BLASTOCYSTIS: INVESTIGATION OF PARASITE ADHESION AND ITS ROLE IN MEDIATING ...

280 Pages·2014·6.35 MB·English

by  Wu Zhaona

BLASTOCYSTIS: INVESTIGATION OF PARASITE
ADHESION AND ITS ROLE IN MEDIATING
INTESTINAL BARRIER COMPROMISE

WU ZHAONA
(Bachelor of Sciences)

A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

DEPARTMENT OF MICROBIOLOGY
NATIONAL UNIVERSITY OF SINGAPORE
2014
“In quietness and trust is your strength.” Isaiah 30:15
DEDICATED WITH LOVE TO MY PARENTS &
FAMILY
Wu Zhaona
01.08.2014
ACKNOWLEDGEMENTS
Firstly, I would like to extend my deepest gratitude to my supervisor A/Prof
Kevin Tan, who has been highly supportive all along my PhD journey. He was
a strict professor, holding onto guidelines when training students or staff in the
way they should go. Sometimes I did not understand why he was strict, but
soon it turned out to be extremely beneficial to me as a research student being
trained. It would have been impossible for the completion of this project
without his patient, timely, and kind help and encouragements, as well as
wisdom which could not be understood in the beginning, but always speaks
for itself later on. He encourages us to pursue projects we are passionate about,
on condition that it comes with a good theoretical basis. It is such a blessing to
pursue my PhD with his guidance.
We are very blessed to have a very good lab environment to work in and enjoy.
All the kind help has been given selflessly to me from my loving labmates,
including past and present members of the Tan Lab. Specially, I would give
thanks to Junhong, a brother-in-Christ, who has helped with every step of
those experiments I used to have trouble with. His patient help has also been a
great support. Also, for Chuu Ling, for her love which is not often verbally
expressed but always abundantly given; Joshua, for his selfless support in
experimental materials and devices; Yan Quan, for sharing lots of insights on
projects in our discussions. My mentor, Haris, who is now at Yale University,
has always been a good model for me in doing research. His passion towards
research has been inspiring to me as a young research student. He also has
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shared a lot about his professional knowledge and insights in medicine which
lends the research work clinical relevance.
I would also like to thank A/Prof Lee Yuan Kun for his kind help with the
adhesion project, not only in knowledge input but also in advising on future
directions; Dr Zhang Yongliang, for his valuable suggestions on various
aspects of my project. His resourceful mind has provided new insights and
approaches to tackle difficult questions. I highly appreciate their time and help
given to me during the TAC meetings.
Many thanks be to Geok Choo, for her constant support and kindness which
has made me feel at home in a foreign land; Mr. Rama, for his “harsh” words
which urged me to have a good habit in experiment; Auntie Zina, for the so
much love from her in treating us like her own children. I would like to thank
Mdm Siti Masnor for her patient and professional administrative assistance. I
also want to thank all the lecturers of the Yong Loo Ling School of Medicine,
for making the four-year PhD study a truly memorable journey. Speical thanks
to Shuying and Wei an from Confocal Microscopy Unit, NUS, for their kind
and timely assistance in my numerous bioimaging experiments.
I would like to thank National University of Singapore for granting me the
scholarship to pursue this project.
Lastly, I extend my deepest gratitude to my dear Mommy and Daddy,
and my dear brother for their unconditional love and support.
Wu Zhaona
2014
ii
CONTENTS

ACKNOWLEDGEMENTS i
TABLE OF CONTENTS iii
SUMMARY ix
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF SYMBOLS xvii
LIST OF PUBLICATIONS xxii

CHAPTER 1: INTRODUCTION 1-34
1.1 Introduction
1.2 Classification
1.3 Cell biology
1.4 Life cycle
1.5 Clinical presentation
1.6 Laboratory diagnosis
1.7 Epidemiology and prevalence
1.8 High risk population
1.9 Treatment
1.10 Pathogenesis
1.11 Objectives of the present study

CHAPTER 2: ADHESION OF BLASTOCYSTIS TO 35-53
ENTEROCYTES AND DEVELOPMENT OF AN
ADHESION ASSAY
2.1 Introduction
2.2 Materials and methods
2.2.1 Culture of Caco-2 colonic epithelial cell line
iii
2.2.2 Parasite culture
2.2.3 Confocal microscopy
2.2.4 Scanning electron microscopy
2.2.5 Fluorescent labeling of live Blastocystis parasites
with CFSE
2.3 Results
2.3.1 Apical attachment of Blastocystis to epithelial cells
2.3.2 Determination of Blastocystis attachment to Caco-
2 cell monolayer by Scanning Electron Microscopy
2.3.3 Development of an adhesion assay with CFSE
staining for Blastocystis
2.4 Discussion

CHAPTER 3: FACTORS INVOLVED IN BLASTOCYSTIS 54-81
ADHESION TO ENTEROCYTES
3.1 Introduction
3.2 Materials and methods
3.2.1 Culture of Caco-2 colonic epithelial cell line
3.2.2 Parasite culture
3.2.3 Coincubation with different modulators and
attachment assay
3.2.4 Statistical analysis
3.3 Results
3.3.1 Time dependent variations in attachment
3.3.2 Dose response studies
3.3.3 Effects of temperature, rotational shaking,
fixation and trypsinization on Blastocystis
attachment
3.3.4 Effects of inhibition of cytoskeletal
components, parasite cysteine proteases and
lipid raft on attachment
3.3.5 Effects of saccharides on Blastocystis
adhesion to Caco-2 cells
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3.4 Discussion

CHAPTER 4: HOST RESPONSES PART I: EFFECTS OF 82-111
BLASTOCYSTIS ON APICAL JUNCTIONAL
COMPLEX AND EPITHELIAL BARRIER
FUNCTION
4.1 Introduction
4.2 Materials and methods
4.2.1 Culture of Caco-2 colonic epithelial cell line
4.2.2 Parasite culture
4.2.3 Epithelial permeability
4.2.4 Western blot and cellular fractionation
4.2.5 Confocal microscopy
4.3 Results
4.3.1 Blastocystis induced time- and dose-
dependent permeability increase in Caco-2
4.3.2 Effect of Blastocystis on the overall expression
of the apical junctional complexes
4.3.3 Effect of Blastocystis on the distribution of the
tight junction proteins by cellular fractionation
4.3.4 Effect of Blastocystis on the distribution of the
tight junction proteins
4.3.5 Loss of co-localization between tight junction
proteins and F-actin after exposure to
Blastocystis
4.3.6 Possible association of host tight junction
protein Claudin-1 with adhering Blastocystis
parasites
4.4 Discussion

CHAPTER 5: HOST RESPONSES PART II: ROLE OF 112-133
BLASTOCYSTIS-INDUCED HUMAN
ENTEROCYTE APOPTOSIS IN MEDIATING
BARRIER DYSFUNCTION
5.1 Introduction
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5.2 Materials and methods
5.2.1 Culture of Caco-2 colonic epithelial cell line
5.2.2 Parasite culture and lysate
5.2.3 Epithelial permeability
5.2.4 Flow cytometry
5.2.5 Confocal microscopy
5.2.6 Western blot
5.2.7 Statistical analysis
5.3 Results
5.3.1 ST-4 (WR-1) strain could not establish
enteroadhesion and cause disruption of tight
junction, in contrast to the enteroadhesive
strain of Blastocystis ST-7 (B)
5.3.2 Blatocystis ST-7 (B) induces characteristics of
apoptosis in Caco-2 cells; ST-4 (WR-1) does
not
5.3.3 Blastocystis ST-7 (B) induces increase in
permeability to FITC-conjugated Dextran; ST-
4 (WR-1) does not
5.3.4 Inhibition of caspase prevented Blastocystis
ST-7-induced ZO-1 rearrangement and
epithelial barrier dysfunction in Caco-2
5.4 Discussion

CHAPTER 6: STRAIN-DEPENDENT VARIATIONS IN 134-176
ADHESION AND ITS POSSIBLE ROLE IN
MEDIATING BARRIER DYSFUNCTION
6.1 Introduction
6.2 Materials and methods
6.2.1 Parasite culture
6.2.2 Culture of Caco-2 colonic epithelial cell line
6.2.3 Parasite viability assay
6.2.4 Growth curve
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