University of South Bohemia Faculty of Science Functional analysis of Salp25D, a homologue of peroxiredoxin, from castor bean tick Ixodes ricinus MASTER THESIS Bc. Radka Hobizalová, BSc. Supervisor: Nataliia Rudenko, PhD. Co-supervisor: Maryna Golovchenko, MSc. Faculty guarantee: Prof. RNDr. Libor Grubhoffer, CSc. České Budějovice, 2012 Hobizalova, R., 2012: Functional analysis of Salp25D, a homologue of peroxiredoxin, from castor bean tick Ixodes ricinus. Mgr. Thesis, in English – 64 p., Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic. Annotation: Antioxidant enzymes play an important role in detoxification of reactive oxygen species, thus protecting the organism from oxidative damage. The blood-feeding lifestyle of ticks is a source of oxidative stress that needs to be balanced by an appropriate antioxidant defense. In this study, a homologue of one-cysteine peroxiredoxins named Salp25D from the tick I. ricinus is described, and its antioxidant activity in vitro is confirmed. Due to similarity with protein from I. scapularis, it is assumed that I. ricinus Salp25D might be involved in processes at tick-host-pathogen interface, facilitating acquisition and potentially also transmission of B. burgdorferi spirochetes. Financial sources: GAČR P302/11/3133 I hereby declare that I have worked on my master thesis independently and used only the sources listed in the bibliography. I hereby declare that, in accordance with Article 47b of Act No. 111/1998 in the valid wording, I agree with the publication of my master thesis, in full to be kept in the Faculty of Science archive, in electronic form in publicly accessible part of the STAG database operated by the University of South Bohemia in České Budějovice accessible through its web pages. Further, I agree to the electronic publication of the comments of my supervisor and thesis opponents and the record of the proceedings and results of the thesis defense in accordance with aforementioned Act No. 111/1998. I also agree to the comparison of the text of my thesis with the Theses.cz thesis database operated by the National Registry of University Theses and a plagiarism detection system. Date: Signature: Acknowledgement: I would like to thank Professor Libor Grubhoffer for giving me the opportunity to work in his laboratory. Further, my thanks go to my supervisors, Natasha Rudenko and Maryna Golovchenko for their scientific guidance. They were always willing to help and answer my questions. Thanks go to our lab group members, for creating a nice atmosphere in the lab as well as for their valuable advice, and to the people from the neighboring labs, namely Gábina Loosová and Ondra Hajdušek, Martina Kovářová, Helča Langhansová and Radek Šíma, for their help with specialized methods. Last, but not least, I would like to thank my boyfriend Martin for standing by me all the time, and my parents for supporting me throughout my whole studies. LIST OF ABBREVIATIONS 1-Cys Prx - one cysteine peroxiredoxin 2-Cys Prx - two cysteine peroxiredoxin AA – amino acid CAT - catalase cDNA - complementary DNA FACS - fluorescence activated cell sorting G - gut GFP - green fluorescent protein GPx - glutathine peroxidase GR - glutathione reductase Grx - glutaredoxin GSH/ GSSG - reduced/oxidized glutathione MCO - metal catalyzed oxidation NADPH/NADP+ - reduced/oxidized nicotinamide adenine dinucleotide phosphate OV - ovaries PCR - polymerase chain reaction PMA - phorbol 12-myristate 13-acetate Prx – peroxiredoxin qRT-PCR - quantitative reverse transcription-polymerase chain reaction RNAi - RNA interference ROS - reactive oxygen species rSalp25D - recombinant salivary protein (mass 25kDa) rxn - reaction SAT - saliva activated transmission SDS-PAGE - sodium dodecylsulphate - polyacrylamide gel electrophoresis SG - salivary glands SGE - salivary gland extract SOD - superoxide dimutase ssRNA/dsRNA - single-stranded/double-stranded RNA Trx - thioredoxin TrxR - thioredoxin reductase TABLE OF CONTENTS 1 INTRODUCTION ............................................................................................................. 1 1.1 Reactive oxygen species (ROS) and oxidative stress ................................................ 1 1.1.1 Sources of ROS .................................................................................................. 2 1.1.2 ROS-induced oxidative damage ......................................................................... 2 1.2 Antioxidant defense ................................................................................................... 3 1.2.1 Non-enzymatic antioxidants ............................................................................... 3 1.2.2 Enzymatic antioxidants....................................................................................... 4 1.2.2.1 Catalase (CAT) ............................................................................................ 4 1.2.2.2 Superoxide dismutase (SOD) ...................................................................... 4 1.2.2.3 Glutathione peroxidase (GPx) ..................................................................... 5 1.2.2.4 Peroxiredoxin (Prx, Prdx) ........................................................................... 6 1.2.2.5 Thioredoxin and glutathione/glutaredoxin systems .................................. 11 1.3 Ticks ......................................................................................................................... 12 1.4 Oxidative stress and antioxidant defense in ticks .................................................... 13 1.4.1 Blood digestion-induced oxidative stress ......................................................... 14 1.4.2 Oxidative stress within tick-host interface ....................................................... 16 1.5 Anti-tick vaccines .................................................................................................... 19 1.6 Salp25D from Ixodes ricinus ................................................................................... 20 2 GOALS OF THE WORK ............................................................................................... 22 3 MATERIALS .................................................................................................................. 23 3.1 Chemicals, buffers, media........................................................................................ 23 3.2 Kits ........................................................................................................................... 27 3.3 Primers ..................................................................................................................... 27 3.4 Ticks ......................................................................................................................... 27 4 METHODS...................................................................................................................... 28 4.1 Bioinformatic analysis ............................................................................................. 28 4.2 Verification of Salp25D insert in the expression vector .......................................... 28 4.3 Production of recombinant protein in a bacterial system......................................... 28 4.3.1 Transformation of competent E. coli cells with the expression vector ............ 28 4.3.2 Pilot expression – time course monitoring ....................................................... 28 4.3.3 Optimization of induction temperature ............................................................ 29 4.3.4 Large-scale expression of Salp25D .................................................................. 29 4.4 Purification of recombinant protein ......................................................................... 29 4.4.1 Preparation of E. coli lysate .............................................................................. 29 4.4.2 Purification on Ni-NTA column ....................................................................... 29 4.5 SDS-PAGE analysis ................................................................................................ 30 4.6 Dialysis, concentration ............................................................................................. 30 4.7 Western blotting ....................................................................................................... 30 4.8 Functional assays ..................................................................................................... 30 4.8.1 DNA nicking assay ........................................................................................... 30 4.8.2 Borrelia viability assay – LIVE/DEAD ............................................................ 31 4.8.3 Borrelia viability assay – FACS ....................................................................... 32 4.8.4 Assay for glutathione peroxidase (GPx) activity .............................................. 32 4.9 RNA interference ..................................................................................................... 33 4.9.1 Preparation of dsRNA ...................................................................................... 33 4.9.1.1 PCR with primers for RNAi ...................................................................... 33 4.9.1.2 Restriction of PCR product and plasmid pLL10 ....................................... 33 4.9.1.3 Ligation into pLL10 vector ....................................................................... 33 4.9.1.4