Functional characterization of the mevalonate- isoprenoid biosynthesis pathway genes in Mucor circinelloides Ph.D. dissertation Dileep Kumar Supervisors: Prof. Dr. Csaba Vágvölgyi Dr. Árpád Csernetics Ph.D. School of Biology Department of Microbiology Faculty of Science and Informatics University of Szeged 2018 Szeged TABLE OF CONTENTS 1. ABBREVIATIONS ........................................................................................................... 4 2. OVERVIEW ...................................................................................................................... 6 3. INTRODUCTION ............................................................................................................. 7 3.1. Characterization and taxonomy of Mucoromycotina fungi ................................................ 7 3.2. Economic and clinical importance of Mucoromycotina fungi .......................................... 9 3.3. Antifungal agents applied in clinics to treat fungal infections ....................................... 11 3.4. The mevalonate-isoprenoid biosynthesis pathway in M. circinelloides ...................... 15 3.5. Genetic background of the mevalonate-isoprenoid pathway in M. circinelloides ................................................................................................................................ 19 3.6. Genetic modification of Mucormycotina fungi ................................................................... 21 4. AIMS OF THE STUDY .................................................................................................. 27 5. MATERIALS AND METHODS .................................................................................... 29 5.1. Strains used in the experiments ............................................................................................... 29 5.2. Applied media and growth conditions ................................................................................... 29 5.3. Primers used in this study .......................................................................................................... 31 5.4. Plasmid constructs used in this study ..................................................................................... 33 5.5. Experimental methods ................................................................................................................ 36 5.5.1. Extraction of genomic DNA from M. circinelloides ................................................. 36 5.5.2. Agarose gel electrophoresis and isolation of DNA from agarose gel .................. 37 5.5.3. Applied PCR techniques and reaction conditions ...................................................... 38 5.5.4. Restriction digestion and ligation ................................................................................... 38 5.5.5. Determination of sequences ............................................................................................. 39 5.5.6. Preparation and transformation of competent E. coli cells and plasmid DNA purification .............................................................................................................................. 39 5.5.7. Total RNA purification from M. circinelloides and cDNA synthesis .................. 40 5.5.8. Quantitative real-time PCR .............................................................................................. 40 5.5.9. Protoplast formation and PEG/CaCl2-mediated transformation of M. circinelloides .......................................................................................................................... 41 5.5.10. Microscopy and examination of mitotic stability of the mutants ........................ 41 5.5.11. Determination of the carotenoid and ergosterol content in M. circinelloides . 42 5.5.12. Antifungal susceptibility test ......................................................................................... 43 5.5.13. Phagocytosis assay ............................................................................................................ 44 5.5.14. Statistical analyzes ............................................................................................................ 45 6. RESULTS AND DISCUSSION ..................................................................................... 46 6.1. Transcription of six genes under different cultivation conditions involved in the mevalonate-isoprenoid biosynthesis in M. circinelloides ................................... 46 6.1.1. Comparison of relative transcription level of selected genes ................................. 46 6.1.2. Effect of environmental conditions on the transcription of selected M. circinelloides mevalonate-isoprenoid pathway genes ................................................ 47 6.1.3. Effect of medium composition on the transcription of the M. circinelloides six mevalonate-isoprenoid pathway genes..................................................................... 55 6.1.4 Effect of statin treatment on the transcription of the M. circinelloides six mevalonate-isoprenoid pathway genes ........................................................................... 63 6.2. Construction of plasmids for overexpression and silencing of M. circinelloides six genes involved in the mevalonate-isoprenoid biosynthesis and transformation experiments ..................................................................... 64 2 6.3. Characterization of the mevalonate-isoprenoid biosynthesis pathway mutant M. circinelloides strains .............................................................................................. 66 6.3.1 Examination of plasmid copy number and relative transcription level change in the mutants ......................................................................................................................... 66 6.3.2 Investigation of macro- and micromorphology of the transformants .................... 68 6.4. Carotenoid and ergosterol content of the M. circinelloides strains mutant in mevalonate-isoprenoid pathway ........................................................................................ 76 6.5. Antibiotic susceptibility of the M. circinelloides strains mutant in mevalonate-isoprenoid pathway ............................................................................................. 81 6.6. Interaction of the mevalonate-isoprenoid pathway mutant M. circinelloides strains with MH-S macrophages ................................................................. 83 7. CONCLUSIONS ............................................................................................................. 85 8. REFERENCES ................................................................................................................ 86 9. ACKNOWLEDGEMENT ............................................................................................. 109 10. SUMMARY ................................................................................................................ 110 11. ÖSSZEFOGALÁS ...................................................................................................... 115 12. SUPPLEMENTARY MATERIALS ........................................................................... 120 3 1. ABBREVIATIONS AmB amphotericin B ATMT Agrobacterium tumefaciens-mediated transformation CBS Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands cDNA complementary DNA CFU colony forming unit CLO clotrimazole DHA dihydroxyacetone DMAPP dimethylallyl pyrophosphate DMSO dimethyl sulfoxide EDTA ethylenediamine tetraacetic acid EMBL European Molecular Biology Laboratory FLU fluvastatin FPP farnesyl pyrophosphate GHMP galacto-, homoserine-, mevalonate- and phosphomevalonate kinases GGPP geranylgeranyl pyrophosphate GPP geranyl pyrophosphate HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A HPLC high performance liquid chromatography hpRNA hairpin RNA IPP isopentenyl pyrophosphate ITR itraconazole LB Luria – Bertani medium Mal maltose MEA malt extract agar medium MEP methylerythritol phosphate miRNA micro interfering RNA MOPS 3-(N-morpholino) propanesulfonic acid MVA mevalonate pathway Na-ac sodium acetate NCBI National Center for Biotechnology Information 4 OD optical density PBS phosphate buffered saline PCR polymerase chain reaction PEG polyethylene glycol PI phagocytic index PMC PEG − sorbitol − MOPS − calcium chloride (buffer) qPCR quantitative real-time PCR RPM revolutions per minute SMC sorbitol − MOPS − calcium chloride (buffer) SIM simvastatin siRNA small interfering RNA SZMC Szeged Microbiology Collection, Hungary TAE tris − acetic acid − disodium EDTA (buffer) TF transcription factor Treh trehalose TRIS tris(hydroxymethyl)aminomethane YNB yeast nitrogen base YPG yeast extract − pepton − glucose medium Mucor circinelloides genes and encoded proteins frequently occur in the thesis: hmgS HMG-CoA synthase mvk mevalonate kinase dmd diphosphomevalonate decarboxylase (pyrophosphomevalonate decarboxylase or mevalonate 5- pyrophosphate decarboxylase) ipi isopentenyl pyrophosphate (IPP) isomerase isoA farnesyl pyrophosphate (FPP) synthase carG geranylgeranyl pyrophosphate (GGPP) synthase leuA α-isopropylmalate isomerase pyrG orotidine 5’-monophosphate decarboxylase gpd1 glyceraldehyde-3-phosphate dehydrogenase 1 zrt1 ZIP zinc transporter 5 2. OVERVIEW Members of the subphylum Mucoromycotina, order Mucorales (such as Lichtheimia, Mucor, Rhizomucor and Rhizopus species) are saprotrophic fungi, which also have medical,