proteomic investigations of the group b streptococcus

274 Pages·2017·2.71 MB·English

by  Ellen Cole

Citation: Yang, Qian (2011) Proteomic investigation of the group B streptococcus. Doctoral
thesis, Northumbria University.
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PROTEOMIC INVESTIGATION OF
THE GROUP B STREPTOCOCCUS

QIAN YANG
A thesis submitted in partial fulfilment of
the requirements of the University of
Northumbria at Newcastle for the
degree of Doctor of Philosophy

Research undertaken in the School
of Life Science
April 2011

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Abstract
The Group B Streptococcus (GBS) is a Gram-positive opportunistic pathogen which is a
leading cause of neonatal disease globally. In 2000-2001, the general incidence of
neonatal GBS infection was 0.72 per 1000 live births in U.K. and the mortality rate is
about 10%, because of which neonatal GBS disease is a significant burden on society.
GBS is part of the commensal flora, colonising the vagina and gastrointestinal tract of
women. Vertical transmission is the main cause of early onset GBS disease. During the
process of GBS neonatal disease, GBS must be able to survive in several very different
host environments, including the vagina, amniotic fluid, the neonate‟s lung and blood. The
vagina is normally acidic, low oxygen and with limited nutrients while the neonate‟s lung
and blood are neutral, high oxygen and with abundant nutrient. Proteomic investigations
of GBS protein expression under conditions representing those associated with benign
maternal colonisation and foetal exposure may help us understand the molecular basis of
GBS virulence.
GBS growth characteristics, long term survival, acid adaptation, viable but non-culturable
state and biofilm formation were investigated to help us understand how GBS survives in
different environments and also help us to develop an in vitro model to reflect in vivo
conditions during GBS disease development.
An in vitro model of GBS growth under conditions reflecting maternal vaginal carriage
(low pH, low oxygen, nutrient stress) and exposure to body fluids during invasive disease
(neutral pH, aeration, nutrient sufficient) was established. Proteins expressed under each
growth conditions were separated by two dimensional electrophoresis. Individual proteins
were subjected to in-gel trypsin digestion and identified using liquid chromatography-
mass spectrometry with peptide mass fingerprinting followed with bioinformatic research.
A total of 76 proteins were identified and 16 of these were expressed differentially. The
putative virulence factor C protein β antigen and proteins involved in responses to
oxidative stress were up-regulated under the conditions reflecting neonatal exposure.
Another in vitro model of GBS growth on Todd Hewitt agar in the presence or absence of
10% human serum was established and followed by proteomic investigation of proteins
differentially expressed under these two conditions, as this model reflects GBS neonatal
septicaemia (exposure to serum). A total of 84 proteins were identified and 11 of which
were expressed differentially. The putative virulence factor C protein β antigen, arginine
deiminase, an ABC transporter substrate-binding protein and glyceraldehyde-3-
phosphate dehydrogenase were up-regulated in the presence of human serum.
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List of contents

Abstract..............................................................................................................1
List of contents..................................................................................................2
List of Tables....................................................................................................11
List of Figures..................................................................................................14
Acknowledgements.........................................................................................16
Declaration.......................................................................................................17
Chapter 1. Introduction...................................................................................18

1.1. Epidemiology of Group B Streptococcus disease......................................18
1.1.1. Morbidity and mortality of neonatal Group B Streptococcus disease.....18
1.1.2. Risk factors of GBS neonatal disease.....................................................22

1.2. The pathogenesis of GBS disease.............................................................26
1.2.1. GBS virulence factors that affect adherence to epithelial surfaces.........28
1.2.2. GBS virulence factors affecting penetration of host cellular barriers......32
1.2.3. GBS virulence factors that help avoidance of immunological clearance.38
1.2.4. GBS virulence factors that activate inflammatory reactions....................42

1.3. Environmental factors affecting GBS growth and virulence factor
expression.........................................................................................................43
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1.3.1. GBS survive in different host environments............................................43
1.3.2. Oxygen affects GBS metabolism and virulence factor expression..........47
1.3.3. pH affects GBS adherence......................................................................50

1.4. Theories of GBS long term survival in relation to colonisation...................50
1.4.1. Acid tolerance responses........................................................................51
1.4.2. Biofilm formation contributes to acid tolerance........................................57
1.4.3. Long term survival in stationary phase by streptococci and other
reference organisms..........................................................................................58
1.4.4. Viable but non-culturable hypothesis......................................................60

1.5. Proteomics..................................................................................................62

1.6. Aims of the present study...........................................................................66

Chapter 2. Materials and Methods.................................................................67

2.1. GBS growth experiments...........................................................................67
2.1.1. Bacterial strains, media and microbiology techniques............................67
2.1.1.1. Bacterial strains....................................................................................67
2.1.1.2. Media....................................................................................................67
2.1.1.2.1. Todd Hewitt Broth medium................................................................68
2.1.1.2.2. LB broth.............................................................................................68
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2.1.1.2.3. Vagina simulative medium................................................................68
2.1.1.2.4. GBS growth in human serum............................................................70
2.1.1.2.5. Comparison of GBS A909 growth with and without 10% human
serum………………………………………………………………………………….70
2.1.1.2.6. M17 agar and M17 with Heme/Menaquinone supplementation........70
2.1.1.3. Microbiology techniques.......................................................................72
2.1.1.3.1. Bacterial stocks and storage.............................................................72
2.1.1.3.2. Gram staining of GBS.......................................................................72
2.1.1.3.3. Lancefield typing of streptococci.......................................................73
2.1.1.3.4. Phosphate buffered saline (PBS)......................................................74
2.1.2. GBS growth.............................................................................................74
2.1.2.1. GBS growth curves measured by microtiter plate reader.....................74
2.1.2.2. Quantification of growth at pH 4 measured by colony forming unity
formation...........................................................................................................74
2.1.3. GBS long term survival experiments.......................................................76
2.1.3.1. GBS long term survival experiments....................................................76
2.1.3.2. Measuring survival of multiple GBS strains in stationary phase using a
spot plate method..............................................................................................76
2.1.4. GBS Live-Dead staining..........................................................................78
2.1.5. Regrowth of extended stationary phase cells..........................................80
2.1.6. Acid tolerance experiments.....................................................................81
2.1.6.1. Auto acidification measurements.........................................................81
2.1.6.2. Adaptation experiments - strategy for exposure to acid.......................81
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2.1.7. Biofilm experiments.................................................................................82
2.1.7.1. Survival of biofilm grown GBS..............................................................82
2.1.7.2. Crystal violet staining assay for biofilm formation................................83
2.1.7.3. Assay for improving biofilm formation by coating plates with
extracellular matrix molecules (hyaluronic acid and heparin)............................83
2.1.7.4. Alternative models for biofilm formation...............................................85
2.1.8. Statistic methods……………………………………………………………..86

2.2. Methods of proteomics...............................................................................86
2.2.1. GBS A909 biomass culture.....................................................................86
2.2.1.1. Biomass for proteomic investigation GBS grown under conditions
associated with neonatal exposure...................................................................88
2.2.1.2. Biomass for proteomic investigation of GBS exposure to human
serum................................................................................................................88
2.2.2. Protein extraction and purification...........................................................90
2.2.2.1. Protein extraction.................................................................................90
2.2.2.2. Protein purification................................................................................91
2.2.2.2.1. Protein purification using a 2D clean up kit.......................................91
2.2.2.2.2. Protein purification using acetone precipitation methods..................92
2.2.3. Protein separation using 2D SDS-PAGE................................................92
2.2.3.1. Loading proteins on Immobiline DryStrip gels.....................................92
2.2.3.2. Isoelectric focusing..............................................................................93
2.2.3.3. Equilibration.........................................................................................94
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2.2.3.4. Resolving gel and stacking gel preparation.........................................95
2.2.3.5. Casting 2D gels....................................................................................95
2.2.3.6. Running the second electrophoresis dimension...................................98
2.2.3.7. Staining gels with colloidal coomassie blue.........................................99
2.2.3.8. 2D gel documentation........................................................................101
2.2.4. Analysis of 2D gels using PDQuest software........................................101
2.2.5. Protein in-gel trypsin digestion..............................................................102
2.2.5.1. Stock solutions...................................................................................102
2.2.5.2. Protein in-gel trypsin digestion...........................................................102
2.2.5.3. Freezing drying protein samples........................................................103
2.2.6. Protein identification using Liquid Chromatography/Electrospray
Ionization-Mass Spectrometry (LC/ESI-MS)...................................................103
2.2.7. Mascot search for protein identification.................................................106
2.2.8. NCBI Blast and UniprotKB search.........................................................106

2.3. Methods of one dimensional SDS-PAGE and Western blotting...............107
2.3.1. Preparation of protein samples for one dimensional SDS-PAGE.........107
2.3.2. One dimensional SDS-PAGE................................................................108
2.3.3. Staining gels and photographing gels...................................................110
2.3.4. Western blotting....................................................................................110

Chapter 3. GBS growth characteristics in vitro..........................................115

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3.1. Background..............................................................................................115

3.2. Results of GBS growth characteristics.....................................................116
3.2.1. GBS growth in different media...............................................................116
3.2.1.1. GBS growth in pH 4.3, 5, 6 and 7 THB medium measured by microtitre
plate reader.....................................................................................................116
3.2.1.2. Quantification of GBS growth at different pH by measurement of colony
forming units....................................................................................................120
3.2.1.3. Comparing GBS growth in TH/YE and human serum........................120
3.2.1.4. GBS growth in pH 5 TH supplemented with 10% human serum........122
3.2.2. Long term survival of GBS....................................................................125
3.2.2.1. GBS survival in extended stationary phase measured by plate
counting...........................................................................................................125
3.2.2.2. GBS long term survival measured using „spot‟ plates........................127
3.2.3. Possible „viable but non-culturable‟ survival of GBS.............................127
3.2.3.1. GBS A909 percentage survival in stationary phase determined by Live-
Dead staining..................................................................................................127
3.2.3.2. Regrowth of extended stationary phase cells into broth.....................130
3.2.4. Acid adaptation......................................................................................134
3.2.4.1. Auto-acidification measurements.......................................................134
3.2.4.2. Acid adaptation experiments: a revised strategy for exposure to
acid..................................................................................................................137
3.2.5. Alternative media for GBS growth.........................................................138
3.2.5.1. GBS A909 growth on THA plates at different pH................................138
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3.2.5.2. GBS growth in vagina simulative medium.........................................141
3.2.5.3. GBS growth on M17 agar versus M17 with heme/MQ supplementation
plates...............................................................................................................141
3.2.6. Biofilm experiments...............................................................................143
3.2.6.1. Biofilm culture in extended stationary phase......................................143
3.2.6.2. Biofilm survival following „feeding‟ with nutrient broth........................145
3.2.6.3. Assay for improving biofilm formation by coating culture plates with
extracellular matrix molecules (hyaluronic acid and heparin)..........................146

3.3. Discussion of GBS growth characteristics................................................148
3.3.1. GBS long term survival in stationary phase..........................................148
3.3.2. Possible „viable but non-culturable‟ survival of GBS.............................150
3.3.3. Acid adaptation......................................................................................152
3.3.4. Biofilm formation contributes to GBS survival.......................................154

Chapter 4. Results and discussion of proteomic investigation of GBS
grown under conditions associated with neonatal exposure...................157

4.1. Background..............................................................................................157

4.2. Results.....................................................................................................158
4.2.1. An in vitro model of GBS growth under conditions associated with
neonatal exposure...........................................................................................158
4.2.2. 2D SDS-PAGE and gel analysis............................................................160
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