Bacteriolytic and anticoagulant proteins in the saliva and intestine of blood sucking bugs (Triatominae, Insecta) [Elektronische Ressource] / submitted by Christian Karl Meiser

Meiser, Christian Karl

Sujets

• Biologie

Informations

• Publié par: ruhr-universitat_bochum
• Publié le: 01 janvier 2010
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Bacteriolytic and anticoagulant proteins in the
saliva and intestine of
blood sucking bugs (Triatominae, Insecta)


Dissertation to obtain the degree
Doctor Rerum Naturalium (Dr. rer. nat.)
at the Faculty of Biology and Biotechnology
Ruhr-University Bochum



Research Group Zoology/Parasitology



submitted by
Christian Karl Meiser

from
Bochum, Germany

Bochum, November 2009 Bakteriolytische und antikoagulante Proteine des
Speichels und Verdauungstraktes von
blutsaugenden Wanzen (Triatominae, Insecta)


Dissertation zur Erlangung des Grades
eines Doktors der Naturwissenschaften
an der Fakultät für Biologie und Biotechnologie
der Ruhr-Universität Bochum


angefertigt in der
AG Zoologie/Parasitologie



vorgelegt von
Christian Karl Meiser

aus
Bochum, Germany

Bochum, November 2009
ERKLÄRUNG

Hiermit erkläre ich, dass ich die vorliegende Arbeit selber verfasst und bei keiner
anderen Fakultät eingereicht und dass ich keine anderen als die angegebenen
Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten
Dissertation um sechs in Wort und Bild völlig übereinstimmende Exemplare.
Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten
und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.


Bochum, den 25.11.2009




C. Meiser
Contents

Contents
1. Introduction 1
1.1. Chagas disease 1
1.1.1. Impact and geographical distribution of Chagas disease 1
1.1.2. The aetiological agent: Trypanosoma cruzi 1
1.1.3. The disease 2
1.1.4. Control of Chagas disease 3
1.2. Triatominae 4
1.2.1. Systematic classification and distribution of Triatominae 4
1.2.2. Development cycle of Triatominae 5
1.2.3. Host finding and feeding 6
1.2.4. Digestion 9
1.2.5. Microorganisms in the digestive tract of triatomines 11
1.3. The immune system of insects 15
1.3.1. Cellular immune response 15
1.3.2. Humoral immune response 16
1.5. Food source blood 18
1.5.1. The complement system and inhibitors 18
1.5.2. The blood coagulation and inhibitors 20

2. Aims 26

3. Feeding-induced changes of bacteriolytic activity and of the pattern 27
of bacteriolytic compounds in the stomach and small intestine of the
haematophagous Hemiptera Triatoma infestans
3.1. Introduction 27
3.2. Materials and methods 30
3.2.1. Insect origin, maintenance and sample preparation 30
3.2.2. Determination of bacteriolytic activity 30
313.2.3. Electrophoresis and zymography
3.3. Results 32
3.3.1 Weight and concentration of soluble proteins of stomach and small 32
intestine
3.3.2. Bacteriolytic activity 32
353.3.3. Protein banding after electrophoresis and bacterial lysis in gels
3.4. Discussion 38

4. Bacteriolytic activity in the saliva of the haematophagous bug 44
Triatoma infestans, sequence of a new lysozyme and variations in the
cDNAs from the salivary glands and in genomic DNA encoding
lysozymes
4.1. Introduction 44
4.2. Material and Methods 45
4.2.1. Insects and saliva collection 45
4.2.2. Bacteriolysis assay 46
4.2.3. SDS-polyacrylamide gel electrophoresis and zymography 46
4.2.4. Nucleic acid techniques 47
4.3. Results 48
4.3.1. Volume and protein content of released saliva 48
4.3.2. Bacteriolytic activity of the saliva 49
4.3.3. Protein pattern and bacteriolytic activity of the saliva after SDS- 50
PAGE
4.3.4. Expression of genes encoding lysozymes in the salivary glands 51Contents

4.3.5. Sequences of genomic DNA encoding lysozymes 54
4.4. Discussion 57

5. Kazal-type inhibitors in the stomach of Panstrongylus megistus 62
(Triatominae, Reduviidae)
5.1. Introduction 62
5.2. Materials and methods 64
5.2.1. Maintenance of triatomines and sample collection 64
5.2.2. Coagulation inhibition assays 64
5.2.3. Nucleic acid techniques 65
5.2.4. Purification of serine protease inhibitors 66
5.2.5. Analysis of protease inhibition 67
5.2.6. Electrophoresis and reverse zymography 67
5.2.7. MALDI-TOF-MS 68
5.3. Results 69
5.3.1. Influence of stomach contents on the coagulation time 69
5.3.2. Characteristics of the Kazal-type inhibitor cDNA 69
5.3.3. Trypsin inhibition after 2D-electrophoresis 71
5.3.4. Characteristics of purified Kazal-like inhibitors 73
5.3.5. Molecular masses of two Kazal-type inhibitors 75
5.4. Discussion 76

6. A salivary serine protease of the haematophagous reduviid 81
Panstrongylus megistus: sequence characterization, expression
pattern and characterization of proteolytic activity
6.1. Introduction 81
6.2. Materials and methods 82
6.2.1. Insects maintenance and saliva collection 82
6.2.2. Nucleic acid techniques 83
6.2.3. Sequence analysis 84
6.2.4. Saliva collection 85
6.2.5. Characterization of proteolytic activity 85
6.2.6. SDS-PAGE and zymography 86
6.2.7. Chromatographic isolation of proteolytic activity 87
6.2.8. In-gel digestion 88
6.2.9. Nano-HPLC/ESI-MS/MS 88
6.2.10. Mass spectrometric data analysis 89
6.3. Results 89
6.3.1. Characterization of the serine protease cDNA 89
6.3.2. Local and temporal expression pattern of serine protease gene 90
6.3.3. Effects of feeding on the release of saliva 92
6.3.4. Characteristics of the proteolytic activity in the saliva 93
6.3.5. Purification of proteases 94
6.3.6. Fibrinolytic activity of the saliva and the purified serine protease 95
6.3.7. Protein profile and proteolytic activity after electrophoretic 96
separation
6.3.8. Sequences of peptides obtained by mass spectrometry 97
6.4. Discussion 98

7. General discussion 103
7.1. Methodological problems 103
7.2. Comparison of the bacteriolytic activities in the saliva and the digestive
tract. 104Contents

7.3. Serine proteases and serine protease inhibitors 110

8. Summary/Zusammenfassung 113
8.1. Summary 113
8.2. Zusammenfassung 116

9. References 119
10. Appendix 157
11. Abbreviations 168
Curriculum vitae 169
Bibliography 170
A: Publications 170
B: Abstracts 170
Acknowledgements 173

1. Introduction 1

1. Introduction

1.1. Chagas disease

1.1.1. Impact and geographical distribution of Chagas disease

Chagas disease is one of the “Big Five”, i.e. one of the five most important tropi-
cal parasitic diseases selected by the World Health Organization (WHO) in
1975/76 as major topic in the campaign against tropical diseases (Schaub &
Wülker, 1984). The aetiological agent of the disease, Trypanosoma cruzi, was de-
scribed for the first time in 1909 by Dr. Carlos J.R. Chagas. During the investigation
of a malaria epidemic in Minas Gerais, a Federal State in southeast Brazil, he
found the flagellate first in the intestine of a blood-sucking assassin bug, Pan-
strongylus megistus, later in the blood of a cat and then in the blood of a girl
(Chagas, 1909; 1922). Chagas disease occurs mainly in Latin America, but also in
the south of North America. While Colombia, Bolivia and Paraguay, with about 30,
24 and 21% have the highest infection rates, in the southern United States only
sporadic infections are reported (WHO, 2002).

1.1.2. The aetiological agent: Trypanosoma cruzi

The causative agent, T. cruzi, belongs to the family Trypanosomatidae and the
order Kinetoplastida. During the development cycle of this protozoon, there is a
host change between mammals and insects and a change of forms between a-,
trypo-, epi- and spheromastigotes (Schaub & Wunderlich, 1985; Schaub, 1988a;
1989; Schaub and Lösch, 1988; Tanowitz et al., 1992; Schaub & Pospischil, 1995;
Tyler & Engmann, 2001). After the flagellate has entered the mammalian host as
metacyclic trypomastigote via mucous membranes or small lesions of the skin,
caused by the mouthparts of the vector for blood ingestion or by scratching as an
reaction to the saliva of the vector (Schuster & Schaub, 2000), it first infects cells in
the area of the entrance, especially macrophages. In the phagosome of a
macrophage, the parasite begins to transform to the amastigote form, and after
secretion of pore-forming proteins it evades into the cytosol, before phagosome
and lysosomes fuse (Burgleigh & Andrews, 1995; Contreras et al., 2002). In the
1. Introduction 2

cytosol of the host cell, the amastigotes divide repeatedly, and cystic nests, called
pseudocysts, are formed in the tissue (Sacks & Sher, 2002). When the resources
of the host cell are depleted, the amastigotes develop via pro- and epimastigote
stages to trypomastigotes and are released after bursting of the host cell. The
blood-trypomastigote can be detected only a short time in the blood where it is
protected against the immune system of the host by a surface coat of glycoproteins
(Hall & Joiner, 1991). Via the blood, these trypomastigotes get access to cells of
other organs, in which the flagellate changes