Proteomic analysis of excretory/secretory proteins from parasitic and free-living stages of ...

192 Pages·2009·4.63 MB·English

Proteomic analysis of excretory/secretory proteins from
parasitic and free-living stages of Strongyloides ratti

Dissertation

zur Erlangung des Doktorgrades des Department Chemie der Universität Hamburg

vorgelegt von
Hanns Soblik
aus Fahrdorf

Hamburg 2009
The present thesis was carried out between January 2006 and Febuary 2009 at the Bernhard
Nocht Institute for Tropical Medicine, Hamburg, Germany and at the Proteomics Center at
Children’s Hospital, Boston, USA.

1. Reviewer: PD Dr. Norbert Brattig
2. Reviewer: Prof. Dr. Peter Heisig

th
Disputation date: 10 of July 2009
Für meine Eltern
Table of Contents I

1 Introduction ................................................................................................ 1
1.1 The life cycle of Strongyloides spp. ................................................................................. 1
1.2 Pathology of Strongyloides infections ............................................................................. 3
1.3 Treatment of strongyloidiasis .......................................................................................... 4
1.4 Diagnosis of strongyloidiasis ........................................................................................... 5
1.5 Immune response to Strongyloides .................................................................................. 6
1.6 Strongyloides ratti as a laboratory model......................................................................... 7
1.7 Protein secretion of nematodes ........................................................................................ 8
1.8 Objectives ..................................................................................................................... 11
2 Animals, Materials and Methods............................................................. 12
2.1 Animals ........................................................................................................................ 12
2.2 Materials ....................................................................................................................... 12
2.2.1 Devices .............................................................................................................. 12
2.2.2 Kits .................................................................................................................... 13
2.2.3 Solutions............................................................................................................ 14
2.2.4 Culture Media and supplements ......................................................................... 16
2.2.5 Plasmids ............................................................................................................ 17
2.2.6 Oligonucleotides ................................................................................................ 17
2.2.7 Enzymes ............................................................................................................ 18
2.3 Methods ........................................................................................................................ 19
2.3.1 Working with Strongyloides ratti ....................................................................... 19
2.3.1.1 Infection of hosts ............................................................................... 19
2.3.1.2 Charcoal culture and Baermann apparatus .......................................... 19
2.3.1.3 Preparation of iL3 .............................................................................. 20
2.3.1.4 Preparation of the free-living stages ................................................... 21
2.3.1.5 Preparation of parasitic females ......................................................... 21
2.3.1.6 Preparation of E/S products ................................................................ 21
2.3.1.7 Preparation of worm extracts ............................................................. 22
2.3.1.8 Whole worm analysis ......................................................................... 22
Table of Contents II

2.3.2 Molecular biological methods ............................................................................ 23
2.3.2.1 E. coli culture .................................................................................... 23
2.3.2.2 Generation of competent bacteria and transformation ......................... 23
2.3.2.3 Plasmid preparations .......................................................................... 23
2.3.2.4 Expression of S. ratti galectin-3 in E. coli .......................................... 24
2.3.2.5 Purification of recombinant proteins by affinity chromatography ....... 24
2.3.2.6 Total RNA isolation ........................................................................... 25
2.3.2.7 Reverse transcription ......................................................................... 25
2.3.2.8 Polymerase chain reaction .................................................................. 26
2.3.2.9 Purification of DNA fragments .......................................................... 26
2.3.2.10 5’- and 3’-cDNAs amplification ......................................................... 26
2.3.2.11 Agarose gel electrophoresis ............................................................... 27
2.3.2.12 Determination of nucleic acid concentrations ..................................... 27
2.3.2.13 Ligating of DNA ................................................................................ 27
2.3.2.14 Restriction analysis ............................................................................ 28
2.3.2.15 DNA sequencing ................................................................................ 28
2.3.3 Biochemical methods ......................................................................................... 29
2.3.3.1 Determination of protein concentration by Bradford assay ................. 29
2.3.3.2 SDS-polyacrylamide gel electrophoresis (PAGE) .............................. 29
2.3.3.3 Coomassie staining of polyacrylamide gels ........................................ 29
2.3.3.4 Silver staining of polyacrylamide gels ................................................ 30
2.3.3.5 Substrate gel electrophoresis - zymogram .......................................... 30
2.3.3.6 Gelatin gel overlay ............................................................................. 30
2.3.3.7 Lactose affinity separation ................................................................. 31
2.3.3.8 One dimensional-electrophoresis and band excision ........................... 31
2.3.3.9 Tryptic digestion ................................................................................ 31
2.3.3.10 Liquid chromatography - tandem mass spectrometry (LC-MS/MS) .... 32
Table of Contents III

2.3.4 Bioinformatic procedures ................................................................................... 37
2.3.4.1 Database searches .............................................................................. 37
2.3.4.2 Phylogenetic analysis ......................................................................... 38
2.3.5 Immunological tests ........................................................................................... 38
2.3.5.1 Western Blot analysis ........................................................................ 38
2.3.5.2 ELISA (Enzyme-linked immuno-sorbent assay) ................................. 38
3 Results ....................................................................................................... 40
3.1 Establishing the S. ratti life cycle .................................................................................. 40
3.2 Optimising and processing of E/S products ................................................................... 44
3.2.1 Dependence on the number of larvae ................................................................. 44
3.2.2 Detection of the metalloprotease ........................................................................ 45
3.2.3 Size determination of the metalloprotease .......................................................... 45
3.2.4 Dependence on the incubation times .................................................................. 46
3.2.5 Dependence on the incubation temperature ........................................................ 47
3.2.6 Inhibition of protein synthesis ............................................................................ 48
3.2.7 Differences in protein secretions and crude extracts ........................................... 49
3.2.8 Differences in protein secretion among various stages ....................................... 50
3.2.9 Antibody recognition of E/S products ................................................................ 51
3.3 Mass spectrometry ........................................................................................................ 52
3.3.1 Comparison of proteins secreted from different stages ....................................... 53
3.3.2 Abundant proteins in E/S products shared by all stages ...................................... 56
3.3.3 Stage-related proteins ........................................................................................ 60
3.3.3.1 Proteins enriched in infective larvae ................................................... 60
3.3.3.2 Proteins enriched in parasitic females ................................................ 62
3.3.3.3 Proteins enriched in the free-living stages .......................................... 64
3.3.4 Demonstration of differentially expressed proteins applying PCR
analysis .............................................................................................................. 65
Table of Contents IV

3.4 Selected candidate functional proteins ........................................................................... 66
3.4.1 Identification and analysis of S. ratti galectins ................................................... 66
3.4.1.1 Completion of galectin sequences ...................................................... 69
3.4.1.2 Sequence analysis of galectins ........................................................... 70
3.4.1.3 Phylogenetic analysis of galectins ...................................................... 73
3.4.1.4 Isolation of native galectins ................................................................ 74
3.4.1.5 Prokaryotic expression of Sr-Gal-3 .................................................... 76
3.4.1.6 Antibody recognition of Sr-Gal-3 ....................................................... 76
3.4.1.7 Sugar-binding assay of galectins ........................................................ 77
3.4.2 Identification and analysis of a S. ratti prolyl oligopeptidase .............................. 80
3.4.2.1 Completion of the Sr-POP-1 sequence ............................................... 80
3.4.2.2 Mass spectrometric analysis of Sr-POP-1 ........................................... 82
3.4.2.3 Sequence analysis of Sr-POP-1 .......................................................... 83
3.4.2.4 Phylogenetic analysis of Sr-POP-1 ..................................................... 87
3.4.2.5 Inhibition of the Sr-POP-1 enzyme activity ........................................ 88
4 Discussion ................................................................................................. 92
4.1 Comparison of results with published EST data from Strongyloides ssp. ....................... 92
4.2 Comparison with data from other parasites .................................................................... 97
4.3 S. ratti galectins ............................................................................................................ 99
4.3.1 Role of galectins in immune responses ............................................................... 99
4.3.2 Galectins identified in S. ratti E/S products and extracts .................................. 101
4.4 The S. ratti POP .......................................................................................................... 104
4.4.1 Role and classification of POPs ....................................................................... 104
4.4.2 Sr-POP identified in parasitic female E/S products and extracts ....................... 105
5 Abstract................................................................................................... 107
6 Zusammenfassung .................................................................................. 109
7 Acknowledgements ................................................................................. 111
Table of Contents V

8 References ............................................................................................... 112
9 Appendices .............................................................................................. 118
9.1 Protein Lists ................................................................................................................ 118
9.1.1 Table 1a: List of Strongyloides EST cluster numbers found in E/S
products from the parasitic, the infective and the free-living stages .................. 118
9.1.2 Table 1b: Nematode RefSeq proteins found in supernatants from the
parasitic, the infective and the free-living stages .............................................. 123
9.1.3 Table 2a: List of Strongyloides EST cluster numbers found only in
E/S products from infective larvae ................................................................... 124
9.1.4 Table 2b: Nematode RefSeq proteins only found in E/S products from
the infective larvae ........................................................................................... 131
9.1.5 Table 3a: List of Strongyloides EST cluster numbers found only in
E/S products from parasitic females ................................................................. 132
9.1.6 Table 3b: Nematode RefSeq proteins only found in E/S products from
the parasitic females ........................................................................................ 135
9.1.7 Table 4a: List of Strongyloides EST cluster numbers found in E/S
products from infective larvae and parasitic females ........................................ 136
9.1.8 Table 4b: Nematode RefSeq proteins only found in E/S products from
infective larvae and parasitic females ............................................................... 139
9.1.9 Table 5a: List of Strongyloides EST cluster numbers found only in
E/S products from the free-living stages ........................................................... 140
9.1.10 Table 5b: Nematode RefSeq proteins only found in E/S products from
the free-living stages ........................................................................................ 141
9.1.11 Table 6a: List of Strongyloides EST cluster numbers found in E/S
products from infective larvae and free-living stages. ....................................... 142
9.1.12 Table 6b: Nematode RefSeq proteins found in E/S products from
infective larvae and free-living stages .............................................................. 143
9.1.13 Table 7a: List of Strongyloides EST cluster numbers found in E/S
products from parasitic females and free-living stages ..................................... 144
Table of Contents VI

9.1.14 Table 8a: List of Strongyloides EST cluster numbers found in extracts
from the parasitic, the infective and the free-living stages ................................ 145
9.1.15 Table 8b: Nematode RefSeq proteins found in extracts from the
parasitic, the infective and the free-living stages .............................................. 147
9.1.16 Table 9a: List of Strongyloides EST cluster numbers found only in
extracts from infective larvae ........................................................................... 148
9.1.17 Table 9b: Nematode RefSeq proteins found only in extracts from
infective larvae ................................................................................................ 149
9.1.18 Table 10a: List of Strongyloides EST cluster numbers found only in
extracts from parasitic females ......................................................................... 150
9.1.19 Table 10b: Nematode RefSeq proteins found only in extracts from
parasitic females .............................................................................................. 153
9.1.20 Table 11a: List of Strongyloides EST cluster numbers found in
extracts from infective larvae and parasitic females ......................................... 154
9.1.21 Table 11b: Nematode RefSeq proteins found in extracts from infective
larvae and parasitic females ............................................................................. 155
9.1.22 Table 12a: List of Strongyloides EST cluster numbers found only in
extracts from the free-living stages .................................................................. 156
9.1.23 Table 12b: Nematode RefSeq proteins found only in extracts from the
free-living stages ............................................................................................. 164
9.1.24 Table 13a: List of Strongyloides EST cluster numbers found in
extracts from infective larvae and free-living stages ......................................... 165
9.1.25 Table 13b: Nematode RefSeq proteins found only in extracts from
infective larvae and free-living stages .............................................................. 166
9.1.26 Table 14a: List of Strongyloides EST cluster numbers found in
extracts from parasitic females and free-living stages ....................................... 167
9.1.27 Table 14b: Nematode RefSeq proteins found in extracts from parasitic
females and free-living stages .......................................................................... 169
9.2 Galectin sequences ...................................................................................................... 170
9.2.1 Sr-Gal-1 ........................................................................................................... 170
Table of Contents VII

9.2.2 Sr-Gal-2 ........................................................................................................... 171
9.2.3 Sr-Gal-3 ........................................................................................................... 172
9.2.4 Sr-Gal-5 ........................................................................................................... 173
9.2.5 Lactose-agarose bead isolation: Identified galectin peptides ............................. 174