Investigating the Functional Role of Tick Antioxidants in Hematophagy and Vector Competence

130 Pages·2017·2.02 MB·English

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Dissertations
Fall 12-2016
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Deepak Kumar
University of Southern Mississippi
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INVESTIGATING THE FUNCTIONAL ROLE OF TICK ANTIOXIDANTS IN
HEMATOPHAGY AND VECTOR COMPETENCE
by

Deepak Kumar
A Dissertation
Submitted to the Graduate School
and the Department of Biological Sciences
at The University of Southern Mississippi
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
Approved:
_________________________________________
Dr. Shahid Karim, Committee Chair
Associate Professor, Biological Sciences
_________________________________________
Dr. Glen Shearer Jr, Committee Member
Professor, Biological Sciences
_________________________________________
Dr. Yanlin Guo, Committee Member
Professor, Biological Sciences
_________________________________________
Dr. Robert C Bateman Jr, Committee Member
Professor Emeritus, Chemistry and Biochemistry
_________________________________________
Dr. Faqing Huang, Committee Member
Professor, Chemistry and Biochemistry
_________________________________________
Dr. Karen S. Coats
Dean of the Graduate School
December 2016
COPYRIGHT BY
Deepak Kumar
2016

Published by the Graduate School
ABSTRACT
INVESTIGATING THE FUNCTIONAL ROLE OF TICK ANTIOXIDANTS IN
HEMATOPHAGY AND VECTOR COMPETENCE
by Deepak Kumar
December 2016
Ticks are obligate hematophagous arthropods and harbor several
pathogens which transmit various diseases to humans and their domesticated
animals. Host blood- digestion in a tick midgut (MG) generates several reactive
oxygen species (ROS), which are extremely toxic to essential macromolecules
(e.g. DNA, proteins, and lipids) within the cell, resulting in high oxidative stress.
Thus, this dissertation focuses on the questions of how tick homeostasis
responds to high oxidative stress, and how ticks and their harbored pathogens
survive the high surge of oxidative stress during blood digestion. We are
specifically interested in the tick-pathogen, Rickettsia parkeri (R. parkeri, Rp),
harbored by Gulf Coast ticks, which has medical and veterinary significance.
Recent literature has suggested the role of redox-switches in tick-pathogen
interaction (vector competence). However, the actual mechanism is not well
understood. The overall purpose of this study is to investigate the roles of tick
antioxidants in tick physiology and their role in facilitating R. parkeri survival
during blood feeding. Previous work from our lab has indicated that the
manipulation of antioxidants by R. parkeri is required for its survival. Reverse
genetics approach was used for the characterization of antioxidants, including
selenogenes (SelO and SelS), in uninfected and Rp-infected ticks. The tissue-
ii
specific roles of SelO and SelS were demonstrated in Rp colonization. SelO,
hypothetically a mitochondrial kinase, likely exploits its antioxidant property for
the colonization and survival of Rp in tick MGs. ER stress sensitivity of R. parkeri
and tick microbiota were also demonstrated in tick midgut. Prior to this work,
exogenous and endogenous oxidative stress were induced into separate groups
of ticks in order to investigate the response of tick antioxidant machinery as well
as the oxidative stress tolerance of ticks. The antioxidant genes, which
demonstrated a high shift in their expression, are currently being characterized
by our lab. Our results reveal the robustness of the tick antioxidant machinery by
exhibiting a high fold upregulation of the antioxidant genes, and as a result,
oxidative stress remains very close to control. As a part of this work, antioxidant
catalase in ticks is characterized and its role in tick reproductive fitness is
demonstrated.

iii
ACKNOWLEDGMENTS
I am extremely grateful to my advisor Dr. Shahid Karim for giving me an
opportunity to work with him, and always being willing to discuss science. I would
like to thank all the committee members (Dr. Robert Bateman, Dr. Glen Shearer,
Dr. Faqing Huang and Dr. Yanlin Guo) who agreed to serve despite of their busy
schedule. I would like to thank all the past and present lab members (Steve
Adamson, Rebecca Browning, Khemraj BC, Nabanita Mukherjee, Rebekah
Bullard, Jaclyn Williams, Gary Crispell and Nicholas Rinderer) for helping me in
making my research better. I would also like to thank all the undergraduates for
being supportive and helpful. I would also take this privilege to thank all the
supporting staffs from the Biological Sciences for answering all my questions
regarding rules and regulations of the department and USM. To all whom I have
mentioned above, and to all whom I couldn’t mention here but helped me in
some or other way, my heartfelt thanks to all of them for their contributions in
making my science better and helped me in becoming a better person.

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TABLE OF CONTENTS
ABSTRACT ...........................................................................................................ii
ACKNOWLEDGMENTS .......................................................................................iv
LIST OF TABLES ................................................................................................. x
LIST OF ILLUSTRATIONS ...................................................................................xi
CHAPTER I - STATE OF KNOWLEDGE ............................................................. 1
Importance of Tick Study ............................................................................... 1
Tick Biology ................................................................................................... 2
Biology of Tick Blood Feeding (Hematophagy) ............................................. 3
Biological Significance of ROS and Antioxidant Machinery ........................... 5
Superoxide Dismutase (SOD) .................................................................... 7
Catalase and its H2O2 Detoxification ........................................................ 7
Glutathione (GSH) and Glutathione Peroxidase (GPx) .............................. 9
Glutathione-S-Transferase (GST) .............................................................. 9
Selenoproteins ......................................................................................... 10
Hematophagy, ROS and Pathogen Transmission (Vector Competence) .... 12
Impact of Blood Digestion on Tick-Pathogen Transmission ........................ 16
Redox-Dependent Responses during Host-Pathogen Interaction ............... 17
Surge of Oxidative Stress when Rickettsia Interacts with Human
Endothelial Cells ...................................................................................... 18
v
Tick–Host–Pathogen Interactions ............................................................ 19
Gulf Coast Ticks .......................................................................................... 24
Life Cycle of Gulf Coast Ticks .................................................................. 25
Medical and Veterinary Significance of Gulf Coast Ticks ......................... 26
Effects of Spotted Fever Group Rickettsiae on Ticks ............................... 27
Rickettsia Parkeri ..................................................................................... 30
Impact of Selenoproteins selS and selO on R. Parkeri-Tick Interaction ... 31
Statement of Problem, Experimental Approach and Hypothesis ................. 34
Hypothesis1 ............................................................................................. 34
Experimental Approach............................................................................ 34
To Induce Optimum (sub-lethal) Oxidative Stress within the Gulf Coast
............................................................................................................. 34
Acquire a Gene Expression Profile of Tick Antioxidant Genes for Sub-
Lethal Oxidative Stress ........................................................................ 35
Select 5-10 Tick Antioxidant Genes, which Demonstrate the Highest
Upregulation at a Sub-Lethal Level of Oxidative Stress ....................... 35
Characterize those Selected Genes by Using the Strategy of Reverse
Genetics (Knocking Down Gene by using an RNA Interference and
Studying the Change in Phenotype). .................................................... 35
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How does R. Parkeri Colonize and Survive Inside the Gulf-Coast Ticks
Under the High Oxidative Stress (Blood Feeding Conditions)? ............... 36
CHAPTER II - ASSESSMENT OF TICK ANTIOXIDANT RESPONSES TO
EXOGENOUS OXIDATIVE STRESSORS AND INSIGHT INTO THE ROLE OF
CATALASE IN THE REPRODUCTIVE FITNESS OF THE GULF COAST TICK,
AMBLYOMMA MACULATUM ............................................................................. 38
Abstract ....................................................................................................... 38
Introduction .................................................................................................. 39
Materials and Methods ................................................................................ 42
Ticks ........................................................................................................ 42
Exogenous Induction of Oxidative Stress ................................................ 42
Endogenous Induction of Oxidative Stress through CAT RNA Interference
and Inhibition ........................................................................................... 43
Tick Tissue Dissection ............................................................................. 45
Quantification of Oxidative Stress ............................................................ 48
Statistical Analysis ................................................................................... 49
Results ........................................................................................................ 50
Bioinformatics Analysis ............................................................................ 50
Antioxidant Response to Exogenous Oxidative Stressors ....................... 52
Impact of CAT Knockdown ...................................................................... 56
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CAT Involvement in Tick Reproductive Fitness ....................................... 59
Discussion ............................................................................................... 60
Conclusions ............................................................................................. 64
CHAPTER III – INVESTIGATE THE ROLE OF SELO AND SELS IN TICK
PHYSIOLOGY AND VECTOR COMPETENCE IN GULF COAST TICKS
(AMBLYOMMA MACULATUM) .......................................................................... 66
Abstract ....................................................................................................... 66
Introduction .................................................................................................. 67
Materials and Methods ................................................................................ 70
Ethics Statement ...................................................................................... 70
Ticks and Other Animals .......................................................................... 70
Tick MG and SG Preparation ................................................................... 71
RNA Preparation, cDNA Synthesis and Quantitative Reverse
Transcriptase (qRT)-PCR ........................................................................ 71
Double-Stranded RNA (dsRNA) Synthesis, Tick Injections and
Hematophagy .......................................................................................... 72
Quantification of Total Bacterial Load ...................................................... 73
Quantification of R. Parkeri Copy Numbers in Tick Tissues..................... 74
Quantification of Oxidative Stress ............................................................ 74
Quantification of ER Stress ...................................................................... 74
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