO (component 3 promoter of RISC) is found to be responsible for nicking the passenger strand, which is similar to QDE-2 activity in N. crassa [76]. RNAi in Drosophila also requires a set of Argonaute proteins (Ago1 and Ago2) and Dicer proteins (Dcr-1 and Dcr-2) [77].

The RNAi pathway is also conserved in mammals. Although less studied than in other organisms like N. crassa and C. elegans, it is known to be triggered by siRNAs or short hairpin RNAs (shRNAs) of 20 – 23 nucleotides. Similar to other eukaryotes, the pathway contains the core components Dicer and Argonaute proteins [78, 79]. Interestingly, mammalian cells do not have the RdRP which suggests that only dsRNA can induce target mRNA degradation [80]. It also suggests that mammalian RNAi does not have an amplification step, because synthesis of transitive RNA requires an RdRP. Consistent with these findings, RecQ homologs (Werner and Bloom DNA helicases) in mammalian cells are not involved in the RNAi pathway [79].

The RNAi mechanism is known to be active in several pathogenic fungi opening up the possibility of treating fungal infections by sequestering the pathway with the introduction of synthetic siRNA to silence essential genes. For example, C. albicans pathogenicity driven by the factor EFG1, relies on host cell invasion via dimorphic shift as well as hyphal and biofilm formation [81]. Transfection of C. albicans with 1 μM Efg1-siRNA resulted in a seven-fold decrease of Efg1 mRNA levels compared to untreated C. albicans. This result revealed that also C. albicans has a RNA interference mechanism which is activated after the addition of dsRNA to the cells [81]. Moreover, Efg1-siRNA is also used to indirectly down-regulate ALS3 which is regulated by Efg1. Adherence of C. albicans to host substrates is mediated by ALS3. Down-regulation of ALS3 after incubation of Efg1-siRNA was more significant

25 than down-regulation of the Efg1 gene suggesting an effective strategy to reduce the virulence C. albicans utilising siRNA targeting transcription factors [82].

Another fungus, Cryptococcus neoformans, can cause serious diseases in individuals with an immune deficiency. This organism also uses the RNA interference mechanism and can be silenced via this mechanism. Liu et al. inhibited the expression of two genes, CAP59 and ADE2, via RNAi. CAP59 is required for the polysaccharide capsule and ADE2 encodes phosphoribosylaminoimidazole carboxylase. These specific genes were selected because their deletion results in clear phenotypes; CAP59 deletion results in cells lacking a capsule and ADE2 deletion results in pink C. neoformans colonies. The genes were individually placed in a hairpin construct and transformed into C. neoformans. The transformation had only a 7-10% success, but nevertheless as shown by qRT-PCR, CAP59 expression was reduced by 23% [83].

RNA interference mediated by siRNA is also active in different Aspergillus species. In 2004, Mouyna et al. described RNAi in A. fumigatus as a method to study essential gene function. Homologs of N. crassa genes qde-1, qde-2 and qde-3 were identified and hairpin constructs were used to activate the RNAi mechanism with duplicate sequences of the gene of interest, ALB1 or FKS1. ALB1 is involved in the melanin biosynthesis which results in a white phenotype compared to the green wildtype. FKS1 is an essential gene that is involved in the synthesis of β(1-3) glucan and down- regulation would result in a loss of growth. Both genes were expressed four-fold less in the transformed strains compared to the wildtype, giving clear phenotypes [84]. siRNA has also been used in Aspergillus niger as a tool to analyse gene function. In a study of Barnes et al., protoplasts were transformed with the E. coli gene uidA which codes the protein β-glucuronidase (GUS). A pool of siRNAs targeting uidA was added to growth medium inoculated with A. niger protoplasts. Protoplasts with the siRNA and a transfection reagent were incubated for 72 hours after which RNA was extracted and RT-PCR performed to measure uidA transcript levels. Results showed uidA expression was down-regulated by 59%, suggesting siRNA can be used as a tool to down-regulate genes of interest in A. niger.

These findings of similar RNAi pathways in a variety of organisms indicate that the RNAi pathway is derived from a common ancestral mechanism [75].

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1.7 Different small RNAs (sRNAs) in fungal gene silencing pathways

Not only are siRNAs produced in fungi, but many more types of functional small RNAs have been discovered that require Dicer and interact with Argonaute proteins to silence RNA expression (Figure 1.3) [85]. New insights have revealed that the RNAi pathway is not only a defence mechanism, but also regulates a variety of cellular functions. siRNA was the first type of small RNA identified in N. crassa after introduction of external sequences and results in silencing the matching mRNA sequence [67].

Figure 1.3. Overview of fungal small RNAs (sRNAs) involved in gene silencing

Small RNAs can function in host defence mechanisms and in endogenous gene regulation. sRNAs, besides siRNAs, involved in host defence are MSUD-associated small interfering RNAs (masiRNAs) and sex induced silencing siRNAs (SIS siRNAs). In addition to siRNAs, QDE-2-interacting sRNAs (qiRNAs) and miRNA-like RNAs (milRNAs) are also involved in endogenous gene regulation. Adapted from [85].

A type of host defence silencing mechanism is meiotic silencing by unpaired DNA (MSUD). MSUD is a mechanism where genes that are unpaired during meiosis are silenced [53]. In addition to the presence of MSUD-associated small interfering RNAs (masiRNAs) in N. crassa, masiRNA is also present in the fungal pathogen of cereal crops Gibberella zeae [86]. Another type of host defence siRNAs are the sex induced siRNAs (SIS siRNAs) and to-date they have only been identified in the pathogenic fungus C. neoformans. The sex induced silencing pathway (SIS) is activated when a transgene with a repeat sequence is present. This type of silencing is approximately

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250 times higher in the sexual growth phase as in the vegetative growth phase, thus it is a silencing pathway induced during the sexual stage [87].

Host defence is not the only function of sRNAs. A variety of sRNAs are also important for the regulation of endogenous genes. These endogenous short RNAs (esRNAs) are similar to the host defence sRNAs, but triggered by endogenous molecules instead of invading or duplicated nucleic acid [85]. QDE-2-interacting sRNAs (qiRNAs) are a type of interference esRNA produced after occurrence of DNA damage. The majority of qiRNAs are mapped to ribosomal DNA and small portions are found to target intergenic regions, open reading frames (ORFs) or tRNA [88]. Homologous recombination is a form of DNA repair and this is required for the synthesis of qiRNA [89].

Another class of esRNAs are (miRNAs) and they are around 21 nucleotides long and derived from hairpin-structure ssRNAs [48]. miRNAs are present in animals and plants and recently discovered to be present in N. crassa. Due to their differences in biogenesis mechanism, miRNAs in N. crassa are called microRNA-like RNAs (milRNAs). 4 different classes of milRNAs are described in N. crassa, ranging from milR-1 to milR-4, because of the difference in proteins required for their biosynthesis. The proteins involved in gene silencing are Dicer, QDE-2, QIP and MRPL3, but the production of milRNA from each class depends on a different combination of one of these four proteins. MRPL3 is a key protein required for the production of dicer- independent mil-RNAs. It is a RNAse III domain-containing protein which is important for the digestion of dsRNA into smaller sRNAs. The classification of milRNA is distinct as follows: milR1 requires Dicer, MRPL3, QDE-2 and QIP; milR-2 only requires QDE- 2; milR-3 only requires Dicer activity; and milR-4 requires MRPL3 and depends partly on Dicer [90].

Moreover, there is a class of sRNAs that does not depend on dicer protein activity. In mammals, these sRNAs are called piwi-interacting RNAs (piRNAs) as they depend on piwi-related proteins and not on Dicer proteins. Piwi proteins belong to the family of Argonaute proteins and are responsible for piRNA binding. piRNAs have the size of approximately 23 – 31 nucleotides and are important in germline and adult stem cells [91]. In N. crassa the sRNAs that do not depend on Dicer protein activity, are called dicer-independent small interfering RNAs (disiRNAs) and they are approximately 23 nucleotides long. It appears that disiRNAs are generated independent of dicer, QDE- 1, QDE2 and QDE-3. Thus, another pathway for the production of this type of sRNAs has yet to be discovered [90].

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1.8 Uptake of siRNA by fungi

Although RNAi mediated silencing via transformation of fungal cells with the DNA of a gene of interest is working in Aspergillus species and N. crassa, it is time consuming and inefficient. In the study of Mouyna et al., described earlier in this chapter where they studied the RNAi by using hairpin constructs, only 1% of the transformants had the FKS1 silenced phenotype [84]. In addition to its inefficiency, this method that includes the production of protoplasts and of fungal cell is highly impractical, if not impossible, for the use of siRNAs as antifungals for human fungal infection. Interestingly, several studies reveal the possibility of RNAi-mediated silencing via uptake of external RNA from medium. The first study that describes uptake of RNA via the medium was reported by Khatri et al. The target for siRNA in this study was ornithine decarboxylase (ODC) in the fungus Aspergillus nidulans. ODC is the precursor of the biosynthesis of polyamines. Polyamines are present in all living organisms and in fungi they are essential for spore germination, growth and development. Reduction of spore germination, inhibition of germ tube length with 50%, smaller diameter of colonies and a 33% reduction in growth were observed after incubating 8 hour-old germinated spores in 25 nM ODC-siRNA for 12 hours. Significant reduction in odc mRNA levels was seen after 18 hour treatment with 25 nM ODC-siRNA. Addition of the polyamines putrescine or spermidine to the medium rescued conidia development, germ tube length and growth caused by ODC-siRNA. Together these data suggest the results were due to the inhibition of polyamine biosynthesis [92].

In another study, Kalleda et al. reported reduced mRNA levels of rasA by 93.53% and rasB by 96.07% in A. nidulans by using siRNAs in the growth medium [93]. Their success is based on a pool of diced-siRNAs (d-siRNAs) targeted to a variety of short sequences in the target mRNA compared to a single siRNA targeted to one specific sequence in the target mRNA. To make d-siRNAs, dsRNA identical to the mRNA of the target gene was incubated with RNase III family enzymes (Dicer). The enzymes cut the dsRNA into smaller RNA fragments, called d-siRNAs. In this study, synthesised siRNAs were also made. RasA is essential for conidial germination in both A. nidulans and A. fumigatus. The ability of the chemically synthesised siRNAs to silence rasA was compared to the d-siRNAs and results showed down-regulation of rasA mRNA levels to 44.38% when treated with chemically synthesised siRNA whereas the diced- siRNA dramatically silenced the mRNA levels of rasA to 5.30%. The pooled d-siRNAs method has also been used by Wang et al. where transcripts encoding Dicer-like proteins of the fungal plant pathogen Botrytis cinerea were used as targets [94]. Since B. cinerea double dicer deletion (dcl1dcl2) shows reduced virulence on flowers, fruits and vegetables, 20 ng/µL d-siRNAs targeted to dcl1 and dcl2 were sprayed on

29 a variety of host tissues and subsequently infected with wildtype B. cinerea spores. The virulence of B. cinerea was visibly inhibited: lesion size and relative biomass were significant reduced compared to water and unrelated d-siRNAs controls [94]. These results suggest that B. cinerea can take up external dsRNAs from the environment.

It has also been reported that A. fumigatus can take up RNA from growth medium [95]. In this study a cDNA library of small RNA species, resulting from different stages of development, was generated [95]. Surprisingly, within this library sequences of the yeast Saccharomyces cerevisiae were present. Further experiments showed that A. fumigatus mycelium is able to scavenge the yeast RNAs from the medium. The mycelial cell wall is positively charged whereas the conidial cell wall is negatively charged. It is thought that the negatively charged nucleic acids are attracted by the positively charged cell wall of hyphae and this causes accumulation of nucleic acids in the cell wall. Although accumulation has been shown, it is thought that small amounts of the siRNAs are transported into the where they bind to target mRNA. This was shown using siRNA targeting odcA (ornithine decarboxylase). OdcA is an important enzyme for growth and development of A. fumigatus. pyrG (orotidine- 5’-monophosphate decarboxylase), which codes for an enzyme involved in uridine monophosphate biosynthesis, was also used as a target [96]. odcA and pyrG siRNAs in this study reduced the target mRNA levels, measured by qRT-PCR, by 30 – 60% respectively.

1.9 Challenges with the use of siRNA as therapeutic agents

1.9.1 Stability of siRNA

Preventing siRNA degradation is one of the major challenges to be overcome if siRNA is to be a useful drug. Since RNA can be rapidly degraded by RNases, chemical modifications to the backbone or the nucleotide sugars must be incorporated to stabilise the siRNA and optimise its activity [97]. One strategy is modification of the nucleotide backbone (Figure 1.4A). The backbone is formed of phosphodiester linkages which can easily be degraded by RNases. An example of backbone modification which is useful for the construction of siRNA is the replacement of the oxygen molecule to sulphur [98]. This does not affect the negative charge of the backbone, but the advantage of a phosphorothioate (PS) linkage is its resistance to degradation [99]. One other promising backbone modification is the substitution of both the ribose sugar and the internucleotide linkages with N-(2- aminoethyl)-glycine (PNA). PNA is neutrally charged, but still obeys the Watson-Crick base pairing rules [100]. Other great properties of using PNAs are its resistance against and proteases and improved binding affinity to complementary mRNA [98].

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Sugar modifications to the nucleotides are a good method to improve binding affinity of complementary mRNA strands (Figure 1.4B). A promising sugar modification for siRNA that is often used, 2’-O-Methyl modification of the ribose in nucleotides, has great potential as it increases siRNA stability and binding strength. The 2’-fluoro-RNA (Fluoro) modification is also widely used and siRNAs with this modification have increased binding affinity compared to unmodified RNAs. The 2’deoxy-2’-fluoro-β-D- arabino (2’F-ANA) modification is comparable to the 2’F-RNA modification only the ribose is replaced with arabinose. It is used in siRNAs as it increases stability against RNases and therefore increases silencing [101].

Figure 1.4. Selection of oligonucleotide chemical modifications Chemical modifications are used for siRNA construction to increase nuclease resistance, stability and silencing function. A) The internucleotide backbone of RNA consists of phosphodiester links, but it can be modified into phosphorothioate (PS) or Peptide Nucleic Acid (PNA) to enhance nuclease resistance. B) RNA stability and the binding affinity to the complementary mRNA can be improved by modifying the nucleotide sugars. In this project we have modified the 2’ sugars of RNA with either an O-Methyl group or a fluoro group.

It is also important to note that siRNAs can have off target effects when the passenger strand is guiding the RISC instead of the guide strand or, when the guide strand recognises short sequences found in multiple mRNAs. In addition, siRNA can cause nonspecific innate immune responses when used as a therapeutic [101]. The immune system recognizes and responds to exogenous nucleic acids such as synthetic siRNAs.

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For example, exogenous dsRNA is recognized by the Toll-like receptor 3 (TLR3). TLR7 and TLR8 recognize ssRNA. This recognition up-regulates the interferon proteins IFN- α and IFN-β and therefore activates the human immune system [102]. Some sugar modifications like LNA or 2’-O-Me modification can reduce this immune response.

1.9.2 Delivery of siRNA

The activation of RNAi in fungi is mostly performed by the use of siRNA expression systems or by transformation of siRNA into protoplasts [84, 103-105]. This is an efficient method when siRNAs are used as a tool, but less practical when siRNAs are used as a drug. In mammalian cells, one of the barriers of cytoplasmic delivery is its inefficient diffusion across the cell membrane [106]. An extra barrier in fungal cells is the cell wall [107]. These barriers can theoretically be overcome with the use of delivery systems such as cell-penetrating peptides (CPPs), lipid vesicles or nanoparticles.

Many different peptides are known to deliver molecules into mammalian cells. However, testing 20 well-studied mammalian cell-penetrating peptides (CPPs) on the fission yeast S. pombe resulted in internalisation of only six [108]. This included Erns, MAP, penetratin, pVEC, TP and TP10. Erns and TP10 had the most efficient uptake, but TP10 was distributed equally in the yeast cell whereas Erns showed localised internalisation [108]. The minimum inhibitory concentration (MIC) of TP10 in the fungus C. albicans was 4 μM and 8 µM for the budding yeast S. cerevisiae [109]. Another CPP study in S. cerevisiae and C. albicans showed efficient internalisation of pVEC and (KFF)3K, although pVEC was most stable [110]. (KFF)3K is efficient to be used as a PNA-peptide conjugate, tested in [111]. pVEC was also tested on the green alga Chlamydomonas reinhardtii where pVEC was clearly internalised after 15 min incubation and no toxicity was measured [112]. The D- isomer of pVEC (all-D-pVEC) internalised almost 7 times more at the peptide concentration of 10 μM in C. albicans, but this phenomenon can be species specific [110]. Moreover, the CPP penetratin was rapidly degraded when incubated in S. cerevisiae, but degradation is not or hardly seen in incubation with C. albicans. The opposite is true for the peptide (KFF)3K. However, internalisation of penetratin in C. albicans was very low (0.35 nmol per mg protein) and therefore penetratin is less interesting to use as CPP.

Another class of peptides, PAF (Penicillium AntiFungal), are derived from the paf gene of Penicillium chrysogenum. The hexapeptides PAF26, PAF95 and PAF96 are derived from the original protein of 54 amino acids [113]. PAF26 has a N-terminal cationic and C-terminal hydrophobic motif and therefore only this hexapeptide, PAF26, is translocated into N. crassa and A. fumigatus and has an antifungal activity at around

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1 μM [114, 115]. Both motifs are important for antifungal activity as both PAF95, lacking the hydrophobic property, and PAF96, lacking the cationic property, lost this ability. PAF95 only internalises in the intracellular organelles of N. crassa and PAF96 was not able to penetrate either N. crassa or A. fumigatus.

The pH-Low-Insertion-Peptide (pHLIP) can translocate molecules into the cytoplasm by spontaneous insertion and folding across a lipid bilayer. Tested in HeLa cells and mammalian cancer cells, it can translocate molecules with MW up to 800 such as PNA, phalloidin and fluorescent dyes FITC, Alexa 488-, Alexa 546-, Alexa 647- maleimide and IR680-maleimide into the cytoplasm [116-118]. Molecules attached to pHLIP by disulphide bond are translocated into the cytoplasm most effectively when the environmental pH changes from 8 to 3.6. The disulphide bond is then cleaved in the environment of the cytoplasm. The unfolding and exit of the peptide takes place at a pH between 6 and 8, therefore washing the cells with a buffer at these pH values leaves the cleaved molecule inside and removes the peptide [119]. pHLIP is studied widely in mammalian cells and the low pH property is a great advantage to target cancer cells where the environment is at a lower pH than healthy cells. However, pHLIP is not yet studied in yeast or fungal cells.

Delivery of drug molecules can also be achieved by the use of lipid-based nanoformulations such as liposomes or lipid nanoparticles. The nanoformulations come in different size, surface structure and chemical properties, but they all have the ability to hide unfavourable properties of encapsulate molecules. Unfavourable properties of siRNAs are for example its hydrophilicity, negative charge and susceptibility to nucleases. Cationic formulations have the most potential as their positive charge causes the effortless encapsulation of negatively charged siRNAs. The cationic activity is also important to pass the different barriers such as the negatively charged cell wall of the fungus. Promising nanoparticles for siRNA delivery are the biodegradable mesoporous silica nanoparticles (MSNs). MSNs have a size of 20 nM and are taken up effectively by the roots of maize plants. The uptake was shown using red fluorescing MSNs that accumulated in the xylem [120]. Moreover, lipid- based nanoformulations are also used in filamentous fungi. For example in A. flavus and A. parasiticus, where a mixture of different lipids has been used for efficient siRNA delivery [121, 122]. Indeed, some antifungal drugs use lipid-based nanoformulations as encapsulation lowers the toxic side effect to its host cells and results in more effective treatments [123-125]. These reports suggest that lipid- based nanoformulations are translocated across the cell wall and could be useful to deliver siRNAs into N. crassa or A. fumigatus.

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1.10 Aims

This PhD project has the following three aims:

1. Defining the effect of al-1 siRNA treatment on N. crassa and the effect of odcA and pptA siRNA treatment on A. fumigatus

The first aim of the project is to assess the effect of siRNA treatment in N. crassa and A. fumigatus. The effect of treatment with unmodified and modified siRNAs on phenotype and target mRNA levels will be determined by microscopy, qRT-PCRs and phenotypic assays. The efficacy of a pool of siRNAs targeted to a variety of short sequences in the target mRNA will be reported, compared to the effect of a single siRNA targeted to one specific sequence.

2. Optimising delivery of siRNA

The second aim is to overcome the difficulty of delivering siRNA into the fungi. Fluorescently labelled siRNAs will be used to visualise siRNA localisation by the use of confocal microscope. siRNA up-take from medium, encapsulated in lipid-based carriers and attached to cell penetrating peptides will be assayed.

3. Characterisation of the small RNA transcriptome in A. fumigatus

The last aim is to determine the function of RNAi in A. fumigatus and characterise naturally occurring small RNAs in A. fumigatus. Small RNA libraries generated from wildtype and RNAi mutant strains of A. fumigatus, with a single gene deletion of either qde-2 or dicer-1 or with deletion of both dicer-1 and dicer-2, will be generated. Comparative analysis of the generated strains and molecular phenotypes will be reported.

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Chapter 2

Materials and Methods

2. Materials and Methods

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2.1 Strains and conditions

Strains used in this study are listed in Table 2.1. Aspergillus strains were maintained at 37°C in the dark on Sabouraud dextrose (SAB) agar (oxoid). Spores were collected in 1x Phosphate Buffer Saline with 0.1% Tween-80 (PBS-T) and filtered through glass wool. A1160p+ served as the parental strain for all gene deletion mutants and is therefore used as the A. fumigatus wildtype control. N. crassa strains were maintained at 30°C in the light on Vogel’s minimal medium (VMM) with additional biotin (50 ng/mL) [126]. Spores were collected and washed three times in ddH2O. For liquid growth, strains were cultured at either 30°C or 37°C up to the indicated time-point in VMM containing 1% glucose (VMM+) as a carbon source. ΔodcA was supplemented with 10 mM putrescine, whereas ΔpptA was supplemented with 10 mM

Lysine and 1.5 mM FeSO4. Spore concentrations were calculated with the Improved Neubauer haemocytometer (Hawksley) and resuspended in suitable medium to use for further experiments.

Table 2.1. Fungal strains used in this study Species Strain Description Source

Isolated from an invasive aspergillosis A. fumigatus CEA10 MFIG [127] patient. Used as wildtype standard.

Derived from CEA10, Ku80Δ, pyrG+. A. fumigatus A1160p+ Used as parental strain for gene MFIG [128] deletions.

Gifted by N. A. fumigatus A1160p+h+ Derived from A1160p+, hph+, aft4- van Rhijn

Derived from A1163, pyrG+, β- Gifted by D. A. fumigatus AfGFP tubulin::GFP McDonald

Gifted by M. A. fumigatus ΔodcA Derived from A1160p+, hph+, odcA- Bromley

Gifted by M. A. fumigatus ΔpptA Derived from A1160p+, hph+, pptA- Bromley [129]

A. fumigatus Δqde2 Derived from A1160p+, hph+, qde-2- This study

A. fumigatus Δdcr1 Derived from A1160p+, hph+, dicer-1- This study

Derived from A1160p+, hph+, ptrA+, A. fumigatus Δdcr1Δdcr2 This study dicer-1-, dicer-2-

Isolated from human clinical specimen, Gifted by P. A. lentulus CBS117885 USA. Used as wildtype standard. Dyer [130]

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Isolated from straw from Nottingham. Gifted by P. A. niger 8-171 Used as wildtype standard. Dyer [131]

Gifted by P. A. nidulans FGSC A4 Used as wildtype standard. Dyer

N. crassa 2489A Used as wildtype standard. FGSC [132]

Gifted by M. N. crassa H1-GFP Derived from 2489, Histone-1::GFP Freitag [133]

N. crassa 11267 Derived from 2489, hph+, hsp30- FGSC [134]

2.2 Polymerase Chain Reaction (PCR)

PCR reactions were carried out using a VeritiTM thermal cycler (Applied Biosystems) and according to the following reaction, unless stated otherwise. A typical PCR reaction with a total volume of 25 µL consisted of 100 ng DNA, 1x MyTaq buffer (Bioline), 1.5 U LongAmp Taq DNA polymerase (NEB), 0.4 µM reverse primer and 0.4 µM forward primer. DNA was denatured for 2 minutes at 95°C and 35 cycles were used for amplification. Denaturing and annealing during amplification were 30 seconds and the elongation time was dependent on the size of the product as LongAmp Taq DNA polymerase anneals 1 kb per 50 seconds. The primers used and annealing temperatures per product are described in Table 2.2. PCR products were checked on 0.8 – 2 % agarose gels.

Table 2.2. PCR, RT-PCR and sequencing primers used in this study

Annealing Product Description Forward 5’- 3’ Reverse 5’- 3’ temp (°C) size CCCAAGCTTTACTCT CCGGAATTCCCCA N. crassa al-1 CGTCTCCAAAGCCC TCAACGCCACCAA 70 530 bp DNA product GT CAAAG N. crassa actin AGCTGTTTTCCCTTC ATACCACGCTTGG 55 108 bp qRT-PCR CATCGT ACTGAGC N. crassa al-1 TGGAGTTTGTCGGG GGGGACGTGGATG 55 135 bp qRT-PCR TATGGC TAGAAGC N. crassa qde-2 CCGGATGTCTACAA TCCACCGGATGTC 56 115 bp qRT-PCR GGGAAT TACAAGG TAATACGACTCACTA TAATACGACTCACT N. crassa al-1 TAGGGAGAGAGACT ATAGGGAGATAGC DNA for siRNA 57 2001 bp CAGAGACCACGAAG AAGAAATACAGCA pool C CCCCC A. fumigatus pptA TAATACGACTCACTA TAATACGACTCACT DNA for siRNA TAGGGAGAGGCTCT ATAGGGAGAGTAC 57 1107 bp pool GCACAAAACGAGAG

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ACTGACAGACACC CGT TAATACGACTCACTA TAATACGACTCACT hph DNA for TAGGGAGAGCGACG ATAGGGAGATATT 57 1050 bp siRNA pool TCTGTCGAGAAGTT CCTTTGCCCTCGG ACG A. fumigatus β- CGACAACGAGGCTC CAACTTGCGCAGA 54 152 bp tubulin qRT-PCR TGTACG TCAGAGTTGAG A. fumigatus odcA AGACCGGCATCGAT GCGCAGATAGGAC 52 90 bp qRT-PCR CCTA TTGGTCT A. fumigatus pptA ACCAAGCATCTCTC GCTAGGGTTGGCG 54 92 bp qRT-PCR GTAGCC AATACC

A. fumigatus qde- AGACAAGAGACAGG TAGTTCTGTTACCG 2 3’ flank (P1 & GCTCCA AGCCGGAGGAAGA 55 1200 bp P2) TTGGGTTAAGCGC

A. fumigatus qde- GCTCTGAACGATAT AGTGAAGTCGAAA 2 5’ flank (P3 & GCTCCAACTGTTGTT CCCGGTC 55 1060 bp CGCTACACGCCTA P4) A. fumigatus qde- ATGGGAGAGACCAT AACTCCTCCCGTG 2 fusion PCR (P5 CCAGAA AAACCTT 55 5539 bp & P6)

A. fumigatus CCGCGAGAGAACCA TAGTTCTGTTACCG dicer-1 3’ flank TTGAAC AGCCGGGCTGGGT 55 1116 bp TGGAAAGGATGGA (P1 & P2) GCTCTGAACGATAT GCGACGATATGGG A. fumigatus GCTCCAACCCTCTG AAGCGTA dicer-1 5’ flank 55 1137 bp GAGGTATGGACGAA (P3 & P4) G A. fumigatus GCCGTTGTTTCAGC GTTCCCTCTGAAG dicer-1 fusion PCR TCTTTC CGACAAG 55 7677 bp (P5 & P6)

A. fumigatus CTGAACCGCATAGC TAGTTCTGTTACCG dicer-2 3’ flank TGTTGC AGCCGGGTGACAA 55 1200 bp CCCGAGTAAGGCA (P1 & P2)

A. fumigatus GCTCTGAACGATAT GGCCCAGTCCTGT dicer-2 5’ flank GCTCCAACATGCCA ACGAAAA 55 1097 bp TTTGACGACGGTGA (P3 & P4) A. fumigatus AATTTTCAGGCACA CCATCGAGAAGCT dicer-2 fusion PCR GGCAAG CAAGGTC 55 6803 bp (P5 & P6) CCGGCTCGGTAACA GTTGGAGCATATC GAACTAACGGCGTA GTTCAGAGCTCTT hph fragment 55 2698 bp ACCAAAAGTCAC GACGACCGTTGAT CTG CCGGCTCGGTAACA GGGAGCATATCGT GAACTATCGTCTTCG TCAGAGCGCCTAG pyrithiamine CAATGCGCTGATGG ATGGCCTCTTGCA 55 2112 bp fragment CACTCAGGCCAATT TC G

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A. fumigatus AGACAAGAGACAGG AGTGAAGTCGAAA 55 5671 bp Δqde-2 validation GCTCCA CCCGGTC A. fumigatus CCGCGAGAGAACCA GCGACGATATGGG 55 7792 bp Δdcr-1 validation TTGAAC AAGCGTA A. fumigatus CTGAACCGCATAGC GGCCCAGTCCTGT 55 7067 bp Δdcr-2 validation TGTTGC ACGAAAA

2.3 RNAi mutant strains

2.3.1 Generation of RNAi mutant strains by two step fusion PCR

RNAi mutant strains were generated by the use of a two-step fusion PCR protocol (Figure 6.2A). This method consists of multiple rounds of PCR and combining the products together into a target specific fusion product. All primers used are listed in Table 2.2. The qde-2, dicer-1 and dicer-2 fusion products contain a hygromycin resistance (hph) cassette that was amplified from pAN7-1. To generate a double dicer deletion strain, another dicer-2 fusion product was generated containing a pyrithiamine resistance (ptrA) cassette amplified from pPTRII. Primers P1 and P2 were used to amplify the upstream flanking region (3’ flank) and the primers P3 and P4 were used to amplify the 5’ downstream flanking region (5’ flank) of the target genes. The 3’ flank, 5’ flank and either the hph or ptrA cassette were purified using a Qiagen PCR purification kit and fused using the conditions previously described by Fraczek et al. [128]. The fusion products are transformed into protoplasts of A1160p+ using a polyethylene glycol (PEG)-mediated transformation protocol [128]. All single- deletion transformants were picked and purified at least 3 times on SAB agar supplemented with 200 µg/mL hygromycin. The double dicer deletion (Δdcr1Δdcr2) transformants were picked and purified at least 3 times on Aspergillus minimal medium supplemented with 0.5 μg/mL pyrithiamine (Takara).

2.3.2 Verification of RNAi mutant strains

DNA was extracted from spores using the cetyl trimethyl ammonium bromide (CTAB) method [128]. The RNAi mutant strains were screened for hph or pyrithiamine gene replacement by PCR using primers listed in Table 2.2. PCR products of the RNAi mutants were compared to the PCR products of the parental strain A1160p+.

2.3.3 Phenotypic screening of RNAi mutant strains

Phenotypes of the RNAi mutant strains were tested on RPMI agar and SAB agar plates. Conidia collected from the validated RNAi mutant strains Δqde2, Δdcr1, Δdcr1Δdcr2, the impure strain Δdcr2 and their parental strain A1160p+ were diluted to a final concentration of 4x105 spores/mL and 10 µL was spotted in the centre of

39 the plates. The radial growth was measured in triplicate every 24 hours and the plates were imaged (Canon Powershot S5 1S) at 72 hours of growth.

Growth assays were performed in either 1x Rosswell Park Memorial Institute (RPMI) 1640 medium [pH 7.0] (1% RPMI-1640 powder (Sigma), 0.165mol/L MOPs, 3.6% glucose), 1x RPMI1640 medium [pH 8.0], Aspergillus minimal medium (AMM) or Aspergillus complete medium (ACM) [135]. RNAi mutant strains (Δqde2, Δdcr1, Δdcr1Δdcr2) and their parental strain (A1160p+) were diluted in the tested media and added to a 96-well plate (StarLab) at a final concentration of 105 spores/mL. The static cultures were incubated at 37°C and the optical density (OD) was measured by the PowerWare X-1 plate reader (MTX Lab Systems) at 600 nm after 48 hours. Analyses were done using excel and GraphPad Prism 7. Relative fitness was calculated using the OD values of 8 replicates per strain and quantified relative to the parental strain.

Histidine spot tests were performed by spotting 10 µL of a 107 to 104 spores/mL dilution series per strain in the centre of VMM+ plates supplemented with histidine ranging in concentrations from 0 to 10 mg/mL. Plates were incubated for 24 hours at 37°C and images were taken by digital camera (Canon Powershot S5 1S).

2.4 Generation of siRNAs

2.4.1 Unmodified synthetic siRNAs

The pptA synthetic siRNAs were designed using the corresponding mRNA sequence (AFUA_2G08590) downloaded from the fungal genomic database FungiDB (http://www.fungidb.org). The design of odcA synthetic siRNAs were taken from Jöchl et al. [96], whereas the al-1 synthetic siRNAs were designed using the unpublished RNAseq data of Ran Wang (see section 3.2). Unmodified RNA oligos were synthesised by Eurofins Genomics (Table 2.3). In order to visualise pptA siRNA by confocal microscopy, another pptA antisense primer was synthesised with a 3’ fluorescent dye carboxy-tetramethyl-rhodamine (TAMRA, Applied Biosystems). All RNA oligos were synthesised with a phosphate group at the 5’ end. The sense and anti-sense oligos (50 µM) were annealed by heating to 90°C for 1 minute in annealing buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl and 5 mM EDTA) and cooling down (0.02°C/s) using a thermal cycler (Biometra®). The siRNA products were checked on 20% polyacrylamide gels.

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Table 2.3. Synthetic unmodified siRNAs synthesised by Eurofins Genomics

Complementary Organism Target gene Sequence 5’- 3’ Length to UAUCACCGAAGAA Antisense Phytoene desaturase AUCGUCAACA N. crassa 23 (al-1) UUGACGAUUUCUU Sense CGGUGAUAAG CUGCUAAAGAAGC Antisense CACUAUUCUU N. crassa Unrelated 23 GAAUAGUGGCUUC Sense UUUAGCAGUU UAGGACUUGGUCU Antisense Ornithine UGCAGGUU A. fumigatus 21 decarboxylase (odcA) CCUGCAAGACCAA Sense GUCCUAUU UAUUUCAGCAGGU Antisense Phospho-pantetheinyl UUGAGGCGdTdT A. fumigatus 23 transferase A (pptA) CGCCUCAAACCUG Sense CUGAAAUAdTdT UGUCACUGUUGAG Antisense AUGCAGUGdTdT A. fumigatus Unrelated 23 CACUGCAUCUCAA Sense CAGUGACAdTdT

2.4.2 Modified synthetic siRNAs

Modified siRNAs were synthesised by Eurofins Genomics, TriLink BioTechnologies or Cambridge Research Biochemicals. The modifications are listed in Table 2.4 where the antisense primer is used as an example, but sense primers were designed using the same modifications.

All RNA oligos were synthesised with a phosphate group at the 5’ end. The sense and anti-sense oligos (50 µM) were annealed by heating to 90°C for 1 minute in annealing buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl and 5 mM EDTA) and gradiently cooling down (0.02°C/s) using a thermal cycler (Biometra®). The siRNA products were checked on 20% polyacrylamide gel.

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Table 2.4. Modifications of antisense synthetic RNA oligos

Modifications Abbreviation Target Sequence 5’ - 3’ Synthesised by

USUSGSASCSGSASUSUS al-1 USCSUSUSCSGSGSUSGS Phosphorotioate ASUSASASG Eurofins PS (S) backbone USASUSUSUSCSASGSCS Genomics pptA ASGSGSUSUSUSGSASGS GSCSGSdTSdT UUOGACGAOUUUCUO O Phosphorotioate al-1 UCGGUG AUASASG (S) 3’ overhang Eurofins PS OMe and 2’-O-methyl UAOUUUCOAGCOAGGU Genomics O O sugar ( ) pptA UUGA GGCGSdTSdT

UUGAFCGAFUFUUFCUU al-1 FCGGUGAFUAAG TriLink 2’-fluoro sugar Fluoro BioTechnologies, (F) F F F F UAU UU C AGCAGGU Inc. pptA UUFGAGGCFGdTdT

TTGACGATTTCTTCGG al-1 TGATAAG Cambridge Peptide nucleic PNA Research acid backbone TATTTCAGCAGGTTTG Biochemicals pptA AGGCGTT

2.4.3 Preparation of diced-siRNAs pool

T7 promoter sequences were introduced at both ends of the DNA primers and PCR was performed to amplify the target gene with the primers that are listed in Table 2.2. After purification, the T7 containing DNA fragments were transcribed into RNA and annealed to dsRNA using MEGAscript T7 kit (Invitrogen) according to manufacturer’s protocol. The long dsRNA was digested with shortcut® RNAse III (NEB) for 20 minutes at 37°C and purified according to manufacturer’s protocol. Quantity of the pool of siRNAs was measured by spectrophotometer (Synergy2, BioTek) and product quality was confirmed on 20% polyacrylamide gel.

2.5 Gel electrophoresis

2.5.1 Agarose gels

Depending on product sizes, a 0.8% - 2% agarose gel was made with 1x TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA [pH 8]) and dissolved by heating. When cooled down to approximately 60°C, safeview was added to a final 1x concentration. The agarose gel was run in 1x TAE buffer for approximately 1 hour at 80 – 100 Volts. Gels were imaged using geldoc (Bio-Rad).

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2.5.2 Polyacrylamide gels

Polyacrylamide gels were made with 30% acrylamide/bis-acrylamide, 37.5:1 (2.7% crosslinker) solution (Bio-Rad) to a final concentration of 20% polyacrylamide in 1x TBE buffer (89 mM Tris-borate and 2 mM EDTA [pH 8.3]) substituted with 0.1% freshly prepared ammonium persulfate (Sigma-Aldrich) and 0.05% tetramethylethylenediamine (TEMED, Sigma-Aldrich). Samples were run at low voltage (40 – 60 Volts) for approximately 4 hours. After running, the gel was stained with 10x SYBR gold nucleic acid stain (Life Technologies) in 1x TBE for 15 minutes. Gels were images using geldoc (Bio-Rad).

2.6 Amplification of al-1 DNA

N. crassa wildtype genomic DNA, 200 nM al-1 primers and 2x MyTag™ Red Mix (Bioline) was used in a 50 μl reaction. The PCR was performed with the primers listed in Table 2.2 to generate a 530bp al-1 DNA product. The PCR started with 5 minutes at 95°C and continued with 40 cycles of 30 sec. at 95°C, 1 minute with the annealing temperature of 70°C and 1 minute at 72°C. The PCR was extended for 10 minutes with 72°C to generate a 530 bp al-1 DNA product. PCR products were pooled to one sample of 100 μl and 15 μl was loaded on a 0.8% agarose gel next to a 1 KB ladder (New England Biolabs) for 50 min. at 80 volts to check the al-1 product size of 530 bp.

2.7 Analysis of siRNA treated cultures

2.7.1 Growth assay

Wildtype A. fumigatus spores (cea10) at a total concentration of 6x104 spores/mL were inoculated in 2x RPMI medium [pH 7.0] (2% RPMI-1640 powder (Sigma), 0.165 mol/L MOPs, 3.6% glucose) and 0.01% Tween80 in a 96 well plate, 200 μl/well. Unrelated or odcA siRNAs were added to the cultures at a final concentration of 25 nM. The OD was measured by the PowerWare X-1 plate reader (MTX Lab Systems) at 600 nm every 10 minutes over a period of 48 hours in RPMI medium. Analyses were done using excel and GraphPad Prism 7. The threshold of OD600 0.05 was set to check for any delayed growth.

2.7.2 Phenotype assay

A. fumigatus cea10 wildtype spores and ΔodcA spores at a final concentration of 107 spores/mL were germinated in liquid VMM+, in presence or absence of 10 mM putrescine, for 6 hours at 37°C and shaken at 100 rpm. Germinated spores were incubated for another 6 hours using the same conditions supplemented with 25 nM

43 of odcA siRNA. Following the siRNA treatment, 3 µL of each sample was spotted in the centre of VMM+ agar plates and VMM+ agar plates supplemented with 10 mM putrescine. Plates were incubated at 37°C for 2 days and imaged by digital camera (Canon Powershot S5 1S).

Experiments with siRNA coated agar medium used corning Costar 12-well plates (Sigma-Aldrich) and VMM+ for A. fumigatus cultures and VMM for N. crassa cultures. The solid medium was coated with 25 nM siRNA or nuclease-free water (control), spread thoroughly and dried in a sterile environment. Spores were diluted in water at a final concentration of 5x106 spores/mL and 2 µL was spotted in the centre of the wells. N. crassa wildtype strain 2489A and A. fumigatus strains cea10 wildtype, ΔodcA and ΔpptA were used. ΔpptA was also grown on VMM+ supplemented with 10 mM Lysine and 1.5 mM FeSO4. A. fumigatus cultures were incubated at 37°C in dark and N. crassa cultures were incubated at 30°C in constant light or dark up to 3 days. Images were taken using a digital camera (Canon Powershot S5 1S).

2.7.3 Confocal microscopy

Spores were incubated at a final concentration of 2.5x104 spores/mL in VMM+ for 8 hours to generate young hyphae and treated for 10 hours with 25 nM of Alexa Fluor® Red-labelled or fluorescein-labelled 21 bp dsRNA (Block-ITTM, Life Technologies) in an 8-chamber coverglass (NUNC Lab-Tek). Images were taken by confocal microscope (Leica SP8) at the specific timepoint using the white light laser at 555 nm. The fluorochrome CalcoFluor White (CFW) was used in a concentration of 50 µg/mL to stain the cell wall and septa and imaged using a diode 405 nm laser. Images were analysed using FIJI.

2.7.4 Live cell imaging

For time-lapses, fungal cultures at a concentration of 107 spores/mL in VMM+ were loaded in the chamber of a CellAsic ONIX Y04D microfluidic plate at 8 psi for 5 seconds. Non-trapped cells were washed by flowing clean VMM+ for 5 seconds at 10 psi. After 4 hours of incubation at 30°C for N. crassa and 37°C for A. nidulans, 25 nM Block-ITTM siRNAs (B-siRNAs) were continuously distributed into the chamber at 3 psi and images were taken every 30 minutes using a confocal microscope (Leica SP8). Images were analysed using FIJI.

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2.7.5 RNA extraction and qRT-PCR

Either A. fumigatus or N. crassa spores at a concentration of 107 spores/mL were incubated in VMM+ for 8 hours and incubated for another 10 hours in the presence or absence of 25 nM siRNAs (unrelated or targeted to the gene of interest). VMM+ was supplemented with 10 mM Lysine and 1.5 mM FeSO4 in experiments where pptA was used as a target. Each sample was performed in 3 biological replicates. Fungal tissue samples were harvested and grinded in liquid nitrogen. Tissue from untreated samples served as a control. Total RNA was extracted using RNeasy plant mini kit (Qiagen) with on column DNase treatment. Levels of target mRNA were measured by quantitative real time PCR (qRT-PCR) using Superscript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies) according to manufacturer’s protocol. The housekeeping genes actin (NCU04173.2) in N. crassa and β-tubulin (AFUB_086810) in A. fumigatus were used to normalise results. Samples were controlled for primer efficiencies and measured in triplicate using 7500 Fast Real-Time PCR system (Applied Biosystems). The primers used for qRT-PCR are listed in Table 2.2. No RNA and no reverse transcriptase controls were run in every qRT-PCR for each primer set. Products were checked on 2% agarose gels for sizes and analyses were done using excel and GraphPad Prism 7.

2.8 Minimal inhibitory concentration

2.8.1 Hygromycin

Hygromycin B drug stock solution (500 mg/mL) was diluted to 40 mg/mL in nuclease free water. A serial dilution was prepared in nuclease free water by halving the concentrations, starting at 40 mg/mL and ending at 312.5 µg/mL. The dilution series were made in triplicate and two series were enriched with either 25 nM unrelated d- siRNAs or 25 nM hph d-siRNAs. 10 µL of the drug serial dilution series were added to designated columns of 96-well plates (CytoOne, Starlab). Column 10 served as a no- drug control where 10 µL nuclease-free water was added and column 11 served as a blank control with 100 µL RPMI (no drug, no fungus). Spores were diluted to a concentration of 2.5x104 spores/mL and 90 µL of A. fumigatus wildtype (A1160p+), A. fumigatus hygromycin resistant (A1160p+h+), N. crassa wildtype (2489A) and N. crassa hygromycin resistant (11267) spore solutions were added to the designated wells. The A. fumigatus static cultures were incubated at 37°C and the N. crassa static cultures were incubated at 30°C. The OD was measured after 24 hours and 48 hours at 600 nm by the microplate reader spectrophotometer (Synergy2, BioTek). Analyses were done using excel and GraphPad Prism 7. The MIC was set as the lowest concentration that inhibited the fungal growth by 50% (MIC50).

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2.8.2 Amphotericin B

Amphotericin B drug stock solution (5000 mg/L) was diluted in 100% DMSO to 1600 mg/L. Further dilutions were obtained in nuclease free water to a 10x final desired concentration. 10 µL of the drug serial dilution series were added to columns 1-10 of two 96-well plates (CytoOne, Starlab). Columns 1-11 were infected with 90 µL of A. fumigatus wildtype (cea10) at a concentration of 2.5x104 spores/mL, resulting in final Amphotericin B concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/L. Column 11 served as a no-drug control where 10 µL nuclease-free water was added and column 12 served as a blank control with 100 µL RPMI. The different rows were used to test the variety of siRNAs in triplicate. Unrelated siRNAs, pptA siRNAs, PS OMe pptA siRNAs and ppta PNAs were added to the designated wells at a final concentration of 25 nM. The static cultures were incubated at 37°C and the OD was measured after 48 hours at 600 nm by the microplate reader spectrophotometer (Synergy2, BioTek). Analyses were done using excel and GraphPad Prism 7. The MIC was set as the lowest concentration that inhibited the fungal growth by 80% (MIC80).

2.9 Statistical analysis

Statistical analyses were done using GraphPad Prism 7. Groups were first tested for a normal distribution by performing a Shapiro-Wilk normality test. Comparison between normally distributed groups was done by performing one-way anova and the Tukey's honestly significant difference (HSD) post hoc multiple comparisons test, unless stated otherwise. Comparison between non-normal datasets was done using the nonparametric Kruskal-Wallis H test. Confidence interval was set at 95% and the significance of the p values were determined as: ** = p<0.01 and * = p <0.05.

2.10 Protoplast transfection

A1160p+ spores at a concentration of 106 spores/mL were inoculated in liquid SAB and divided into three 9 cm petri dishes (Sigma-Aldrich). Static cultures were incubated overnight at 37°C for 14-16 hours. Mycelia was harvested and transferred into a 50 mL sterile falcon tube containing 10 mL of freshly made 5% VinoTaste pro (novozymes) solution. The solution was mixed thoroughly and incubated in a flat position at 30°C and 80 rpm for approximately 4 hours until most cell wall material was degraded. Protoplasts were harvested using sterile lens tissue and centrifuged for 10 min at 4°C and 3000 rpm. The pellet was resuspended in 1 mL ice-cold KCl/CaCl2 solution and the concentration of protoplasts was determined using the Improved Neubauer haemocytometer (Hawksley). 200 µL of 106 protoplasts/mL were incubated in an 8-chamber coverglass (NUNC Lab-Tek) with B-siRNAs at a final

46 concentration of 2.5 µM for 1 hour. Images were taken at 0 minutes and 60 minutes incubation by confocal microscope (Leica SP8) and images were analysed using FIJI.

2.11 Labelling cornea with siRNA

Pig eyes were donated by Mettrick’s Butchers Glossop. Eyes were frozen after dissection and before use, eyes were defrosted in warm Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS, Sigma-Aldrich) and 1 µg/mL of the antibiotics streptomycin (Sigma-Aldrich) and ampicillin (Sigma-Aldrich). The cornea was surgically removed from the eyeball then transferred to a sterile 6-well CytoOne® plate (Starlab) with 3 mL fresh DMEM++

(DMEM with above supplements) and cultured overnight at 37°C with 5% CO2 in dark.

Before infection, cornea were wounded by scratching with a surgical blade. Cornea were then infected with 25 µL of A. fumigatus CEA10 spores at a concentration of 106 spores/mL and incubated at 37°C with 5% CO2 in fresh DMEM++ medium. After 8 hours of infection, medium was refreshed and B-siRNA at a final concentration of 100 nM was added. The cornea were incubated for another 16 hours and transferred to a 2-well glass slide (ThermoFisher Scientific) with fresh 1 mL DMEM++ prior to imaging. Images were taken using a confocal microscope (Leica SP8) and images were analysed using FIJI.

Cornea without infection and infected cornea without B- siRNA but with the Alexa Fluor 555 dye only were used as controls. Both incubated in DMEM++ at the same temperatures and times as infected samples.

2.12 Mammalian cell cultures

® For all experiments, cells were maintained at 37°C with 5% CO2 in Gibco DMEM without phenol red (Life technologies), supplemented with 10% FBS.

2.12.1 Transfection of A549 cell line

105 cells of the human pulmonary carcinoma epithelial cell line A549 were seeded per well of a 2-well µ-slide chambered coverslip (Ibidi) and incubated to ≥90% confluence. B- siRNAs were incubated with LipofectamineTM 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. 25 nM of siRNA-lipofectamine complex was incubated with the A549 cells overnight. Following co-incubation with siRNA, cultures were washed with DMEM without phenol red and infected with 105

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AfGFP spores for 16 hours. Images were taken using a confocal microscope (Leica SP8) and images were analysed using FIJI.

2.12.2 Generation of exosomes

The monocytic cell line THP-1 (ATCC® TIB-202™) was used for the generation of exosomes. THP-1 cells were harvested and centrifuged at 2000 x g for 30 minutes. The supernatant was transferred to a new tube with 0.5 volumes of the Total Exosome Isolation (from media) reagent (Invitrogen) and incubated overnight at 4°C. After incubation, cells were centrifuged at 10,000 x g for 1 hour at 4°C and the pellet was resuspended in 2 mL of Gibco® 1x Dulbecco’s Phosphate Buffer Saline (DPBS, Life Technologies).

The concentration of exosomes was determined by measuring protein standards and creating a standard curve of protein concentrations. Protein determination reagent was prepared by adding 1 mL of copper sulphate pentahydrate 4% solution (Sigma- Aldrich) to 49 mL of bicinchoninic acid solution (Sigma-Aldrich). Protein standards were prepared from a 100 mg/mL BSA stock. Protein solutions were incubated for 30 minutes at 37°C in a 1:8 ratio with determination reagent. Standards and samples were then measured in a microplate reader spectrophotometer (Synergy2, BioTek) at 562 nm. The standard curve was calculated using excel and a linear equation was used to calculate protein concentration of exosome samples.

Exosomes were centrifuged and resuspended in IT NucleofectorTM solution (Lonza) and B-siRNA was added to the exosomes in a 2:1 exosome:B-siRNA ratio. The mixture was electroporated with 2 pulses of 400 mV, 125 µF using a NucleofectorTM Device (Lonza). After transfection, the mixture was transferred to a new Eppendorf with 10 volumes of 1x DPBS and centrifuged at 4000 x g for 1 hour at 4°C. The pellet was resuspended in 200 µL 1x DPBS. siRNA uptake by exosomes was checked using a confocal microscope (Leica SP8).

A. fumigatus CEA10 spores at a concentration of 106 spores/mL were incubated at 37°C for 8 hours in VMM+ until germination occurred. siRNA-captured exosomes were incubated at 37°C with germinated CEA10 spores for 16 hours at a final concentration of 25 nM. Incubation of germinated spores with B-siRNAs without exosomes and wildtype CEA10 spores only were used as controls. Images were taken using a confocal microscope (Leica SP8) and images were analysed using FIJI.

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2.13 Lipid fungal transfections

2.13.1 Transfection reagents

The B-siRNAs were captured into lipid vesicles of the transfection reagents Viromer Blue (Lipocalyx) and Lipofectamine 2000 (ThermoFisher Scientific) according to manufacturer’s protocols. A. fumigatus CEA10 spores at a concentration of 106 spores/mL were incubated for 8 hours at 37°C in VMM+. A final concentration of 25 nM siRNA-lipids mixture was added to the germinated A. fumigatus CEA10 spores and incubated at 37°C for another 10 hours in VMM+. Incubation of germinated spores with B-siRNAs only and wildtype CEA10 spores only were used as controls using the same conditions. Images were taken using a confocal microscope (Leica SP8) and images were analysed using FIJI.

2.13.2 Liposomes

Liposomes were prepared by adding B-siRNAs diluted in 1x PBS to the lyophilised powder in the kit (Sigma-Aldrich) at a final B-siRNA concentration of 250 nM, resulting in a 2:1 liposome:B-siRNA ratio. To promote incorporation of the B- siRNA into the liposomes, the mixture was mixed well and agitated for 30 minutes at room temperature. 106 spores/mL of A. fumigatus CEA10 was incubated for 8 hours at 37°C in VMM+. The siRNA-liposome mixture was added to the germinated spores at a final B-siRNA concentration of 25 nM and incubated at 37°C for another 10 hours in VMM+. Incubation of germinated spores with B-siRNAs only and wildtype CEA10 spores only were used as controls using the same conditions. Images were taken using a fluorescence microscope (Nikon Eclipse TE2000-E) and images were analysed using FIJI.

2.14 Cell penetrating peptides (CPP) PAF

PAF26 (RKKWFW), TMR-labelled PAF26, PAF95 (AAAWFW), PAF96 (RKKAAA) and Peptide8 (cyclo(RKKW(C2-BODIPY)FWG)) were gifted by dr Can Zhao (MFIG, University of Manchester). PAF26 with a cysteine residue (PAF26-C, RKKWFWC) was synthesised by Peptide Protein Research Ltd.

2.14.1 Synthesis of CPP-siRNA

To promote unspecific binding, the sense RNA oligo (25 μM) was incubated with 33 pmol γ32P[ATP] and 20 U T4 Polynucleotide Kinase (NEB) in reaction buffer for 1 hour at 37°C. RNA was purified by Micro Bio-Spin P-30 Tris Chromatography Columns (Bio-Rad) according to the manufacturer’s protocol. The labelled sense RNA oligo was annealed to its antisense RNA oligo to obtain a radiolabelled siRNA construct. The

49 labelled siRNA was incubated with either 25 μM PAF26 or PAF95 or 75 μM PAF26. Incubation with TE buffer (10 mM Tris-HCl [pH 8] and 1 mM EDTA) only was used as a control. The products were run on a 20% polyacrylamide gel (30% acrylamide/bis- acrylamide (Sigma), 5x TBE buffer, 10% ammonium persulfate, and TEMED) for 1 hour at 4°C and 60 V. The gel was wrapped in cling film and autoradiograms were prepared with Kodak MR Film with exposure times of 30 seconds, 2 minutes, 5 minutes and 20 minutes.

To covalently link siRNA to PAF26-C, the sense pptA RNA oligo was synthesised by Eurofins with an amine functionality incorporated at the 3’ terminus and the antisense RNA oligo was synthesised by Eurofins with the fluorophore TMR incorporated at the 3’ terminus. The sense and anti-sense oligos (50 µM) were annealed by heating to 90°C for 1 minute in annealing buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl and 5 mM EDTA) and gradiently cooling down (0.02°C/s) using a thermal cycler (Biometra®). The siRNA was conjugated to the succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker via its amine group and SMCC is linked to the cysteine residue of PAF26-C according to the method of Williams and Chaput (Figure 2.1) [136]. The construct was checked on a 20% polyacrylamide gel using the Alexa 546 dye laser on the geldoc system (Bio-Rad).

Figure 2.1. Schematic overview of linking siRNA to PAF26-C. The covalently linked siRNA – PAF26-C construct was made using the method of Williams and Chaput [136]. A) Sense pptA RNA oligo with a 3’ amine was annealed to the antisense RNA oligo with a 3’ TMR fluorophore. B) The siRNA was linked to succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). C) The siRNA-SMCC construct was linked to PAF26-C.

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2.14.2 Co-cultures of siRNA-SMCC-PAF26 and B-siRNAs with peptide8

Wildtype cea10 A. fumigatus spores were incubated at a final concentration of 2.5x104 spores/mL in VMM+ for 8 hours to generate young hyphae and treated for 10 hours with 250 nM of siRNA-SMCC-PAF26 or 25 nM of B-siRNAs in an 8-chamber coverglass (NUNC Lab-Tek). Peptide8 was incubated 10 minutes before imaging at a concentration of 20 µM. Images were taken by confocal microscope (Leica SP8). Images were analysed using FIJI.

The internalisation of cell membrane counterstain PAF96 was tested using cea10 A. fumigatus young hyphae in VMM+ at a final concentration of 2.5x104 spores/mL in an 8-chamber coverglass (NUNC Lab-Tek). PAF95 was incubated for 15 minutes prior to imaging at a final concentration of 1 µM. Images were taken every 30 seconds by confocal microscope (Leica SP8), starting at the time when 20 µM peptide8 was added to the hyphae. Images were analysed using FIJI.

2.15 Small RNA library construction

The RNAi mutant strains Δqde2, Δdcr1, Δdcr1Δdcr2 and its parental wildtype strain A1160p+ were grown in 1x RPMI [pH 7.0] at a final concentration of 106 spores/mL in triplicate for 24 hours at 37°C and 180 rpm. Tissue was harvested using Whatman filter paper (Sigma-Aldrich) and a vacuum pump and samples were snap frozen in liquid nitrogen. The frozen tissue samples were ground in liquid nitrogen and the small RNAs (sRNAs) were extracted using the NucleoSpin miRNA extraction kit (Macherey-Nagel). The concentration of sRNAs was measured using spectrophotometer (Synergy2, BioTek) and the integrity was checked on Novex TBE gel. The sRNA library was prepared using 0.5 µg of each sample with the TruSeq® Small RNA Library Preparation Kit A (Illumina) according to the manufacturer’s protocol. During this protocol the sRNA samples were reverse transcribed into cDNA, amplified with designated indices and checked on a Novex TBE gel. The primers and specific indices per sample used in the generation of the sRNA libraries are listed in Table 2.5. Concentrations of cDNA samples were determined by using a QubitTM RNA HS Kit (Invitrogen) on the Qubit 4 Fluorometer (Invitrogen). As each sample has a specific index, all samples were pooled in equal quantities to form one small RNA library before the purification. The library was purified through the use of AMPure XP beads, removing any DNA below 100 bp.

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Table 2.5. Primers and indices used to generate the small RNA library Description Sample Sequence (5’ – 3’) RNA 5’ Adapter (RA5) All GUUCAGAGUUCUACAGUCCGACGAUC RNA 3’ Adapter (RA3) All TGGAATTCTCGGGTGCCAAGG Stop Oligo (STP) All GAAUUCCACCACGUUCCCGUGG RNA RT Primer (RTP) All GCCTTGGCACCCGAGAATTCCA RNA PCR Primer (RP1) All AATGATACGGCGACCACCGAGATCTAC ACGTTCAGAGTTCTACAGTCCGA RNA PCR Primer Index 1 Wildtype 1 ATCACG RNA PCR Primer Index 2 Wildtype 2 CGATGT RNA PCR Primer Index 3 Wildtype 3 TTAGGC RNA PCR Primer Index 4 Δqde2 1 TGACCA RNA PCR Primer Index 5 Δqde2 2 ACAGTG RNA PCR Primer Index 6 Δqde2 3 GCCAAT RNA PCR Primer Index 7 Δdcr1 1 CAGATC RNA PCR Primer Index 8 Δdcr1 2 ACTTGA RNA PCR Primer Index 9 Δdcr1 3 GATCAG RNA PCR Primer Index 10 Δdcr1Δdcr2 1 TAGCTT RNA PCR Primer Index 11 Δdcr1Δdcr2 2 GGCTAC RNA PCR Primer Index 12 Δdcr1Δdcr2 3 CTTGTA

2.16 Small RNA sequencing and bioinformatics analysis

The small RNA library was processed by the Genomic Technologies Core Facility (GTCF) at the University of Manchester. Firstly, quality control was performed by running the library pool on DNA 1000 ScreenTape with the Agilent 4200 TapeStation system. The exact concentration of the library was determined by qPCR and diluted to a final concentration of 10 nM. The library was then sequenced on the Illumina 4000 system and the data was sent to the bioinformatics facility at the University of Manchester. The paired-end sequencing data was processed using trimmomatic and aligned to the A. fumigatus Af293 reference genome using bowtie2. HTseq was used as counting method. Groups were tested for differential expressed genes by performing principal component analysis (PCA) using DESeq2. Confidence interval was set at 95%.

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Chapter 3

Defining the effect of siRNA treatment on N. crassa and A. fumigatus

3. Defining the effect of siRNA treatment on N. crassa and A. fumigatus

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3.1 Introduction

Targeting essential genes by siRNA could be a useful tool to control fungal disease. In mammalian cells, down-regulation of essential mRNA by the use of siRNAs is intensively researched to control a number of mammalian diseases such as cancer and metabolic diseases [137, 138]. For example in diabetic patients, up-regulation of the proinflammatory cytokine tumor necrosis factor α (TNF-α) plays an important role in the progression of the disease by inducing insulin resistance. Inhibitor of nuclear factor-kB kinase β (IKKβ) is used as a RNAi target as it is indirectly one of the regulators of TNF-α and its down-regulation can control insulin resistance. siRNA treatment of human myotubes resulted in reduction of IKKβ protein expression by 73% and IKKβ mRNA levels by 55% [139]. Moreover, a variety of virus infections can be inhibited by the use of siRNAs such as HIV, dengue virus and Hepatitis C. [140-142]. To date, no siRNA-based therapeutics are on the market yet, but around 22 clinical trials are running at the moment using miRNA and siRNA-based therapeutics [143, 144].

In genetically transformed fungal cells, siRNAs derived from a dsRNA-expressing are effective in down-regulating target mRNA [103, 105]. siRNA targeting essential genes can lead to growth inhibition or . This shows the potential of siRNAs as an antifungal drug. In certain fungal species, siRNA added to the growth medium is transported across the fungal cell wall without the help of a carrier. Examples of this are the studies carried out by Moazeni et al. in the dimorphic fungus Candida albicans and in the fungus A. nidulans [82, 145]. Wang et al. showed the uptake of external siRNAs by Botrytis cinerea. siRNA targeting dicer-like genes were sprayed on the surface of the fungus which caused RNAi activation and reduced growth of B. cinerea [94]. The siRNAs were targeted against one gene, but a range of different short sequences was pooled together. The siRNA pool was derived from long target dsRNA and generated by RNase-III in vitro. The pooled siRNA method was shown to be more efficient than the use of chemical siRNA in A. nidulans [93].

In A. fumigatus, Jöchl et al. describes the uptake of siRNA in germinating conidia and hyphae, but also shows the accumulation of siRNA in the fungal cell wall. The research indicates that although much of the siRNA is stuck to the cell wall, some of it is transported across the cell wall and leads to a down-regulation of 30% of the target mRNA levels. The target gene odcA is a precursor of polyamine biosynthesis and is essential for growth and development. However, the mRNA down-regulation of odcA was insufficient to affect the growth of A. fumigatus. There are no reports in the literature of studies on the uptake of siRNA by N. crassa.

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In this research, I determined the effect of synthesised siRNAs on the pathogenic fungus A. fumigatus and the model filamentous fungus N. crassa. Initially I used the odcA siRNA designed by Jöchl et al. (2009). Besides odcA, pptA is used as a siRNA target for A. fumigatus. pptA encodes a sfp-type 4’-phosphopantetheinyl transferase. This essential enzyme is involved in lysine biosynthesis and secondary metabolism (e.g. melanin biosynthesis) and is therefore a suitable target for growth and phenotype assays. The target used in N. crassa is the polyketide synthase ALBINO- 1. Al-1 null mutants have a defect in the carotenoid pigment biosynthesis leading to a characteristic white spore phenotype. Moreover, I compare the efficacy of using a single siRNA targeted to one specific sequence to a pool of siRNAs targeted to different short sequences in the target mRNA. To test the pool of siRNAs, hygromycin B phosphotransferase (hph) is also used as a universal target for the fungal species studied in this research. For these experiments, I used fungal strains expressing hph that results in resistance against the antibiotic hygromycin B. Successful down- regulation is measured by phenotype assay, growth assay or qRT-PCR.

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3.2 The generation of siRNAs used in this study

Previous RNA sequencing data from N. crassa al-1 hairpin transgenic strains (Ran Wang, unpublished data) was used to design siRNA primers targeting al-1. Induction of the al-1 hairpin was achieved by the use of 0.01M quinic acid to activate the qa-2 promoter placed upstream of al-1. This dataset showed increased reads mapping to al-1 in strains with induced expression of the al-1 hairpin, suggesting this is an effect of RNAi. The sense siRNA primer used in this study is derived from the al-1 region with the most mapped sRNA reads in the al-1 hairpin induced strain.

The sequences of the siRNA primers targeting odcA were taken from literature [96] (see Table 2.3). SiDirect 2.0 Web server was used to design siRNA targeted against Aspergillus fumigatus pptA. Parameters were set on Max Tm of 21.5°C and siRNAs with nearly perfect complementarity to human mRNA were excluded to reduce cytotoxicity [146]. The following parameters were set to improve siRNA efficiency; GC content was set on 30 – 65%, no GC stretches longer than 7, less stability of 5’ end of guide strand, guide strand should start with A or U where the passenger strand starts with either G or C [147]. This search resulted in 4 possible target sequences. Choosing a siRNA targeting the middle of the coding sequence should be avoided which roughly excluded 2 possible targets [148]. Finally, the choice between the 2 possible siRNA sequences was based on which sequence was the closest to the and unpublished RNAseq data showed expression of this region. SiDirect 2.0 adds a 3’ 2 nucleotide overhang to the siRNA within the target, but to protect the siRNA from RNases it is better to have a 3’ dTdT overhang. For that reason, the passenger siRNA sequence is slightly improved by deleting the 3’ CC overhang, adding CG at the 5’ end to make it 23 bp long, adding dTdT to the 3’ end and attaching a 5’ phosphate group. The dTdT overhang and the 5’ phosphate group were also attached to the guide siRNA sequence, resulting in the following siRNA sequences for pptA:

Guide (antisense) = 5’ P-UAUUUCAGCAGGUUUGAGGCGdTdT-3’ Passenger (sense) = 5’ P-CGCCUCAAACCUGCUGAAAUAdTdT-3

All antisense and sense strands were purchased from Eurofins genomics and annealed to form siRNA duplexes. The siRNA duplexes for al-1 and odcA are shown on polyacrylamide gel (Figure 3.1). All other siRNA duplexes are generated in the same way and checked on polyacrylamide gel.

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Figure 3.1. Polyacrylamide gel confirming primer annealing into siRNA Sense and antisense primers of al-1 and odcA were annealed to form al-1 or odcA siRNA duplexes. The annealing was confirmed by a polyacrylamide gel where the single-stranded (ss) RNA primers are used as a control. al-1 primers are 23 nucleotides and odcA primers are 21 nt.

3.3 The effect of synthetic al-1 siRNA treatment on N. crassa

As described in the introduction, the RNAi pathway is present in N. crassa. However, RNAi activation by external means has only been reported after transformation of siRNA expression vectors. To test whether or not the RNAi pathway can be activated without transformation of N. crassa, asexual spores were harvested and grown in the presence and absence of siRNAs targeting albino-1 mRNA.

3.3.1 N. crassa phenotypes after siRNA treatment

Activation of the RNAi pathway and down-regulation of al-1 will cause a defect in carotenoid biosynthesis leading to a white phenotype. Spores were inoculated on Vogel’s minimal medium (VMM) agar impregnated with a 25 nM siRNA layer and grown in either constant dark or constant light for a total of 2 days at 30°C (Figure 3.2). Cultures showed a slightly enhanced orange colour when spores were incubated in light. However, phenotypic changes between cultures treated with unrelated siRNA or siRNA targeted to al-1 were not observed by eye.

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Figure 3.2. No observable effects were seen in N. crassa phenotypes on siRNA coated agar medium N. crassa spores were inoculated on top of Vogel’s minimal medium (VMM) agar coated with a layer of 25 nM siRNA. Plates were incubated in either constant dark (left panel) or constant light (right panel) at 30°C for 2 days. Images were taken after 24 hours and 48 hours.

3.3.2 No significant changes in mRNA levels after al-1 siRNA treatment in N. crassa

Previously, phenotypic changes were not observed after al-1 siRNA treatment. However, it is conceivable that if al-1 is only slightly down-regulated there would be no significant impact on carotenogenesis. Thus, al-1 mRNA levels were measured. To determine siRNA-mediated knockdown of al-1 mRNA, germinated spores were cultured in liquid medium containing 25 nM al-1 siRNA for 10 hours and the mRNA levels were measured by qRT-PCR. Two types of al-1 siRNA, one with a 5’ phosphate group on both strands of the siRNA duplex and one without the 5’ , were used. The RNaseIII-like enzyme dicer produces 5’P siRNAs and the 5’P is anchored in the mid-domain of the Ago2 protein which activates the RNAi pathway [149]. Furthermore, a 530 bp segment of an al-1 PCR product was also used to treat germinated spores as it has been shown that spores transformed with this DNA have an albino phenotype (See section 3.1.3.3). Results show that no significant changes in gene expression were observed in all samples (Figure 3.3).

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Figure 3.3. al-1 mRNA levels did not change significantly after siRNA treatment in N. crassa Germinated spores were grown in the presence of oligonucleotides for 10 hours after which mRNA levels were measured by qRT-PCR. Oligonucleotides tested are a 23 nt al-1 siRNA, 23 nt al-1 siRNA with a 5’ Phosphate group on both strands and a 530 nt al-1 DNA PCR product. Fold expression of three biological replicates of albino-1 (al- 1) in N. crassa were calculated with the 2-ΔΔCt method, normalised against actin and quantified relative to unrelated siRNA. No significant differences were measured in mRNA levels after treatment with the tested oligonucleotides.

3.4 The effect of synthetic odcA siRNA treatment on A. fumigatus

As was pointed out in the introduction to this thesis, Jöchl et al. demonstrated the use of odcA siRNA in A. fumigatus to down-regulate its target mRNA by 30% [96]. However, this reduction was not sufficient to affect fungal growth. Hence, without optimisation the likelihood that siRNA could be used as an antifungal drug is low. To address this a series of experiments was carried out to test the effect of siRNA on A. fumigatus and the published odcA siRNA sequence was used in the first experiments of this chapter to set the baseline.

3.4.1 A. fumigatus phenotypes after siRNA treatment

To determine the effect of siRNA treatment on A. fumigatus phenotype, germinating spores were grown in liquid in the presence or absence of odcA siRNA (Figure 3.4). If the siRNA is taken up and activates RNAi, it would be expected that mRNA levels of odcA will be down-regulated and this should result in either reduced or no growth. To provide a control for 100% inhibition of odcA expression, an odcA knockout mutant was used. Loss of odcA leads to a putrescine auxotrophic phenotype. The gross

59 phenotype of the wildtype A. fumigatus strain (A1163) was unaffected by siRNA treatment.

Figure 3.4. Phenotype of A. fumigatus in presence of odcA siRNA is not affected Wildtype A. fumigatus on minimal Vogel’s medium with 1% glucose (VMM+) with or without additional 10 mM putrescine after 25 nM odcA siRNA treatment. Growth phenotype of ΔodcA is rescued in the presence of 10 mM putrescine.

The previous results show the growth phenotype of A. fumigatus after siRNA treatment in liquid (Figure 3.4). To test whether a solid medium has an effect on the siRNA uptake, 25 nM odcA siRNA was spread onto VMM supplemented with 1% glucose (VMM+) as a carbon source and germinated spores were inoculated on the surface. After a 48 hour incubation at 37°C, the wildtype A. fumigatus phenotype appears to have a slightly larger radius, but no differences were observed between spores grown on unrelated siRNA and odcA siRNA (Figure 3.5).

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Figure 3.5. Phenotype assay of A. fumigatus on siRNA coated agar medium Wildtype A. fumigatus spores were inoculated in the centre of VMM agar medium coated with a layer of either unrelated siRNA or 5’P siRNA targeted against odcA. Plates were incubated at 37°C for 48 hours.

3.4.2 Growth of A. fumigatus is not affected in the presence of siRNA

Minor differences in growth arrest are difficult to visualise by eye, therefore the effect of odcA siRNA on the growth rate of A. fumigatus was assessed. Spores were incubated with 25 nM of odcA siRNA. Growth was measured every 10 minutes over 48 hours using a plate reading spectrophotometer and growth curves were obtained (Figure 3.6A-C). To identify a delay in the early stages of growth, a threshold is set and the times when the growth of samples pass the threshold is obtained (Figure 3.6D). No significant differences in growth rate or the time at which the culture exited the lag phase of growth were seen. Interestingly, cultures treated with unrelated siRNA show a slightly higher OD after 24 hours.

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Figure 3.6. odcA siRNA treatment in A. fumigatus did not affect growth

OD600 nM readings of A. fumigatus cultures were taken every 10 minutes. The average of 6 replicates per sample was plotted in a graph to obtain a growth curve (A-C where A is untreated wildtype, B is treatment with unrelated siRNA and C is treatment with odcA siRNA). The threshold of OD 0.05 was set to check for any delayed growth (D). No significant differences were observed in the time at which the culture exited the lag phase.

3.4.3 No significant changes in odcA mRNA levels after siRNA treatment in A. fumigatus

Although no significant change in phenotype or growth was detected, small amounts of siRNA could enter the fungal cell and have an impact on mRNA levels. To measure the odcA mRNA levels after siRNA treatment, germinated spores were incubated for 10 hours with 25 nM odcA siRNA. As explained earlier, siRNA is normally synthesised with a 5’ phosphate group. Therefore, odcA siRNA is also tested with and without a 5’ phosphate group at both RNA strands. Three biological replicates were performed and the fold expression of three technical replicates was calculated (Figure 3.7). Remarkably no significant differences in odcA mRNA levels after siRNA treatment were measured.

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Figure 3.7. odcA mRNA levels did not change after siRNA treatment in A. fumigatus Germinated spores were treated with odcA siRNA for 10 hours and mRNA levels were measured by qRT-PCR. Fold expressions of at least two biological replicates of odcA in A. fumigatus were calculated with the 2-ΔΔCt method, normalised against β-tubulin and quantified relative to unrelated siRNA. No significant differences were measured using one-way ANOVA.

3.5 The effect of a pool of siRNAs (d-siRNAs) against one target

The synthetic siRNAs were carefully designed according to a variety of parameters (see section 3.1.2). However, it is not known whether this specific siRNA can activate the RNAi pathway. To test the efficacy of a pool of siRNAs targeted to a variety of short sequences in the target mRNA, siRNAs were generated in vitro by RNase III and checked on a polyacrylamide gel (Figure 3.8). Pools of diced siRNAs (d-siRNAs) were generated to target al-1 in N. crassa, pptA in A. fumigatus and hph as unrelated control in both species.

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Figure 3.8. Generation of d-siRNAs confirmed on 20% polyacrylamide gel Long dsRNAs were generated by in vitro transcription of the target DNA. The d- siRNAs, ranging from 18 – 25 bp, were generated in vitro by incubation of long dsRNA with RNase III. Long dsRNA and the d-siRNA products of every target (pptA, hph, and al-1) were run on polyacrylamide gel. The synthetic ssRNA primer (antisense al- 1) and synthetic siRNAs (al-1) of 23 bp were used for comparison.

3.5.1 The effect of a pool of diced al-1 siRNAs (d-siRNAs) on N. crassa

To determine the effect of al-1 d-siRNAs on N. crassa, the same experiments were performed as in sections 3.1.3.1 and 3.1.3.2 only with a pool of different siRNAs. N. crassa phenotypes were imaged after 24 hours and 48 hours incubation at 30°C in either constant dark or constant light when grown on agar with a layer of al-1 d- siRNAs (Figure 3.9). An observable difference is seen between the wildtype and cultures arising from the d-siRNAs treated spores grown for 48 hours in the light. Wildtype cultures seem to have a more orange phenotype than cultures incubated d- siRNAs. Differences are more difficult to see between cultures treated with the control (unrelated d-siRNAs) and treated with al-1 d-siRNAs. Therefore, mRNA levels of al-1 were measured to study any differences in RNA abundance. Germinated spores were treated in liquid media with 25 nM al-1 d-siRNAs for 10 hours and mRNA levels of al- 1 were assayed by qRTPCR. As before, there are no significant differences between the samples (Figure 3.10).

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Figure 3.9. d-siRNA coated agar medium does not change the phenotype of N. crassa N. crassa spores were inoculated at the centre of VMM agar plates. The plates were coated with a layer of annealing buffer as control (top panel), 25 nM unrelated d- siRNAs (middle panel) or 25 nM al-1 d-siRNAs (lower panel). Plates were incubated in either constant light or constant dark at 30°C for 2 days. Images were taken after 24 hours and 48 hours.

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Figure 3.10. d-siRNA treatment did not affect al-1 mRNA levels in N. crassa A pool of siRNAs (d-siRNAs) targeted to the whole region of the al-1 mRNA was tested for RNAi activation in N. crassa. Germinated spores were treated with unrelated or al-1 d-siRNAs for 10 hours and mRNA levels were measured by qRT-PCR. Fold expressions of three biological replicates of al-1 in N. crassa were calculated with the 2-ΔΔCt method, normalised against actin and quantified relative to unrelated siRNA. No significant differences were measured using one-way ANOVA.

3.5.2 The effect of pptA d-siRNAs on A. fumigatus

As explained in the introduction, pptA is a suitable drug target in A. fumigatus because its deletion is lethal. pptA null-mutants grown in media with additional iron and lysine also have a defect in melanin biosynthesis and therefore have a white instead of green phenotype. Wang et al. demonstrated uptake of external RNAs by the fungus B. cinerea from plants and fruits coated with d-siRNAs [94]. Fungal biomass was significantly less after coating the organisms and this was also observed by eye. To determine whether a similar phenomenon would occur when agar was coated with d-siRNA targeted to pptA in A. fumigatus, spores were inoculated on the surface of VMM+ agar with 25 nM d-siRNAs and compared to spores inoculated on non-coated agar after 3 days of growth (Figure 3.11). The growth phenotype of the pptA null-mutant (ΔpptA) is rescued with additional iron and lysine and shows a white phenotype. If RNAi is activated by the pptA d-siRNAs, reduced growth is expected on VMM and a white phenotype is expected on VMM+ with additional iron and lysine. Results show that there is no growth defect or white phenotype in the presence of pptA d-siRNAs, in fact contrary to expectations at 24 h in the presence of iron and lysine slightly enhanced growth is visible.

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Figure 3.11. pptA d-siRNAs do not cause a change in phenotype VMM+ agar was impregnated with a coating of 25 nM d-siRNAs on half of the plates. Images of the phenotypes were taken at 24 hours (left) and 48 hours (right) after inoculation of A. fumigatus wildtype or ΔpptA spores on medium with and without d- siRNAs. Additional iron (1.5 mM) and lysine (10 mM) rescues the ΔpptA phenotype.

Next, pptA mRNA levels were measured after exposure to pptA siRNAs in liquid medium to test for any effects of pptA siRNAs at the molecular level (Figure 3.12). Iron and lysine were added to the medium to prevent any growth defects. Germinated spores were treated for 10 hours and mRNA levels were measured by qRTPCR. Results were quantified relative to unrelated siRNA meaning cultures treated with unrelated siRNAs have fold expressions around 1.0. mRNA fold expression ranging from 1.5 to 8.3 was measured after treatment with the pool of pptA siRNAs, but no significant differences in mRNA levels were detected.

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Figure 3.12. No significant differences in pptA mRNA levels after d-siRNAs treatment A pool of siRNAs (d-siRNAs) targeted to the whole region of the pptA gene was tested for RNAi activation in A.fumigatus. Germinated spores were treated with unrelated or pptA d-siRNAs for 10 hours and mRNA levels were measured by qRT-PCR. Fold expression of three biological replicates of pptA in A. fumigatus were calculated with the 2-ΔΔCt method, normalised against β-tubulin and quantified relative to unrelated siRNA. No significant differences were measured using one-way ANOVA.

3.5.3 The effect of hph d-siRNAs on N. crassa and A. fumigatus

The antibiotic hygromycin B, purified from the bacterium Streptomyces hydroscopicus, is commonly used as a selection marker. Fungal strains transformed with the E. coli hygromycin resistance gene (hph) can be selected on medium with 200 µg/mL hygromycin B as this concentration is lethal to wildtype cultures [150]. In this experiment, siRNAs are used to target the hph in hygromycin resistant strains of N. crassa (NCU11267) and A. fumigatus (ΔaftA). If d-siRNAs targeted against hph successfully activates the RNAi pathway, hph should be down-regulated and the hygromycin resistant strains should become susceptible to hygromycin B.

First the minimum inhibitory concentration (MIC) of hygromycin was determined. Experiments were performed with a range of hygromycin concentrations to test the efficacy of hph d-siRNAs. The cultures were imaged after 24 hours (Figure 3.13A) and 48 hours (Figure 3.14A). The OD600nm of the fungal cultures grown with hygromycin B in presence or absence of d-siRNAs was measured after 48 hours and plotted in a graph (Figure 3.13B and Figure 3.14B).

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N. crassa grew much faster than A. fumigatus and the wells were overgrown after 48 hours. Therefore, OD measurements after 24 hours are shown here (Figure 3.13). The MIC was determined as the lowest concentration of hygromycin B needed for a growth reduction of 50%. The MIC of wildtype N. crassa is less than 31.25 µg/mL whereas the MIC of the hygromycin resistant strain is more than 4 times higher at a concentration of 125 µg/mL. When incubated with hph d-siRNAs, the MIC of the hygromycin resistant strain remained the same i.e. it did not become more susceptible to hygromycin B. This suggests that hph d-siRNAs do not result in down- regulation of hph.

Figure 3.13. The effect of hph d-siRNAs on N. crassa N. crassa hygromycin resistant strain (11267) was grown in the presence and absence of hph d-siRNAs. N. crassa wildtype strain was used as a control for hygromycin susceptibility. Different concentrations of hygromycin B were tested descending from 4000 µg/mL to 0 µg/mL and OD600nm was measured after 24 hours. The phenotypes were imaged (A) and growth rates were plotted (B). The wildtype MIC is lower than 31.25 µg/mL and the MIC for 11267 is 125 µg/mL.

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The MIC of hygromycin for A. fumigatus was determined after 48 hours growth. The MIC of a clinical wildtype strain A. fumigatus (CEA10) is 125 µg/mL and the MIC of the hygromycin resistant strain A1160p+h+ is 4000 µg/mL (Figure 3.14). The MIC of A1160p+h+ could not be determined and is at least above 4000 µg/ml when hph d- siRNAs are present. Remarkably, the OD is significant higher compared to the growth of A1160p+h+ in absence of hph d-siRNAs. This is also true for A1160p+h+ in presence of unrelated d-siRNAs. This suggests that the hph d-siRNAs might be used as nutrients, but are not able to activate the RNAi pathway.

Figure 3.14. The effect of hph d-siRNAs on A. fumigatus A. fumigatus wildtype and hygromycin resistant strain (A1160p+h+) were grown in presence and absence of hph d-siRNAs. Different concentrations of hygromycin B were tested descending from 4000 µg/mL to 0 µg/mL and OD was measured after 48 hours. The phenotypes were imaged (A) and growth rates were plotted (B). The wildtype MIC is 125 µg/mL and the A1160p+h+ is resistant to hygromycin B as the MIC is above 4000 µg/mL.

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3.6 Discussion

In contrast to reports in the literature, siRNAs used in this study did not activate the RNAi pathway and therefore did not down-regulate target mRNA in N. crassa or A. fumigatus treated in liquid media or grown on siRNA-coated agar for up to 2 days.

No significant differences were detected in A. fumigatus phenotype or growth after treatment with synthetic odcA siRNA, which is in agreement with the study of Jöchl et al. However, no significant differences in mRNA levels were measured in our study, which is contrary to the results of Jöchl et al. where a down-regulation of 30% in the odcA mRNA levels was observed. Besides this study, studies on other fungi including A. nidulans demonstrated significant down-regulation of the target genes sidB, odc and rasA when the organisms were treated with synthetic siRNAs targeting transcripts encoded by these genes [92, 93, 145]. The efficacy of a pool of diced siRNAs (d-siRNAs) is reported to be higher than treatment with single sequence synthetic siRNAs in A. nidulans [93]. After ras-A d-siRNA treatment levels of rasA mRNA were reduced to 5.3% of levels in untreated controls and to 44.38% after single sequence synthetic siRNA treatment. However, data reported in this chapter provide no evidence of a reduction in target gene expression after treatment of A. fumigatus with either d-siRNAs or single sequence siRNA.

Although the experimental protocols followed in the experiments described in this chapter were similar to the protocols reported in literature, there were small differences that might account for the different results. In this work germinated spores were treated for longer with a slightly higher concentration siRNA (25 nM rather than 20 nM) and scrambled odcA siRNA was used as unrelated control whereas Jöchl et al. used a siRNA directed against Homo sapiens. Moreover, the minimal medium used in our study consists of Vogel’s salts [151] and 1% glucose whereas the referenced studies use a variety of media. An overview is given of all studies performed on Aspergillus spp. that state that siRNA in the medium are taken up by the fungi and results in successful RNAi activation (Table 3.1). All studies mentioned showed a significant difference in either growth or mRNA levels when treated with siRNA compared to wildtype and unrelated siRNA controls. We have chosen a minimal medium for the work in this chapter to rule out any other side effects and to have consistency throughout the experiments.

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Table 3.1. Summary of methods of previous siRNA studies on Aspergillus spp.

Spores siRNA Incubation Extra Species Medium Ref. /mL (nM) method information Transfection 6hr germlings Czapek- reagent A. flavus 106 5 – 50 incubated for 24h [121] Dox broth lipofectamine with siRNA 3000 is used Protoplasts 20 °C for 24 h to A. flavus YES are used with allow transfection and medium the 103 5 – 25 to proceed. Then [122] A. with 1.2M transfection 5 days at 25 °C parasiticus sorbitol reagent in the dark lipofectin 6hr germlings Czapek- A. fumigatus 104 15 – 50 incubated for 12h [152] Dox broth with siRNA 6hr germlings Aspergillus incubated for 6h minimal with siRNA or 16h A. fumigatus 108 20 [96] medium hyphae incubated (AMM) for 16h with siRNA 8hr germlings This A. fumigatus VMM+ 107 25 incubated for 10h study with siRNA Potato 6hr germlings A. nidulans dextrose 108 25 incubated for 12h [145] agar with siRNA Synthetic Aspergillus 6hr germlings siRNAs are complete A. nidulans 108 25 incubated for 12h compared to a [93] medium with siRNA pool of diced (ACM) siRNAs Czapek- 6hr germlings 104 or A. nidulans dox 5 – 25 incubated for 6h [92] 108 medium with siRNA Protoplasts are used with ACM with the A. niger 0.8M 106 266 Up to 72 hours [104] transfection sorbitol reagent GeneSilencer

Instead of fresh spores, in liquid cultures, young hyphae were incubated with siRNA. This was because previous research on A. fumigatus, showed that hyphae were able to scavenge labelled siRNAs, but that spores could not [96]. When comparing all studies on filamentous fungi referenced in this chapter, only germinated spores and hyphae are used in most siRNA treatment experiments. Interestingly, incubation of A. nidulans ungerminated spores with siRNAs targeting odc, which is essential for growth and development, resulted in a 12% decrease of spore germination compared

72 to the control [92]. However, this decrease is minimal compared to the results of treated germinated spores where germ tube lengths were reduced by 50% compared to the control spores. This same study showed that target mRNA levels of odc siRNA in treated samples, after an initial reduction, increased over time. mRNA levels were measured after 18h, 24h, 36h, 48h and 72h of incubation with odc siRNA. After 72 hours, the odc mRNA levels were not significantly different compared to the controls, suggesting the siRNA was not amplified over time. Thus, in the experiments reported here the maximum treatment was 48 hours.

The advantage of using al-1 as a target in N. crassa is the change in phenotype from orange (wildtype) to white (al-1 null mutant) in the absence of al-1 expression. However, minor changes of phenotype that might occur if al-1 expression is reduced rather than abolished can be difficult to observe by eye. al-1 is induced by light and slight differences in light exposure times or intensity can cause large changes in al-1 mRNA levels [153]. Since qRT-PCR is sensitive and will detect even the smallest variation of mRNA levels, the cultures were grown and harvested in dark. No significant differences in the N. crassa experiments using al-1 siRNAs were found and this can have several reasons: 1) siRNAs did not make it into the cell, 2) siRNAs did not activate the RNAi pathway or 3) siRNAs did activate the RNAi but half-life is short and the signal might not have been amplified. The latter however is less likely as RdRPs are involved in the RNAi pathway of N. crassa that are responsible for siRNA amplification [154].

During growth assays, the presence of siRNAs (either target or unrelated) repeatedly caused an increase in fungal growth (see Figure 3.6 and Figure 3.14). This might suggest that siRNAs are taken up by the fungus, but instead of incorporation into the RNAi machinery they are used as nutrients. It is not clear if they are digested outside of the fungus and subsequently taken up. To investigate this, RNAs could be extracted from the medium and either sequenced or run on a gel. Comparison between fungal samples with and without siRNAs in the medium and medium with siRNAs only would answer this question. Off-target effects might also be the cause of an increase in growth, depending on which off-target genes have an increase or decrease in mRNA levels. This phenomenon is also reported in a study with mammalian cells where 10 different siRNAs were designed to target one gene (MEN1), but some siRNAs also caused significant differences in the protein levels of two genes other than the target ( and p21) [155]. Another possibility is that the mRNA is not cleaved and degraded by the siRNA when it enters the cell, but only prevents translation of the protein. If this occurs, it can trigger an increase in transcript levels.

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One of the reasons why no phenotype, decrease in growth or down-regulation of mRNA levels is seen after treatment of naked siRNAs could be the rapid degradation of naked siRNAs by nucleases [154]. If the siRNA is not present long enough in the media to be taken up by fungal cells, no effect will occur. To address the possibility that the RNA is rapidly degraded, modified siRNAs were designed and experiments using these are described in the next chapter.

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Chapter 4

The effect of modified siRNA on fungi

4. The effect of modified siRNAs on fungi

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4.1 Introduction

Naked siRNAs were not taken up readily by the fungal cell and therefore had no effect on the RNAi pathway (Chapter 3). There are several possible explanations why treatment with siRNA had no measurable effect. Naked siRNAs might need a transporter for efficient delivery in the cell which will be discussed in the next chapter. Another possible explanation is that the siRNAs are not stable enough and are being degraded by nucleases before any effect can take place. Chemical modifications of siRNAs such as modifications to the nucleotide backbone or sugar modifications can improve RNA stability, uptake and binding affinity to complimentary mRNA strands [98, 154, 156-158].

RNA and DNA have phosphodiester linkages between their nucleotides which are very susceptible to nucleases. Modifying the RNA backbone is a useful tool to prevent degradation by nucleases. The phosphate internucleotide linkages can be modified by replacing the oxygen molecule with sulphur resulting in a phosphorothioate linkage [98]. This does not affect the negative charge of the backbone, but the advantage of a phosphorothioate (PS) linkage is its resistance to nuclease degradation [99]. It has been shown by X-ray crystallography that PS modifications between G - C of a hexanucleotide do not cause a substantial alteration in its structure [159].

Modification of the 2’ position of the ribose is mostly used in gapmers where the sugars at the termini of the oligonucleotides are modified and therefore protect the internal nucleotides from exonuclease activity. Incorporating 2’ sugar modifications into PS modified siRNA further enhances stability and efficacy of the siRNA as shown by Wu et al. In this study, the combination of 2’-OMe modification with phosphorothioate linkages at both available oxygens (MePS2) showed enhance loading of siRNA into the RISC as well as enhanced stability. Knockdown of a target protein was 50% after treatment with either unmodified siRNAs or 2’-F or 2’-OMe modifications. The combination of 2’-OMe and PS2 modifications improved the target protein knockdown by a further 35% [156]. This improvement can be due to the increased Ago2 protein binding of the MePS2 and its enhanced serum and intracellular stability compared to unmodified siRNA.

The incorporation of such modifications play an important role in the efficacy of RNAi- based drugs. For example, the first RNAi-based drug, Fornivirsen, approved in 1998 by the Food and Drug Administration (FDA) in the USA is a phosphorothioate 21-base oligodeoxynucleotide used against the cytomegalovirus retinitis virus (CMV) [160]. Another RNAi-based drug, Mipomersen, was approved by the FDA in 2013. Mipomersen is phosphorothioate 20-base oligodeoxynucleotide, it includes five 2′-O-

76 methoxyethyl (MOE) sugar modifications at each end and is targeted against the human apoliprotein B (apoB)-100 [161]. This protein is an essential component of low-density lipoprotein cholesterol (LDL-C) and patients with high cholesterol (hypercholesterolemia) benefit from mipomersen therapy as it reduces LDL-C levels to the right plasma tolerance level. Although these drugs are based on anti-sense oligonucleotides (ASO) and consists of DNA rather than RNA, these are good examples for the improvement of nuclease stability and the efficacy of the drug by oligonucleotide modifications.

Although many studies are performed with antisense oligonucleotides in which all internucleotide linkages are phosphorothioated, it has been shown by Haung et al. that only the sense strand of the siRNA needs to be modified with PS linkages to achieve resistance against serum nucleases [162]. Interestingly, only the 3’ overhang of the strand was modified to show that RNase A recognises, besides single strands RNA, the thermodynamic asymmetry of the siRNAs. This modification, 3’ overhang PS linkages, are also tested in a siRNA-albumin conjugate for stability in mouse serum and delivery in mice target cells [163]. The efficient siRNA molecule in this study consisted of 2’-OME sugar modifications besides the 3’ overhang PS linkages in both sense and antisense strands.

Another modification that is useful is the change of the ribose sugar backbone to a N-(2-aminoethyl)glycine backbone linked by peptide bonds which is called peptide nucleic acid (PNA). These molecules display interesting properties such as high resistance to nucleases and proteases and high binding affinity to complementary oligonucleotides [98]. Moreover, peptide nucleic acid mimics the base-pair recognition of oligonucleotides and is neutrally charged. The latter might be a reason why the stability of a PNA-DNA duplex is higher than a normal double-stranded DNA molecule [100].

In this chapter, modified siRNAs were tested to determine whether they could enter fungal cells and activate RNAi. The siRNA modifications used in this research are PS modifications, PS modifications combined with the sugar modifications 2’-O-methyl (2’-OMe) or 2’-fluoro (2’-F) and PNA. The effect of modified siRNAs on N. crassa and A. fumigatus was tested by phenotype assay, growth assay and measurement of target mRNA levels by qRT-PCR. Moreover, the uptake of fluorescently labelled modified siRNAs by the pathogenic fungus A. fumigatus and the model N. crassa was assessed by confocal microscopy.

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4.2 Modified siRNA treatment does not affect N. crassa

Modified siRNAs targeted against al-1 were carefully designed (Table 2.4) and tested for RNAi activation. The enzyme phytoene desaturase encoded by al-1 is light- sensitive and involved in carotenoid biosynthesis giving rise to the orange wildtype phenotype. Defects in al-1 can be easily detected as the phenotype will be white. Modifications include siRNAs with a phosphorothioate backbone (PS), with a phosphorothioate backbone at the 3’ overhang and 2’-O-methyl sugar modifications (PS OMe), with 2’-fluoro sugar modifications (Fluoro) and with a peptide nucleic acid backbone (PNA).

To observe any phenotypical changes of N. crassa after incubation with modified al- 1 siRNAs, N. crassa spores were inoculated in the centre of siRNA-coated agar medium. Images were taken after 24 hours and 48 hours incubation at 30°C in either light or dark (Figure 4.1). Cultures conveyed a slightly more orange colour after 48 hours when incubated in constant light. However, no phenotype differences between the controls (untreated cultures and cultures incubated with unrelated siRNA) and the cultures incubated with any modification of siRNA were observed.

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Figure 4.1. Phenotype of N. crassa on modified siRNA coated agar medium was not affected N. crassa spores were inoculated in the centre of VMM agar coated with a layer of 25 nM modified siRNAs. Plates were incubated in either constant light or constant dark at 30°C for 2 days. Images were taken after 24 hours and 48 hours.

Although no phenotypic changes were seen after treatment, the possibility remains that small amounts of siRNA enter the fungal cells and activate RNAi. However, if enough mRNA escapes silencing, sufficient enzyme (phytoene desaturase) may be produced to maintain the wildtype phenotype. To test this hypothesis, germinated spores were treated with 25 nM of modified al-1 siRNAs for 10 hours in the dark and mRNA levels of al-1 were measured by qRTPCR (Figure 4.2). Surprisingly, the al-1 mRNA levels of PNA treated samples were significantly (p<0.04) higher than al-1 RNA levels in all other samples, including the controls. In contrast, significant down- regulation was measured (p=0.024) between samples treated with PS Ome modified

79 al-1 siRNA and the untreated samples, but importantly not between PS Ome modified al-1 siRNA and unrelated siRNA.

Figure 4.2. al-1 mRNA levels in N. crassa after modified siRNA treatment Germinated spores of N. crassa wildtype were treated with unrelated or modified siRNAs targeted against al-1 for 10 hours and mRNA levels were measured by qRT- PCR. Fold expression of three biological replicates of al-1 mRNA levels in N. crassa were calculated with the 2-ΔΔCt method, normalised against actin and quantified relative to al-1 levels in samples exposed to unrelated siRNA. Standard deviations are represented by the error bars. Significant (p<0.05) differences (indicated by a star) were determined using one-way ANOVA.

As stated in paragraph 3.6, al-1 is light sensitive and even the smallest fluctuations in light can change the mRNA levels of al-1 independently of any siRNA treatment. Therefore, mRNA levels of an important component in the RNAi pathway was measured to see if changes in al-1 mRNA levels are due to regulation of al-1 by light or that RNAi is activated by siRNA. Proteins of the Argonaute family (AGO) play essential roles in RNAi mechanisms. In N. crassa, RISC mainly consists of the Argonaute protein QDE-2 and it was shown that qde-2 is up-regulated in response to dsRNA and RNAi activation [67]. Thus, the qde-2 mRNA levels were measured in the samples treated with modified al-1 siRNAs. N. crassa transformed with a plasmid containing an al-1 hairpin under the control of the inducible qa-2 promoter was used as a positive control. This hairpin is transcribed when the strain is grown on medium containing quinic acid (QA). Expression of the al-1 hairpin leads to siRNA-mediated silencing of endogenous al-1 and elevated levels of qde-2 mRNA. If the modified al- 1 siRNA activates RNAi, qde-2 levels will increase and the mRNA levels of qde2 should

80 be comparable with the qde-2 mRNA levels of the positive control. However, measurements of qde2 mRNA levels showed that none of the samples are similar to the positive control (Figure 4.3). Interestingly in PS OMe treated samples, down- regulation of al-1 mRNA levels was measured however no significant difference in qde2 mRNA levels was measured.

Figure 4.3. qde2 mRNA levels in N. crassa after modified siRNA treatment In samples treated with unrelated or modified siRNAs targeted against al-1 fold expression of qde2 mRNA in three biological replicates were calculated with the 2-ΔΔCt method, normalised against actin and quantified relative to unrelated siRNA. An RNAi expression vector for al-1 attenuation was used as a positive control for qde-2 up- regulation. Standard deviations are represented by the error bars. Significant (p<0.05) differences (indicated by a star) were determined using one-way ANOVA.

Statistical analysis showed a significant difference between the positive control and all other samples. The qde2 mRNA fold expressions of all tested samples did not exceed 1.3, whereas the mRNA fold expression of qde2 in the positive control is beyond 6.

Altogether, these results suggest that the RNAi pathway in N. crassa was not activated after treatment with modified siRNAs targeted against al-1.

4.3 Modified siRNA treatment does not affect A. fumigatus

Comparable to the experiments in N. crassa, A. fumigatus was also tested for RNAi activation after treatment with modified siRNAs. The target pptA is used for A.

81 fumigatus as deletion of this gene is lethal and with additional iron and lysine it shows a white melanin deprived phenotype [129]. Modifications include siRNAs with a phosphorothioate backbone (PS), with a phosphorothioate backbone at the 3’ overhang and 2’-O-methyl sugar modifications (PS OMe), with 2’-fluoro sugar modifications (Fluoro) and with a peptide nucleic acid backbone (PNA).

To test the effect of modified siRNAs on A. fumigatus, a phenotype assay was carried out. Spores were spotted on siRNA coated Vogel’s minimal agar medium and grown for 3 days. Images were taken after 2 and 3 days (Figure 4.4). At each time point, no differences in phenotype were observed between the untreated wildtype control (Untreated), the scrambled unrelated siRNA control (Unrelated) and samples treated with modified siRNAs.

Figure 4.4. Phenotypes of A. fumigatus in presence of modified siRNAs did not show observable differences A. fumigatus spores were inoculated on top of siRNA coated Vogel’s minimal agar medium in petri dishes and grown up to 3 days at 37°C. Images were taken after 48 hours (top) and 72 hours (bottom). No phenotypic differences were observed after modified siRNA treatment. Abbreviations: Untreated wildtype (WT), scrambled unrelated siRNA (Unrelated), Phosphorothioate backbone (PS), phosphorothioate backbone and 2’-O-methyl sugar modifications (PS OMe), 2’-fluoro sugar modifications (Fluoro) and with a peptide nucleic acid backbone (PNA).

To confirm the results of the phenotype assay, we also extracted RNA from cultures after treatment with modified siRNAs targeted against pptA for 10 hours. The pptA mRNA levels were measured in three biological replicates, fold expressions were calculated using the 2-ΔΔCt method and plotted with the standard deviations (Figure 4.5). Consistently, we found no significant differences in the mRNA levels after modified pptA siRNA treatment compared to the controls. Thus, there is no evidence that the RNAi pathway is triggered in A. fumigatus cultures grown in the presence of naked modified siRNAs.

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Figure 4.5. pptA mRNA levels were not affected in A. fumigatus after modified siRNA treatment Germinated spores were treated with unrelated or modified siRNAs targeted against pptA for 10 hours and mRNA levels were measured by qRT-PCR. Fold expression of at least two biological replicates of pptA mRNA levels in A. fumigatus were calculated with the 2-ΔΔCt method, normalised against β-tubulin and quantified relative to unrelated siRNA. The error bars represent standard deviations and significant differences were determined using one-way ANOVA.

4.4 B-siRNAs are concentrated at the cell wall and septa of N. crassa

Since contrary to published work no changes in phenotype or target mRNA levels were observed after germination and growth of N. crassa or A. fumigatus cultures in the presence of siRNA, experiments were initiated to determine where the siRNA is located. In the following experiments commercially available BLOCK-iTTM siRNA labelled with Alexa FluorTM 555 (BLOCK-iTTM Red) was used. BLOCK-iTTM siRNAs (B- siRNAs) produced by Invitrogen are not targeted to any known gene in either mouse, rat or human and have chemical modifications to the siRNA for a more persistent and stable signal [164]. It is tested and used in mammalian cells as a transfection efficiency control [165].

First, germinated N. crassa spores were incubated in liquid medium for 10 hours with B-siRNAs at 30°C or Alexa Fluor alone and/or CalcoFluor white. CalcoFluor White (CFW), a fluorescent stain that binds to chitin in the fungal cell wall, was added 10 minutes before imaging of cultures under the confocal microscope. As expected, images show the clear outline of the fungal cell stained by CFW, but the dye on its own does not enter the fungal cell nor does it label any components of the fungal cell

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(Figure 4.6A). Interestingly the B-siRNAs are seen mostly in the septa of the fungal hyphae (Figure 4.6B).

Figure 4.6. Labelled siRNAs accumulated at N. crassa septa A) N. crassa germinated spores were incubated with the dye Alexa Fluor 555 as a control. CalcoFluor White (CFW) was used to stain the chitin in the septa and cell wall. B) BLOCK-iTTM siRNAs (B-siRNAs) labelled with Alexa Fluor 555 were added to N. crassa germinated spores and incubated for 10 hours at 30°C. Scale bar, 5 µm.

This phenomenon was not rare and was observed at different time points in experiments performed with N. crassa and B-siRNAs. N. crassa strain with fluorescently labelled nuclei (H1-GFP) was used and stained with CFW to visualise the chitin in the septa and cell wall [133]. Germinated spores were incubated with B- siRNAs for only 1 hour and CFW was added 10 minutes before imaging. Imaging was carried out using the differential interference contrast (DIC) modus of a fluorescence microscope which altogether makes it easier to see the specific compartments of the hyphal cell (Figure 4.7A). Consistent with the previous results, we detected B-siRNAs in the septa. To visualise the septa labelling in more detail, the imaged Z-stack was compressed into a 3D model using the software of the confocal microscopy Leica SP8 (Figure 4.7B). This software allows the user to change the viewpoint which revealed the accumulation of B-siRNAs on the outside of the septa.

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Figure 4.7. B-siRNAs concentrate on the outside of the septa in N. crassa A) The H1-GFP N. crassa strain, expressing fluorescently labelled nuclei (green), was incubated with B-siRNAs (red). The cell wall and septa are stained with CFW (blue) and the DIC modus of the fluorescence microscope was used in order to improve visualisation of specific compartments of the fungal cell. Scale bar, 5 µm. B) 3D model of the Alexa555 signal using the Leica SP8 software.

These results raise the question of how the siRNA ends up in the septa. One possibility is that it enters the cytoplasm and gets stuck in the septa. Alternatively siRNA in the cell wall or cell membrane may be drawn into the septa as they develop. To investigate this, we followed the fungus in time after addition of the B-siRNAs and checked whether or not the B-siRNAs move in the cytoplasm or are stable in one location. To do so, we used a microfluidic system called CellASIC ONIX. Spores were loaded into the platform of the CellASIC plate which has a ceiling of 5-7 microns and were continuously perfused with fresh medium. Due to the platform ceiling, cells are restricted and grow in a single focal plane and therefore can be easily followed in time. Live-cell imaging revealed that one of the septa was labelled after one hour of exposure to B-siRNA. Under high magnification some fluorescence in the outer wall of the fungus was observed (Figure 4.8). However, only after 4 hours the other septum was labelled. A possible explanation for these results may be the age or position of the septa. The older septa were always labelled first. Moreover, the fluorescent signal was stationary during the observed periods whereas the cytoplasmic contents were actively moving through the hyphae.

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The data reported here support the assumption that the B-siRNAs remain on the outside of N. crassa and concentrate at the septa.

Figure 4.8. Live-cell imaging of N. crassa reveal accumulation of B-siRNAs to septa and cell wall N. crassa young hyphae were followed in time at 30°C while incubated with B-siRNAs up to 20 hours. Images were taken every hour from the start of B-siRNAs incubation using a confocal microscope and representative images are shown for 1, 2, 4, 8, and 16 hour timepoints.

4.5 B-siRNAs are concentrated at the cell wall of A. fumigatus

According to Jöchl et al., DNA oligonucleotides bind to the outer cell wall of A. fumigatus and are mostly not able to penetrate into the cytoplasm [96]. To determine whether RNA oligonucleotides can penetrate the cell wall, a range of experiments using unrelated siRNA labelled with Alexa FluorTM 555 (BLOCK-iTTM Red) was performed and the location of the siRNA was imaged by confocal microscopy.

Germinated spores were first incubated with BLOCK-iTTM-siRNAs (B-siRNAs) for 10 hours then imaged by confocal microscopy (Figure 4.9). The fluorescent dye, Alexa Fluor 555, was used alone as a control to analyse if the B-siRNAs are responsible for the signal.

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Figure 4.9. B-siRNA accumulation in A. fumigatus A. fumigatus germinated spores were incubated with either the dye Alexa Fluor 555 (A) or Alexa Fluor 555 labelled siRNAs (B) for 10 hours at 37°C. 10 minutes before imaging under the confocal microscope, CFW was added to the fungal hyphae. Fluorescence was quantified and showed an overlay of CFW and B-siRNAs on the outside of the fungal cell (C). Top: plot of fluorescence levels across one hypha. Bottom: fluorescence intensity plotted was measured along the indicated line. Images show the accumulation of B-siRNAs on the outer layer of the hyphae. Scale bar, 5µm.

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As expected, Alexa Fluor 555 alone was not transferred into the cytoplasm nor did it accumulate on the cell wall/cell membrane or in any cellular compartments. B-siRNAs were found to accumulate on the outside of the fungus, overlapping the signal of CFW (Figure 4.9C). However, in A. fumigatus while CFW stains septa, this was not the case for B-siRNAs.

The fungal cell wall is important for its strength, allowing fungi to cope with osmotic stresses, and the plasticity of the cell wall is needed for fungal growth. The B-siRNAs accumulate outside the fungus and as it seems from the overlap with CFW that it is likely the accumulation occurs at the cell wall. However, more research is needed to confirm this. To gain more insight of the exact location of B-siRNAs, the cell membrane or cell wall can be disrupted. The fungal specific cell-penetrating peptide labelled with a BODIPY fluorophore, peptide8, is known to interact with the A. fumigatus cell membrane and internalises after maximum 10 minutes by disrupting the cell membrane [166]. Therefore, peptide8 was used to promote internalisation of the cell membrane counterstain PAF96. Incubation of A. fumigatus hyphae with peptide8 showed the internalisation of the cell membrane counterstain PAF96 after 5 min and 30 sec. This confirmed the theory that peptide8 promotes the uptake of molecules accumulated to the cell membrane as the cell membrane is disrupted (Figure 4.10A). If B-siRNAs are accumulating at the cell membrane, similar to PAF96, then in the presence of peptide8 they may be seen to enter the cytoplasm. To test this, we incubated the B-siRNA labelled fungal hyphae with peptide8 and imaged the cells after 10 minutes (Figure 4.10B). B-siRNAs remained on the outside of the cell, suggesting that they are not accumulated at cell membrane but at the cell wall.

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Figure 4.10. A. fumigatus co-cultured with peptide8 did not improve B-siRNA uptake A) Germinated spores were incubated for 15 minutes with the cell membrane stain PAF96. Time-lapse imaging shows the binding of peptide8 to the cell membrane after 2 minutes, the internalisation of the cell membrane stain PAF96 after 5 minutes and 30 seconds and after 10 minutes peptide8 is internalised. B) Germinated spores incubated with B-siRNAs (Alexa555) for 10 hours are co-cultured with peptide8 (BODIPY) for 10 minutes to test for B-siRNA uptake during cell membrane disruption of peptide8. Scale bar, 5µm.

These results led to the hypothesis that B-siRNAs attach to the cell wall of A. fumigatus. To investigate this hypothesis, the cell wall was removed by the enzyme Glucanex. This enzyme hydrolyses the poly(1,3)-glucose of the cell wall glucan and results in the formation of protoplasts. Protoplasts were incubated with B-siRNAs for one hour and visualised by confocal microscopy (Figure 4.11A). Remarkably, even though the medium was saturated with the B-siRNAs the B-siRNAs did not accumulate in any compartment of the fungal cells. This data was quantified by measuring the fluorescence inside of the protoplasts and in the medium (Figure 4.11B). The fluorescence in the medium was significantly higher than in the protoplasts, even after incubation with B-siRNAs for 60 minutes, which suggests that the B-siRNAs are mainly in the medium. The fluorescence in the protoplasts was not significantly different over time. Protoplasts are generally not very stable due to the loss of the cell wall and therefore incubation for longer than 1 hour causes the protoplasts to burst.

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Figure 4.11. B-siRNAs are excluded from protoplasts The fungal cell wall was digested by the enzyme Glucanex and the resulting protoplasts were incubated with B-siRNAs. A) Protoplasts were monitored for one hour by confocal microscopy. B) Relative fluorescence units (RFU) of 10 individual protoplasts were compared to the corresponding environment using a Student’s T- test (** P <0.01). Scale bar, 20µm.

4.6 A variety of Aspergillus spp are labelled by B-siRNAs

As was pointed out in the introduction to this thesis, various pathogenic fungi have an active RNAi mechanism. Research performed on fungi such as A. niger, C. albicans, C. neoformans and A. nidulans show the down-regulation of target genes after RNAi activation. Especially pertinent for our research are the studies on Aspergillus spp. In A. nidulans it is stated that siRNA can have an effect on the level of target mRNA after incubation with naked siRNAs [92, 93]. For this reason, we wanted to investigate the translocation of B-siRNAs in several Aspergillus spp. As it is reported that A. nidulans can scavenge siRNA from the medium and this causes

90 down-regulation of the target mRNA, A. nidulans B-siRNA treatment was followed over time in the microfluidic cellASIC system. After 6 hours, germinated spores were treated with B-siRNAs for up to 10 hours and images were taken by confocal microscopy (Figure 4.12). Live cell imaging showed that a selection of A. nidulans young hyphae were already labelled with B-siRNAs after 15 minutes of treatment. However, despite continuous perfusion with B-siRNAs, after 7 hours the germinated spores in the original view seem to be fluorescently labelled whereas almost all the surrounding elongated hyphae are not labelled. It is clear that the B-siRNAs are not translocated into the spore heads and after 4 hours and 7 hours there is an outline of B-siRNAs at the outside of the hyphae. The labelling of B-siRNAs on the cell wall is however not as distinguished as in A. fumigatus.

Figure 4.12. Time lapse of A. nidulans B-siRNAs treatment Germinated A. nidulans spores were followed over time using the cellASIC system with continuously perfused VMM and 25 nM of B-siRNAs. Images were taken every 15 minutes by confocal microscope and a representation of the time lapse is shown here. Scale bar, 10 µm.

Germinated spores of Aspergillus lentulus and Aspergillus niger were also incubated with B-siRNAs overnight and imaged by fluorescence microscopy. Similar to A. fumigatus and N. crassa, B-siRNAs label the fungi A. lentulus and A. niger (Figure 4.13A). More detailed images show the accumulation of B-siRNAs on the outside of the hyphae, mainly at the conidial head and the older parts of the hyphae (Figure 4.13B). CalcoFluor White was added 10 minutes before imaging to stain chitin in the fungal septa and cell wall.

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Figure 4.13. Aspergillus lentulus and Aspergillus niger are labelled with B- siRNAs A) Germinated spores of Aspergillus lentulus and Aspergillus niger were incubated for 10 hours with 25nM B-siRNAs. Images were taken using a 20x objective on an inverted microscope. Scale bar, 20 µm. B) CalcoFluor White was added 10 minutes prior to imaging to label chitin in the septa and fungal cell wall. Images were taken using a 40x objective on an inverted microscope. Scale bar, 10 µm.

Accumulation of B-siRNAs is obvious, but maybe when it is not accumulated and small portions are distributed throughout the cell, it is less visible and hence the appearance of unlabelled hyphae.

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4.7 B-siRNAs can be used as a diagnostic tool for fungal keratitis

The results so far indicate that B-siRNAs do not cross the cell wall and therefore are unable to activate the RNAi pathway. This has been tested in A. niger, A. lentulus, A. nidulans and A. fumigatus. A. niger and A. fumigatus are the major causes of aspergillosis however A. nidulans is also found in aspergillosis patients although less frequently [167]. Besides aspergillosis, Aspergillus spp. are one of the major causes of eye infections. The recent findings on B-siRNA labelling of Aspergillus spp can be an advantage as B-siRNAs could be of use for the detection of fungal keratitis. To test this, pig cornea were incubated with A. fumigatus for 8 hours and treated with B-siRNAs for another 16 hours. The cornea was imaged by confocal microscopy to see if labelling was clearly visible and specific to Aspergillus hyphae (Figure 4.14).

Figure 4.14. Labelling of A. fumigatus infection in pig cornea Pig cornea without infection, but with siRNA treatment and cornea with infection, but without siRNAs were used as controls (Left panel). Pig cornea were infected with 106 conidia/mL of A. fumigatus wildtype for 8 hours and treated with 100 nM of B-siRNAs for another 16 hours (Right panel). Corneas were imaged by confocal microscopy. Scale bar, 10 µm.

Despite the use of antibiotics in the medium, bacteria were still present on the corneas. This was however useful as cornea treated with B-siRNAs, but without a fungal infection, did not show any fluorescence. This suggests that the binding of B- siRNAs is fungal specific. Moreover, the lack of fluorescence on cornea infected with A. fumigatus but not treated with B-siRNAs suggests that there is no auto fluorescence of mammalian cells at this wavelength. The A. fumigatus hyphae are clearly labelled with B-siRNAs after 16 hours. Incubation of B-siRNAs without a fungal infection on the cornea resulted in no fluorescence, suggesting the B-siRNAs do not label corneal cells. Also no fluorescence was seen when cornea were incubated with

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A. fumigatus only or when incubated with the Alexa Fluor 555 dye. This suggests that the fluorescence seen on infected cornea is a result of the B-siRNAs labelling the A. fumigatus hyphae. This method is promising for the detection of fungal keratitis.

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4.8 Discussion

In summary, it has been shown in this chapter that naked modified siRNAs are not readily taken up by the fungal cell and consequently do not trigger the RNAi pathway. confirming the results of chapter 3.1. It seems most likely that uptake is unsuccessful due to the accumulation of B-siRNAs on the fungal cell wall. It can be thought that the 23 bp siRNAs are too large to cross the fungal cell wall and therefore attach to the outside. However, the cell wall is known to be permeable to certain molecules up to 200 kDa e.g. and siRNAs have a molecular weight of approximately 15 kDa [168].

For N. crassa the results suggest that the B-siRNAs are accumulated on the outer layer of the fungal cell wall, but have a preference to accumulate at the septa. Depending on the orientation of the fungus during the imaging experiments a round tunnel is visible. When the imaged Z-stack data was compressed into a 3D model, it is further confirmed that the cytoplasm does not contain fluorescence as it is a hollow tube when rotating and visualising the Z dimension.

The accumulation of B-siRNAs is only seen in the older septa close to the spore, not in the tip or newly formed hyphae. This excludes the accumulation to proteins that are involved in septa formation only. It is known that the presence of chitin increases as the cell wall matures [169]. It could be that RNA is attaching to chitin and that is why we see the accumulation more frequently near the spore and not at the apical side. One of the advantages of using N. crassa as a model is the availability of knock- out strains. It would be interesting to use N. crassa strains lacking cell wall or septa components and examine the B-siRNAs translocation compared to wildtype. This could reveal what the siRNAs are binding to and what prevents access to the cell membrane.

In general, the cell wall of fungi consists of a core layer of β-(1,3) glucan and chitin [170]. Besides the core components, the A. fumigatus hyphal cell wall consists of among others α-(1,3) glucan which is not present in Candida albicans, Saccharomyces cerevisiae and Botrytis cinerea. α-(1,3) glucan is found in other pathogenic fungal species such as Cryptococcus neoformans, Histoplasma capsulatum and Blastomyces dermatidis [171]. Interestingly, uptake of siRNA from the medium is reported in the species named above that lack α-(1,3) glucan, but it is not reported for the species that contain α-(1,3) glucan besides Aspergillus spp. However, the microscopy data on the uptake of fluorescently labelled siRNA in A. nidulans and A. fumigatus is poor and we were not able to repeat these results in this research [93, 152].

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Our research showed that B-siRNAs did not internalise A. fumigatus and instead accumulated on the outer layer of the hyphae. The data reported here appear to support the hypothesis that the B-siRNAs are accumulating in the cell wall. First of all, incubation of A. fumigatus hyphae with B-siRNAs and CFW reveal the binding in similar compartments of the fungal cell. As it is known CFW binds to chitin in the septa and cell wall, we can hypothesise that B-siRNAs binds to chitin. Moreover, incubation with a cell-membrane disruptive peptide did not cause the internalisation of the B-siRNAs where it did cause the internalisation of the membrane binding peptide PAF96. This observation on its own cannot rule out the possibility that the B- siRNAs are intensely attached to the membrane and therefore cannot diffuse into the cytoplasm. However, the B-siRNAs were found predominantly in the medium when the cell wall was digested. Protoplasts do still have a membrane and therefore the assumption that it is attached to the cell membrane can be ruled out. Correspondingly, protoplasts lack cell-wall material including chitin and have a chitin synthase activity increasing in time [172]. Interestingly, only germinated spores and hyphae appear to attract B-siRNAs, which can be due to the positive charge of the cell wall [96]. Looking at the cell wall composition of conidia and hyphae, the hyphal cell wall contains almost 8 times more chitin compared to the conidia. Besides, the outer cell wall of conidia, including the chitin, is covered by a layer of hydrophobins such as RodA [173].

The fungal keratitis diagnosis is limited and most cases are reported from developing countries where traditional diagnosis is even more limited due to expenses and resources. Currently, the approach in patients with suspected eye infection is to scrape material from the cornea or performing confocal microscopy in vivo [174]. Samples removed from the eye are first tested by Gram staining for bacteria and KOH or CalcoFluor White staining for fungi. Although this initial step is not time consuming, setting up a culture remains a necessary step to distinguish between species. Early diagnosis is important, but impossible when waiting for cultures to grow which takes at least 2 days. Moreover, scraping is done by a surgical blade or platinum spatula which is painful and can also cause permanent damage to the eyes [175]. The confocal microscopy in vivo approach is rapid and accurate with a sensitivity of 88.3%, but is cost effective [176]. However, especially in developing countries, this equipment is too expensive and clinicians are not experienced in confocal microscopy which is necessary for a reliable diagnosis. Therefore, labelling fungi on the cornea with B-siRNAs could be a cost-effective and time-limited solution. Experiments performed in this chapter indicate that B-siRNAs have a high prospective value in the diagnosis of fungal keratitis.

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The perfect protocol for the diagnosis of fungal keratitis would be: 1) Labelling the infection with eye drops containing B-siRNAs with incubation times less than 1 hour 2) The use of a torch-like laser that can visualise the B-siRNAs by eye or by using a small magnifier. 3) The labelling pattern will reveal if the infection is bacterial or fungal and ideally distinguish between species. However, the results shown in this chapter are preliminary and the method needs to be optimised. At the specific concentration used in this study (100 nM), overnight incubation gave the best results. More studies need to be carried out to reduce the incubation time by for example increasing the B-siRNAs concentration. Besides, eyes were not fresh as they came from the local butcher and were stored in the fridge for at least a day until collection. Antibiotics in the medium removed most bacteria, but the eyes still seemed to have some bacterial infections. This might be due to the unsterile way of removing the eyes from its host. It is best to use fresh human cornea for consistent and clean eyes and this could be achieved via a collaboration with the human eye bank (Manchester Royal Eye Hospital).

In addition to the use of B-siRNAs as a tool to detect fungal keratitis, siRNAs have a potential as a therapeutic to silence essential genes. Thus far, the evidence indicates that siRNAs are not able to internalise into fungal cells and silence essential genes by the use of the RNAi mechanism. There would therefore seem to be a definite need for siRNA delivery systems. In the next chapter the principal findings on the use of several delivery systems in A. fumigatus will be presented.

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Chapter 5

Development of siRNA delivery systems in

A. fumigatus

5. Development of siRNA delivery systems in A. fumigatus

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5.1 Introduction

RNA interference has previously only been studied in A. fumigatus after transformation of plasmid constructs that express hairpin RNA molecules or when naked siRNAs are added to the growth medium [84, 96, 103, 152, 177-179]. However, the addition to the growth medium does not give optimal silencing results. In partial accordance with our research where naked siRNA do not have an effect on A. fumigatus gene silencing, these studies suggest optimisation of the siRNA uptake for efficient down-regulation. However, it is clear that fungi can take up extracellular siRNAs from transgenic plants expressing an RNAi construct targeting fungal virulence genes [180]. On maize expressing hairpin transcripts targeting alpha- amylase, A. flavus contamination and pathogenicity is reduced [181]. More recently, fungal infections can be controlled by coating plants with a solution containing siRNAs [94]. It is only recently discovered that transgenic Arabidopsis plants expressing hairpin RNAs excretes siRNAs in extracellular vesicles [182]. Therefore, experiments described in this chapter test the hypothesis that the uptake of siRNAs by Aspergillus fumigatus can be promoted by extracellular vesicles (EVs). There are three classes of EVs: apoptotic bodies, shedding microvesicles and exosomes. Exosomes are small lipid-like vesicles with a size between 50 to 90 nm that are capable of transporting genetic material between cells [183, 184]. Vesicle lipids protect their content from degradation by RNAses and facilitate endocytosis into cells [185].

It seems that lipid-containing delivery systems have an important role in drug delivery. The advantages for these types of delivery systems include; the shielding of vesicle contents from degradation, effective delivery of contents due to interactions between vesicle lipids and the target cell envelope, and the wide range of biocompatible lipid-based carriers from which vesicles can be generated [186]. A range of different lipid-like molecules, lipidoids, demonstrated successful silencing in three animal models and proved its utility in both local and systemic delivery of siRNA drugs [187].

Few studies have been performed on siRNA delivery systems in fungi. In the filamentous fungi A. niger and A. flavus lipid-based transfection reagents, GeneSilencer and Lipofectamine respectively, were used a siRNA delivery method [104, 121]. These commercially available transfection reagents, mainly used in mammalian cells, consist of cationic lipid formations where anionic siRNAs can be trapped and fused with the cell membrane. Endocytosis occurs and although the release from the is not efficient, it does result in down-regulation of the target gene by the siRNA molecules [188].

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Most drug delivery systems are based on liposome formations, which contain a combination of various lipids. For example, Amphotericin B, a frequently used antifungal drug, is captured in liposomes (AmBisome) for a more effective and efficient drug delivery which results in significant less renal toxicity [189]. AmBisome is able to pass through the fungal cell wall intact, even though the liposome size is much larger than the ~6nm radius of cell wall pores [190]. Liposomes are also used with numerous azole antifungal drugs such as fluconazole, croconazole and itraconazole. Fluconazole is significantly more effective in treating Candida albicans eye infections when it is entrapped in liposomes [124, 191]. Research on fluconazole- resistant C. albicans used a novel liposome formation with thymoquinone, a compound found in the seed oil of the plant Nigella sativa, which was demonstrated to be effective in both fluconazole-resistant and susceptible C. albicans infection in mice [192]. Moreover, liposomal itraconazole formulations were found to have an increased antifungal activity in A. flavus compared to non-capsulated drugs [125].

Another promising delivery method is the use of cell-penetrating peptides (CPPs). CPPs can be targeted to specific tissues or cells and most enter the cell through endocytosis [193]. Cationic cell penetrating peptides have the advantage that they easily bind to negative charged siRNA via electrostatic attachment. Promising fungal specific CPPs are the Penicillium AntiFungal (PAF) peptides, derived from the paf gene of Penicillium chrysogenum. The hexapeptides PAF26, PAF95 and PAF96 derived from the original protein of 54 amino acids [113]. PAF26 is known to internalise in N. crassa and A. fumigatus and has an antifungal activity at around 1 μM [114]. PAF95 and PAF96 are derived from PAF26, but either the first three amino acids or the last three amino acids are changed to Alanine which results in the loss of antifungal activity. Moreover, PAF95 lacks the N-terminal cationic property of PAF26 and PAF96 lacks the C-terminal hydrophobic property. PAF96 was not able to penetrate either N. crassa or A. fumigatus and PAF95 was shown to internalise N. crassa. Once inside the cell the hexapeptides are susceptible to degradation [194]. This property can be useful once siRNA is delivered in the cell as they can move freely to the target without being attached to a peptide.

To date, there are no reports of delivery systems being used to translocate siRNAs into A. fumigatus. In this chapter, we firstly tested if concentrations of siRNA 100x higher than previously used would change the outcome and then continued to test different delivery methods such as exosomes, lipid vesicles and cell penetrating peptides for siRNA uptake by A. fumigatus.

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5.2 Optimising the concentration of siRNA treatment

All previous experiments on siRNA in this project were performed under the same conditions to reduce variability. The specific condition used to test the effect of siRNA in either liquid or solid fungal cultures, which was a treatment with 25 nM siRNA in Vogel’s minimal medium with 1% glucose, is a combination of conditions used in various successful siRNA experiments [92, 93, 96, 145, 152].

The experiments were designed to obtain an optimal and cost effective result and hence the concentrations used in this research (25 nM) are consistent with literature. The lowest concentration of siRNA proven to result in silencing in mammalian cells (20 nM) has also been found to reduce the nonspecific effects of siRNA [195, 196]. Correspondingly, concentrations used in siRNA treatment on Aspergillus spp. range from 25 nM in Aspergillus fumigatus to 10 nM in Aspergillus nidulans [92, 96]. The concentration of siRNA in Candida albicans treatment is remarkably higher, ranging from 50 nM to 1000 nM, which lead to the idea to test our samples at higher siRNA concentrations [81, 82].

To test the effect of a range of different pptA siRNA concentrations, a phenotype assay was performed and cultures exposed to labelled siRNA were visualised by confocal microscopy. Beside the altered siRNA concentrations, all other conditions were identical to previous experiments (Chapter 3). Phenotypes of A. fumigatus cultures were tested on siRNA coated VMM+ plates (Figure 5.1). In these experiments unrelated siRNA was used as a control and 3 different types of pptA siRNAs (that were previously used in this research at lower concentrations) were tested; unmodified siRNA, PS Ome modified siRNA and a pool of diced siRNAs. As pptA is essential, targeting this gene will result in reduced or impaired growth. However, fungal cultures grown up to 3 days show no observable effect in the presence of all types of siRNA at all concentrations tested.

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Figure 5.1. Concentrations of pptA siRNA up to 250 nM did not affect A. fumigatus phenotypes A. fumigatus spores were inoculated on top of siRNA-coated VMM+ agar in 12-well plates and grown up to 3 days at 37°C. Images were taken after 24 hours (left) and 72 hours (right). 3 different concentrations, 25 nM, 100 nM and 250 nM, were tested for a variety of previously used pptA targeted siRNA including unmodified, PS Ome modified and pooled d-siRNAs.

Next, visual observation of A. fumigatus cultures grown in the presence of high concentrations fluorescently labelled siRNA was carried out. Spores were incubated for 8 hours to allow germination then treated with fluorescently (TMR) labelled pptA siRNA for 10 hours. The liquid cultures were imaged using a fluorescence microscope (Figure 5.2).

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Figure 5.2. Treatment of A. fumigatus with high concentrations of labelled pptA siRNA cause morphological differences Germinated A. fumigatus spores were treated for 10 hours with fluorescently (TMR) labelled siRNA targeted against the essential gene pptA. Spores without any siRNA treatment (Untreated) were used as a negative control whereas the treated spores were incubated with either 25 nM, 250 nM or 2500 nM of TMR-pptA siRNA. Cultures treated with the higher concentrations of siRNA show hyperbranching and shorter hyphae. Scale bar, 10 µM.

A. fumigatus cultures showed a morphological change after treatment with higher concentrations of TMR-pptA siRNA. The hyphae were shorter and hyperbranching compared to the untreated control (WT) and the culture treated with the standard siRNA concentration of 25 nM of previous experiments (Figure 4.9). To test whether this is due to direct effects on the target pptA, scrambled pptA siRNA was tested that is unrelated to any known gene in A. fumigatus. The A. fumigatus wildtype strain and the pptA knockout strain were incubated for 8 hours in VMM+ to allow spores to germinate before exposure to 2500 nM scrambled pptA siRNA (unrelated) or pptA targeted siRNA for 10 hours. Images were taken using a 40x objective on an inverted microscope (Figure 5.3). The A. fumigatus strain A1160+, the parental strain of the knock out library (Bromley et al. unpublished), was used as a positive control for morphology. As discussed before, pptA is an essential gene that requires additional iron and lysine for growth. Therefore the pptA knockout strain is used in VMM+ as a negative control for fungal growth and development.

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Unrelated siRNAs do not target pptA and control for any observable changes not due to the direct effects of pptA siRNA on pptA transcript levels. Treatment of fungal cultures with 2500 nM unrelated or pptA siRNA showed the same morphology, but a different morphology than wildtype or pptA KO. Therefore, the morphological changes seen in 2500 nM treated cultures are most likely not due to successful targeting of pptA.

Figure 5.3. Morphological changes in A. fumigatus cultures treated with 2500 nM siRNA Germinated spores of wildtype A. fumigatus were treated for 10 hours with 2500 nM unrelated siRNA or siRNA targeted against the essential gene pptA. Non-treated wildtype A1160+ A. fumigatus spores were used as a positive control for the growth and development. The pptA knock out strain was used as a negative control. Images were taken with the 40x objective of an inverted microscope. Scale bar, 20 µM.

These results show that increasing the concentration of siRNA, even to an amount that has morphological effects for the cells, does not result in better uptake of siRNA. Besides the toxicity, using high concentrations of siRNA is costly. In the following experiments the concentration of siRNA treatment was set at 25 nM, the most commonly used concentration of siRNA in literature.

Transporting molecules into the fungal cell is important for the efficacy of drugs such as siRNAs and therefore in the next part of this chapter, different delivery methods are discussed.

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5.3 Disrupting the cell membrane using the antifungal drug amphotericin B

Amphotericin B is known to interact with ergosterol and forms pores in the fungal cell membrane [36]. Thus when the fungal cell is treated with amphotericin B, siRNAs might have a greater chance to be taken up. To test for siRNA uptake, the minimum inhibitory concentration (MIC) of Amphotericin B can be measured. In the case of Amphotericin B, the MIC is measured as the lowest concentration needed to reduce fungal growth by 80%. In this study, the MIC80 of wildtype A. fumigatus treated with Amphotericin B in RPMI was found to be around 1 mg/Liter in this study which is in agreement to clinical studies [151]. If pptA siRNA is taken up by the fungus in presence of Amphotericin B, the fungus should show reduced growth which is correlated to the MIC of Amphotericin B. Fungal cultures were incubated with Amphotericin B only as control and in the presence of either unrelated siRNA or different types of pptA siRNA (unmodified, PS OMe modified and PNA) (Figure 5.4). Remarkably, cultures incubated with unrelated or pptA targeted siRNA show slightly enhanced growth however the MIC is not changed. Therefore, treatment of A. fumigatus with amphotericin B gives no indication of siRNA uptake and RNAi activation.

Figure 5.4. Amphotericin B MIC did not change in presence of pptA siRNA

The OD600 of A. fumigatus was measured when grown in presence of the antifungal drug amphotericin B in RPMI as a control. A. fumigatus was incubated with a combination of amphotericin B and either unrelated siRNAs or pptA targeted unmodified siRNA, PS Ome modified siRNA or PNA to test for differences in growth. The minimum inhibitory concentration (MIC) of 1 mg/Liter is measured as the lowest concentration to reduce fungal growth by 80% in all tested conditions.

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5.4 Testing extracellular vesicles of mammalian cells for siRNA transfection

Mammalian cells excrete extracellular vesicles that contain genetic information, including microRNA [184]. As discussed before, extracellular vesicles excreted from plants can be taken up by some plant pathogenic fungal species [182]. Because A. fumigatus is a human pathogen, we tested whether mammalian EVs could deliver a cargo of siRNAs into the cytoplasm of the fungus.

To test any vesicle trafficking from mammalian cells towards the fungus, mammalian cells were incubated with fluorescently labelled siRNAs overnight and infected the next day with GFP-expressing A. fumigatus spores. The human lung epithelial cell line A549 was incubated with siRNA captured in lipofectamine. Lipofectamine is a high efficiency lipid-based reagent for siRNA transfection in mammalian cells [197]. siRNA was transfected into the mammalian cells overnight. Then, before infection with A. fumigatus, cells were washed to remove any siRNAs that had not been taken up. If the siRNA is excreted from the mammalian cells in extracellular vesicles, it might be taken up by fungal cells. Confocal microscopy showed that siRNA loaded in lipofectamine internalises into the mammalian cells, but it is not clear if it is subsequently exported in vesicles or taken up by the fungal cells (Figure 5.5). However, siRNA was seen surrounding A. fumigatus hyphae as some parts of the hyphae were labelled with siRNA on the outside (Figure 5.5B).

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Figure 5.5. Transfection of mammalian cells with labelled siRNA and A. fumigatus A) Human lung epithelial cells (A549) (top panel) were transfected with B-siRNA loaded lipofectamine (bottom panel) during an overnight incubation and checked by confocal microscopy. B) A549 mammalian cells, transfected with B-siRNA loaded lipofectamine, were infected for 24 hours with A. fumigatus spores that express GFP in its cytosol. White arrows indicate labelled A. fumigatus hyphae. Scale bar, 10 µm.

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From this data, it was not clear if the siRNA was excreted into vesicles before reaching the fungal cells, or whether the fungal cells were available to the siRNA because they were internalised in the mammalian cells. To test more specifically if extracellular vesicles could enter the fungal cells, exosomes were extracted from mammalian monocytic cells (THP-1 (ATCC® TIB-202™)). B-siRNAs were then electroporated into the exosomes and loading checked by confocal microscopy (Figure 5.6A). Next, the B-siRNA-loaded exosomes were incubated with germinated A. fumigatus spores for 10 hours. Confocal microscopy images indicated that there were no differences in uptake between incubation of A. fumigatus with B-siRNAs only or with B-siRNAs captured in mammalian exosomes (Figure 5.6B).

Exosomes have a lipid like membrane and because the lipid-based reagent lipofectamine is intensively used in siRNA transfection of mammalian cells, the next step was to test lipid-based formulations for siRNA transfection into A. fumigatus.

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Figure 5.6. A. fumigatus treated with B-siRNA loaded mammalian exosomes Exosomes were collected from THP-1 mammalian monocytes and loaded with fluorescently labelled siRNAs (B-siRNAs) by electroporation. A) Exosomes were checked for successful siRNA loading before added to germinated A. fumigatus spores. B) Germinated A. fumigatus spores were incubated with B-siRNA-loaded exosomes for 10 hours and images were taken by confocal microscope. Scale bar, 5µm.

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5.5 Testing lipid-based formulations for siRNA transfection

Lipid-based formulations are widely used in research to deliver siRNA molecules [123, 186, 187]. The advantages of this delivery method is that the siRNA is captured into vesicles and therefore the disadvantages of the siRNA molecules (high molecular weight, hydrophilicity and negative charge) are masked. Moreover, lipids facilitate an active uptake mechanism by fusing with the cell envelope.

Lipofectamine is an efficient lipid-based reagent for siRNA transfection and is widely used in mammalian cells [198]. So far, lipofectamine is not tested in A. fumigatus, but a recent study showed that lipofectamine-based delivery of siRNA into A. flavus resulted in down-regulation of the targets genes [121]. To test the transfection ability of lipofectamine in A. fumigatus, labelled siRNAs were incubated with lipofectamine and added to A. fumigatus germlings. Images were taken by fluorescence microscope after 10 hours of treatment. Results showed the successful capturing of labelled siRNA into lipid vesicles and the diffusion of the vesicles into the medium (Figure 5.7).

Figure 5.7. B-siRNAs captured in lipofectamine lost their ability to accumulate to the cell wall of A. fumigatus B-siRNAs were incubated with the transfection reagent lipofectamine and the B- siRNA-lipofectamine mixture was added to A. fumigatus germlings. After 10 hours, Z-stacks were taken using a fluorescence microscope. The top panel shows the Z- stack where most of the fungus is in focus, whereas the lower panel shows the Z- stack where B-siRNAs are focussed. Scale bar, 10 µm.

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A clear difference is seen compared to previous results where B-siRNAs labelled the fungal cell wall. The vesicles seem to be located randomly in the medium and not aggregated in or on the fungal cell as the Z position of images showed either a focussed fungal hyphae or focussed siRNA-containing vesicles, but never both. Washing the samples after lipofectamine treatment released the medium from any fluorescence. This data suggests that lipofectamine does not improve siRNA uptake, but it does stop siRNAs from accumulating to the fungal cell wall. Lipofectamine consists of positively charged complexes and this could cause the complexes to be repelled from the anionic cell wall [197].

The commercially available transfection reagent Viromer Blue (lipocalyx) contains neutral complexes that prevents aggregation with the positively charged cell wall and with positively charged proteins. Viromer blue mimics a viral infection process by utilising an active endosome escape mechanism that leads to cytosolic delivery of Viromer Blue encapsulated-siRNA. A mixture of multi-lamellar vesicle liposomes, commercially available from Sigma Aldrich was also tested. Liposomes containing amphotericin B (AmBisome) are used in antifungal treatment as they deliver amphotericin B more efficiently and have fewer toxic effects than treatment with amphotericin B alone [199]. To test the multi-lamellar liposome mixture and Viromer blue, siRNA was captured in either of the reagents according to the manufacturer’s protocols. The siRNA-containing mixtures were incubated with A. fumigatus germlings for 10 hours and images were taken using a confocal microscope (Figure 5.8). Consistent with all other data in this thesis, treatment of B-siRNAs only in A. fumigatus results in concentrated signal of B-siRNAs on the cell wall. B-siRNAs captured in Viromer Blue did not show any differences to incubation of A. fumigatus with naked B-siRNAs. Interestingly, incubation of germlings with liposomes containing B-siRNA did not show a strong fluorescent signal associated with the cell wall. B-siRNAs did not accumulate at the cell wall and hence the fluorescent signal was much less even though the germlings were incubated with the same concentration of B-siRNAs. The brighter fluorescent spots were not corresponding to the fungal hyphae, however whether the indistinct B-siRNA signal is seen in or on the hyphae has to be studied in detail in future experiments by for example visualising the fungal hyphae and the moving cytoplasm during siRNA treatment over time.

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Figure 5.8. Treatment of A. fumigatus with B-siRNA encapsulated in Viromer Blue or multi-lamellar liposomes B-siRNAs were captured in lipid formulations, either the non-charged transfection reagent Viromer Blue or liposomes. The B-siRNA containing complexes were incubated with A. fumigatus germlings for 10 hours and uptake was monitored by confocal microscopy. Scale bar, 5µm.

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5.6 Testing cell penetrating peptides for siRNA transfection

As previously discussed, the hexapeptide PAF26 is fungal specific and demonstrates cell-penetrating activities in A. fumigatus. The hypothesis that PAF peptides might translocate bound siRNA into fungal cells was tested. PAF26 was bound to labelled siRNA using two different methods and evidence of uptake was visualised by fluorescence microscopy.

Muñoz et al. described the membrane translocation capability of PAF26 and showed that PAF26 and yeast tRNA can bind after incubation for 30 minutes on ice in TE binding buffer [115]. This protocol was performed using radio-labelled siRNA. The 5’ end of the RNA oligos were labelled with 32P and incubated with either PAF26 or PAF95 (Figure 5.9). Incubation of 25 µM PAF95 with 250 ng radiolabelled siRNA showed only free siRNA, as in the siRNA control, suggesting PAF95 is not able to bind to siRNA. The same conditions using PAF26 resulted in partial binding of RNA to the peptide. Incubation of siRNA with 75 µM PAF26 resulted in a stronger signal of bound RNA to the peptide. PAF26 has a cationic motif which PAF95 lacks and is therefore not able to bind non-specifically to negatively charged siRNAs.

Figure 5.9. Binding assay of radio-labelled siRNA to PAF peptides Autoradiogram of radio-labelled siRNA alone or siRNA incubated with 25 µM or 75 µM PAF26 or 25 µM PAF95. Samples were separated through a 6% polyacrylamide gel.

Next, germinated fungal spores were incubated with the bound peptide-siRNA complexes for 10 hours. In this experiment, a complex with fluorescein labelled BLOCK-iT siRNA (B-siRNAs) and TMR labelled PAF26 was used to visualise the location of both siRNA and peptide. Confocal microscopy revealed the presence of PAF26 in the vacuoles, but interestingly, also a part of the PAF26 stayed on the outside of the

113 fungus (Figure 5.10). B-siRNAs were only seen on the outside of the fungus, which suggests that under these conditions PAF26 cannot translocate B-siRNAs into A. fumigatus.

Figure 5.10. Treatment of A. fumigatus with B-siRNA-PAF26 complex did not change uptake of B-siRNA Fluorescein-labelled BLOCK-iT siRNAs (green) are bound to the cell penetrating TMR- labelled hexapeptide PAF26 (red) and incubated with germinated A. fumigatus spores for 10 hours. Confocal microscopy revealed the partial uptake of PAF26 by the vacuoles and partial binding on the cell wall of the fungus. B-siRNAs were only seen on the outside of the fungus. Scale bar, 5 µm.

These results indicate that the nonspecific binding of the siRNA-PAF26 complex might not be strong enough to translocate the siRNA into the cell. As it is clear that PAF26 is able to enter the cell, siRNA was bound to PAF26 by a different technique. This technique covalently links amine-modified siRNA to cysteine-modified peptides using a heterobifunctional crosslinker [136]. For this method, the sense strand of pptA

114 siRNA containing an amine functionality incorporated at the 3’ terminus was used and an antisense strand with the fluorophore TMR incorporated at the 3’ terminus. These sense and antisense pptA RNA oligos were annealed and the amine-modified siRNA was conjugated to a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker. The siRNA-SMCC complex was then coupled to the cysteine residue of the PAF26-C peptide and binding checked on polyacrylamide gel. The polyacrylamide gel was exposed to the Alexa 546 dye laser to detect the position of TMR and a small shift is seen between the TMR-labelled siRNA only sample and the TMR-labelled siRNA-SMCC-PAF6 complex suggesting the binding was successful (Figure 5.11). The siRNA has a molecular weight of 14949 g/mol, the SMCC of 334 g/mol and the peptide of 1095 g/mol. The siRNA-SMCC-PAF26 has therefore a molecular weight of 16360.835 and compared to the siRNA only, this is seen as a small shift in the gel.

Figure 5.11. Polyacrylamide gel confirming the generation of siRNA-SMCC- PAF26 complex The TMR-labelled siRNA-SMCC-PAF26 complex was run through a polyacrylamide gel next to the TMR-labelled siRNA and TMR-labelled antisense oligonucleotide. PAF26 does not have a fluorophore attached and therefore is not visible when exposed to the Alexa 546 dye laser. A small shift is observed between the complex and the siRNA suggesting a successful binding of SMCC-PAF26 to the siRNA.

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The covalently linked PAF to amine-modified siRNA was then used to test for uptake by the fungal cell. Germinated A. fumigatus spores were treated for 10 hours with the TMR-labelled siRNA-SMCC-PAF26 complex and imaged using confocal microscopy (Figure 5.12). Even though siRNA was covalently attached to PAF26 via the SMCC linker, the TMR labelled siRNA was seen at the cell wall suggesting that PAF26 is not able to translocate the siRNA into the fungal cell. To confirm that the siRNA is localised at the cell wall as seen in previous experiments, peptide8 was added. Peptide8 interacts with the cell membrane and promotes uptake of molecules bound to the cell membrane [166]. Confocal microscopy revealed the uptake of peptide8 and the binding of TMR-labelled siRNA-SMCC-PAF26 to the cell wall as the TMR signal did not change after cell membrane disruption and uptake of peptide8.

Figure 5.12. Treatment of A. fumigatus with siRNA covalently linked to PAF26 in the presence and absence of peptide8 Germinated A. fumigatus spores were treated with TMR-labelled siRNA targeted against pptA covalently linked to PAF26. Peptide8, a cell membrane interacting peptide labelled with a BODIPY fluorophore (green), was added 10 min prior to imaging. After cell membrane disruption and internalisation by peptide8, the signal of TMR-labelled siRNA is still accumulated at the cell wall. Scale bar, 5µm.

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5.7 Discussion

The characteristics of siRNA – high molecular weight, negative charge and hydrophilicity – prevent, even at high concentrations, its direct uptake by Aspergillus fumigatus. Therefore, delivery needs to be facilitated. In this chapter some of the widely used methods in siRNA delivery research were tested. The combination of amphotericin B and pptA siRNA did not change fungal viability and using PAF peptides, mammalian exosomes or the neutral transfection reagent Viromer Blue did not improve siRNA uptake. In all these methods siRNA accumulated at the fungal cell wall. However, two promising delivery methods are presented. Both lipid-based methods, utilising the cationic transfection reagent lipofectamine and a mixture of multilamellar vesicle liposomes, overcome the problem of siRNA accumulating to the cell wall.

Effective down-regulation can be obtained with siRNA concentrations ranging from 1 nM to 10 nM in the mammalian HeLa cell line [200]. However, in studies focussing on fungi siRNA, concentrations of around 25 nM are used [92, 96]. This concentration also resulted in more easily observed fluorescence during confocal microscopy experiments compared to treatment with lower concentrations of labelled siRNA. However, exposing A. fumigatus to 25 nM siRNAs did not result in down-regulation of target transcripts (Chapter 3 and 4). One possible reason for this could be that such a small percentage of the siRNAs are taken up, that they have only a small and local effect on target RNA. Thus, the effect of higher siRNA concentrations was tested. Treating A. fumigatus with a 250 nM and 2.5 µM siRNA concentration caused morphological defects in the hyphae. Hyphae were shorter and seemed to hyperbranch at high siRNA concentrations. This was not a specific response to the siRNA targeting pptA RNA since the same morphology also presented when germlings were incubated with unrelated siRNA. Studies showed that using siRNA in higher concentrations resulted in increased nonspecific effects in mammalian cells even when the siRNA was not targeted to any human genes [195]. Microarray analysis revealed that a nonspecific effect was already seen after incubation with siRNA concentrations higher than 20 nM and when using 100 nM, a large number of non- targeted genes showed to be 4-8 fold upregulated [201].

Having confirmed that the siRNA concentration should not be higher than 25 nM, the siRNAs were tried to be delivered into the fungal cell by alternative means. Numerous cell-penetrating peptides (CPPs) show great potential to translocate siRNAs into the cell [108-110, 202, 203]. In this research Penicillium AntiFungal (PAF) peptides PAF26 and PAF95, derived from the paf gene of Penicillium chrysogenum were tested. PAF26 is known to penetrate A. fumigatus, but has no affinity towards human red

117 blood cells, the clinically relevant bacterial strains Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa, and limited affinity towards the human fungal pathogens Candida albicans and Candida glabrata [166, 204]. The A. fumigatus specific uptake of PAF26 is an advantage for the development of siRNA therapeutics. Moreover, the properties of PAF26 being small and linear, make it susceptible to degradation by proteases in the fungal cell and theoretically this would release the siRNA in the cell. siRNA binding was only successful when using the cationic PAF26, and not the neutral PAF95, confirming that the cationic property of PAF26 is important for nonspecific binding to anionic molecules. However, confocal microscopy experiments showed that the PAF26-siRNA complex was not strong enough to also translocate siRNA molecules into the cell. Moreover, covalent binding of PAF26 to TMR-labelled amine-modified siRNA using the linker SMCC did not result of uptake of siRNA. A possible explanation for the latter result might be that the siRNA is too big and restricts the efficient uptake of PAF26. Uptake of the unlabelled PAF26 used in this experiment could not be confirmed. Ideally both siRNA and peptide would be labelled with different fluorophores however the cost of obtaining labelled PAF26 was prohibitive.

Another delivery method explored in this chapter was the use of siRNA in combination with the antifungal drug Amphotericin B. Amphotericin B is targeted to ergosterol in the cell membrane and results in disruption of membrane integrity. When the membrane is disrupted the drug is transported into the fungus. The hypothesis tested was that siRNA incubated together with Amphotericin B could take advantage of the lack of membrane integrity and translocate into the hyphae. This was tested by measuring the OD600nm of cultures in presence of Amphotericin B and/or pptA siRNAs. If the siRNA is taken up and targets pptA, a decrease in viability is expected in addition to decrease caused by Amphotericin B. The minimum inhibitory concentration of Amphotericin B did not change after co-incubating with pptA siRNA, suggesting that either siRNA did not translocate into the cell or did not have an effect on pptA. One explanation could be that the siRNA is still not able to cross the cell wall and therefore cannot take advantage of the disrupted membrane. Although it is known that Amphotericin B forms trans-membrane channels [205], the precise action of amphotericin B and how it crosses the cell wall is not known. Interestingly, liposomal amphotericin B (AmBiosome) is reported to be more effective in delivering the antifungal drug and therefore results in less toxicity to human cells [199]. This contributed to the idea to test lipid formations for siRNA delivery in A. fumigatus.

As plant vesicles are known to deliver siRNA molecules into plant pathogens, mammalian exosomes were tested if they have the same property in A. fumigatus as it is a human pathogen [182]. Mammalian exosomes contain the lipid

118 phosphatidylcholine and are enriched in cholesterol [206]. The liposome formulation of the antifungal drug AmBisome also contains cholesterol and L-α- phosphatidylcholine and is successful in transporting the drug across the fungal cell wall [189]. However, results showed that there was no sign of siRNA uptake by the fungal cell after incubation with mammalian cells or mammalian exosomes. We continued testing other lipid formations such as the commercially available liposome mixture that consists of cholesterol, L-α-phosphatidylcholine and stearylamine. The addition of stearylamine in multilamellar liposomes increased the loading efficiency of the antifungal fluconazole [191]. There are also many lipid-based transfection reagents commercially available and in this research the neutral charged complexes of Viromer Blue and the positive charged complexes of Lipofectamine were tested. Lipofectamine and the liposome mixture turned out to be a promising delivery method in A. fumigatus. It suppressed the disadvantages of siRNAs by capturing it in vesicles and they were the only methods that got rid of the accumulation of B-siRNAs to the fungal cell wall. Imaging A. fumigatus hyphae after siRNA-lipofectamine incubation clearly showed the capturing of B-siRNAs in the vesicles, but there was no evidence that the vesicles fused with the fungal cell membrane nor that the siRNA entered the hyphae. It might be possible to deliver siRNA-lipofectamine into the fungal cell when combined to another delivery method. For example liposome-siRNA-peptide complexes are shown to be efficient deliveries of siRNAs into the central nervous system across the blood-brain barrier in mice [207]. On the other hand, the liposome mixture showed more potential as the images obtained by confocal microscopy suggest that there may be fluorescence signal inside of the fungus (Figure 5.8). To address the difficulties of determining whether a fluorescent signal is inside or outside of the cell wall, experiments can be done using nucleases that digest the RNA on the outside. If the signal stays the same after enzyme digestion and a wash, siRNAs have most likely crossed the cell wall. Another way of determining if siRNAs have entered the cell is to follow the cytoplasm over time. When the fluorescent signal is not stationary and moving with the cytoplasm it is definite that the siRNA has crossed the cell wall. If these experiments show that the siRNA is actually translocating into the fungus, target mRNA levels should be measured to test if this delivery method could result in RNAi activation and down-regulation of target genes.

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Chapter 6

Characterisation of the small RNA transcriptome in A. fumigatus

6. Characterisation of the small RNA transcriptome of A. fumigatus

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6.1 Introduction

In general, siRNAs function as gene expression regulators by down-regulating their complementary mRNA. The RNAi pathway contributes to multiple biological processes such as the development of stem cells, metabolism and heterochromatin formation. A non-functional RNAi mechanism could therefore have disastrous effects on biological processes of an organism. For example in mice, mutation of dicer-1 results in unviable [208]. The lethality of dicer deletions might be due to the inability of processing endogenous miRNAs that are important for development. The plant pathogen B. cinerea shows compromised virulence in single Dicer mutant strains [94]. When both Dicers were knocked out, no visible lesion of infection was seen on fruits, vegetables and flower petals, suggesting that the Dicer proteins are essential for virulence in B. cinerea. Therefore, the RNAi pathway might be a useful target in the development of therapeutics. In addition to using the RNAi pathway as a target, the identification of siRNAs made by A. fumigatus may be useful in a therapeutic approach. Once the delivery method is optimised, the identified siRNAs can be used to define the optimal siRNAs to use in the treatment of fungal disease.

Multiple studies on A. fumigatus have reported a working RNAi mechanism resulting in target gene silencing [84, 96, 103, 152, 178, 179]. However, little is known about siRNAs in A. fumigatus and it is not clear what properties they possess. In a very generalised form of RNAi silencing, long dsRNA are cleaved by the ribonuclease III (RNase III) endonuclease Dicer into siRNAs. The siRNAs are then loaded into the RNA inducing silencing complex (RISC), consisting of an Argonaute protein, and guided to the target transcript to cleave and down-regulate the target mRNA. The small RNA transcriptome has been defined in a variety of eukaryotic species. Sequence length of siRNA duplexes range from 19 – 25 nt, but research on D. melanogaster showed that 21 bp is the most efficient trigger for RNA silencing [209]. In fungi, the sequence length range from 21 – 25 nt in N. crassa and 20 – 22 nt in P. striiformis [49, 210]. Looking at other properties of fungal endogenous siRNAs, siRNA sequences have a preference for uracil or at the 5’ position and contain a 3’ two nucleotide overhang [210, 211]. This is consistent with studies on siRNA characteristics in other eukaryotic siRNAs such as human, nematode and fly siRNAs [212-214]. Human Argonaute protein was tested for its preference in 5’ nucleotide and not surprisingly the structural data indicated that only U and A provide the tight essential interaction with the Argonaute MID domain [214]. The small RNA transcriptome of A. fumigatus is characterised in various conditions, with sizes ranging from 15 nt to 500 nt [95]. However, this study reported to be unsuccessful in detecting any small ncRNAs with siRNA characteristics. Conversely, siRNAs were detected and validated in virus-free and dsRNA virus-infected A. fumigatus isolates by Coutts et al. [215]. However, both

121 of these studies did not test the effect of gene deletions on silencing by using RNAi mutants.

Assessments of mutants in the siRNA synthesis pathway in N. crassa revealed the importance of QDE2 in guiding siRNAs to its complementary mRNA by showing that the target mRNA in Δqde2 is not degraded [67]. Δqde2 does contain the same amount of small RNAs, as the QDE2 is involved downstream of Dicer. Deleting the two Dicer-like genes in N. crassa resulted in the loss of RNAi-mediated silencing as dsRNAs were not digested into siRNAs. The two Dicer proteins were shown to be redundant, as the single dicer mutants maintained the silencing function [49]. Dicer independent mechanisms of small RNA synthesis have been defined in N. crassa, and one of them was identified as MRPL3. In a previous study, QDE2 associated small RNAs were sequenced and it was shown that not all small RNAs derived from dsRNA were cleaved by Dicer proteins [90]. MicroRNA-like RNAs (milRNAs), a class of small RNAs, were cleaved by MRPL3 and are also involved in post transcriptional silencing, suggesting not all RNAi mechanisms are dicer-dependent. In A. nidulans, deleting Dicer and Argonaute resulted in the loss of RNA silencing, but did not cause any phenotypic changes [216].

In this chapter, I confirm the functional components of the RNAi pathway in A. fumigatus and test the requirement for the RNAi pathway by generation of knock out strains that lack essential RNAi components. To assess if Dicer dependent RNAi biosynthesis is essential for viability, the RNAi mutants strains were grown in environments that mimic in vivo conditions. The small RNA transcriptome of the wildtype was then compared to small RNA transcriptome of the RNAi mutant strains to find out: 1) If losing core components such as Argonaute or Dicer results in the loss of or a less functional RNAi, 2) if endogenous A. fumigatus siRNAs can be identified and which properties they have and 3) which sRNAs are mapped to the target gene pptA.

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6.2 Conservation of RNAi proteins in fungi

The key proteins involved in RNAi are Argonaute, Dicer and RNA-dependent RNA polymerase (RdRP), but there is a great variability in their number and other proteins involved. This part of the thesis provides an overview of the conservation of Argonaute, Dicer and RdRP proteins in eukaryotic species relevant to the project. As the RNAi pathway is intensively researched in N. crassa, in silico analysis was used to predict the biosynthetic pathway in A. fumigatus using the N. crassa amino acid sequences of functional RNAi proteins previously discussed in Figure 1.2. NCBI BLASTp analysis showed the identification of orthologues of all N. crassa RNAi proteins, with percentage identities (BLAST ID) ranging from 27% to 62% and alignment scores (BLAST cover) ranging from 39% to 99%.

Table 6.1. Characterisation of conserved N. crassa RNAi proteins in A. fumigatus N. crassa N. crassa A. fumigatus BLAST ID BLAST cover protein gene ID gene ID

RPA-1 NCU03606 62% 86% AFUA_2G06320

RPA-2 NCU07717 38% 99% AFUA_6G11130

RPA-3 NCU01460 34% 94% AFUA_5G12895

Qip NCU00076 27% 74% AFUA_3G11950

QDE-3 NCU08598 39% 39% AFUA_2G04960

QDE-2 NCU04730 36% 85% AFUA_8G05280

QDE-1 NCU07534 32% 70% AFUA_5G09430

Dicer-2 NCU06766 32% 95% AFUA_4G02930

Dicer-1 NCU08270 44% 90% AFUA_5G11790

Protein conservation was predicted via computational gene ontology (http://uniprot.org and http://www.aspergillusgenome.org) in combination with research found in literature, as RNAi is not extensively studied in all species and not all genes are characterised. To predict the occurrence of RNAi components, search criteria were set on specific characteristics of the RNAi components. Argonaute proteins are defined by the presence of a PIWI, Argonaute and Zwille (PAZ) domain, which is responsible for the 3’ binding of siRNA to the protein, and a PIWI domain,

123 which mostly has a slicer function [217, 218]. Dicer proteins are defined by N- terminal helicase domain, multiple dsRNA-binding domains, PAZ domain and two RNAse III domains. For a functional slicing activity, the two RNAse III domains are necessary as they are individually responsible for the cleavage of one strand, resulting in the formation of siRNA products with 3’ nt overhangs [219]. RdRP seem to share a common origin with the beta subunit of DNA-dependent RNA polymerase (DdRP) as they both contain a conserved double-psi beta-barrel (DPBB) domain [220]. The predicted occurrence of Argonaute, Dicer and RdRPs is summarised in Figure 6.1. Several eukaryotic species have a different number of paralogous silencing proteins, suggesting that RNAi has evolved in response to environmental conditions. It is interesting to note that A. nidulans only carries one functional Dicer, compared to the existence of two functional Dicers in closely related species including A. fumigatus. In some organisms, for example the closely related S. cerevisiae and C. glabrata, it was beneficial to completely lose the function of RNAi. In these species, no RNAi silencing proteins were found. In K. polysporys and S. castellii, no RdRPs were found, but RNAi was still functional with the remaining Argonaute and Dicer proteins [211].

Figure 6.1. Cladogram showing the conservation of RNAi components in several eukaryotes The core components of RNAi have been conserved throughout most of the discussed species. The predicted occurrence of the RNAi components Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), is indicated.

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These results confirm conservation of RNAi in A. fumigatus. For the reason that RdRP null mutants are likely to have a functional RNAi mechanism, the RNAi mechanism was further studied by generating null mutants for the two other key proteins Argonaute and Dicer.

6.3 Dicer and QDE-2 mediated siRNA synthesis are dispensable for viability in A. fumigatus.

A. fumigatus has been shown to have a functional siRNA biosynthetic pathway. To assess if the core RNAi components Dicer and QDE-2 are required for viability in A. fumigatus, five attempts were made to generate null mutants. The A1160p+ strain was chosen as a host for mutagenesis [221]. This strain has enhanced levels of homologous recombination as it lacks the ability to repair DNA by non-homologous end joining. Gene knockout (KO) cassettes were generated by amplifying 1-1.2kb regions immediately flanking the genes of interest and combining these with a selectable marker (either hph for hygromycin resistance or ptrA for pyrithiamine resistance) by fusion PCR (Figure 6.2A) [128].

The qde-2 (Δqde2), dicer-1 (Δdcr1) and dicer-2 (Δdcr2) null mutants were generated by transforming protoplasts with KO cassettes harbouring the hygromycin resistance marker. The dicer-1/2 double KO mutant (Δdcr1Δdcr2) was generated by transforming the dicer-1 null mutant with a dicer-2 KO cassette incorporating the ptrA selectable marker. Two to three transformants per strain were obtained and spores from all transformed colonies were picked and streaked on selection media for at least 4 times to purify the deletion strains.

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Figure 6.2. Generation of gene KO cassettes for qde-2, dicer-1 and dicer-2 A) Schematic overview of the two-step fusion PCR method. Upstream (5’) and downstream (3’) flanks of the target genes were amplified by PCR using either P1-P2 and P3-P4 respectively (1). The 5’ and 3’ flanks were fused to the selectable marker hph or ptrA (2) to generate the gene specific KO cassette (3). B) The 5’ and 3’ flanks of qde-2 (Q2), dicer-1 (D1) and dicer-2 (D2) are amplified, resulting in products between 1 kb and 1.2 kb. B) The 5’ and 3’ flanks are combined with the selection marker hygromycin to generate fusion product of approximately 5.2 kb. To generate a double dicer knockout, the pyrithiamine selection marker is used in combination with D2 flanks to generate a 4.3 kb fusion product.

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To confirm precise replacement of the target genes, a PCR assay was performed that was able to discriminate between wildtype and recombinant loci. Primers shown as P1 and P4 in the schematic overview of the method (Figure 6.2A) amplify gene products in the wildtype strain with a size of 5671 bp, 7792 bp and 7067 bp when using Q2, D1 and D2 primers respectively. P1-P4 amplification products in all mutant strains should show a single band at the same size of the fusion products, approximately 5.2 kb for hygromycin and 4.3 kb for pyrithiamine. The generated single band amplification products indicated the successful insertion of the hygromycin cassette and deletion of qde-2 and dicer-1 (Figure 6.3A). However, the amplification products in Δdcr2 using the P1-P4 D2 primers showed two distinct bands around 5 kb and 7 kb. Repeating the purification more than 10 times with Δdcr2 transformants from multiple transformation rounds did not change the result. This confirmed that the generation of a dicer-2 null mutant was unsuccessful. Remarkably, deleting the dicer-2 gene in the single dicer-1 null mutant was effective as amplification of dicer-2 DNA with D2 P1-P4 primers did not show a 7 kb wildtype band (Figure 6.3B). In summary, two single deletion strains Δqde2 and Δdcr1 and a double dicer deletion strain Δdcr1Δdcr2 were generated during this study. These results indicate that the deleted genes are not required for the viability of A. fumigatus.

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Figure 6.3. Validation of RNAi mutant strains To validate the RNAi mutant strains, the gene of interest in the wildtype or the selection cassette in the mutant strain was amplified using the P1 and P4 primers. A) Replacement of the wildtype gene with the hph knockout cassette was confirmed for Δqde2 and Δdcr1 strains as amplification resulted in a 5.1 kb product. Amplification of wildtype DNA with Q2, D1 and D2 primers resulted in a 5.6 kb, 7.8 kb and 7.1 kb product respectively. B) Replacement of the wildtype genes dicer-1 and dicer-2 with the knockout cassettes was confirmed as amplification of the D1 hph cassette resulted in 5.1 kb product and amplification of the D2 ptrA cassette resulted in a 4.3 kb product.

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6.4 RNAi mutants are dispensable for growth and stress adaptation

To assess if deleting genes involved in the RNAi synthesis pathway have any phenotypic effects, growth of RNAi mutant strains were compared to the parental wildtype strain on solid defined media (RPMI1640) and a solid complete media (SAB).

Figure 6.4. Phenotypic analysis of RNAi mutant strains The validated RNAi mutants and its parental strain A1160p+ were tested for any phenotypic differences. A) Images of colonies were taken after 72 hours of growth on either RPMI or SAB solid medium. The impure Δdcr2, indicated with an asterisk, was also included. B) The radial growth of colonies cultivated on SAB agar was measured after 24, 48 and 72 hours. Data represent the mean values of three replicates and error bars signify the standard deviation. No significant growth differences were measured using one-way anova.

No morphogenic differences were observed for Δqde2, Δdcr1 and Δdcr1Δdcr2 (Figure 6.4A). The impure Δdcr2 was also included in the cultivation on plates and

129 interestingly, the impurity was shown in its morphology on SAB agar. All other strains did not show any obvious colour or morphology differences. The radial growth of cultures on SAB agar plates was determined at 24, 48 and 72 hours and no significant differences were observed (Figure 6.4B).

The growth rate was assessed in various commonly used liquid media. Spores were inoculated in either RPMI [pH 7.0], RPMI [pH 8.0], VMM or ACM and the OD600nm was measured after 2 days of incubation at 37°C. Under these conditions, A. fumigatus experiences various stressors. RPMI is a low iron containing media and the static cultures will experience a slight hypoxic environment. The optimal pH for growth in A. fumigatus is 7.0, thus the alkaline environment of RPMI [pH 8.0] also induces a stress response [222]. The fitness of the mutant strain relative to the wildtype in the specific condition was calculated and no significant differences were measured in all tested media (Figure 6.5).

Figure 6.5. Relative fitness of RNAi mutant strains Growth values of the RNAi mutants were quantified relative to the parental strain (WT). The means and standard deviations of the relative fitness per strain and condition were plotted. No significant differences were measured using one-way anova.

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Recently, reports have demonstrated that small RNAs play an important role in DNA damage repair (DDR) [223, 224]. In N. crassa, QDE-2 and QDE-2 interacting RNAs (qiRNAs) are induced after DDR and the production of qiRNAs requires QDE-1, QDE- 3 and the dicer enzymes [88]. We hypothesised that if this is also true for A. fumigatus, mutants in the RNAi synthetic pathway would show increased sensitivity to DNA damage. Histidine is known to cause an accumulation of single strand nicks in the DNA of N. crassa at concentrations of 2.5 mM which equals approximately 0.4 mg/mL [223]. In A. fumigatus the susceptibility to histidine is unknown, therefore we tested the wildtype ( A1160p+) against a range of concentrations from 0 mg/mL to 10 mg/mL (Figure 6.6). Fungal cultures showed increased sensitivity to histidine at a concentration of 10 mg/mL. The RNAi mutants were also tested for their responses in DDR however no differences were observed between the RNAi mutant strains and its parental strain. As no significant differences were seen in the tested conditions, conditions mimicking the human host infection environment (RPMI, 37°C, pH 7.0) are used in the following experiments.

Figure 6.6. DNA damage assay reveals similar responses in WT and RNAi mutants RNAi mutant strains and their parental strain were spotted on VMM agar plates supplemented with concentrations of the mutagen histidine ranging from 0 mg/mL to 10 mg/mL. The plates were imaged after 24 hours of growth at 37°C in the dark. All strains showed increased sensitivity to histidine at a concentration of 10 mg/mL.

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6.5 Variable sizes of sRNAs are detected in sRNA libraries

Previous studies on RNAi in A. fumigatus have tended to focus on the functional and molecular aspects rather than having a closer look at the transcriptome of the fungal cell. The experimental work presented here provides one of the first investigations on the role of Argonaute and Dicer proteins in A. fumigatus sRNA productions. We aimed to identify transcripts that look like siRNAs by sequencing the small RNAs. In order to do so, a small RNA library was created from the RNAi biosynthesis mutants generated in this study. The RNAi mutants and its parental strain A1160p+ were grown in triplicate in RPMI for 24 hours at 37°C and a cDNA library was generated from small RNAs isolated from these cultures using the TruSeq Small RNA library kit (Illumina) (Figure 6.7). cDNAs amplified using the TruSeq kit incorporate 5’ linkers and 3’ linkers, therefore adapter-ligated sRNA fragments, corresponding to 20 – 30 nt siRNAs, would be 140 bp and 160 bp. Fragments consistent with siRNAs were clearly evident in the amplified libraries from the wildtype isolate, suggesting the protocol of the sRNA library preparation was successfully completed. However, fragments corresponding to all mutant strains showed different sizes of cDNA compared to wildtype. Each sample has a specific index and therefore all samples were pooled in equal quantities to form one sRNA library before purification and sequencing.

Figure 6.7. sRNA libraries of RNAi mutants reveal the variability in cDNA sizes Small RNA libraries were created from wildtype and RNAi mutant cultures grown in triplicate in RPMI for 24 hours at 37°C. The parental strain A1160p+ was used as a wildtype control. Triplicate samples were pooled and assessed on Novex TBE gel. 20 to 30 nt small RNAs ligated with adapters correspond to cDNA with a size between 140 to 160 bp. Unligated adapters and indexes correspond to a band of 100 bp and below.

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6.6 Sequencing analysis of small RNA library

The small RNA library, consisting of RNAs up to 200 bp of the RNAi deletion strains Δqde2, Δdcr1 and Δdcr1Δdcr2, was processed by The Genomic Technologies Core Facility (GTCF) at the University of Manchester. The small RNA sequencing data analysis was done by Ping Wang (University of Manchester). The paired-end data was first processed using trimmomatic, a tool that removes technical sequences such as barcodes and filters the data to improve the quality [225]. The sequencing reads were aligned to the A. fumigatus Af293 reference genome using the bowtie2 software. Finally, principal component analysis (PCA) of the differential expressed (DE) genes was performed with DESeq2. Using this method, no significant differences were measured in fold change of the annotated gene expressions. However, the quality of the data is not up to standard as there is a high level of variable reads in the replicates of all strains (Table 6.2). One replicate of the wildtype is classified as an outlier as it lacks sufficient reads, probably due to degradation of the sample, which most likely imbalances the normalisation of all samples. In addition, tRNAs are most likely produced independently of the RNAi system and the abundance should be similar in all RNAi mutant strains. This is not the case for Δdcr1Δdcr2, where very low reads were measured in all annotated sRNAs, suggesting that these samples have poor quality. Due to these limitations, quantitative evaluation of the data is not possible. In this chapter we assess the qualitative aspects of the data.

Table 6.2. Total sRNA sequencing reads per replicate Replicate 1 Replicate 2 Replicate 3 Wildtype 65260 599 79210 Δqde2 70664 157009 475 Δdcr1 95009 848 366 Δdcr1Δdcr2 624 276 597

The classification of annotated sRNAs, with an abundance of >20 in one replicate of the parental strain A1160p+, presented a variety of sRNA categories such as tRNAs, degraded fragments of mRNA or , and intergenic sRNAs (Table 6.3). The IDs of the sRNAs summarise the start location, for example chr1F1704991 is located on the forward strand of chromosome 1 at nucleotide 1704991. The targets

133 of the high abundance sRNA were accounted to intergenic regions for 36%, 30% to intronic regions, 4% to exonic regions and 30.6% was accounted to tRNAs. The total reads per strains were calculated which shows the low abundance of the double dicer knockout in all targets including tRNAs. Interestingly, no sRNA described in this table exhibits the size property of a potential siRNA and for that reason we characterised sRNAs between 19 and 25 bp from all the reads in the DESeq2 analysis data. Total reads of these sRNAs revealed a peak at 21 bp and the characteristic of the individual sRNAs showed that only four out of the total seven sRNAs between 19 and 25 bp were targeting coding regions of the annotated genes (Figure 6.8). From these two sRNAs, only one (chr1F4382634) is antisense to its target mRNA as the gene is encoded on the reverse strand and the sRNA is mapped to the forward strand. Although this sequence does not contain a 5’ Adenine, this feature is only a preference but not a necessity to function as a guide strand for RNAi-mediated gene silencing. The potential siRNA chr1F4382634 targets an uncharacterised protein and this protein does not have high similarity in other species. The highest query percentage of only 56.39% was found in another uncharacterised protein in the species Aspergillus fischerianus. However, the target has 18.06% similarity with ANIA_10579, a putative intermediate filament associated protein in A. nidulans, and a paralogue was found on chromosome VIII of A. fumigatus that also encodes for a putative intermediate filament associated protein.

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Table 6.3. Classification of high-abundance annotated sRNAs Δdcr1 Size WT Δqde2 Δdcr1 ID Location Target Target description Δdcr2 (bp) reads reads reads reads

1704991- Intergenic, closest to chr1F1704991 48 Putative uncharacterised protein 52 96 190 3 1705038 AFUA_1G05910

2486502- chr1F2486502 71 of AFUA_1G09620 Protein kinase family protein 71 58 13 0 2486572

3698790- chr1F3698790 96 AFUA_1G13835 tRNA 24377 44879 21313 260 3698885

394769- Intergenic, closest to chr1F394769 56 Fungal specific transcription factor 36 133 4 1 394824 AFUA_1G01100

4157768- chr1F4157768 90 AFUA_1G15425 tRNA 6636 9293 5956 60 4157857

86288- Intergenic, closest to chr1F86288 18 Class V chitinase, putative 20 10 4 2 86305 AFUA_1G01100

1126541- Intergenic, closest to chr1R1126541 31 Spo7-like protein 27 46 8 1 1126571 AFUA_1G03910

1469464- Intergenic, closest to chr1R1469464 94 PH domain protein 52 30 13 1 1469557 AFUA_1G05130

1595891- chr1R1595891 72 Intron of AFUA_1G05560 GTP binding protein, putative 24 55 8 0 1595962

3432096- chr1R3432096 51 Intron of AFUA_1G12950 Avl9 protein, putative 32 77 21 1 3432146

1290542- Intergenic, closest to Ubiquitin C-terminal hydrolase, chr2F1290542 90 44 59 31 1 1290631 AFUA_2G04720 putative

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142946- chr2F142946 99 AFUA_2G00655 tRNA 280 160 119 3 143044

2321608- DNA replication licensing factor chr2F2321608 35 Intron of AFUA_2G09060 25 60 25 1 2321642 Mcm4, putative

4740491- chr2F4740491 79 Intron of AFUA_2G17820 O-methyltransferase 59 98 18 1 4740569

937469- chr2F937469 92 AFUA_2G03518 tRNA 64 75 61 0 937560

2871735- Intergenic, closest to chr2R2871735 75 Developmental regulator FlbA 36 124 27 0 2871809 AFUA_2G11180

1669094- Intergenic, closest to chr3F1669094 49 ThiJ/PfpI family protein 193 420 141 0 1669142 AFUA_3G06720

2087856- Intergenic, closest to PWI domain mRNA processing chr3F2087856 47 436 597 277 6 2087902 AFUA_3G08120 protein, putative

2351409- Intergenic, closest to chr3F2351409 91 Putative uncharacterized protein 26 85 12 0 2351499 AFUA_3G09260

2792267- Intergenic, closest to chr3F2792267 91 tRNA 188 544 137 1 2792357 AFUA_3G10743

2822038- chr3F2822038 46 Intron of AFUA_3G10840 Branchpoint-bridging protein 23 56 18 0 2822083

3583443- chr3F3583443 97 AFUA_3G13565 tRNA 23 8 15 0 3583539

1012377- Intergenic, closest to chr3R1012377 91 Putative uncharacterized protein 15137 29133 9326 202 1012467 AFUA_3G03720

2165973- Intergenic, closest to chr3R2165973 94 Putative uncharacterized protein 35 214 26 0 2166066 AFUA_3G08450

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2342071- chr3R2342071 97 AFUA_3G09225 tRNA 6176 9292 3878 59 2342167

2345005- chr3R2345005 99 AFUA_3G09235 tRNA 8664 19672 6620 104 2345103

401852- chr3R401852 98 AFUA_3G01595 tRNA 1188 1447 436 7 401949

2090742- Intergenic, closest to chr4F2090742 95 RAB GTPase Ypt5, putative 28 25 16 0 2090836 AFUA_4G08040

1162108- Protein kinase activator Bem1, chr4R1162108 78 Intron of AFUA_4G04120 71 57 6 0 1162185 putative

1345828- Inositol kinase kinase (UvsB), chr4R1345828 64 Intron of AFUA_4G04760 32 83 24 0 1345891 putative

1820461- chr4R1820461 96 AFUA_4G07025 tRNA 1047 1319 401 9 1820556

2412525- chr4R2412525 97 AFUA_4G09213 tRNA 26814 43609 25204 352 2412621

2561682- chr5F2561682 98 AFUA_5G09895 tRNA 11458 22092 9659 124 2561779

2654345- chr5F2654345 98 AFUA_5G10365 tRNA 2735 2002 805 22 2654442

1804318- Intergenic, closest to chr5R1804318 81 DUF300 domain protein, putative 57 117 41 0 1804398 AFUA_5G07250

2398695- Signal transduction protein Syg1, chr5R2398695 68 Intron of AFUA_5G09320 138 354 75 1 2398763 putative

3125052- chr5R3125052 99 intron of AFUA_5G12060 C2H2 transcription factor, putative 26 62 4 0 3125150

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3565034- Intergenic, closest by chr5R3565034 97 Putative uncharacterized protein 30 62 11 0 3565130 AFUA_5G13570

3821253- Intergenic, closest by Heat shock trehalose synthase, chr5R3821253 99 98 348 83 2 3821351 AFUA_5G14780 putative

1901569- chr6F1901569 55 Intron of AFUA_6G08170 DNA polymerase V, putative 29 42 1 1 1901624

660792- chr6F660792 82 Intron of AFUA_6G03120 Putative uncharacterized protein 176 491 87 5 660874

1578955- Intergenic, closest by chr6R1578955 47 Na+/H+ antiporter, putative 24 58 6 0 1579002 AFUA_6G07050

2103894- chr6R2103894 75 Intron of AFUA_6G08860 Sugar isomerase, KpsF/GutQ 25 24 1 0 2103969

2661868- Tetratricopeptide repeat protein 1 chr6R2661868 92 Intron of AFUA_6G10770 30 30 13 0 2661960 (TTC1), putative

632327- chr7F632327 94 AFUA_7G02317 tRNA 29274 24634 4860 138 632421

670775- Extracellular serine-rich protein, chr7R670775 71 of AFUA_7G02460 32 17 7 0 670846 putative

1016432- chr8F1016432 91 AFUA_8G04485 tRNA 1941 2603 1559 19 1016523

1099431- chr8F1099431 81 Intron of AFUA_8G04800 Valyl-tRNA synthetase 38 47 28 0 1099512

1265348- Mitochondrial ATPase subunit chr8R1265348 92 Exon of AFUA_8G05440 35 47 26 0 1265440 ATP4, putative

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Figure 6.8. Identification of siRNA-like transcripts in A. fumigatus Annotated sRNAs were filtered on sizes between 19 and 25 bp. A) Total reads of WT, Δqde2, Δdcr1 and Δdcr1dcr2 were plotted. B) The filtration revealed seven unique transcripts that were summarised including their location, target and sequence.

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The lack of potential siRNAs in the DESeq2 analysis data lead to the evaluation of siRNA like molecules in the annotated reads using The University of California Santa Cruz (UCSC) Genome Browser. The focus was on the target gene of this thesis, pptA, as mimicking endogenous siRNAs might be an useful therapeutic once the obstacle of siRNA delivery is overcome. sRNAs annotated upstream of or targeted to the gene pptA were characterised by viewing mapped reads on the reverse strand using the UCSC Genome Browser (Figure 6.9). The gene is located on the forward strand of chromosome II and therefore potential antisense siRNA sequences are mapped to the reverse strand. As expected, the double dicer deletion strain did not show any sRNAs, whereas the WT and the single deletion strains did. The dicer-1 deletion strain still produced sRNAs and this observation suggests that the dicer-1 gene is redundant. The length of the total 17 pptA sRNAs range from 88 – 21 bp. The properties of the annotated pptA sRNAs that do not exceed the proposed siRNA length of 25 bp are summarised in Table 6.4. The ideal candidate would, in addition to the 19 – 25 bp size and a sequence antisense to the mRNA, cover all the siRNA characteristics such as a preference for 5’ Uracil or Adenine and targeted to the . Sequences 9, 11 and 15 contain all the above features which suggests that these sequences can be classified as endogenous pptA antisense RNAs.

Table 6.4. Properties of 19 – 25 bp sRNAs mapped to pptA Target Size ID Location 5' base Strain Sequence 5’- 3’ region (bp) 2205469- GGAGCUGGAGUUUA 3 Exon 25 Guanine WT 2205445 CGGAUGUUAUU 2205771- CGACGGGUGUCUGU 4 Exon 25 Cytosine WT 2205747 CAGUGUACAAA 2204822- GAGGCCCUCCAACCA 8 Exon 22 Guanine Δqde2 2204801 UCGGACC 2205100- AUCCCACAGUCCCAU 9 Exon 21 Adenine Δqde2 2205080 GGCGCA 2205600- UUGAGGUAGUCGCG 11 Exon 22 Uracil Δqde2 2205579 UUUGAGAC 2205770- CGGGUGUCUGUCAG 12 Exon 21 Cytosine Δqde2 2205750 UGUACAA 2204775- AUAGACACGCGUCAA 15 Exon 23 Adenine Δdcr1 2204753 UUGACAGU 2205783- AGGAGAGUCUAUAU 17 Intergenic 22 Adenine Δdcr1 2205804 GAGCGUUC

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Figure 6.9. Overview of sRNAs annotated to the target gene pptA sRNA expression data using The University of California Santa Cruz (UCSC) Genome Browser. The annotated sRNAs upstream of or targeted to the A. fumigatus gene CADAFUAT00004371 (pptA) is shown. Properties of the numbered sRNAs with a sequence length between 19 and 25 bp are summarised in Table 6.4.

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6.7 Discussion

In this chapter, we defined the RNAi components in A. fumigatus and shown that the gene deletion strains of Argonaute (Δqde2) and Dicer (Δdcr1 and ΔdcrΔ1dcr2) were not required for viability. In the first instance, it seems that these genes are not useful targets to control fungal diseases, but this might change under different conditions as the role of the RNAi pathway in pathogenicity of A. fumigatus still has to be investigated. Moreover, we provided an overview of the sRNA transcriptome in A. fumigatus and identified siRNA-like sequences that might be useful in the development of therapeutic siRNAs.

Searching the genome for proteins with characteristics described in the introduction of this chapter, resulted in a list where a selection of proteins is not involved in RNAi. For example in C. elegans, four RdRPs are found and only one RdRP is associated with RNAi. Besides, twenty-seven Argonaute proteins were found however only one (RDE-1) engaged with primary siRNAs. Secondary siRNAs, siRNAs that were amplified by RdRPs and not derived from the original dsRNA trigger, interact with several other Argonaute proteins [226]. Another interesting finding in literature was the lack of a canonical Dicer gene in C. albicans, S. castellii and K. polysporus, but conversely it was proven that these species are able to cleave introduced dsRNAs into siRNAs. A RNAse III domain-containing gene found with a different domain architecture than other known Dicers, was predicted to be the Dicer of budding yeasts and explains the functioning RNAi of the species lacking the canonical Dicer [211].

The null-mutants Δqde2, Δdcr1 and ΔdcrΔ1dcr2 were validated, but a pure dicer-2 null mutant with either a hygromycin or pyrithiamine cassette was not generated. Generally, the transformation efficiency in filamentous fungi is low [227]. For example, the transformation efficiency does not exceed 0.0003% per µg DNA when using 107 protoplasts in N. crassa [228]. Therefore, multiple transformation rounds were followed where all six individual isolated transformants with the pyrithiamine cassette and two with the hygromycin cassette were isolated and tested. Considering the literature on dicer-2 gene functions in mammalian cells and the fact that the generation of a dicer-2 null-mutant was unsuccessful, would superficially suggest that dicer-2 is essential. However, the double dicer deletion strain was viable in our research, which makes this hypothesis unlikely. For example in , the lack of Dicer resulted in a general developmental growth arrest after 2 weeks and no viable fish after 3 weeks [229]. Most species closely related to A. fumigatus maintain multiple dicer proteins and it is not reported that dicer null-mutants have lethal effects in plants or fungi. In some species dicer mutations are not lethal, but cause severe phenotypes. In the plant Arabidopsis thaliana, mutation of the Dicer

142 homologue caf-1 results in abnormal organ shapes and defects in axillary and floral meristems (the stem cells of plants), suggesting that Dicer also plays a role in plant development [230]. The fungus Mucor circinelloides contains two dicer-like genes, where a dicer-1 deletion strain does not have an impaired gene silencing mechanism but demonstrates developmental defects [231]. Deletion of dicer-2 does display compromised RNA silencing, suggesting only one dicer protein is required for an efficient RNAi mechanism [232].

In this research, the validated RNAi mutants of the filamentous fungus A. fumigatus did not show any significant phenotypic changes from the wildtype strain under the tested conditions. This result is consistent with literature on analysis of Dicer and Argonaute deletion strains in a range of filamentous fungi such as A. nidulans, M. oryzae and Fusarium graminearum [216, 233, 234]. Besides their incapability of sufficient RNA silencing, RNAi deletion strains of these species displayed normal morphology and growth phenotypes. However, in the filamentous fungi M. circinelloides, Metarhizium robertsii and Trichoderma atroviride, the Δdcr2 and Δqde2 mutant strains show a reduction of spore production [235-237]. Although this phenotype is not yet reported in Aspergillus spp., the possibility is not excluded as the production of conidia has not been explicitly tested in RNAi mutants. Therefore, it is important to investigate conidiation in future analysis of the A. fumigatus Dicer and Argonaute gene mutants.

The sRNA expression preliminary data showed the lack of significant differences in the fold change of the annotated sRNAs in the mutant strains compared to the parental strain. It was expected that the Δqde2 shows no significant differences in the production or accumulation of sRNAs, as qde-2 is involved downstream of the production of siRNAs. An interesting finding is the ability of the single dicer deletion strain Δdcr1 to generate sRNAs, suggesting dicer-1 is redundant. Both of these findings are consistent with literature on RNAi mutant strains in N. crassa [49, 67]. The high variability of the sRNA reads resulted in a dataset with a limitation to its analysis, but we were able to characterise potential siRNAs targeted to pptA using the genome web browser UCSC. In siRNA design, it is recommended to avoid GC stretches longer than 7 [238]. Moreover, RNA polymerase III has a preference to terminate the transcription after stretches of U longer than 3, which should be avoided in a siRNA molecule [239]. According to these standards, the most efficient antisense siRNA sequence should be number 9 (Table 6.4). However, most of these features are unique to the tested species and the most effective siRNA molecule is not yet determined for all Aspergillus spp. Once the delivery of siRNAs is optimised, all seven characterised siRNA-like sequences mapped to the coding region of pptA

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(3, 4, 8, 9, 11, 12 and 15) should be tested for efficient gene silencing in A. fumigatus.

Only one potential siRNA, chr1F4382634, was characterised in the DESeq2 analysed dataset, but once the RNAseq data is improved this method can be used to characterise additional potential siRNAs. One of the highest limitations of the data was the wildtype outlier that most likely causes an imbalance in the normalisation of all samples. The total reads of this wildtype sample sums up to 599 compared to the other 2 replicates with a total of 65260 and 79210 reads. When the outlier is not taken into account, the average non-normalised reads of all annotated sRNAs per sample is 72235 in WT, 76049 in Δqde2, 32074 in Δdcr1 and 499 in Δdcr1Δdcr2. The enhanced decrease of sRNA accumulation in the double dicer deletion strain would suggest an important role for sRNA production by both dicer proteins, if it was not for the decrease of all sRNAs including tRNAs. To confirm the hypothesis that deletion of both dicer genes result in the reduced production or lack of siRNAs, new samples have to be sent for sequencing. Future experiments also have to address the generation of a Δdcr2 small RNA library. The preliminary data and the methods used in this chapter show potential for the characterisation of sRNAs involved in the RNAi mechanism of A. fumigatus and potential targets for siRNA therapeutics.

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Chapter 7

Conclusions and

Future Work

145

7. Conclusions and future work

The disastrous consequences of fungal infections and the increase of antifungal drug resistance makes the need for new antifungal drugs inevitable. An emerging method for controlling diseases is targeting the essential genes that regulate those diseases by the use of siRNAs. This method is currently being tested to treat a range of mammalian diseases such as rheumatoid arthritis, different types of cancer and cardiovascular disease, but is not yet extensively used in fungi [240-242]. This thesis strives to promote the development of siRNAs to treat fungal disease by tackling the obstacles it faces such as siRNA stability and delivery. In addition, identifying the RNA interference components and endogenous sRNAs in A. fumigatus might be useful to define the optimal siRNAs for the treatment of fungal disease.

Taken together, in the third and fourth chapter of this thesis I have shown that incubation with naked unmodified, modified, or pooled siRNAs did not change the phenotype of either A. fumigatus or N. crassa nor did it change target mRNA levels. From the results, it is clear that the majority of fluorescently labelled siRNAs accumulate on the outside of the fungal cell. In A. fumigatus the B-siRNAs were mostly seen in the cell wall, overlapping the signal of the chitin stain CFW. In N. crassa, B-siRNAs are besides the cell wall also seen in the septa. To find out more about the exact location of the B-siRNAs, similar experiments could be carried out in a variety of cell wall or septa mutant strains. For example, the A. fumigatus cell wall mutant Δags1, that has a 50% reduced α-1,3-glucan cell wall content, or Δfks1, that lacks β-1,3-glucan, could be used to find out if B-siRNAs bind to α-1,3-glucan, β-1,3- glucan or neither [243, 244]. Similarly, using chitin synthesis inhibitors (nikkomycin Z) or β-glucan synthesis inhibitors (caspofungin) at concentrations below the MIC could contribute to the discovery of B-siRNA binding location. In addition, to elucidate where the siRNAs accumulate, it is also useful to explore why siRNAs do not enter these specific fungi. Comparative analysis of DNA sequencing data between either A. fumigatus or N. crassa and close relatives, such as A. nidulans and B. cinerea, that have been shown to take up naked siRNAs can be carried out to discover and exploit potential uptake mechanisms [93, 94]. The uptake of siRNAs by B. cinerea is stimulated by extracellular plant vesicles, but how naked siRNAs enter fungal cells is not known and therefore it would be useful to know the genotypic differences of species that are able to translocate siRNAs into the cytoplasm versus those that cannot. Potential candidates are genes encoding transport proteins such as pumps, cotransporters and channel proteins.

In this project, the observation that B-siRNAs accumulate in the cell wall of A. fumigatus is used to investigate the usefulness of fluorescently labelled siRNAs to diagnose fungal infection in corneas. The current diagnostic tests are: 1) time-

146 consuming as it relies on culture growth that can take up to two weeks, 2) cost effective as it involves confocal microscopy and 3) unreliable as it is difficult to distinguish between infected species. Labelling fungi infecting corneas with B-siRNAs is a promising method of diagnosis since the B-siRNAs are not targeted to any mammalian gene, did not label bacteria, are most likely not toxic for mammalian cells and the method did not exceed 24 hours. However, the toxicity of B-siRNAs should be tested for mammalian cells and the highest non-toxic concentration needs to be determined for quicker diagnostics. Incubation of corneas with B-siRNAs for 24 hours showed sufficient labelling of the cornea, but treatment is time-sensitive. More research should define whether increasing the concentration of B-siRNAs on corneas can increase its labelling efficacy and decrease the incubation time required for detection. Different species of pathogenic fungi should also be tested for differentiation between the species.

In chapter five, a range of delivery systems are tested for translocation of siRNA into fungal cells. The tested transfection agents that are optimised for mammalian cells, Viromer Blue and Lipofectamine, and mammalian extracellular vesicles were unsuccessful in the translocation of B-siRNAs into fungal cells. The cell penetrating peptide PAF26 did enter A. fumigatus, but was unable to deliver B-siRNAs across the fungal cell wall. A mixture of lipid vesicles (liposomes) are considered to be a promising delivery method as B-siRNAs lost their ability to accumulate at the cell wall. Liposomes can be combined with cell-penetrating peptides for an optimal siRNA delivery [207]. To confirm this hypothesis, experiments in which the ability of such vesicles to deliver siRNAs into fungi must be examined by phenotype assays and qRT- PCRs to measure target mRNA levels. In this project, only a selection of the array of delivery methods was tested that have been studied in mammalian cells. An abundant variety of peptides, nanoparticles, carbon nanotubes and viruses can also be considered for siRNA delivery into fungi [202, 245-247]. For example, The pH-Low- Insertion-Peptide (pHLIP), can insert across a cell membrane under pH conditions lower than 7.0 by forming a helix. Molecules can be attached to pHLIP by a disulfide bond and are cleaved in the environment of the cytoplasm, leaving the peptide outside of the membrane when washed under pH 7.4 [116]. The intracellular pH of A. fumigatus is 6.4 and after 24 hours as low as 5.4 hence this peptide could be effective in siRNA delivery in A. fumigatus [248]. When exploring these options, it would be wise to remember that most of these delivery systems are merely optimised to cross a cell membrane. Here, no evidence is found that mammalian exosomes are able to deliver siRNAs in fungal cells however it was previously proven that exosomes deliver siRNA in human HeLa cells and human fibrosarcoma cells (HT1080) [249]. Thus, as human EVs can deliver siRNAs into human cells, fungal EVs might be able to deliver siRNAs into fungal cells. Fungal EVs contain a variety of RNA molecules,

147 such as miRNAs, and are most likely excreted to communicate between fungal cells or to activate host immune cells [250]. In addition to fungal EVs, plant EVs could also be exploited as a mechanism to deliver siRNAs into fungal cells. Similar to human and fungal EVs, plants such as sunflower (Helianthus annus) and Thale cress (A. thaliana), communicate through packing molecules into exosome-like vesicles [251, 252]. Moreover, a study of the fungal plant pathogen Blumeria graminis showed that siRNA molecules derived from RNAi expressing constructs in barley (Hordeum vulgare) and wheat (Triticum aestivum) were successful in silencing target genes in the fungus [253]. Recently, these two observations were combined into the conclusion that A. thaliana excretes siRNAs in EVs as a host defence mechanism and these EVs are taken up by B. cinerea, resulting in silencing of fungal target genes [182].

Chapter six examines the function of the RNA interference pathway in A. fumigatus. The conservation of Argonaute and Dicer proteins were confirmed and the results revealed that they are not indispensable for growth and morphology of A. fumigatus under the experimental conditions tested. From this it can be concluded that targeting the RNAi pathway itself would not be a useful approach to treat fungal infections. However, the RNAi pathway might be important for pathogenicity therefore the contribution of RNAi to host-pathogen interactions should be tested. The generation of a dicer-2 deletion strain was unsuccessful however the function of dicer-2 can be tested by other means. Down-regulation of dicer-2 can be achieved by expression of an RNAi construct or by the use of a CRISPR/Cas9 system [103, 254]. If dicer-2 is not essential for development in A. fumigatus, the generation of dicer-2 null-mutant can be achieved by restoring dicer-1 in the double dicer KO. The RNAi mutant strains Δqde2, Δdcr1 and Δdcr1Δdcr2, as well as Δdcr2, should be used in more comprehensive studies to elucidate the function of RNAi in A. fumigatus. For example assays on conidiation, germination and stress responses can be carried out. An initial experiment on conidiation did suggest a lowered sporulation yield in the dicer mutants, but this has to be confirmed.

The small RNA transcriptome of A. fumigatus was also studied. RNAseq data revealed a potential siRNA, chr1F4382634, that maps to a gene encoding an uncharacterised protein that is likely to be involved in the coordination of cytoskeletal elements resembling its paralogue in chromosome VIII and its orthologue in A. nidulans [255, 256]. In addition, multiple potential endogenous siRNA-like sequences were identified targeting the essential gene pptA. The latter information could be useful for the development of siRNA therapeutics. However, the variability of the RNAseq data between replicates and the low sRNA reads of the double dicer KO prevented its quantitative analysis. To make any useful conclusions involving sRNA reads, the data

148 has to be improved or samples need to be repeated. These results show that RNAseq can be a very useful tool for the identification of sRNAs and with improved data quality, quantitative analysis of gene expression in the RNAi deletion strains versus the wildtype strain could be carried out. This might reveal if the dicer-1/dicer-2 null- mutant abolishes the production of siRNAs or if RNAi can function independently of Dicer. Moreover, the analysis should be extended with the combination of total RNA sequencing data to determine the precursors of siRNA-like sequences. Eventually, the siRNA candidates can be aligned to characterised sRNA databases of species with a well-studied RNAi machinery such as N. crassa for insight into probable functions. To study if RNAi is important for host-pathogen interaction, the experiments of chapter six could be performed with strains expressing RNA hairpin constructs or virus-infected strains that activate the RNAi pathway [215].

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