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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

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

Bochum, Germany

Bochum, November 2009

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

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
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
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
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
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 again to the amastigote form and
If the blood-trypomastigote is ingested together with the blood by an assassin
bug of the subfamily Triatominae, it develops in the anterior part of the digestive
tract to epi- and spheromastigotes. These attach to the perimicrovillar membranes
on the wall of the small intestine and multiply. In the rectum of the bug, they trans-
form to the non-replicative metacyclic trypomastigote form. The metacyclic trypo-
mastigote is the infectious form that is excreted in the faeces of the bug, beginning
development in the mammal. Unlike Trypanosoma rangeli, the other human try-
panosomatid which is transmitted via the saliva, T. cruzi colonizes only the diges-
tive tract of the bug (Schaub & Wunderlich 1985; Kollien et al., 1998; Kollien &
Schaub, 2000; Vallejo et al., 2009). In addition to the vectorial transmission, in the
past transmission via contaminated blood transfusions was a second important
transmission pathway (summarized in Wendel, 1998). The transmissions via con-
taminated food, organ transplants or the transmission from mother to child during
pregnancy or childbirth are only of minor importance (WHO, 2008).

1.1.3. The disease

The course of the disease in humans is divided into two or three barely dis-
tinguishable phases (WHO, 2008). After the infection, first an oedema is formed at
the entry side. Later in the acute phase, non-specific symptoms of infections de-
velop, such as fever and the swelling of local lymph nodes. These symptoms re-
main for 1-2 weeks after the infection and are caused by the increased number of
blood stages of the pathogen. In this acute phase, less than 5% of infected people
die, especially children (Guimarães et al., 1968).
1. Introduction 3

In the following latent and chronic phases of the disease, which are now
grouped together as chronic phase, hardly any blood stages can be detected and
the pathogen develops almost exclusively intracellularly. In the initial latent phase,
which can last from several years to several decades, hardly any symptoms are
noticeable. The chronic phase is characterized by the damage caused by the intra-
cellular development of the pathogen. Frequently megaorgan syndromes occur in
large hollow muscular organs, such as the colon or the heart. Damage of the myo-
cardium often results, due to the deteriorated electrical conduction, in pathological
cardiac arrhythmia (Schaub & Wunderlich, 1985; Schaub & Pospischil, 1995;
Coura & de Castro, 2002; Coura, 2007).

1.1.4. Control of Chagas disease

®Chagas disease in humans is treated by chemotherapy with Nifurtimox and
®Benznidazole . Both drugs are primarily used for treatment of the blood stages in
the acute phase and attack the tissue stages of the parasite only weakly, resulting
in a cure of approximately 50% of treated infections. In addition to the low thera-
peutic success both drugs have strong side effects (Schaub & Wunderlich, 1985;
Docampo, 2007).
For the control of Chagas disease in South America control initiatives were
launched, such as the Southern Cone Initiative in 1991, representing Argentina,
Bolivia, Brazil, Chile, Paraguay and Uruguay (Schofield & Dias, 1991). In addition
to the serological surveillance of blood donors and blood banks (Remme et al.,
2006), control campaigns focus mainly on the vector. The aim is the reduction of
domestic triatomine populations, especially the widespread Triatoma infestans.
These campaigns are not only based on the use of insecticides, but also include
the education of the rural populations and improvement of the general hygienic
conditions and quality of house constructions, the latter to provide fewer retreat
possibilities for the bugs close to the people (Schaub & Wunderlich, 1985).
These campaigns resulted in the states of the Southern Cone initiative in a sig-
nificant reduction of the domestic triatomine populations and since 2001 led to the
certification of individual states by the World Health Organization as free of vector
transmissions of Chagas disease (Remme et al., 2006). Thus, for example Brazil
was certified for the elimination of the principal vector T. infestans and the exclu-
1. Introduction 4

sion of infections by blood transfusion in June 2006 by the Pan American Health
Organization (PAHO) and the WHO (Dias 2006; Schofield et al., 2006). By similar
actions in most countries affected by Chagas disease, the infection levels in Latin
America were reduced over the past 16 years by about 50%, according to estima-
tions of WHO from 20 million to 8 million people (WHO, 2007) and according to
PAHO from 30 million to approximately 12 million (Dias, 2007).

1.2. Triatominae

1.2.1. Systematic classification and distribution of Triatominae

Triatomines are only about 0.18 % of the approximately 80,000 species of
Hemiptera (syn. Rhynchota). Hemiptera are characterized by piercing sucking
mouth parts. Most species feed on plant juices or predatate on other insects. In the
suborder Heteroptera, which are characterized by basal hardened forewings, the
hemielytra, all species in the families Cimicidae (bedbugs) and Polyctenidae (bat
bugs) are bloodsuckers, whereas in the family Reduviidae (assassin bugs) only
species of the subfamily Triatominae are obligate haematophagous. The approxi-
mately 140 species within the Triatominae belong to 6 tribes, the Alberproseniini,
Cavernicolini, Bolboderini, Linshcosteusinii, Rhodniini and Triatomini. The Alber-
proseniini and Cavernicolini both contain one genus representing two species. The
Bolboderini include four genera with 14 species and the Linshcosteusinii one ge-
nus with six species. The species-richest tribes are the Rhodniini with the genera
Rhodnius and Psammolestes and 19 species and the Triatomini with nine genera
and nearly 100 species, with the genus Triatoma itself including 67 species
(Galvão et al., 2004; Schofield & Galvão, 2009). Most species of Triatominae can
be found on the American continent between 42 ° N and 46 ° S latitude (Lent &
Wygodzinsky 1979; Schofield, 1994; Gorla et al., 1997). Species of the genus
Linshcosteus occur only in India, and Triatoma rubrofasciata, which is associated
with rats, was spread from South America by sailing ships to many tropical and
subtropical ports (Schaub, 2009).
The Latin American Triatominae mainly settle in sylvatic habitats. They are often
closely associated with their hosts, for example in the burrows of rodents, the
caves of bats or nests of birds (Schaub, 2009). Individual species successfully