DISSERTATION Characterization of intestinal antibacterial factors of Triatoma infestans (Reduviidae, Insecta) and their interaction with Trypanosoma cruzi (Trypanosomatidae, Kinetoplastida) JENNIFER KATHARINA PAUSCH Research Group Zoology/Parasitology Ruhr-Universität Bochum Characterization of intestinal antibacterial factors of Triatoma infestans (Reduviidae, Insecta) and their interaction with Trypanosoma cruzi (Trypanosomatidae, Kinetoplastida) DISSERTATION to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology and Biotechnology Ruhr-University Bochum International Graduate School of Biosciences Ruhr-University Bochum Research Group Zoology/Parasitology submitted by JENNIFER KATHARINA PAUSCH from Bottrop, Germany Bochum, February 2012 Charakterisierung intestinaler antibakterieller Faktoren von Triatoma infestans (Reduviidae, Insecta) und ihre Interaktion mit Trypanosoma cruzi (Trypanosomatidae, Kinetoplastida) DISSERTATION zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie Internationale Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum angefertigt in der Arbeitsgruppe Zoologie/Parasitologie vorgelegt von JENNIFER KATHARINA PAUSCH aus Bottrop Bochum, Februar 2012 This work has been performed between May 2008 and February 2012 by Jennifer Katharina Pausch under the supervision of Prof. Dr. Günter A. Schaub in the Research Group Zoology/Parasitology, Faculty of Biology and Biotechnology of the Ruhr-University Bochum. Author Jennifer Katharina Pausch Title Characterization of intestinal antibacterial factors of Triatoma infestans (Reduviidae, Insecta) and their interaction with Trypanosoma cruzi (Trypanosomatidae, Kinetoplastida) 1st referee Prof. Dr. Günter A. Schaub 2nd referee Prof. Dr. Klemens Störtkuhl Dedicated to my parents Peter & Roswitha Pausch “Nature prefers that neither host nor parasite should be too hard on the other.” (BURNET & WHITE 1972) CONTENTS I CONTENTS I List of figures IV List of tables VI List of abbreviations and nomenclature IX Acknowledgements XII CHAPTER 1 1 1. General Introduction 2 1.1 Chagas disease (American Trypanosomiasis) 2 1.1.1 History, geographical distribution and epidemiology of Chagas disease 2 1.1.2 The etiological agent Trypanosoma cruzi 4 1.1.3 The disease 6 1.2 Triatominae 7 1.2.1 Systematic classification and distribution of Triatominae 7 1.2.2 Developmental cycle of triatomines 8 1.2.3 Host finding and feeding 9 1.2.4 Digestion 11 1.2.5 Microorganisms in the digestive tract of triatomines 12 1.3 Interactions of Trypanosoma cruzi and triatomines 16 1.3.1 Subpathogenicity of Trypanosoma cruzi in triatomines 16 1.3.2 Interactions with digestive enzymes, hemolysins and agglutinins of the 16 vector 1.3.3 Interactions with the mammalian complement system in the vector 17 1.4 The immune system of insects 18 1.4.1 Cellular immune response 18 1.4.2 Humoral immune response 18 1.5 Objectives 22 CHAPTER 2 23 2. General Materials and Methods 24 2.1 Materials: Insect source and maintenance 24 2.2 Methods 24 2.2.1 Measurement of protein concentrations and antibacterial activity 24 2.2.2 SDS-polyacrylamide gel electrophoresis and zymography 25 2.2.3 Enzymatic digestion of proteins for mass spectrometry 25 2.2.4 Nano-HPLC and Electrospray ionization tandem mass spectrometry 26 2.2.5 Mass spectrometric data analysis 27 CHAPTER 3 28 3. Influence of Trypanosoma cruzi on Triatoma infestans : 29 intestinal bacteriolytic activity and expression pattern of lysozymes 3.1 Introduction 29 3.2 Methods 30 3.2.1 Mammal maintenance, parasites and exposure to Trypanosoma cruzi 30 CONTENTS II 3.2.2 In vitro cultivation of Trypanosoma cruzi epimastigotes 31 3.2.3 Establishment of experimental groups 32 3.2.4 Sample preparation 33 3.2.5 qRT-PCR and data analysis 34 3.3 Results 34 3.3.1 Concentration of soluble proteins 34 3.3.2 pH-dependence of bacteriolytic activity 36 3.3.3 Bacteriolytic activities in the small intestine and rectum 40 at the optimal pH 3.3.4 Protein pattern and bacteriolytic activity of small intestine contents after 42 SDS-PAGE 3.3.5 Protein identification by mass spectrometry 44 3.3.6 Temporal expression profile of TiLys1 and TiLys2 in the small intestine 46 of non-infected and short-term T. cruzi -infected T. infestans 3.4 Discussion 48 3.4.1 Concentration of soluble proteins and bacteriolytic activity in the small 48 intestine and rectum of non-infected and T. cruzi -infected T. infestans 3.4.2 Pattern of bacteriolytic activity in the small intestine and identification 51 of responsible proteins by mass spectrometry 3.4.3 Transcript profile of TiLys1 and TiLys2 53 CHAPTER 4 57 4. Pattern of bacteriolytic compounds in the small intestine of the 58 haematophagous Hemiptera Triatoma infestans 4.1 Introduction 58 4.2 Methods 60 4.2.1 Sample preparation 60 4.2.2 Enrichment of intestinal proteins, zymography and mass spectrometry 60 4.3 Results 61 4.3.1 Antibacterial activity of the purified compounds 61 4.3.2 Proteins in the small intestine homogenates 61 4.3.3 Identified peptides after mass spectrometry 62 4.4 Discussion 64 CHAPTER 5 68 5. General discussion 69 5.1 Material and methodological problems 69 5.2 Pattern of bacteriolytic activities of unfed, short-term and long-term 70 T. cruzi - infected T. infestans CHAPTER 6 72 6. Summary/Zusammenfassung 73 6.1 Summary 73 6.2 Zusammenfassung 75 CONTENTS III CHAPTER 7 77 7. References 78 CHAPTER 8 93 8. Supplemental data 94 8.1 Supplemental data for chapter 3 94 8.2 Supplemental data for chapter 4 115 CHAPTER 9 124 9. Appendix 125 9.1 Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous 125 bug Triatoma infestans (Reduviidae): sequence characterization, expression pattern and characterization of proteolytic activity 9.1.1 Abstract 126 9.1.2 Introduction 127 9.1.3 Experimental procedures 128 9.1.3.1 Insect origin, maintenance and dissection 128 9.1.3.2 Nucleic acid techniques 129 9.1.3.3 Sequence analysis 131 9.1.3.4 pH measurement of the midgut 131 9.1.3.5 Pepstatin affinity chromatography 132 9.1.3.6 Sodium dodecyl sulphate polyacrylamide gel electrophoresis 132 (SDS-PAGE) 9.1.3.7 Characterization of proteolytic activity 132 9.1.3.8 Enzymatic digestion of protein bands and nanoLC-ESI-MS/MS 133 analysis 9.1.3.9 MS data analysis 133 9.1.4 Results 134 9.1.4.1 Characterization of the cathepsin D-like protease cDNA s 134 9.1.4.2 Molecular masses and characteristics of purified TiCatD and 138 TiCatD2 9.1.4.3 pH profile of the midgut 140 9.1.4.4 Tissue-specific expression of the cathepsin D-like protease genes 141 TiCatD and TiCatD2 9.1.4.5 Temporal expression profile of TiCatD and TiCatD2 142 9.1.5 Discussion 143 9.1.6 References 147 9.1.7 Supplemental 152 Curriculum Vitae 154 Bibliography 155 Own contribution to the Publications 156 Erklärung 157 LIST OF FIGURES IV CHAPTER 1 1.1 Estimated number of immigrants with T. cruzi infection living in non- 4 endemic countries (RASSI et al . 2010). 1.2 Approximate distributions of T. cruzi lineages. 5 1.3 Digestive tract of fifth instar nymphs of T. infestans at 5 days after 11 feeding. CHAPTER 3 3.1 Protein concentrations in small intestine and rectum of the different 36 groups in the first approach. 3.2 Protein concentrations in small intestine and rectum of the different 36 groups in the second approach. 3.3 Activities/µg protein at different pH in the first approach. 38 3.4 Activities/µg protein at different pH in the second approach. 39 3.5 Activities/µg protein in small intestine and rectum of the different 41 groups in the first approach. 3.6 Activities/µg protein in small intestine and rectum of the different 42 groups in the second approach. 3.7 Protein profiles and zymography of small intestine contents of long- 43 control and short-control under non-reducing conditions. 3.8 Protein profiles and zymography of small intestine contents of short- 44 term groups under non-reducing conditions. 3.9 Temporal expression of TiLys1 and TiLys2 in the small intestine of 47 chicken blood fed T. infestans referring to the amount of transcripts of unfed bugs . 3.10 Temporal expression of TiLys1 and TiLys2 in the small intestine of 48 rat blood fed T. infestans A) referring to the amount of transcripts of unfed bugs and B) referring to chicken blood fed bugs . 3.11 Temporal expression of TiLys1 and TiLys2 in the small intestine of 48 short-term T. cruzi -infected T. infestans referring to the amount of transcripts of A) unfed bugs and B) the non-infected control group . 3.12 Multiple sequence alignment of lysozymes of different triatomines. 55 CHAPTER 4 4.1 First chromatographic separations of bacteriolytic compounds from 62 small intestine homogenates of T. infestans . 4.2 Multiple sequence alignment of TiLys1, TiLys2 and TiLys3. 64 4.3 Putative amino acid sequence of trialysin. 64 LIST OF FIGURES V CHAPTER 8 S8.1.1 Bacteriolytic activities (∆OD) plotted against the respective protein 94 concentration in small intestine and rectum of the different groups in the first approach. S8.1.2 Bacteriolytic activities (∆OD) plotted against the respective protein 95 concentration in small intestine and rectum of the different groups in the second approach. S8.1.3 Multiple sequence alignment of the amino acid sequences of 96 Triatoma infestans cathepsin B and L using CLUSTALX (V2.0). S8.1.4 Multiple sequence alignment of the amino acid sequences of 96 Triatoma infestans cathepsin D and D2 using CLUSTALX (V2.0). S8.2.1 Second chromatographic separation of bacteriolytic compounds from 115 small intestine homogenates of T. infestans to exclude an overload of the column. S8.2.2 Third chromatographic separation of bacteriolytic compounds from 116 small intestine homogenates of T.