AN ABSTRACT OF THE THESIS OF
Mohammad Farhad for the degree of Master of Science in Microbiology presented on
May 21, 2013.
Title: The Role of Salmonella typhimurium Type Three Secretion System (T3SS) and
Effector Proteins in Chromatophore Cell Response.
Abstract approved:
Janine E. Trempy
Bacterial contamination of food poses a great risk to human health worldwide. A
chromatophore cell-based biosensor, utilizing B. splendens erythrophore cells, is an
emerging technology that has shown potential to detect bacterial toxicity based on
function-dependent mechanisms. Previous studies have investigated the response of
erythrophore cells to foodborne pathogens, pesticides, and environmental toxicants.
Numerous studies reveal that the chromatophore cell type, erythrophore, can rapidly
detect, by aggregating their pigment organelles, several food associated bacterial
pathogens, including Salmonella typhimurium. This study describes the Betta splendens
erythrophore cells response to Salmonella typhimurium, including an analysis of the
bacterial mechanism(s) that may be involved in this response.
Previous studies using mutagenesis, identified genes of S. typhimurium that
contributed to a response in erythrophore cells. The results indicated a potential role for
the Type Three Secretion System (T3SS) apparatus coded for by the prgHIJK operon. In
the current study, a mutation was identified in a region different from the mutation that
disrupted the prgHIJK operon. However, both mutantions induced a similar but not
identical response in erythrophore cells.
The first objective of this study was to test the hypothesis that this second S.
typhimurium (ST10) mutant has a mutation residing in either the gene(s) coding for the
process regulating expression of the prgHIJIK operon or the gene(s) coding for the
effector proteins secreted by the PrgHIJK apparatus. The S. typhimurium (ST10) mutant
contained an insertional mutation in the hilD gene, a regulator involved in regulating the
expression of the prgHIJK operon. Failure to express hilD in S. typhimurium impacts
pigment organelle movement in erythrophore cells.
The second objective of this study was to test the hypothesis that the effector
proteins of T3SS (SipA, SopB, SopE2, SptP) are the components of S. typhimurium’s
invading mechanism directly involved in inducing pigment organelles to undergo an
aggregative response in erythrophore cells. Mutations in sopE2, sipA, or sptP did not
change the erythrophore cell response to S. typhimurium as the magnitude and kinetics of
the response was similar to that observed with wildtype S. typhimurium. A mutation in
the sopB gene that encodes the SopB effector protein, which is involved in actin filament
rearrangement, did change erythrophore cell response to S. typhimurium as the magnitude
and kinetics of the response was different to that observed for the wildtype S.
typhimurium. This is the first evidence in support of a direct role for the S. typhimurium
SopB effector protein in pigment organelle aggregation in erythrophore cells.
Results from this research effort suggest that hilD and sopB genes of S.
typhimurium are involved in pigment organelle movement in a neuron-like pigment cell,
an erythrophore cell of the chromatophore cell classification, isolated from the fish
species, Betta splendens. The outcome to this research effort is to increase understanding
of the biological basis of the erythrophore cell response to bacterial pathogens. Increased
understanding will lead to better utility of erythrophore cells as biosensors for bacterial
toxicity testing.
© Copyright by Mohammad Farhad
May 21, 2013
All Rights Reserved
The Role of Salmonella typhimurium Type Three Secretion System (T3SS) and Effector
Proteins in Chromatophore Cell Response.
by
Mohammad Farhad
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented May 21, 2013
Commencement June 2013
Master of Science thesis of Mohammad Farhad
presented on May 21, 2013
APPROVED:
Major Professor, representing Microbiology
Chair of the Department of Microbiology
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Mohammad Farhad, Author
ACKNOWLEDGMENTS
I would like to express my appreciation to my Major Professor, Dr. Janine
Trempy, for her commitment, support and guidance in overcoming obstacles throughout
my undergraduate and graduate academic career. Her contribution was not limited to my
education. She made sure I preserved my culture, kept my long-term goals in sight and
persevered through difficulties in my personal life. Working in her laboratory has been a
life changing experience. I would also like to thank my committee members Dr. Bruce
Geller, Dr. Martin Schuster and Dr. Jerry Heidel.
It was an honor to work with Dr. Karen P. Dierksen. I have been lucky to benefit
from her vast scientific knowledge and technical expertise. She was my primary source
for overcoming experimental and scientific complications in the laboratory. She is kind
and has incredible amount of patience. Her guidance and help was vital in finishing my
graduate studies.
I would like to thank all the Trempy lab members, especially Roberto Garcia and
Holly Roach, with whom I had the honor to work side by side and for their friendship and
support. I also would like to thank a previous graduate student in Dr. Trempy’s research
program, Dr. Stephanie Dukovcic, whose prior research efforts in this laboratory helped
me form new hypotheses, design experiments and interpret results.
I would like to thank the Department of Microbiology and Dr. Janine Trempy for
the financial support in completing my degree. I would like to thank Dr. Garry L. Adams
in the College of Veterinary Medicine at Texas A&M University for providing me with
Salmonella typhimurium effector protein mutant strains.
I would like to thank the people who I love and cherish since I have come to the
United States in pursuit of my education. I would like to say thank you to Mike Manaton
for his unconditional love, support and encouragement. He has been an inspiration for
me. I would not have made it to the United State if it was not for him. I would also like to
thank my best friend, Jolie Drion for her love and support in times of stress and getting
homesick.
Finally, I would like to thank my family: my mother, my father, my sisters and
brothers. I have been blessed to be part of a family so loving, strong, and wise. Thank
you for believing in me. I would never forget your sacrifice. Mom and Dad: thank you for
giving me the permission to come to the United State to study; I can not imagine how
much pain and worry you have endured because of me being away from home. Sisters:
thank you for being the best sisters in the world. Brothers: you are my heroes. You guys
have provided me with everything to ease my journey of getting my education. Words
cannot express my gratitude for all my family has done for me.
CONTRIBUTION OF AUTHORS
Dr. Janine E. Trempy and Dr. Karen P. Dierksen collaborated in experimental
design, interpretation of results and editing of this manuscript. Dr. Karen P. Dierksen
assisted in the laboratory preparation for experiments. Dr. Stephanie Dukovcic created
the Salmonella typhimurium mutants. Holly Roach assisted in fish care as well as
preparation and maintenance of Betta splendens chromatophore cell tissue cultures.
Roberto Garcia assisted in general laboratory procedures necessary for this research
effort. All the experiments in this study were conducted in Dr. Janine E. Trempy’s
research program.
TABLE OF CONTENTS
Page
1. Introduction……………..…........................................................................................ 1
2. Literature Review…..................................................................................................... 3
Part 1: Food Associated Illnesses and Salmonella enterica................................. 3
Foodborne Illness…………….……………................................ 3
Salmonella Associated Foodborne Illnesses……….................... 4
Salmonella enterica……........................…………………….…. 5
Salmonella enterica servovar Typhimurium Pathogenesis…….. 7
Salmonellla Pathogenicity Islands………………………....…....10
Adhesion…………………………………………………….…..12
Invasion...………………………………………………………..13
SCV Maturation………………………………………………....15
Gram-negative Secretion system ……………………………….15
Type III Secretion System………………………………………17
Salmonella T3SS-1 Apparatus…………………………………..18
SPI-1 T3SS Secreted Effector proteins………………………….19
Part 2: Dectection Metohds and Chromatophore Cell-based Biosensors……….22
Detection Methods for Bacterial Pathogesn……………….….…22
Immunology-based Methods……………………………..……...23
Polymerase Chain Reaction-based Methods………………..…...24
Biosensors…………………………………………………..…...25
Chromatophore Cell-based Biosensor……………………….….26
TABLE OF CONTENTS (Continued)
Page
Chromatophore Cells and Pigmentation……………………….. 27
Betta splendens………………………………………………….30
Research Objectives……………………………………………………………..33
3. Materials and Methods…………..…………………………………………………....35
Fish Care…………………………………………………………….…………..35
Cell Culture…………………………………………………………….………..35
Response Assay and Computational Analysis…………………………………..38
Bacterial Growth Conditions…………………………………………………....39
Polymerase Chain Reaction (PCR).……………………………………………..41
Bacterial Transformations……………………………………………………….42
Identifying Transposome™ Insertion Site(s) and Sequencing…………….……46
Complementation…………………………………………………………….….46
4. Results..……………………………………………………………..............….……..49
The Physiological Response of Betta splendens Erythrophore Cells...................49
Betta Splendens Erythrophore Cell Response To Wildtype
Salmonella Typhimurium And Salmonella Typhimurium
ST10 Mutant.............................................................................................52
Identification Of hilD Gene Product And Complementation
of hild Function........................................................................................55
Erythrophore Cell Response To Salmonella Typhimurium Wildtype,
ST10 Mutant, ST10-pTA108 And ST10-pTA108-hilD............................58
TABLE OF CONTENTS (Continued)
Page
Betta Splendens Erythrophore Cell Response To
Salmonella typhimurium Human Isolate And
Salmonella typhimurium Bovine Isolate...................................................62
Betta Splendens Erythrophore Cell Response To
Salmonella typhimurium With Mutations In Effector
Protein SopE2, SipA, Or SptP Genes........................................................64
Betta Splendens Erythrophore Cell Response To
Salmonella typhimurium With Mutations In
Effector Protein SopB Or SipB/SopB Genes.............................................66
5. Discussion…………………………………………………………….……...............68
The Response Betta Splendens Erythrophore Cells To
Salmonella typhimurium ST10 Mutant......................................................68
Salmonella typhimurium ST10 Mutant Complementation....................................73
Betta Splendens Erythrophore Cells Response To
Salmonella typhimurium Effector Protein Mutants...................................75
Future Directions For This Research Effort...........................................................82
6. Conclusion.……………………………………………………….............…….……..83
7. Bibliography…………..………………………………………….............…….……..86
Appendix…………..………………………….…………………….............…….…….109
Biographical Information…………..……………………….............…….…….110
LIST OF FIGURES
Figure
Page
1. Betta splendens erythrophore cell response to
Clonidine, MSH, and LB…...………………………………………………...….51
2. Betta splendens erythrophore cell response to
wildtype Salmonella typhimurium and Salmonella
typhimurium ST10 mutant. …………..….……………………..…….………… 54
3. Identification of the hilD gene……….………….………………………….……..…. 56
4. Cloning complementation of hilD gene ………………………………...….…….….. 57
5. Betta splendens erythrophore cell response to wildtype
Salmonella typhimurium, Salmonella typhimurium
ST10 mutant, ST10 containing pTA108, and ST10
containing pTA108-hilD (60 minutes) ……….……………………....……..….. 59
6. Betta splendens erythrophore cell response to wildtype
Salmonella typhimurium, Salmonella typhimurium
ST10 mutant, ST10 containing pTA108, and ST10
containing pTA108-hilD (90 minutes) ……….………………..…….……..….. 61
7. Betta splendens Chromatophore Cell Response to
Salmonella typhimurium wildtype (human isolate) and
Salmonella typhimurium wildtype (bovine isolate)..……………….……….….. 63
LIST OF FIGURES (Continued)
Figure
Page
8. Betta splendens erythrophore cell response to wildtype
Salmonella typhimurium bovine isolate and specific
effector protein mutants with mutations in sopE2, sipA, or sptP.….……….….. 65
9. Betta splendens erythrophore cell response to wildtype
Salmonella typhimurium bovine isolate and specific effector
protein mutants with mutations in sopB and
sipA/sopB…...........................................................................................................67
10. Salmonella Pathogenicity Island 1 (SPI-1) Genetic Organization...............................70
11. A model of transcriptional regulation of SPI-1............................................................72
12. A hypothetical model as to how S. typhimurium might enter an
erythrophore cell....................................................................................................81
LIST OF TABLES
Table
Page
1. Bacterial Strains and Plasmids used in this study…………………………….………41
2. Primers used in this study……...……………….………………………….…………43
For my Family
Chapter 1
Introduction
Bacterial and environmental toxicant contamination of food has been, and
continues to be a threat to human health worldwide. Developing rapid and accurate
methods to detect bacterial and viral pathogens in contaminated food is important for
human health. In recent years, cell based biosensors, living cells that detect the presence
of biological and chemical toxicants, have become the main focus of researchers (19,
192). The chromatophore cell-based biosensor is an emerging biosensor that has shown
the potential to detect contaminants based on function-dependent mechanisms (202, 204,
207, 208, 231, 237, 238). Function-dependent mechanisms give the chromatophore cellbased biosensor the ability to respond to biological (a viable pathogen or associated
toxins) and environmental stimulants (heavy metals, chemical) in a physiological manner
that can be measured. Thus, this type biosensor overcomes the limitation of other
molecular and immunology based methods.
Chromatophore cell-based biosensors utilize a class of pigmented neuron-like
cells called chromatophores. These cells are capable of moving their intercellular pigment
organelles upon exposure to physiological stimuli. This property has made
chromatophore cells valuable for their use in a detection system solely based on
biological activity. Induction of pigment granule movement in chromatophore cells due
to disrupted intracellular functions in response to different biological stimuli is measured
and analyzed to assess the toxicity of a pathogen, whereas other detection methods can
2 only assess the presence but not toxicity of a pathogen (207, 208, 209). These pigment
movements could be toward the peri-nucleus of the cell called aggregation or outward to
the periphery of the cell called dispersion (208, 209). The degree, magnitude, and kinetics
of chromatophore cell responses (aggregation and dispersion) vary depending on the
different pathogens and toxic stimuli (208, 209). The similarity of chromatophore cells to
neuron cells of other animal makes them of interest in detecting biological substances of
concern to human health (210, 209).
Betta splendens erythrophore cells have been used as cell-based biosensors for the
detection of chemicals, bacterial exotoxins, pesticides, Gram-positive foodborne bacteria
and more recently, Gram-negative foodborne bacteria (204, 207, 209, 231). More studies
are essential to better understand the mechanism by which the Gram-negative bacterium,
Salamonella typhimurium, triggers the response in B. splendens erythrophore cells. The
objective of this study is to further investigate and increase our knowledge of B.
splendens erythorophores for the detection of S. typhimurium.
3 Chapter 2
Literature Review
The literature review chapter consists of two parts. The first part covers a brief
introduction to food associated illness, and a much greater emphasis on the current
understanding of Salmonella typhimurium pathogenesis. The second part briefly
discusses different bacterial detection methods and focuses on the chromatophore cellbased biosensor.
Part1: Food Associated Illnesses and Salmonella enterica
Foodborne Illness
Although detection technology has advanced, food associated illness caused by
bacterial and viral contamination of food poses a significant global health concern. Each
year in the United States, 5000 people die, 128,000 are hospitalized and 48 million report
illness as a result of foodborne illness (232). The reported numbers of illnesses
correspond to 17% of the U.S. population, which is remarkable considering the U.S. food
supply is considered one of the safest in the world. The World Health Organization
recently put forth an initiative to estimate the global burden of foodborne diseases (233).
Not only is it difficult to estimate the global burden of foodborne diseases, it is also
difficult to estimate the global incidence of foodborne diseases. Globally, diarrheal
diseases are the leading cause of mortality and morbidity in children, with bacterial
contaminated food and water being one of the major contributors to global diarrhoeal
4 diseases (234). The bacterial species of prime concern include Salmonella sp., Bacillus
cereus, Campylobacter jejuni, Escherichia coli-producing toxin (including 0157:H7),
Listeria monocytogenes, Clostridium perfringens, Shigella sp., Staphylococcus aureus,
Vibrio parahaemolyticus, and Vibrio vulnificus (232).
Salmonella Associated Foodborne Illnesses
Salmonella enterica subspecies serovars are estimated to cause 1.3 billion cases of
illness worldwide including 3 million deaths due to non-typhoidal Salmonella annually.
Intestinal disease due to non-typhoidal Salmonella species is highest in third world
countries but non-typhoidal Salmonella is also of concern in developing countries (1).
Salmonella enterica subspecies are typically orally acquired pathogens that result in
typhoid fever, diarrhea, bacteremia and chronic asymptomatic carriage (2). In the United
States, Salmonella enterica causes about 1.4 million cases of salmonellosis, 19,586
hospitalizations, 378 deaths among humans and an economic loss of around $3.3 billion
annually (3, 239, 240).
Ingestion of more than 5x104 Salmonella in contaminated water or food will
cause salmonellosis. Acute, crampy, abdominal pain and diarrhea with or without blood
are salmonellosis symptoms that occur between 6 and 72 hours after ingestion. Other
common symptoms are fever, nausea, and vomiting. Salmonellosis usually occurs in the
ileum of the small intestine but it also causes inflammation in the large bowel, with rare
infections in the jejunum, duodenum and stomach (7, 8).
Salmonella is a major concern for the food industry. Salmonella is found in
5 poultry, uncooked meat, fish, shellfish and natural environments. Eradicating Salmonella
from food processing environments and products has been a challenging task (10). Cattle
infected with Salmonella can excrete up to 108 CFU/g of feces, which becomes the main
source of further contamination of the environment and other uninfected animals. Cattle
that are active carriers of Salmonella do not exhibit clinical symptoms, however they are
able to excrete Salmonella in their feces. Contaminated feces is a problem in food
production especially when cattle feces is recycled as manure or is not properly disposed
(11). Studies have shown that swine shed S. typhimurium through their feces for up to 28
weeks after initial infection (12).
Fresh fruits and vegetables contaminated with Salmonella have been associated
with human salmonellosis. Salmonella, mostly from untreated irrigation waters, can
contaminate fruits and vegetables through roots, stem scars, and leaves (14, 15). Pre- and
post- processing food facilities may become contaminated with Salmonella through all
these original sources of Salmonella such as poultry, cattle, fresh fruits and vegetables.
Peanut butter, mangoes, cantaloupe, ground beef, dry dog food, and raw scrap ground
tuna product are some of the most recent contaminated sources of Salmonella that have
been associated with salmonellosis outbreaks in humans in the United States (13).
Salmonella enterica
Salmonella is a Gram-negative, non-spore forming, facultative intracellular
anaerobe that belongs to the family of Enterobacteriaceae. Salmonella is a
chemoorganotrophic organism; it is capable of degrading organic material and
6 compounds for energy. It is oxidase negative, catalase positive and produces hydrogen
sulfide. Salmonella can decarboxylate lysine and ornithine and uses citrate as its sole
carbon source (10). Salmonella is capable of growing in extreme and harsh environments
due to its exceptional tolerance and adaptive characteristics. Salmonella is classified as a
mesophile, optimally reproducing in a temperature range of 30 to 40o C. However,
Salmonella can also reproduce in foods stored at temperatures of 2-4o C, which then
classifies Salmonella as a psychrophile. Salmonella can survive the acidic environment of
the stomach due its wide range tolerance for pH (4.6-9.5), and is thus able to invade the
small intestine. It has a tolerance for salt concentrations of 3-4%; salt tolerance increases
with increasing temperature (10).
To overcome challenges of different environmental conditions, Salmonella
detects changes in its environment and responds by orchestrated gene expression to adapt
and survive. Altering the gene expression profile allows for adaptation to environmental
stresses such as nutrients depletion, pH and temperature extremes, while virulence gene
expression enables Salmonella to acquire resistance, evade, and systematically confront
the elements of innate immunity of the host (16).
Leon Le Minor and Michel Popoff contributed to the current understanding of the
two species of Salmonella genus, Salmonella enterica and Salmonella bongori (9).
Salmonella
species
are
divided
into
subspecies,
which
are
divided
into
serotypes/serovars; so far 2500 serotypes of S. enterica have been identified (1, 2).
Serovars are conventionally distinguished through a species-specific antigenic scheme
7 classification based on somatic (O), flagellar (H), Capsular (K) antigens and virulence
(Vi). In recent years, World Health Organization Center for Reference and Research on
Salmonella has developed serotype-specific 16S rRNA and other gene sequence
comparison schemes for Salmonella typing (4).
The study of Salmonella intestinal infection had been restricted to studies using
bovine ileal loop inoculations, intestinal epithelial cell cultures and oral infections, until a
murine model of Salmonella enteropathogenesis was developed (5). The murine model
made it possible to better understand the mode of Salmonella pathogenesis and different
stages of salmonellosis. These studies revealed that virulent S. enterica localizes to the
apical epithelium, triggers invasion and associated virulence gene expression to elicit
significant inflammatory changes leading to local and diffuse polymorphonuclear
leukocytes infiltrate, crypt abscesses, epithelial necrosis, edema and fluid secretion (1, 6).
Salmonella enterica servovar Typhimurium Pathogenesis
Salmonella enterica servovar Typhimurium infection starts with ingesting
contaminated food or water. Salmonella typhimurium crosses the host’s digestive tract,
and enters the stomach. To survive the low pH of the stomach, S. typhimurium expresses
acid shock and outer membrane proteins through acid tolerance response (ATR). In
addition to low pH, nutrient deprivation sigma factor (δs) can also induce the ATR
response. After reaching the lumen of the small intestine, it expresses the virulence
factors that grant S. typhimurium protection from antimicrobial peptides, bile salts,
digestive enzymes, secretory IgA, and the innate host immune system, facilitating an
8 opportunity to transverse the intestinal mucus layer, and invade intestinal epithelial cells
(22). Salmonella typhimurium expresses and utilizes fimbriae that give the bacterium the
ability to adhere to intestinal epithelial cells (23). Further progression of infection in the
specific host cell has staggering impact depending on which uptake mechanism, active or
passive, is utilized (26).
The following provides an overview of S. typhimurium pathogenesis. Each phase
of S. typhimurium pathogenesis will be discussed in detail later in the literature review.
Salmonella typhimurium is capable of infecting a wide range of cell types such as
dendritic cells, phagocytes, and non-phagocytic epithelial cells of the gut. It can enter
host epithelial cells in two ways: active or passive (25, 26). The active or “trigger”
mechanism initiates rearrangement and ruffling of the host cell membrane by injection of
virulence proteins into the host cell using Type III Secretion System which results in the
internalization of the bacteria (21, 241). Salmonella has been viewed as prototype for
pathogens using the “trigger” mechanism (18, 19, 20, 24). On the other hand, one or more
bacterial surface proteins bind to one or more specific host cell receptors to induce the
passive or “zipper” mechanism, consequently, initiating the internalization of the bacteria
(241). Listeria monocytogenes has been viewed as prototype for pathogens using “zipper”
mechanism (241). The “trigger” and “zipper” mechanisms phenotypically differ from
each other. In addition to trigger and zipper mechanisms, host immune cells that have
phagocytic characteristics can internalize Salmonella typhimurium.
Upon adhesion (preferably) to microfold cells (M cells) in the intestinal
9 epithelium, S. typhimurium stimulates the inflammatory response, induces secretion of
chemokine interleukin-8 (IL-8) and activates polymorphonuclear leukocytes (PMN) to
move across the intestinal epithelial cell layer (27, 28, 31). M cells lack IgA, which
becomes a bridge for Salmonella to get to underling tissues. Salmonella enters M cells
using “trigger” mechanism by deploying SPI-1 T3SS that injects the effector proteins
involved in rearranging the actin cytoskeleton (33, 34). M cells are specialized cells in the
intestinal epithelium, important in inducing mucosal immunity through sampling of
antigens using a pinocytosis mechanism. Using pinocytosis, M cells engulf S.
typhimurium and deliver them to lymphoid cells in the Peyer’s patches beneath the
epithelium (32).
These events result in bacterial internalization and formation of
Salmonella-containing vacuole (SCV), which acts as a protective niche for S.
typhimurium survival and replication (29). Various markers on the vacuole are
responsible for different phases of SCV maturation (30).
The first phase of SCV maturation, biogenesis, occurs in less than 30 minutes and
Salmonella controls most of the different markers on the vacuole (30). SCV moves
through the cell, staying close to the Golgi in the perinuclear region of the host cell for 30
minute to 5 hours post infection. The final phase of SCV maturation is indicated by the
formation of Salmonella-induced filaments (SIFs) (35). SIFs are long membranous
tubules that diffuse away from SCV approximately 8 hours post infection. The tubular
structures are enriched with lysosome-associated membrane protein 1 (LAMP1) and
other markers which facilitates SCV fusion with endocytic vesicles, which act as nutrient
sources vital to Salmonella intracellular replication (36, 37). During the phases of SCV
10 biogenesis, the SCV membrane is loaded with cholesterol, which is an important
component required for Salmonella replication; inhibiting cholesterol production will
stop bacterial growth (38).
Another important component of Salmonella virulence is SPI-2 T3SS required for
S. typhimurium intracellular survival. SPI-2 T3SS injects effector proteins across the
SCV into the host cell cytoplasm, which prevents lysosomal fusion to SCV (39, 40).
Salmonella pathogenesis is the coordinated work of greater than 30 effector proteins
secreted by the T3SS-1 and T3SS-2 apparatus that reside on Salmonella pathogenicity
island- (SPI-1) and Salmonella pathogenicity island- (SPI-2), respectively (18, 41).
Salmonellla Pathogenicity Islands
Pathogenicity islands are clusters of virulence genes in a confined region of the
chromosome. The following three characteristics of pathogenicity islands suggest that
Salmonella pathogenicity islands genes are acquired through horizontal gene transfer: 1)
pathogenicity islands have different GC composition than the rest of the chromosome, 2)
pathogenicity islands contain genes that are closely related and they are not found in nonpathogenic bacteria, and 3) transposon insertions are often localized around the
pathogenicity island (42).
Salmonella pathogenicity islands are essential for invading the host cell,
intracellular survival, and pathogenesis. According to Hensel in a 2004 review article, 12
SPIs (SPI-1 – SPI-10, SGI-1 and HPI) have been described and most of them share
11 common motifs; however, size, structure and function of these SPI and their distribution
in Salmonella subspecies and serovars varies significantly (43). SPI 7, 8, 9 or 10 are not
present in S. typhimurium (43, 53). Another review by Fabian et al. in 2012 presents the
conclusion that 21 SPIs (SPI-1 – SPI-21) have been identified in Salmonella and only
SPI-1 to 6, 9, 11, 12, 13, 14, and 16 are present in S. typhimurium (61). Even though there
is a difference in nomenclature of the SPIs in the two studies, they both agree that SPI-1 –
SPI-6 are significant contributors in S. typhimurium pathogenesis. Only SPI-1 and 2 are
relevant to this research and they will be described in detail.
Salmonella pathogenicity island 1 is responsible for encoding a T3SS, which is an
apparatus for injecting effector proteins into the host cell (43, 34). SPI-1 is 40 kb in size
and is mostly conserved among all Salmonella species. Some of the SPI-1 effector
proteins are involved in the invasion of non-phagocytic cells by Salmonella, through
rearranging the host cell’s actin cytoskeleton. Other SPI-1 effector proteins are involved
in eliciting inflammation of the intestinal epithelium, diarrheal symptoms and other
entero-pathogenesis processes (44).
Salmonella pathogenicity island 2 is 40 kb in size, composed of two distinct
portions: a 25 kb portion that is exclusive to S. enterica and a 15 kb portion that is
observed in S. bongori and S. enterica (45). The first portion has a G+C content of 43%.
It plays a crucial role in systemic pathogenesis and encodes the T3SS-2 in S. enterica that
is induced during intracellular survival and replication. The second portion has a G+C
content of 54% encodes tetrathionate reductase (Ttr) used in anaerobic respiration (46).
12 Any bacterial products that are involved directly or indirectly in Salmonella
pathogenesis are called Salmonella virulence factors. Adhesion, invasion, capsule
formation, immune evasion strategies, and regulation of virulence gene expression are
some of the mechanisms that are associated with Salmonella virulence factors. Although
exhaustive investigations have been done on Salmonella virulence factors, this study will
only review virulence factors that are direct contributors to successful Salmonella
pathogenesis.
Adhesion
The presence of S. typhimurium in the mucosal cells and Peyer’s patches
stimulates the signaling pathways of host cells, resulting in secretion of inflammatory
cytokines, which act like physical barriers. Various adhesion factors of S. typhimurium
such as fimbria, flagella, and lipopolysaccharide (LPS) interact with host-receptors (62,
63).
Salmonella typhimurium uses 13 different fimbriae and 3 nonfimbrial adhesins for
colonization, and their expression are controlled according to time and space (64, 61).
Fimbriae (or pili) are inflexible, straight filamentous proteins, anchored on the outer
membrane surface, and are recognized by the host cell receptor for initiating cell-to-cell
contact, which results in signal transduction. These proteins are assembled through the
Chaperone usher (CU) pathway. Non-fimbrial adhesins are mono- or oligomeric proteins
on the outer membrane surface that function in the same general way as fimbriae
adhesins, however, they are not assembled through the CU pathway (65).
13 Salmonella typhimurium also possess flagella that confer a complete adhesion of
S. typhimurium to M cells. The propulsion and motility of S. typhimurium is due the
chemotactic responses of flagella (73, 74). This flagella structure is assembled by
proteins encoded by 30 genes that are controlled by three classes of promoters (75).
A hydrophobic lipid A, a short non-repeating core oligosaccharide, and a distal
polysaccharide (O-antigen) are the three main domains of LPS in S. typhimurium (66).
The longer chain forms of O-antigen in LPS contribute to S. typhimurium resistance to
neutrophils, phage lysis, and other host responses, adaptation and intestinal colonization
(67-72).
Six of the twelve SPIs present in S. typhimurium are involved in bacterial
adhesion to the host cell prior to the onset of the disease (61).
Invasion
Following adhesion by S. typhimurium, invasion of the host begins. Invasion is
encoded and controlled by SPI-1 that induces rearrangement and ruffling of the host cell
membrane to internalize the bacteria (76). Salmonella typhimurium uses about 29 genes
of SPI-1 to encode virulence factors to initiate invasion. These genes code for: a)
avirulence factor A (Avr A), Salmonella secreted protein (Ssp) SipA, SipB, SipC and
tyrosine phosphatase protein (SptP); b) SPI-1 genes expression regulator, HilA; c) Inv,
Spa, and Prg and other T3SS proteins; d) the chaperone proteins important for injecting
effector proteins into the host cell (77-79). SipA and SipC proteins are crucial to
remodeling and rearrangement of host cytoskeleton to facilitate bacterial entry, which
14 triggers signal transduction to mobilize the polymorphonuclear leukocyte (PMN) through
the intestinal epithelial layer (80, 81). SipB works as an effector protein that binds to
cholesterol, crucial to formation of the translocon for translocating other SPI-1 effector
proteins (82). SopE is responsible for activating the Cdc42 and Rac small G-proteins
inside the host cell resulting in actin rearrangement and induction of inflammatory
cytokines (78, 83-85).
Salmonella pathogenicity island 1 is activated and controlled by HilA, a locus that
resides within SPI-1 (86). HilA belongs to the OmpR/ToxR transcriptional activators
family and binds to prg, inv/spa, and sip operon promoters (87). In response to an
unsuitable intestinal lumen environment, hilA expression is induced through a BarA/SirA
two component system (TCS). HilA expression is positively controlled by HilD, HilC
and the RstA/RstB TCS (87). HilA is also responsible for inducing T3SS-1 expression
and expression of encoded effector proteins (88).
To insure its survival inside the host cell, S. typhimirum takes steps to keep the
host cell alive. After S. typhimurium is internalized, the SptP-mediated process reverses
the SopE related activity to repair the host cytoskeleton (78). HilA expression is blocked
through PhoP/PhoQ system due to the low Mg2+ concentration level inside the vacuole
(SCV) (89). In addition, RcsCDB phosphorelay system is also known to be responsible
for repression of hilA, invF and invG SPI-1 gene transcription (90). Along with inhibiting
SPI-1 gene expression and their activity through RcsB and the PhoP mechanism, the
PhoP activates SPI-2 genes expression for commencing the next stage of Salmonella
15 systemic infection (87).
SCV Maturation
SopE and SopB (T3SS-1 effector proteins) and SpiC (T3SS-2 effector protein) are
used as markers to prevent SCV fusion with endosomes or lysosomes, allowing SCV to
go through the maturation phase (91, 92). SsrA/SsrB TCS (Ssr regulatory operon),
T3SS/SPI-2 (Ssa operon), the secretion system chaperones (Ssc operon) and the secretion
system effectors (Sse operon) are some of the 40 genes that reside and are encoded by
SPI-2 in S. typhimurium (93,77, 94). The signal that activates SsrA/SsrB TCS to regulate
sse, ssa, and ssc operons is unknown, however, it is well known that SsrA/SsrB TCS is
regulated by pH and osmolarity change in OmpR/EnvZ-dependent pathway (95-97).
The Salmonella-containing vacuole moves to the peri-nuclear region in close
proximity of Golgi. This movement gives SCV the opportunity to obtain nutrients using
SPI-2 effectors, SifA, SseG and SseF, from endocytic/exocytic transport vesicles (98).
Following the localization of SCV and nutrient acquisitions, S. typhimurium replication
commences (61).
Gram-Negative Secretion Systems
Gram-negative bacteria have evolved numerous secretions systems for
transporting proteins to the outer membrane surface, extracellular space, or into the host
cytoplasm (99). Some of these secretion pathways are complex and involve
macromolecular structures to overcome the limitations of having an outer membrane. So
16 far, eight different secretion systems have been discovered and are classified in two
categories: a) sec-dependent and b) sec-independent systems.
The type II secretion system (T2SS), type V secretion system (T5SS),
chaperone/usher system (CU), and the extracellular nucleation-precipitation system
(ENP) are classified as Sec-dependent secretion systems. Type II secretion system
secretes hydrolytic enzymes, toxins and type IV pili to the outer membrane (100-102). In
T5SS, substrates facilitate their own secretion into the outer membrane after being
targeted by inner transmembrane protein complexes (103, 104). The CU system, beststudied system in E. coli, uses an assembly foundation on the outer membrane that is
created by a periplasmic chaperone, FimC and an usher, FimD (105, 106). In E. coli,
ENP system is essential in secretion of curli, adhesive fibers, to the outer membrane.
ENP is also important for the formation of biofilm (107, 102).
Type I secretion system (T1SS), type III secretion system (T3SS), type IV
secretion system (T4SS) and the twin-arginine secretion system (Tat) are classified as
sec-independent secretion systems. Type I secretion system secretes proteins with high
molecular weight that possess a C-terminal signal sequence directing them outside of the
cell. The transport of these proteins occurs without the cleavage of the C-terminal
sequence and it is best described for the exotoxin, α-hemolysin (HlyA), in pathogenic
Echerichia coli (108). In contrast, Tat secretory system transports folded proteins with an
N-terminal peptide signal composed of two arginines through an active transport system,
proton-motive force (109). Type IV secretion systems are secretory systems that can
17 transport single-stranded-DNA-protein complexes in addition to proteins in two different
ways: cell contact-dependent or cell contact-independent (110-112). Since T3SS is part of
this study and it is induced in Salmonella sp. pathogenesis, it will be discussed in more
detail.
Type III secretion system
Type III secretion systems are evolved specialized virulence devices that directly
inject bacterial virulence proteins/effector proteins into the host cytoplasm in order to
control and modify host cell functions (113). Delivery of these effector proteins into the
host cell cytoplasm to alter host-cell cytoskeleton, membrane trafficking, cytokine gene
expression, and signal transduction is a complex task that is carried out by a needle-like
apparatus that projects outward from the bacterial outer membrane (114). This apparatus
is comprised of 20 proteins divided into substructures consisting of: base, inner rod,
needle and translocon. Base is a multi-ring structure that spans the inner and outer
bacterial membranes. In addition, base functions as an anchor for the inner rod and
hollow needle structure. The inner and outer membrane structures of the needle-like
apparatus are evolutionarily related to the bacterial flagellar secretion apparatus and PulD
family of type II secretion system, respectively (114-116). T3SS secretes translocon
proteins that are embedded in the host cell membrane. Translocon proteins assemble a
pore, and act as a needle apparatus target (117). The structures of the T3SS are highly
conserved among different species of Salmonella, however, the effector proteins secreted
by the T3SS varies across Salmonella species (118). The effector proteins are encoded
18 by sets of genes that reside on the same pathogenicity island (SPI-1) encoding the T3SS
or somewhere else in the chromosome (113).
Salmonella T3SS-1 Apparatus
The base structure of Salmonella is composed of equal molar amounts of InvG,
PrgH and PrgK, which are sec-dependent proteins. InvG is inserted into S. typhimurium
outer most membrane. PrgH and PrgK linked to each other form a structure that is
inserted into the inner most cytoplasmic membrane of S. typhimurium. PrgJ and PrgI
proteins form the inner rod and hollow needle structures, respectively. Numerous other
proteins such as InvC, InvA, SpaP, SpaQ, SpaR and SpaS lie within the PrgH/PrgK inner
member ring that make up the effector protein export machinery. InvC is an ATPase that
ensures an efficient translocation of the PrgJ and PrgI proteins by recognizing and
unfolding them to form the inner rod and the hollow needle, respectively. Several
chaperones and stabilizing proteins play critical roles in the formation of the apparatus
but they are not part of the fully assembled apparatus. InvJ is not a permanent part of
T3SS apparatus but it ensures the proper formation of the needle complex (119, 120).
Another non-permanent T3SS related lipoprotein, InvH, translocates the InvG protein to
assemble the outer most membrane ring in the base structure (121, 122). Type III
secretion system uses a “substrate switching” mechanism that specifies what kinds of
proteins are allowed to be delivered through the needle. The substrate switching is
possible because InvJ plays a critical role in causing conformational changes in the
secretion apparatus that allows PrgJ and PrgI to pass through the needle and block other
19 effector proteins (123). Other chaperones guide effector proteins to the T3SS apparatus.
For example InvB escorts SipA, SopE, and SopE2 effector proteins (124, 125).
SipB, C, and D are proteins that are secreted through the T3SS apparatus and
form the translocon pore in the host cell membrane (126). According to recent studies,
SipD is thought to be located at the tip of the needle complex, initiating a translocon
assembly by associating itself with the host cell membrane upon contact (127, 128). SipB
and SipC play a critical role in the assembly and proper function of the translocon pore;
however, they were not observed immediately after the initial contact between the needle
apparatus and host cell membrane (128). Since SipB has a high affinity for binding
cholesterol, the location of translocon pore on the host membrane is most likely to be the
cholesterol rich regions called the lipid rafts. SipB can also recruit cholesterol to the
assembly site of the translocon pore on the host cell membrane (129).
SPI-1 T3SS Secreted Effector Proteins
The internalization of S. typhimurium into the host cell is facilitated by the
collaborative work of SPI-1 T3SS effector proteins. SipA and SipC work in concert to
remodel the host cell membrane (130). SipC binds and bundles microfilaments called Factins to trigger the formation of actin at the bacterial adhesion site. SipA enhances SipC
activity by binding the actin filaments to induce polymerization and stabilize the actin
filaments by up-regulating T-plastin activity, a localized actin bundling protein (131134). In addition SipC blocks host cell factors to prevent actin filament disassembly (135,
136). Other effector proteins such as SopE and SopE2, do not bind actin filaments
20 directly; they mimic the host cell guanine-nucleotide exchange factors (GEFs) to trigger
Rho GTPase, Rac-1 and Cdc42 that ultimately cause host cytoskeleton remodeling and
membrane ruffling through the Arp2/3 pathway (133, 137). SopB is an inositol
phosphatase that contributes to extensive actin remodeling by targeting host GEFs to
activate RhoG GTPase (138, 133). After internalization and formation of the SCV, SopB
and SopD work together to seal the host membrane infolding (139, 133). SptP restores
changes in the host membrane by reversing the activities of SopE, SopE2 and SopB
effectors. SptP mimics the GTPase-activation proteins (GAPs) to deactivate Rho GTPase,
Rac-1 and Cdc42 by hydrolysis of bound GTP to GDP (140-143). SptP also promotes
host cytoskeleton recovery using its tyrosine phosphatase activity targeting tyrosine
Kinase (ACK), a downstream effector of vimentin (an intermediate filament protein) and
Cdc42 (144).
A number of SPI-1 T3SS effector proteins that are involved in nonphagocytic
cellular invasion, also induce the host immune system which results in acute intestinal
inflammation. SopE activates Rac-1, and SopE, SopE2 and SopB work in a concert to
activate Cdc42. The activation of Cdc42 and Rac-1 result in Raf1-dependent upregulation
of the Erk, Jnk and p38 mitogen-activated protein kinase (MAPK) signaling pathways,
which leads to activation of AP-1 and NF-κB transcription factors (150, 138, 133, 137,
152, 151). Subsequently, the activation of these pathways induces the transcription,
translation and secretion of proinflammatory cytokine, IL-8 (also know as CXCL8), by
the invaded host epithelial cells (138, 153). IL-8 act as a chemoattractant to recruit PMNs
to the infection site. SopB, SopE, SopE2 and SipA facilitate the migration of PMNs
21 across the epithelium by disrupting tight junctions (154). In addition, SipA, and SopA
trigger the Arf6 and Phospholipase D signaling cascades resulting in release of
hepoxilllin A3 that contributes to PMNs recruitment and its migration across the
intestinal epithelial layer (155, 156, 157, 158, 150, 132). The migration of PMNs coupled
with fluid flux and secretion of chloride by the invaded epithelial cell contributes to
diarrhea (154, 150, 133). SipB binds and activates caspase-1, which then triggers the
production of other inflammatory cytokines such as IL-18 and IL-1β.
In addition, Salmonella strives to block the infected cell from going through
apoptosis by deploying effector proteins in order to allow bacterial survival and
replication inside SCV (171). Salmonella effectors block apoptosis of the host cell by
suppressing NF-κB-dependent gene expression in numerous ways.
SspH1 effector
protein binds PKN1 to stop NF-κB-dependent gene expression (172). Another effector
protein, AvrA, makes host cell survival possible by blocking the JNK signaling pathway
(173). SptP mimics ATPase activity to inactivate Raf1 that would downregulate MAPK
signaling in order to reverse actin rearrangement by GAP activity (174). SopB is involved
in preventing host cell apoptosis by activating Akt (175,176). SpvC, Phospho-threonine
lyase, inactivates MAPK via beta-elimination reaction. It also reduces the mRNA level of
proinflammatory cytokines by dephosphorylating ERK (177). SseL, a SPI-2 effector
protein, decreases inflammatory cytokines by protecting Iκ-Bα, a NF-κB inhibitor, from
degradation (178).
It can be seen from the overview described in Part 1 that Salmonella pathogenesis
22 in a natural host system is highly complex and involves numerous effector proteins.
Hutchison and Dukovcic have demonstrated that a Betta splendens (Siamese fighting
fish) chromatophore based biosensor can detect and differentiate between pathogenic
bacteria and closely related non-pathogens (204, 213, 231). What remains to be
elucidated are the mechanisms by which cells that are not natural hosts for pathogenic
bacteria respond to pathogens. The remainder of this review will discuss detection
methods for bacterial pathogens in general, and the Betta splendens chromatophore based
biosensor used in this research.
Part 2: Detection Methods and chromatophore cell-based biosensor
Detection Methods for Bacterial Pathogens
Rapid and accurate detection methods for bacterial pathogens contaminating food
and water are essential to identification, treatment and prevention of diseases that
continue to plague humans and animals worldwide. Approximately two thirds of research
efforts have been invested in developing pathogen detection applications for the food
industries, clinical diagnosis, and water and environmental quality control (192). Most
efforts in this field have been focused on developing rapid pathogen detection methods.
Culturing and colony counting, immunology-based methods and Polymerase chain
reaction (PCR), are three well established and most commonly used methods for
pathogen detection. These methods are quite time consuming, however, and can only
report on the presence, not toxicity, of the contaminating microorganism (192).
23 Immunology-based Methods
Immunology-based methods provide various ways to analyze and detect a wide
range of bacterial pathogens. One of the most well established immunology-based
methods is Enzyme-linked immunosorbent assay (ELISA) that uses specially designed
antibodies to bind specific target antigens (195). ELISA is used to detect cell surface
antigens of target bacteria. Many companies provide antibodies used in ELISA for
detecting toxins or spores of B. anthracis (196), extracellular lipopolysaccharides of
Salmonella serovars (197), outer membrane proteins of Salmonella serovars (198), and
other various bacterial antigens.
In a typical ELISA, samples are added to a microtiter plate that contains
immobilized antigen-specific antibodies that binds the antigens from the samples and
then allows the rest of the sample to be washed away. This is followed by the addition of
a second antibody tagged with an enzyme that will induce a color change upon binding.
The color is then quantified by colorimetric methods. Although this is a very visible
method that is easily quantified, this method can only report on the presence, not viability
or toxicity, of contaminating bacteria.
Another immunology-based method is immunomagnetic separation (IMS) that is
used in concert with other detection methods. IMS is used to separate the target bacterial
pathogen from a pool of bacteria using specific antibody coated magnetic beads that bind
to the target bacteria (199). Optical, magnetic force microscopy, and magnetoresistance
are some of the methods that can be used for further detection after the target bacteria is
24 captured and extracted by IMS (200, 201).
Polymerase Chain Reaction-based Methods
Polymerase chain reaction (PCR) methods are used for bacterial pathogen
detection. PCR is based on the isolation, amplification and quantification of short DNA
or RNA sequences of target bacteria. Real-time PCR, multiplex PCR and reverse
transcriptase PCR are other PCR methods that have been developed for bacterial
detection. These methods require a known nucleic acid template, specific primers, and
other essential thermostable polymerization enzymes. PCR consists of a denaturation
cycle of the purified DNA by heat, an extension cycle using specific primers, and DNA
polymerase, followed by the exponential amplification of each new double stranded
DNA. Then, gel electrophoresis is used to verify the presence of the new amplified DNA
sequence (192).
Even though PCR is a valuable method in detecting different types of bacterial
pathogens, it has some limitations. For example, PCR does not provide information about
the viability of the bacterial cells due the presence of DNA in both, dead and living cells.
Additionally PCR cannot report directly on the toxicity of the contaminating bacteria.
Also in some cases, PCR needs purified DNA or RNA as template, which often requires
more than 24 hours to acquire an appropriate amount because culturing is still part of the
process. RT-qPCR (reverse transcriptase-quantatative PCR) addresses some of these
limitations.
25 Biosensors
Biosensors are detection devices that utilize biological materials such as cells,
tissues, organelles, microorganisms, cell receptors, antibodies, nucleic acids or enzymes.
Biosensors also utilize biologically derived materials like engineered proteins and
antibodies, or biomimic materials like synthetic catalysts, imprinted polymers and ligands
(192). These materials are attached to a physiochemical transducer/transducing
microsystem that can be electrochemical, optical, piezoelectric, magnetic thermometric or
micromechanical and are used for biochemical interactions with targets for detection
(192, 202). Biosensors have two main steps in detecting the target: immobilization of the
target and analysis of the target (202). Biosensors have been very important in the
pathogen detection field due to their ability to detect a wide range of microorganisms and
assess their toxicity. However, cell-based biosensors have become the ideal detection
method due to their distinguishable physiological behavior toward pathogenic and nonpathogenic bacteria (203-205). Unlike other detection methods that require DNA
homology, antibody-antigen specificity, or structure recognition, cell-based biosensors’
physiological responses and their reaction to the environment are used as the detection
tool (202).
Cell-based biosensors can be divided into two categories: engineered cell-based
biosensors and innate cell-based biosensors. Engineered cell-based biosensors are cells
that are altered/engineered for a specific pathogen or target. The rat basophilic leukemia
mast cell and the B-lymphocyte cell are the two examples of engineered cell-based
26 biosensors to detect foodborne pathogens that show high specificity (206). Even though
the high specificity of engineered cell-based biosensors is preferred, it takes years of
research to develop it. In contrast, innate cell-based biosensors are biosensors that do not
need any modification; their use as a detection tool is reliant upon the quantifiable signal
of their inherent physiological characteristics. These biosensors use their natural
receptors to trigger an intracellular signal transduction pathway that can be measured. A
prominent example of innate cell-based biosensors is the chromatophore cell-based
biosensor.
Chromatophore Cell-based Biosensor
Chromatophore cell-based biosensors utilize a class of pigmented neuron like
cells called chromatophores. These cells are capable of moving their intercellular pigment
organelles upon exposure to physiological stimuli. This property has made
chromatophore cells valuable in their use in a detection system solely based on biological
activity.
Induction of pigment granules movement in chromatophore cells due to
disrupted intracellular functions in response to different biological stimuli are measured
and analyzed to assess toxicity of a pathogen, whereas other detection methods can only
assess the presence but not toxicity of a pathogen (207, 208, 209). These pigment
movements could be toward the peri-nucleus of the cell called aggregation or outward to
the periphery of the cell called dispersion (208, 209). The degree, magnitude and kinetics
of chromatophore cell responses (aggregation and dispersion) vary depending on the
different pathogens and toxic stimuli (208, 209). The similarity of chromatophore cells to
27 neuron cells of other animal makes them of interest in detecting biological substances of
concern to human health (210, 209).
Chromatophore Cells and Pigmentation
Amphibians, crustaceans, fish and reptiles are poikilothermic animals that have
pigmented cells, called chromatophores, located under their dermis layer. This class of
neuron like cells is responsible for changing the animal’s overall body pigmentation and
color intensity in response to environmental stress. Chromatophore cells are used as an
integumentary system in Poikilothermic species, participating in survival, visual
communication and mating processes (214-217). Chromatophore cells are named based
on their intracellular pigment organelles: cyanophores (blue), erythrophores (red),
iridophores (iridescent), leucophores (white), melanophores (brown), and xanthophores
(yellow). Chromotaphore cells are subdivided into two major categories, light-absorbing
and light-reflecting, based on their optical properties. Erythrophore, cyanophore,
melanophore and xanthophore cells are light-absorbing and have a dendritic morphology.
In contrast, iridophore and leucophore are light-reflecting and have non-dendritic, round
shaped cells (218, 219).
Except iridophores, the remaining five types of chromatophore cells contain
motile pigment organelles called chromatosomes. These chromatosomes have the
capability to exhibit different colors based on the absorption, or reflection in the case of
leucophores, of different light wavelengths. Leucophores have (unidentified) whitish
fibrous materials that scatter all the wavelengths, thus, deemed as light-reflectors (216).
28 Iridophores are the only chromatophore cells that do not contain chromatosomes, instead,
they have stacked platelets of guanine crystals in the cytoplasm (216). Due to the high
refractive index of these platelets, they give an iridescent color to many Poikilothermic
species. Unlike other chromatophore cells, iridophores are not involved in changing
color intensity through intracellular motile pigment organelles (216).
Specialized cytoskeletal microtubules that spread from the center of the cell to
dendrites, facilitates a movement route for chromatosomes through the activity of
ATPase motor proteins; giving chromatosomes the ability to aggregate or disperse.
Aggregation refers to movement of chromatosomes toward the center of the cell and
dispersion refers to movement of chromatosomes toward the periphery of intracellular
space. Aggregation is caused by the coupling activities of the first signaling messenger,
neural or hormonal ligands, binding to receptor and the activation of G-protein, which in
turn activates dynein, a eukaryotic microtubule motor protein. The resulting signal
cascade is translated into the movement of pigment organelles toward the peri-nucleus of
the cell along the microtubules (220).
Chromatophore cells are generated in the neural crest of poikilothermic animals.
The chromatosomes in chromatophore cells are controlled by the endocrine and neural
systems via different hormones (216, 218). For example: the pituitary gland secretes
melanophore stimulating hormone (MSH) that causes dispersion, movement of
chromatosomes toward the cell periphery, in light-absorbing chromatophore cells.
Interestingly, MSH causes aggregation in light-reflecting iridrophore and leucophore
29 chromatophore cells by means other than causing translocation of the chromatosomes.
The hypothalamus secretes melanin concentrating hormone (MCH) and melatonin that
cause aggregation, movement of chromatosomes to the peri-nucleus region of the cell, in
light-absorbing chromatophore cells (218). Betta splendens possess sympathetic nervous
system that is also involved in regulation of chromatosome motility in the chromatophore
cells. Changes in the level of different intracellular ions concentration can cause a signal
transduction, which affect the nervous system. Thus, the nervous system releases
neurotransmitters to cause aggregation or dispersion (218).
Physiological studies have shown that environmental stress is also responsible for
the overall body color changes in fish, amphibians and crustaceans. The color changes in
these animals are characterized as hyper – or hypopigmentation (225). The dermis layer
of Poikilothermic animals that contain chromatophore cells act as the integumentary
system of the animal and thus, is exposed to a variety of different environmental
conditions (226). Toxicants, chemicals (e.g. industrial waste), and heavy metals are
associated with depigmentation in fish, amphibians and crustaceans. For example: some
studies have shown that mercuric chloride is responsible for hypopigmentation in
rainbow trout (154), Rohu carp (226) and estuarine crab larvae (170). Environmental
stress related depigmentation is also caused by bacterial pathogens or bacterial
pathogens’ exotoxins. The depigmentation in numerous species of fish is exhibited as a
primary symptom after exposure to bacterial toxin (198).
30 Betta splendens
The use of Betta splendens (Siamese fighting fish) erythrophore cells to detect
chemical and bacterial toxicity is a good example of a chromatophore cell-based
biosensor. Positive controls, clonidine hydrochloride (Cl) and α-melanocyte simulating
hormone (MSH), for aggregation and dispersion, respectively, were established in
erythrophore cells from Betta splendens by Mojovic et al (209, 211). In other studies,
erythrophore cells from B. splendens have been exposed to different pathogenic bacteria
(e.g Bacillus cereus, Clostridium perfringens, C. botulinum and Salmonella enteritidis)
and nonpathogenic bacteria (e.g. B. subtilis, B. cereus nonpathogenic mutant).
Interestingly, erythrophore cells respond by aggregating in the presence of the pathogenic
bacteria, yet remain nonresponsive to nonpathogenic bacteria (204, 213). In addition,
erythrophore cells have been instrumental in detecting water contaminants, chemical
agents and purified bacterial toxins (207, 209). Erythrophore cell-based biosensor is an
emerging technology that exhibits the potential to detect toxic conditions not only caused
by the presence of foodborne pathogens, but also toxic chemicals.
Betta splendens have acquired worldwide popularity as ornamental fish due to
their brilliant coloration. Rice farmers in Thailand domesticated and bred this species of
fish for ornamental and sports purposes. B. splendens with long fins and bright colors are
used as household ornamental fish. The brilliantly colored phenotype of the ornamental
form of B. splendens no longer resembles the phenotype of wild B. splendens living in
rice puddles or the B. splendens with short and round fins for sport fighting in Thailand
31 (183, 184 and 185). Betta splendens is of a great commercial value in Southeast Asia.
Thailand alone exports about 3.6 million male B. splendens fish each year (184). Wild B.
splendens are naturally found in Thailand and other Southeast Asian countries in the
freshwater ponds, rice puddles and marshes (186, 183). These ponds and puddles make a
perfect niche for these fish because of their shallow, high temperature (about 29.9 0C) and
low in dissolved oxygen water (187).
Genus Betta belongs to the family Osphronemidae and is classified as a
freshwater ray-finned fish. Betta species possess lung-like organs that allow them to gulp
air at the water surface, putting them under suborder Anabantoidei (the labyrinth fish)
(188).
Parental care characteristics, mouth-brooder or bubble-nest builders, are the
distinguishing factors among different species of wild-type Betta. Betta splendens are the
bubble-nest builders, a reproduction strategy that sets them apart from other fish species.
Betta splendens coloration plays important roles in reproduction and protection of the
offspring. The male B. splendens builds a bubble nest by gulping air at the water surface
and starts initiating courtship with females (186). Males protect the bubble nest by
projecting a territorial behavior and aggression toward the intruder or unwanted female
by blazing its opercula (gill covers), spreading fins, and deepening his overall color (189,
186).
The courtship and spawning process is divided into five phases: prespawning
preparatory phase, courtship phase, clasp, swimming inhibition and postspawning phase
32 (190). Coloration is the main signal of prespawning for triggering courtship. The male B.
splendens switches from being pale and dull color to a bright and uniformed body color
to display for female. Female B. splendens changes from a pale color to a phenotype of
deeply colored vertical bars on the body and a gold patch on the belly to signal the male
that she is ready for courtship and spawning (190, 191). The female B. splendens work is
done in the reproduction process after spawning. The male B. splendens picks the
fertilized eggs in his mouth and places them in the bubble nest and continues to protect
the eggs and newly hatched fry (190). Any faulty response from either of the two fish,
male or female, in any of the above five phases results in interruption of reproduction
(190).
Betta splendens make an ideal organism for chromatophore cell-based biosensors.
Betta splendens are readily available in any pet store and can be purchased throughout the
year. Due to their small body size, Betta splendens are easy to maintain. Each fish can be
placed in a small beaker and kept in a relatively small facility for the duration of 21 days
before tissue culture. The large flowing fins provide large number of chromatophore cells
that can be used for multiple experiments. In addition, tissue cultures from B. splendens
are easy to maintain. Chromatophore cells are cultured in 6, 12, 24 or 48 well plates and
stored on a flat surface at room temperature. The tissue culture remains viable and
responsive for up to five weeks without addition of hormones or growth factors.
33 Research Objectives
Previous research efforts using the chromatophore cell type, erythrophore, for
detection of bacterial toxicity revealed this cell type could rapidly detect several food
associated bacterial pathogens, including S. typhimurium. Previously tested hypotheses
using mutagenesis to analyze the attributes of S. typhimurium that led to a response in
erythrophore cells revealed a potential role for the T3SS apparatus coded for by prgHIJK.
Although wildtype S. typhimurium rapidly induces pigment organelles to undergo an
aggregative response in erythrophore cells, S. typhimurium containing a mutation in the
prgHIJK operon does not induce the pigment organelles to undergo an aggregative
response in erythrophore cells. A mutation identified in a region different from the
mutation that disrupted the prgHIJK operon also had a similar impact on erythrophore
cells. The first objective of this study was to test the hypothesis that this second mutation
resided in either gene(s) coding for the process regulating expression of the prgHIJI
operon or the gene(s) coding for the effector proteins that traveled through the PrgHIJK
apparatus. The approaches used to test this hypothesis included sequencing the region
containing the mutation and identifying the gene(s) impacted by this mutation through
comparative sequence analysis, cloning the wildtype gene sequence and using this
wildtype gene sequence to complement the mutant thereby demonstrating that the
mutation is linked to the resulting biological outcome. The second objective of this study
was to test the hypothesis that the effector proteins of T3SS are the components of S.
typhimurium’s invading mechanism directly involved in inducing pigment organelles to
undergo an aggregative response in erythrophore cells. The approach used to test this
34 hypothesis involved testing the response of erythrophore cells to S. typhimurium strains
containing targeted, well characterized mutations in genes coding for T3SS effector
proteins. The outcome to this research effort increased understanding of the role of the
T3SS in chromatophore cell response. This understanding lead to better utility of
chromatophore cells as biosensors for bacterial toxicity.
35 Chapter 3
Materials and Methods
Fish Care
Red male Betta splendens were purchased from the local Petco or Animal House
(pet store), depending on availability. Fish were maintained individually in 1000 ml
beakers that contained 900 ml conditioned tap water (3.5 L autoclaved tap water, 460 µL
Stress Coat, 2.3 g Aquarium Salt, pH∼7.0) at 26 oC (80.6 oF) with alternating light and
dark cycles for 14 and 10 hours, respectively. Fish were fed New Life® Spectrum Betta
Formula pellets six days a week and blood worms on the seventh day. Once a week, they
were dipped for 30 seconds in a 3% aquarium salt solution and transferred to a new
beaker containing fresh water. These husbandry conditions were implemented in
accordance with regulations of Oregon State University’s Institutional Animal Care and
Use Committee (approval #4409). Fish were held at least 21 days before use in
chromatophore cell extraction.
Cell Culture
Fish were euthanized in an ice water bath for approximately 20 minutes prior to
chromatophore cell extraction. After discarding the ice water, the fish was briefly rinsed
with phosphate buffered saline (PBS) [128 mM NaCl, 2.7 mM KCL, 10 mM Na2HPO4,
1.46 mM KH2PO4, supplemented with 5.6 mM glucose and 1% antibiotic/antimycotic
(Invitrogen Cat# 15140122), in tissue culture grade H2O, pH 7.3] before transfer with
sterile forceps to a petri dish containing 10mL of PBS. Anal, caudal, and dorsal fins were
36 clipped using sterile scissors and forceps and transferred to a second petri dish containing
10 mL of PBS. Using sterile forceps, the clipped fins were transferred to a 50 mL Falcon
tube containing 10 mL of skinning solution (SK) [128 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.46 mM KH2PO4, 1 mM EDTA (FW=372.2), supplemented with 5.6 mM
glucose and 1% antibiotic/antimycotic (Invitrogen Cat# 15140122), pH 7.3]. Fins were
washed in 10 mL SK for 1 minute on a rocker plate. Skinning solution was then
discarded carefully, using disposable pipets, and replaced with 10 mL fresh SK. The SK
wash step was repeated nine times. The SK wash removes non-pigmented epithelial cells,
protective mucus, and scales from fin tissues.
After the last 10 mL SK wash was discarded, a digestive enzyme solution (ENZ)
[15 mg collagenase (Worthington Biochemical Cat# LS004196), 1.5 mg hyaluronidase
(Worthington Biochemical Cat# LS002594), 7 mL PBS] was added to the fins in the 50
mL Falcon tube to release chromatophore cells from fin tissue. Batch lots of collagenase
and hyaluronidase were prepared, and aliquots of 15 mg/1 mL PBS and 1.5 mg/1 mL
PBS, respectively, were stored at -200C in 1.5 mL microcentrifuge tubes (epitubes) for
single-use. The exposure time of fins to ENZ varied depending on fish age, fin size, and
the rate of fin digestion. The normal exposure time ranged from 1 minute up to 5 minutes.
After fin digestion, ENZ with released chromatophore cells, was transferred to a 15 mL
Falcon tube using a sterile disposable pipette. Chromatophore cells were then separated
from ENZ by centrifugation for three minutes at 600 rpm in a swinging bucket centrifuge
(IEC Centra CL3). The supernatant ENZ was added back to the remaining fin tissues for
further digestion. The fin digestion and separation of ENZ from released chromatophore
37 cells steps were repeated until most of the chromatophore cells were released and the fin
rays became visible. Usually, 1-4 digestion steps are performed. The first digestion time
is short and typically the cells are discarded due to excessive scales.
The pelleted chromatophore cells from each digestion were resuspended and
washed in approximately 7 mL Leibovitz L15+ medium [L15+(Invitrogen Cat#
15090046), supplemented with 20 mM HEPES buffer (Invitrogen Cat# 15630080) and
1% antibiotic/ antimycotic (Invitrogen Cat# 15140122)] and then centrifuged for three
minutes at 600 rpm in a bucket centrifuge. The supernatant was discarded and the
chromatophore cell pellet was resuspended in 100-400 µL L15+, depending on the size of
the pellet, and transferred to a 1.5 mL epitube. A 5 µL drop from each of the resuspended
chromatophore cell pellets was visualized under the microscope to observe the
concentration and scale presence.
A 5 µL drop of chromatophore cells was placed in the center of each well of a 24well Falcon tissue culture plate. Chromatophore cells were left undisturbed for 15 to 20
minutes in order to attach to the bottom of the wells. One mL of L15+ medium was
added to each well, and after 20 minutes of equilibration, 50 µL of fetal bovine serum
(5%) (FBS, Sigma-Aldrich Cat# F2442) was added. After 24 hours, old media was
replaced with 1.5 mL of L15+ with 5% FBS.
Chromatophore cells were stored at room temperature (220C) and media was
changed weekly to avoid accumulation of cellular waste products and debris.
Chromatophore cells were maintained for 1-2 weeks before using them for any
38 experiments.
Response Assays and Computational Analysis
Red chromatophore cells (erythrophores) from B. splendens were tested to
measure their physiological responsiveness to the control agents α-melanocyte
stimulating hormone (MSH, Sigma- Aldrich Cat# M4135), which induced pigment
dispersion and clonidine (Sigma-Aldrich Cat# C7897), which induced pigment
aggregation prior to conducting each experiment. Frozen single-use aliquot stock
solutions of both clonidine and MSH at a concentration of 1 µM were thawed prior to
use. A 150 µL volume of L15+ medium (10% of well volume) was removed from the
designated chromatophore cell culture well, and replaced by adding 150 µL of either
MSH or clonidine (final concentration of 100nM). Luria-Bertani (LB) broth used for
culturing different strains of Salmonella was also included as a control and added to
chromatophore cells in the same manner as MSH and clonidine.
A viewing field of chromatophore cells was observed for 60 to 90 minutes at
100X through a Leica (Leica, Inc., Wetzlar, Germany) DMIL inverted microscope
(Bartels and Stout, Issaquah, WA, USA). After control agents or bacterial cultures were
added, time-lapsed images were captured at specified time-points during the 60 to 90
minute interval with a SPOT Insight 320 color camera (Diagnostic Instruments, Inc.,
Sterling Heights, MI, USA) mounted on the Leica microscope and SPOT Advanced
software version 3.5.6.2 (Diagnostic Instruments, Inc.). Time-points for capturing images
pictures during the 60 to 90 minute time course for MSH, clonidine, LB and all
subsequent bacterial cultures were as follows: every 15 seconds for the first minute (0,
39 15, 30, 45 seconds), 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes,
7.5 minutes, 10 minutes, and every five minutes thereafter. Using Image Pro Plus 7.01
(Media Cybernetics, Bethesda, MD, USA) software, images captured at each time-point
were analyzed and data were exported to Microsoft Excel for further analysis. The
response of chromatophore cells was measured by calculating area change in pigment
based on pixels using the following equation:
Change in Pigment Area = – [(Ao – At)/Ao] x 100
Ao is the initial chromatophore cell pigment area at time zero, At represents the pigment
area at any given time-point, and multiplying the fraction by 100 will give the change in
the pigment area.
The collected data were represented graphically in Microsoft Excel by plotting
“Change in pigment area (%)” versus “Time (minutes)”. A positive change in pigment
area correlates to a dispersion response as seen with the dispersive control, MSH.
Whereas a negative change in pigment area correlates to a pigment aggregation response,
as seen with the aggregative control, clonidine. Chromatophore cell response assays with
the above specifications were conducted at least twice.
Bacterial Growth Conditions
Salmonella typhimurium strains were grown and maintained in Luria-Bertani
broth [LB, 10 g tryptone, 5 g yeast extract, 5 g NaCl, 1L double distilled water, pH 7.4].
Bacterial stocks were stored at -80 oC in LB supplemented with 20% glycerol. Fresh
bacterial cultures were streaked on LB agar plates and incubated at 37 oC overnight. For
40 every chromatophore cells response assay, 5 mL LB in a 25 mL flask was inoculated with
an isolated colony of each Salmonella strain from the LB agar plate and incubated at 37
o
C in a shaking chamber (150 rpm) overnight (Table 1). Luria-Bertani media was
supplemented with an appropriate 50 µg/mL antibiotic: ampicillin (Amp), kanamycin
(Kan) or nalidixic acid (Nal), when needed. Overnight cultures were back diluted 1:100
(50 µL of overnight culture in 4.95 mL of LB in 25 mL flask) in fresh LB media and
grown at 37 oC in a shaking chamber for 4 hours. One hundred fifty µL of the backdiluted bacterial culture (10% final volume) was added to a well after removing 150 µL
of L15+ medium. Images were captured and analyzed as described previously.
Salmonella strains are Biosafety Level 2 (BSL2) bacteria. Thus, all the chromatophore
cell response assays that involved Salmonella were conducted in a BSL2 facility,
approved by the Oregon State University’s Institutional Biosafety Committee.
Chromatophore cell response assays with each bacterial strain were performed on
chromatophore cells from at least two different fish.
41 Table 1: Bacterial strands and plasmids used in this study.
Strain/plasmid
S. typhimurium ATCC 70020
S. typhimurium ST10
S. typhimurium ST10 pTA108
S. typhimurium ST10
pTA108HilD
S. typhimurium ATCC 14028
S. typhimurium ZA9
S. typhimurium ZA10
S. typhimurium ZA11
S. typhimurium ZA15
S. typhimurium ZA26
E. coli JM101
pTA108 (plasmid)
Relevant Characteristics
LT2 wildtype
Derivative of ATCC 70020,
hilD::EZ-Tn5 Kanr
ST10 with pTA108 Kanr,
Ampr
ST10 with wild-type hilD
gene, Kanr, Ampr
IR715 Nalr
Derivative of ATCC 14028,
SopE2 mutant, Nalr
Derivative of ATCC 14028,
SipA mutant, Nalr
Derivative of ATCC 14028,
sptP mutant, Nalr
Derivative of ATCC 14028,
sopB mutant, Nalr
Derivative of ATCC 14028,
SipA/SopB mutant, Nalr
F´ traD36 proA+B+ lacIq Δ
(lacZ)M15/ Δ(lac-proAB) glnV
thi
lacZΔM15 bla (Ampr), 1-5
copies per cell
Source
ATCC
Stephanie Dukovcic’s
dissertation (231)
This Study
This Study
Adams’s Lab (254)
Adams’s Lab (254)
Adams’s Lab (254)
Adams’s Lab (254)
Adams’s Lab (254)
Adams’s Lab (254)
Lab stock
Lab stock (230)
Polymerase Chain Reaction (PCR)
PCR reactions were run using either a Perkin Elmer Gene Amp PCR system
(model 2400) or Eppendorf Mastercycler® gradient machine. PCR samples contained 50100 ng of DNA, 1-2 µL of (20 µM) each primer (Table 2) and Platinum® PCR Super
Mix HiFi (Invitrogen Cat. No. 11306016) to a final volume of 50µL in a 0.5 ml PCR
epitube. Two controls, a negative control lacking DNA template, and a positive control
with known pertinent products, were used with each PCR performed in this study. PCR
reactions were subjected to 95 oC for 5 minutes to ensure complete initial denaturation,
42 and then went through 30 cycles of denaturation (95 oC for 30 seconds), annealing (set at
Tm (Table 2) for 30 seconds) and extension (72 oC for one minute for each kb product). A
final extension was conducted for 10 minutes to ensure that all products were fully
extended by the polymerase. The PCR products were then analyzed by 1% agarose gel
electrophoresis in TAE buffer (0.8 M tris-base, 0.35 M glacial acetic acid, 20 mM
EDTA) to verify the presence of an amplified product. DNA bands were visualized by
ethidium bromide staining.
PCR products were purified using QIAquick® PCR
Purification Kit (Qiagen, Cat. No. 28104) or DNA bands were excised from the agarose
gel using a Qiagen Gel Extraction Kit (Cat. No. 28704).
Bacterial Transformations
Escherichia coli JM101 was transformed following a chemical competency
protocol (228). Briefly, an overnight culture of E. coli in 5 mL LB media in a 25 mL
flask was incubated at 37 oC with aeration. The overnight culture was back diluted
(1:100), the next day, in 5 mL LB in a 25 mL flask and grown to mid-log phase (2-3
hours of incubation). One mL of the culture was transferred to a 1.5 mL epitube for each
transformation and control. Each epitube was centrifuged in a microcentrifuge at
maximum speed for 1 minute. Supernatant was decanted and cells were
43 Table 2: Primers used in this study.
44 resuspended in ~50 µL residual medium. Then, 100 µL of cold TSS (10 g PEG 3350 or
8000, 0.5 g MgSO4 7H2O, 88 mL LB, 5 mL DMSO) was added to the cell suspension in
each epitube. Cells were incubated on ice while adding 1µL of plasmid DNA (10-50 ng).
Cells were incubated on ice for 20 minutes and then heat shocked at 42 oC for 1.5
minutes and immediately returned to ice for 3 additional minutes. Nine hundred µL of LB
was added to each epitube and incubated at 37 oC for 1 hour to allow the cells to recover.
The transformed culture was then diluted 1:10 and 1:100. One hundred µL of each
dilution was plated on the appropriate selective media. Plates were incubated overnight at
37 oC. Several well isolated colonies were restreaked to fresh plates and incubated
overnight at 37 oC. Plasmids were extracted from 5 mL overnight cultures using the
QIAprep® Spin Miniprep Kit (Cat. No. 27106). The correct plasmid was verified by
restriction digest of plasmid mini-preps and sequencing.
Salmonella
typhimurium
was
transformed
following
an
amended
electrocompetency protocol (227). A 5 mL overnight culture of ST10 (S. typhimurium
mutant) was diluted into 500 mL LB (1:100) and transferred to a 1 L flask. The culture
was incubated at 370C with heavy aeration (250 rpm) for 2 hours to bring it up to late log
phase. From this point on, every step was performed on ice and all the
materials/equipment and reagents were kept chilled.
The culture was divided into
centrifuge bottles and spun at 5,000 rpm for 15 minutes at 4 oC using a Beckman JA 12
rotor. Supernatant from the culture was discarded and cells pellets were resuspended in
100 mL sterile ice water. The cells were spun at 5,000 rpm for 15 minutes at 4 oC.
Supernatant was discarded and cell pellets were resuspended in 50 mL sterile ice water
45 and combined into on centrifuge bottle. The cells were centrifuged under the same
conditions. The cells were resuspended in 2 mL 10% glycerol and spun at 5,000 rpm for
15 minutes at 4 oC. In the final step, the cells were resuspended in 400 µL 10% glycerol.
The electrocompetent cells were ready for immediate use. However, any left over cells
could be frozen at -80 oC. Forty µL of electrocompetent cells were used for each
electroporation. On ice, 50-100 ng DNA was added to electrocompetent cells and mixed
well by pipetting up and down. The mixture was then transferred to a 0.2 cm prechilled
electrocuvette and tapped to ensure the removal of any air bubbles. Using a BioRad Gene
Pulser set at 1.5-1.8 kV, 25 µF and 200 Ω, a single electric pulse (4-5 ms) was applied.
Immediately after discharge, 1 mL of LB was added to cells and they were incubated at
37 oC for 1 hour with heavy aeration (250 rpm) to allow the cells recover.
The
transformed cells were spread on LB plates containing 50 µg/ml ampicillin for each
transformation sample and control as follows: a 10 µL, 100 µL and a concentrated 100 µL
(900 µL remaining cells were spun and resuspended in 100 µL LB) aliquot. The LB agar
plates were incubated overnight at 37 oC. Several well isolated colonies were restreaked
on fresh LB plates containing 50 µg/ml ampicillin and incubated overnight at 37 oC. A
5mL overnight culture was used for mini prep using the QIAprep® Spin Miniprep Kit
(Cat. No. 27106). The correct plasmid was verified by PCR of plasmid mini-preps, and
sequencing. A 1mL glycerol stock of each confirmed transformant was prepared and
stored at -80 0C.
46 Identifying Transposome™ Insertion Site(s) and Sequencing
All sequencing was conducted by the Center for Genome Research and
Biocomputing (CGRB, Oregon State University) using an ABI 3730 capillary sequence
machine. Templates and primers concentrations were optimized to the specifications of
the CGRB. All the sequences received from CGRB were then aligned and analyzed using
BioEdit Sequence Alignment Editor version 7.0.9.0.
To find the transposome insertion site in ST10 (S. typhimurium mutant), genomic
DNA from ST10 was extracted using an Ultra Clean Microbial DNA Isolation Kit (Cat.
No. 12224-50-SF). The extracted genomic DNA from ST10 was used as template for
Random Amplification of Transposon Ends (RATE) PCR (229). The PCR products were
run on a 1% agorose gel and samples with a visible band were purified by the QIAquick®
PCR Purification Kit. The purified PCR product was then sequenced. The sequence was
analyzed using BioEdit and sequence was identified using the BLAST program at NCBI
(blast.ncbi.nlm.gove/blast.cgi) database.
Complementation
To complement the Tn5::hilD insertion in the S. typhimurium mutant ST10, the
hilD gene including its promoter and part of the Fur box were cloned into a low-copy
number plasmid (1-5 copies per cell), pTA108. Genomic DNA from S. typhimurium was
amplified with specific primers (Table 2), cloned into the TOPO TA cloning vector using
TOPO TA Cloning® Kit (Invitrogen™, Cat. No. K4575-01SC), and transformed into E.
coli JM101. A 60µL and 150µL aliquot of the transformation mixture was plated on LB
plates containing 50 µg/ml ampicillin. Several well isolated colonies were plated on fresh
47 LB plates containing 50 µg/ml ampicillin overnight at 37 oC. An overnight culture of a
single white colony from the LB plates containing 50 µg/ml ampicillin was incubated at
37 oC with aeration. The next day plasmid was extracted from the overnight culture using
QIAprep® Spin Miniprep Kit (Cat. No. 27106). M13 forward and reverse primers
provided with the TOPO Kit were used in a PCR reaction to check for the insert, and the
PCR products were run on a 1% agarose gel. The PCR product was then cleaned up with
QIAuick® PCR Purification Kit (Qiagen, Cat. No. 28104), and sequence confirmed.
Topo::hilD plasmid and pTA108 plasmid were double digested using the restriction
enzymes Sal1-HiFi and EcoR1-Hifi from NEB. The double digested products were run
on an agarose gel, and single bands were extracted from the gel using a Clontech Gel
Extraction Kit. DNA Dephos & Ligation Kit (Roche, Ref. 04898117001) was used to
perform a ligation on the double digested pTA108 and insert. Ligations were carried out
at 16 oC overnight, then heat inactivated at 65 oC for 20 minutes.
The ligated product (pTA108hilD) was chemically transformed into E. coli
JM101, as described previously. An overnight culture of selected colonies from the
transformation were prepared and QIAprep® Spin Miniprep Kit (Cat. No. 27106) was
used to extract the plasmid. pTA108 screening primers were used in a PCR reaction to
screen for pTA108hilD. The PCR product was run on a 1% agarose gel and the right size
single bands were confirmed. The PCR products were then cleaned up and sequenced.
The sequence was analyzed and confirmed pTA108hilD.
The pTA108hilD plasmid was then electroporated into ST10 electrocompent
cells. An empty pTA108 was also electroporated into ST10 as a control. Transformants
48 were selected on LB plates containing 50 µg/ml ampicillin and plasmid was extracted.
The extracted plasmids were screened by PCR using specific hilD forward and reverse
primers and correct products size was confirmed on a 1% agarose gel. The PCR products
were then cleaned up, and sent to CGRB for sequencing, and sequences were confirmed
against the S. typhimurium sequence at NCBI.
A complementation assay on B. splendens erythrophores, as described in the
Response Assay and Computational Analysis sections, was performed to determine
whether or not the ST10-pTA108hilD transformant complemented the ST10 phenotype.
49 Chapter 4
Results
The Physiological Response Betta splendens Erythrophore Cells
The Betta splendens chromatophore cell type, erythrophore, a neuron-like cell
with predominately red pigmented organelles, was used in this study. The erythrophore
cell’s physiological responsiveness in primary tissue culture is well characterized (204,
208, 204, 213, 231, 237). Physiological responsiveness in primary tissue culture is
assessed using the neurotransmitters, Clonidine (Cl) and α-Melanocyte Stimulating
Hormone (MSH). Both neurotransmitters induce pigment organelle translocation in
erythrophore cells in a characteristic fashion.
Erythrophore cells were isolated from the fins of red B. splendens and maintained
in primary tissue culture. Erythrophore cells were exposed to 100 nM Clonidine, 100 nM
MSH and LB medium (Luria broth; used to culture bacterial cells) for 60 minutes.
Erythrophore cell response to the bacterial culturing medium, Luria broth (LB), was
assessed because subsequent studies in this research effort characterize erythrophore cell
response to the bacterium, Salmonella typhimurium, cultured in LB medium. Figures 1A,
B, and C illustrate erythrophore cell response to 100nM Clonidine, 100nM MSH, and LB
at time zero (left panels) and 60 minutes (right panels) after exposure. The data described
in Figure 1 were obtained from two different red B. splendens erythrophore cell
preparations. Erythrophore cell response, upon exposure to 100nM Clonidine, was
50 characterized by the rapid aggregation of pigment organelles to the center of the cell
within one minute, with this response plateauing by 5 minutes (Figure 1A and 1D). The
aggregation response induced by Clonidine, and mathematically described as a negative
percent pigment area change (Figure 1D), has been previously reported (204, 208, 204,
213, 231, 237). Erythrophore cell response, upon exposure to 100 nM MSH, was
characterized by a slow and steady dispersion of pigment organelles to the edge of the
cell, with this response plateauing by 30 minutes, (Figure 1B and 1D). The dispersive
response induced by MSH, and mathematically described as a positive percent pigment
area change (Figure 1D), has been previously reported (204, 208, 204, 213, 231, 237).
Erythrophore cell response, upon exposure to the bacterial culturing medium, Luria broth
(LB), was characterized by slight dispersion, but dispersion was negligible and not at the
magnitude as the response observed upon exposure to MSH (Figure 1C and 1D).
51 A
B
D
Change in pigment area (%) C
40 30 20 10 0 -­‐10 -­‐20 -­‐30 -­‐40 -­‐50 -­‐60 -­‐70 0 10 20 30 40 50 60 Time (minutes) Figure 1: Betta splendens erythrophore cell response to Clonidine, MSH, and LB.
Erythrophore cells before (left panels) and after (right panels) exposure for 60 minutes to:
(A) Clonidine, (B) MSH, and (C) LB. Magnification of erythrophore cells is at 100X. (D)
Graphical representation of erythrophore cell area change (percent) in response to: (u)
Clonidine, (¢) MSH, and (p) LB. A negative area change describes an aggregation
response, and a positive area change describes a dispersion response. Data represent the
mean values of two trials. Error bars represent the standard deviation.
52 Betta Splendens Erythrophore Cell Response To Wildtype Salmonella
Typhimurium And Salmonella Typhimurium ST10 Mutant
Previous reports describe Betta splendens erythrophore cell response to foodassociated bacterial pathogens, such as Bacillus cereus and Salmonella typhimurium, and
nonpathogenic mutants of these bacterial strains (204, 208, 204, 213, 231, 237). In a
previous study examining erythrophore cell response to S. typhimurium, mutants were
generated using the commercially available EZ-Tn5 mutagenic vector system (231).
Several mutants from the pool of EZ-Tn5™ mutagenized S. typhimurium were identified
that appeared not to induce an aggregation response in erythrophore cells (231). One S.
typhimurium mutant, ST10, was examined further.
Erythrophore cells were exposed to wildtype S. typhimurium and S. typhimurium
ST10 mutant. Figures 2A and B illustrate the erythrophore cell response to wildtype S.
typhimurium and S. typhimurium ST 10 mutant at time zero (left panels) and 60 minutes
(right panels) after exposure. The data described in Figure 2 were obtained from two
different red B. splendens erythrophore cell preparations. Erythrophore cell response,
upon exposure to wildtype S. typhimurium, was characterized by aggregation of pigment
organelles to the center of the cell, with this response initiating approximately 30 minutes
after exposure to this bacterial pathogen (Figure 1A and 1D). The aggregation response
induced by S. typhimurium is mathematically described as a negative percent pigment
area change (Figure 1D). In contrast, erythrophore cell response, upon exposure to S.
typhimurium ST10 mutant, was characterized by slight dispersion of pigment organelles
after 20 minutes exposure followed by slight aggregation that resulted in very little
53 change of pigment distribution after one hour of exposure to the ST10 mutant. The slight
dispersion followed by the slight aggregation of pigment organelles upon exposure to the
ST10 mutant, was negligible and not at the magnitude as the response observed upon
exposure to the dispersive agent, MSH, or the aggregative agent, Clonidine, or wildtype
S. typhimurium.
54 A
B
C
Change in pigment area (%) 40 30 20 10 0 -­‐10 -­‐20 -­‐30 -­‐40 -­‐50 0 10 20 30 40 50 60 Time (minutes) Figure 2: Betta splendens erythrophore cell response to wildtype Salmonella
typhimurium and Salmonella typhimurium ST10 mutant. Erythrophore cells before (left
panels) and after (right panels) 60 minute exposures to: (A) wildtype Salmonella
typhimurium, and (B) Salmonella typhimurium mutant ST10. Magnification of
erythrophore cells is at 100X. (C) Graphical representation of Erythrophore cell area
change in response to: () Salmonella typhimurium Wildtype, and (✕) Salmonella
typhimurium mutant, ST10. A negative area change describes an aggregation response,
and a positive area change describes a dispersion response. Data represent the mean
values of two trials. Error bars represent the standard deviation.
55 Identification Of hilD Gene Product And Complementation Of hilD
Function
The S. typhimurium ST10 mutant does not induce, in erythrophore cells, pigment
organelle translocation in a manner similar to wildtype S. typhimurium suggesting a
mutation interrupted a gene whose product is involved in this process.
Molecular
analysis of the ST10 mutant, using RATE PCR and sequencing the products, revealed
that the EZ-Tn5 mutagenic vector interrupted the hilD gene (Figure 3). As described
below, complementation of the mutant phenotype was used to demonstrate that a
mutation in hilD was responsible for the ST10 mutant phenotype. By adding back
wildtype hilD gene to the ST10 mutant, presumably this would add back the ability to
induce an aggregation of pigment organelles in erythrophore cells.
The complete sequence of the hilD gene, including an upstream partial Fur box
(FurB), was amplified from wild-type S. typhimurium genomic DNA using specific
primers (Figure 4). hilD was cloned into TOPO TA cloning vector, using TOPO TA
Cloning® Kit, and transformed into E. coli JM101. The confirmed TOPO::hilD plasmid
and pTA108 plasmid were double digested using the restriction enzymes Sal1-HiFi and
EcoR1-HiFi, followed by ligation.
The pTA108-hilD recombinant plasmid was
electroporated into ST10 mutant. As a control, the pTA108 vector was electroporated into
ST10 mutant. Transformants were selected on LB-Amp25 and successful construction of
pTA108-hilD and pTA108 were confirmed by restriction digest and sequence analysis
(Figure 4).
56 Figure 3: Identification of hilD gene: Illustration of the EZ-Tn5™ transposome
insertion in the hilD gene. EZ-Tn5™ was inserted using EZ-Tn5TM <KAN-2>Tnp
Transposome™ Kit. RATE PCR was used to determine the location of the EZ-Tn5™.
The EZ-Tn5™ was inserted in the middle of the hilD gene.
57 Figure 4: Cloning hilD: The complete sequence of the wildtype hilD gene and an
upstream partial Fur box (FurB) was amplified from wild-type S. typhimurium genomic
DNA. hilD was cloned into TOPO TA cloning vector and transformed into E. coli
JM101. TOPO::hilD plasmid and pTA108 plasmid were double digested with Sal1-HiFi
and EcoR1-HiFi, followed by ligation. The pTA108-hilD recombinant plasmid was
electroporated into ST10 mutant. As a control, the pTA108 vector was electroporated into
ST10 mutant. Transformants containing either pTA108-hilD or pTA108 were confirmed
by restriction digest and sequence analysis of the isolated plasmids.
58 Erythrophore Cell response To Salmonella typhimurium Wildtype, ST10
Mutant, ST10-pTA108 and ST10-pTA108-hilD
Betta splendens erythrophore cells were exposed to wildtype S. typhimurium, S.
typhimurium ST10 mutant, ST10 containing pTA108, ST10 containing pTA108-hilD.
Erythrophore cell response, upon exposure to wildtype S. typhimurium, was characterized
by aggregation of pigment organelles to the center of the cell, with this response initiating
approximately 30 minutes after exposure to this bacterial pathogen (Figure 5). The
aggregation response induced by S. typhimurium is described as a negative percent
pigment area change (Figure 5). In contrast, erythrophore cell response, upon exposure to
S. typhimurium ST10 mutant, was characterized by slight dispersion of pigment
organelles after 20 minutes exposure followed by slight aggregation that resulted in very
little change of pigment distribution after one hour of exposure to the ST10 mutant
(Figure 5). Erythrophore cell response, upon exposure to S. typhimurium ST10 mutant
containing just the vector, pTA108, was similar to the erythrophore cell response to the
ST10 mutant without the vector. Interestingly, Erythrophore cells response to st10
containing copy of wild type hilD gene did not show a response as to wildtype
S.typhimurium, which led me to question my experimental design and let it go longer
than the 60 minutes.
59 40 Change in pigment area (%) 30 20 10 0 -­‐10 -­‐20 -­‐30 -­‐40 -­‐50 0 10 20 30 40 50 60 Time (minutes) Figure 5: Betta splendens erythrophore cell response to wildtype Salmonella
typhimurium, Salmonella typhimurium ST10 mutant, ST10 containing pTA108, and ST10
containing pTA108-hilD. Graphical representation of erythrophore cell area change in
response to: () Salmonella typhimurium Wildtype, (x) Salmonella typhimurium mutant,
ST10, (˜) ST10-pTA108, and (u) ST10-pTA108-hilD. A negative area change
represents an aggregation response, and a positive area change presents a dispersion
response. Data represent the mean values of two trials. Error bars represent the standard
deviation.
60 The experimental design of the previous experiment was modified to ensure that
the result was not due to experimental artifact. Erythrophore cells were exposed to
wildtype S. typhimurium, S. typhimurium ST10 mutant, ST10 containing pTA108, ST10
containing pTA108-hilD for 90 minutes instead of 60 minutes (Figure 6). Erythrophore
cell response, upon exposure to wildtype S. typhimurium, was characterized by
aggregation of pigment organelles to the center of the cell, with this response initiating
approximately 30 minutes after exposure to this bacterial pathogen (Figure 6). The
aggregation response induced by S. typhimurium is described as a
negative percent
pigment area change (Figure 6). In contrast, erythrophore cell response, upon exposure to
S. typhimurium ST10 mutant, was characterized by slight dispersion of pigment
organelles after 20 minutes exp osure followed by slight aggregation that resulted in very
little change of pigment distribution after 90 minutes of exposure to the ST10 mutant
(Figure 6). Erythrophore cell response, upon exposure to S. typhimurium ST10 mutant
ontaining just the vector, pTA108, was similar to the erythrophore cell response to the
ST10 mutant without the vector (Figure 6). Interestingly, after 90 minutes exposure to S.
typhimurium ST10 mutant containing pTA108-hilD, erythrophore cell response appeared
to be more similar to that induced by wildtype S. typhimurium.
61 40 30 Area Change Figure20 5: Betta splendens erythrophore cell response to wildtype Salmonella
typhimurium, Salmonella typhimurium ST10 mutant, ST10 containing pTA108, and ST10
10 containing pTA108-hilD. Graphical representation of erythrophore cell area change in
response0 to: () Salmonella typhimurium Wildtype, (x) Salmonella typhimurium mutant,
ST10, (˜) ST10-pTA108, and (u) ST10-pTA108-hilD. A negative area change
-­‐10 an aggregation response, and a positive area change presents a dispersion
represents
response. Data represent the mean values of two trials. Error bars represent the standard
-­‐20 deviation.
-­‐30 -­‐40 -­‐50 -­‐60 0 10 20 30 40 50 Time (min) 60 70 80 90 Figure 6: Betta splendens erythrophore cell response to wildtype Salmonella
typhimurium, Salmonella typhimurium ST10 mutant, ST10 containing pTA108, and ST10
containing pTA108-hilD. Graphical representation of erythrophore cell area change in
response to: () Salmonella typhimurium Wildtype, (x) Salmonella typhimurium mutant,
ST10, (˜) ST10-pTA108, and (u) ST10-pTA108-hilD. A negative area change
represents an aggregation response, and a positive area change presents a dispersion
response. Data represent the mean values of two trials. Error bars represent the standard
deviation.
62 Betta Splendens Erythrophore Cell Response To Salmonella typhimurium
Bovine Isolate And Salmonella typhimurium Human Isolate
Salmonella typhimurium strains containing mutations in sipA, sopB, sopE2 and sptP genes were created in a S. typhimurium bovine isolate strain. To confirm if there is any difference between the erythrophore cells response to S. typhimurim bovine isolate and S. typhimurium human isolate, erythrophore cells were exposed to each one of the two strains. Figure 7 illustrates erythrophore cell response to S.
typhimurium bovine isolate and S. typhimurium human isolate. The data described in
Figure 7 were obtained from two different red B. splendens erythrophore cell
preparations. Erythrophore cell response, upon exposure to either S. typhimurium human
isolate or bovine isolate, was characterized by aggregation of pigment organelles to the
center of the cell, with this response initiating approximately 30 minutes after exposure to
either isolate of this bacterial pathogen (Figure 7). The aggregation response induced by
both S. typhimurium strains is mathematically described as a negative percent pigment
area change (Figure 7). Erythrophore cell response, upon exposure to the bacterial
culturing medium, Luria broth (LB), was characterized by slight dispersion, but
dispersion was negligible and not at the magnitude as the response observed upon
exposure to MSH (Figure 7).
63 40 Change in pigment area (%) 30 20 10 0 -­‐10 -­‐20 -­‐30 -­‐40 -­‐50 0 10 20 30 40 50 60 70 Time (minutes) Figure 7: Betta splendens erythrophore cell response to Salmonella typhimurium
(human isolate), Salmonella typhimurium wildtype (bovine isolate), and LB. Graphical
representation of erythrophore cell area change in response to: () LB, (u) S.
typhimurium bovine isolate, and (¢) S. typhimurium human isolate. A negative area
change represents an aggregation response, and a positive area change presents a
dispersion response. Data represent the mean values of two trials. Error bars represent the
standard deviation.
64 Betta Splendens Erythrophore Cell Response To Salmonella typhimurium
With Mutations In Effector Protein SopE2, SipA, Or SptP Genes
Erythrophore cells were exposed to wildtype S. typhimurium bovine isolate or
isogenic mutants of this strain containing mutations in specific effector protein genes.
Figure 8 illustrates erythrophore cell response to S. typhimurium bovine isolate and
isogenic mutant strains containing mutations in either sopE2, sipA, or sptP. The data
described in Figure 8 were obtained from two different red B. splendens erythrophore cell
preparations. Erythrophore cell response, upon exposure to S. typhimurium bovine isolate
is characterized by aggregation of pigment organelles to the center of the cell, with this
response initiating approximately 30 minutes after exposure to either isolate of this
bacterial pathogen (Figure 8). Mutations in sopE2, sipA, or sptP genes did not
dramatically change erythrophore cell response to S. typhimurium as the magnitude and
kinetics of the response was similar to that observed by wildtype S. typhimurium.
65 40 Change in pigment area(%) 30 20 10 0 -­‐10 -­‐20 -­‐30 -­‐40 -­‐50 -­‐60 0 10 20 30 40 50 60 70 Time (minutes) Figure 8: Betta splendens erythrophore cell response to wildtype Salmonella
typhimurium bovine isolate and specific effector protein mutants with mutations in
sopE2, sipA, or sptP. Graphical representation of erythrophore cell area change in
response to: () wildtype S. typhimurium bovine isolate, (˜) S. typhimurium sopE2
mutant, (¢) S. typhimurium sipA mutant, and (u) S. typhimurium sptP mutant. A
negative area change represents an aggregation response, and a positive area change
presents a dispersion response. Data represent the mean values of two trials. Error bars
represent the standard deviation.
66 Betta Splendens Erythrophore Cell Response To Salmonella typhimurium
With Mutations In Effector Protein SopB Or SipA/SopB Genes
Erythrophore cells were exposed to wildtype S. typhimurium bovine isolate or
isogenic mutants of this strain containing mutations in specific effector protein genes.
Figure 9 illustrates erythrophore cell response to S. typhimurium bovine isolate and
isogenic mutant strains containing mutations in either in sopB or sipA/sopB. The data
described in Figure 9 were obtained from two different red B. splendens erythrophore cell
preparations. Erythrophore cell response, upon exposure to S. typhimurium bovine
isolate, is characterized by aggregation of pigment organelles to the center of the cell,
with this response initiating approximately 30 minutes after exposure to this bacterial
pathogen (Figure 9). Mutations in sopB or sipA/sopB genes did change erythrophore cell
response to S. typhimurium as the magnitude and kinetics of the response was different to
that observed for the wildtype S. typhimurium. Although slight aggregation of pigment
organelles eventually occurred in the presence of either the sopB or sipA/sopB mutant
strains, this response was not to the magnitude observed by wildtype S. typhimurium.
67 40 Change in pigment area (%) 30 20 10 0 -­‐10 -­‐20 -­‐30 -­‐40 -­‐50 0 10 20 30 40 50 60 70 Time (minutes) Figure 9: Betta splendens erythrophore cell response to wildtype Salmonella
typhimurium bovine isolate and specific effector protein mutants with mutations in sopB
and sipA/sopB. Graphical representation of erythrophore cell area change in response to:
() wildtype S. typhimurium bovine isolate, (¢) S. typhimurium sopB mutant, and (u)
S. typhimurium sipA/sopB double mutant. A negative area change represents an
aggregation response, and a positive area change presents a dispersion response. Data
represent the mean values of two trials. Error bars represent the standard deviation.
68 Chapter 5
Discussion The Response of Betta Splendens Erythrophore To Salmonella
typhimurium ST10 Mutant
Chromatophore cells isolated from Betta splendens exhibit great potential as a
unique cell-based biosensor able to respond to the presence of biological and chemical
toxicants by dispersing or aggregating their pigment organelles (202, 207, 208, 204, 231,
237, 238). Numerous studies reveal that the chromatophore cell type, erythrophore, can
rapidly detect several food associated bacterial pathogens, including S. typhimurium (202,
207, 208, 204, 231, 237, 238). Previously tested hypotheses, using mutagenesis to
analyze the attributes of S. typhimurium contributing to a response in erythrophore cells,
revealed a potential role for the T3SS apparatus coded for by prgHIJK (231). Although
wildtype S. typhimurium rapidly induces pigment organelles to undergo an aggregative
response in erythrophore cells, S. typhimurium containing a mutation in the prgHIJK
operon does not induce pigment organelles to undergo an aggregative response in
erythrophore cells (231). A mutation (ST10 mutant) identified in a region different from
the mutation that disrupted the prgHIJK operon had a similar, but not identical impact on
erythrophore cells. The first objective of this study was to test the hypothesis that S.
typhimurium mutant ST10 has a mutation residing in either gene(s) coding for the process
regulating expression of the prgHIJIK operon or the gene(s) coding for the effector
proteins traveling through the PrgHIJK apparatus. The approaches used to test this
69 hypothesis included sequencing the region containing the mutation and identifying the
gene(s) impacted by this mutation through comparative sequence analysis, cloning the
wildtype gene sequence, and using this wildtype gene sequence to complement the
mutant phenotype thereby demonstrating the mutation is linked to the resulting biological
outcome in erythrophore cells.
Salmonella typhimurium mutant ST10 does not induce an aggregation response in
erythrophore cells similar to that observed with the isogenic wildtype S. typhimurium
strain. This suggested the mutation had interrupted a gene whose product(s) is involved in
induction of aggregation in erythrophore cells. Molecular analysis of the ST10 mutant
using the RATE PCR method revealed that the EZ-Tn5™ transposome had interrupted
the hilD gene. The 1221bp EZ-Tn5™ transposome was inserted in the hilD open reading
frame between nucleotide number 3018490 and 3018491 (NCBI, LT2 sequence
numbering), which corresponds to amino acid number 220 (Serine) (253).
The hilD gene resides between the hilA gene and the prgHIJK operon, with the
hilA gene oriented in the same direction as hilD and prgHIJK in the opposite direction on
Salmonella pathogencity island 1 (SpI-1) (Figure 10). Interestingly, most of other genes
located on the SPI-1 reside in opposite orientation of the hilD gene (Figure 10). The
regulatory region of the hilD gene contains a Fur box (putative Fur Box A at -191 to 163, and putative Fur Box B At -48 to -30) and a RNA polymerase binding site at -35
upstream of the start codon. HilD encodes a 309 amino acid protein with a described
function as an invasion regulatory protein (253). The location of the insertion in the hilD
gene and the orientation and regulatory features of this gene suggests the insertion has
70 only impacted hilD gene function, presumably through loss of hilD expression. A polar
effect on the hilA gene, which is adjecent to the hilD gene and in the same orientation,
was roled out for two reasons. One, hilA has its own promotor (255). Second, the distance
between the two genes is more than one thousand base pair, which is greater than typical
for genes in an operon.
Figure 10. Salmonella Pathogenicity Island 1 (SPI-1) Genetic Organization. This
figure shows the position of hilD on SPI-1. The blue colored arrows indicate the genes
and orientation of the genes encoding structural proteins of Type Three Secretion System
1 apparatus. The red colored arrows indicate the genes and orientation of the genes
encoding for transcriptional regulators. The blue outlined arrows represent the effector
proteins and effector proteins related proteins
The hilD gene product positively controls expression of hilA (87). HilA activates
and controls expression of SPI-1 and is responsible for inducing expression of the T3SS1 apparatus genes, prgHIJK, and/or expression of genes encoding effector proteins (86,
88). With respect to the first tested hypothesis in this study, it appears that S. typhimurium
ST10 mutant has a mutation residing in a gene coding for the process regulating
expression of the prgHIJIK operon, and this mutation impacts pigment organelle
movement. The impact of the hilD gene product on pigment organelle movement in
erythrophore cells raises two intriguing questions especially in light of what is previously
71 known about the role of the hilD gene product in the invasion of mammalian host cells.
First, why does a mutation in hilD result in failure of Salmonella typhimurium to induce
aggregation of pigment organelles in erythrophore cells? And second, does the hilD gene
product play a crucial role in inducing pigment organelle movement in erythrophore
cells?
Given that S. typhimurium has the transcriptional regulators HilC and RtsA, with
similar function as HilD, presumably HilC and RtsA can potentially duplicate HilD
function by positively controlling expression of hilA. The HilC and RtsA regulators are
capable of regulating SPI-1 through hilA or by directly binding to invF, leading to SPI-1
activation (247, 248) (Figure 11). However, this apparent duplication of function is not
enough to overcome the impact of a hilD mutation as it relates to pigment organelle
movement in erythrophore cells. Perhaps, the reason the hilD mutant cannot induce
pigment aggregation in erythrophore cells is due to other functions of the hilD gene
product. For example, the hilD gene product belongs to the AraC/XylS family, which
typically act as derepressors and not as typical activators of transcription (249). In S.
typhimurium, the hilD gene product activates hilA by counteracting the effect of negative
transcriptional regulators (e.g. Hha, HilE, Pag, and RNase E) that may repress hilA by
binding to an upstream site located in the regulatory region of the hilA promoter (Figure
11). Boddicker D. et. al. concluded that hilD gene is required for activating hilA
expression even when known negative regulators are absent (249), suggesting that hilD
was an absolute requirement and its activity could not be replaced. Given this
explanation, then presumably the failure to activate hilA expression through activity of
72 the hilD gene product would result in the failure to express the T3SS-1 apparatus genes,
prgHIJK, and/or expression of genes encoding effector proteins regardless of other
activating pathways. As demonstrated previously through mutational analysis, a mutation
disrupting the prgHIJK operon impacts pigment organelle movement in erythrophore
cells (231).
Figure 11: A model of transcriptional regulation of SPI-1. Green arrows indicate a
direct activation effect. Red lines indicate direct repression. The thick green and green
outlined arrows indicate the pathway leading to SPI-1 activation (248)
Salmonella typhimurium mutant, ST8, containing a mutation in the prgHIJK
operon responsible for encoding the T3SS-1 needle-like apparatus, does not induce
73 pigment organelles to aggregate in erythrophore cells by one hour, but after four hours
exposure to this mutant the pigment organelles do fully aggregate (231). This observation
suggests a potential second pathway, possibly similar to that encoded by prgHIJK, for S.
typhimurium to induce pigment organelle aggregation in erythrophore cells. Interestingly,
S. typhimurium mutant, ST10, containing a mutation in the hilD gene responsible for
regulating expression of hilA gene, does not induce aggregation of pigment organelles
even after four hours. This observation suggests there is no other activity that can
substitute for HilD and that this regulator is crucial to the S. typhimurium mechanism
involved in aggregating pigment organelles in erythrophore cells. HilD positively
regulates and enhances hilA expression, which is responsible for activating the prgHIJK
and invF operons, SPI-1 operons encoding T3SS needle-like apparatus and effector
proteins that lead to invasion, respectively. Similar to HilD, HilC (encoded within SPI-1)
and RtsA can also regulate the expression of hilA. The transcriptional regulators, hilD,
hilC, and RtsA regulate each other and themselves. Alternatively, HilD can directly
activate expression of the SPI-1 operon, invF, without regulating hilA (243). Given the
absolute requirement for the hilD gene product in the erythrophore cell response to S.
typhimurium, and the hypothesis that there might be duplicative activity to backup the
functions encoded by the prgHIJK operon, suggests there are other factors encoded by S.
typhimurium involved in the erythrophore cell response.
Salmonella typhimurium ST10 Mutant Complementation
Complementing the mutant phenotype displayed by the ST10 mutant was used to
demonstrate that a mutation in hilD was responsible for the ST10 mutant phenotype.
74 Presumably adding back a wildtype copy of the hilD gene would add back the ability to
induce an aggregation of pigment organelles in erythrophore cells. The wildtype hilD
gene, with partial Fur box (FurB), was cloned into a low copy number plasmid and
transformed in to the ST10 mutant. As hypothesized, the ST10 mutant containing a
wildtype copy of the hilD gene induced an aggregation response in erythrophore cells
similar to that induced by the isogenic wildtype strain of S. typhimurium. However,
erythrophore cell response to the ST10 mutant containing a wildtype copy of the hilD
gene was delayed and the magnitude of pigment organelle aggregation was reduced
compared to that induced by the isogenic wildtype strain of S. typhimurium. The delay in
the erythrophore cell response, and the reduced magnitude in pigment organelle area
change in erythrophore cells suggest that only partial complementation of the mutant
phenotype was achieved. Partial complementation due to more than one insertional
mutation was ruled out because the EZ-Tn5™ transposome was found only to be inserted
in the hilD gene. Partial complementation might have been due to either the vector used
or design of the insert used to clone hilD gene. Although the vector is a low copy number
vector with an origin of replication estimated to produce 2-3 copies per cell, this copy
number still might not have been low enough to express functional hilD under conditions
not toxic to the complemented mutant. Alternatively, the isolated DNA fragment cloned
into the low copy number vector coded for the full length open reading frame (ORF) of
hilD but only contained a short upstream sequence coding for a partial Fur box (FurB).
Perhaps, the fragment with the complete hilD gene needed to also contain a complete Fur
box (Fur A/B) for full complementation of the mutant phenotype. Recent studies have
75 shown that the Fur (ferric uptake regulator) protein (in its metal form), an iron
homeostasis regulator, binds to an AT-rich region (Fur A box) upstream of hilD promoter
to enhance hilD expression (248). Fur protein activates SPI-1 expression by enhancing
hilD expression (245, 246). Several reports suggest that Fur also negatively regulates HNS, a global regulator that negatively regulates all the transcriptional regulators, hilD,
hilC, RtsA, and hilA (Figure 10) (247, 248). Teixidó et al. concluded that Fur, in its
metal-bound form Fur.Fe2+, is needed to directly activate hilD expression (248).
Therefore, complementing a mutant hilD phenotype as it relates to pigment organelle
movement in erythrophore cells might require multiple layers of interactions between
regulators that might be difficult to perfectly reconstruct through simple complementation
of a mutant gene.
Betta Splendens Erythrophore Cells Response To Salmonella
typhimurium Effector Protein Mutants
The second objective of this study was to test the hypothesis that the effector
proteins of T3SS are the components of S. typhimurium’s invading mechanism directly
involved in inducing pigment organelles to undergo an aggregative response in
erythrophore cells. The approach used to test this hypothesis involved testing the
response of erythrophore cells to S. typhimurium strains containing targeted, well
characterized mutations in genes coding for specific T3SS effector proteins. Because
these targeted mutations were in a S. typhimurium strain isolated from bovine rather than
humans, it was necessary to first determine that erythrophore cells responded similarly to
S. typhimurium regardless of the source of isolation. Erythrophore cells respond to S.
76 typhimurium strains by aggregating their pigment organelles within 60-70 minutes
regardless of the source of isolation. Interestingly, the human isolate induces the
aggregation of pigment organelles slightly faster than the bovine isolate.
Mutations in sopE2, sipA, or sptP genes did not change erythrophore cell response
to S. typhimurium as the magnitude and kinetics of the response was similar to that
observed with wildtype S. typhimurium. Mutations in sopB or sipA/sopB genes did
change erythrophore cell response to S. typhimurium as the magnitude and kinetics of the
response was different to that observed for the wildtype S. typhimurium. Although slight
aggregation of pigment organelles eventually occurred in the presence of either the sopB
or sipA/sopB mutant strains, this response was not to the magnitude observed with
wildtype S. typhimurium.
Salmonella typhimurium sipA mutant induced an aggregation response in
erythrophore cells similar to S. typhimurium wildtype. This result suggests SipA is not
directly involved in pigment organelle movement in erythrophore cells. In leucocytes,
SipA enhances SipC activity by binding actin filaments to induce polymerization and
stabilize the actin filaments by up-regulating T-plastin activity, a localized actin bundling
protein (131-134). Previous studies have shown that S. typhimurium sipA mutant slightly
delay S. typhimurium invasion process of epithelial cells (85, 252).
Similar to the result with the sipA mutant, the S. typhimurium sopE2 mutant
induced an aggregation response in erythrophore cells similar to S. typhimurium wildtype
(Figure 8). This result suggests SopE2 is not directly involved in pigment organelle
77 movement in erythrophore cells. Pigment organelles in erythrophore cells move along
microtubule and actin filaments in an aggregation or dispersion response (250, 251).
Previous reports showed in leucocytes, SopE2 stimulates host Rho GTPase proteins
which are responsible for promoting the actin assembly and polymerization (138, 133).
Presumably SopE2 should be an important effector protein in the aggregation response in
erythrophore cells. Based on this prediction, a mutation in sopE2 might have had a
dramatic impact on the aggregation response in erythrophore cells. However, this
prediction was not realized with this experimental approach. Perhaps, because of the
redundancy between sopE and sopE2, the lack of SopE2 was compensated for by SopE,
thus, the sopE2 mutant’s ability to induce an aggregation response in erythrophore cells
was not impaired.
S. typhimurium sptP mutant also induced an aggregation response in erythrophore
cells similar to S. typhimurium wildtype. This result suggests SptP is not directly
involved in pigment organelle movement in erythrophore cells. SptP is involved in
restoring changes in the host membrane and promoting cell cytoskeleton recovery after
rearrangement of microfilaments and membrane enfolding. It was hypothesized that the
lack of SptP in S. typhimurium would result in an increase in S. typhimurium
invasiveness. In agreement with this hypothesis, we observed a slight increase in the
magnitude of pigment organelle aggregation in erythrophore cells in response to S.
typhimurium sptP mutant as compared to wildtype S. typhimurium.
In contrast to the results observed with the sipA, sopE2, and sptP mutants, S.
78 typhimurium sopB mutant and sipA/sopB double mutant induced a response in
erythrophore cells that was different in magnitude and kinetics to that observed for the
wildtype S. typhimurium. Both mutants induced slight pigment organelle aggregation,
after 55 minutes of exposure, in erythrophore cells; however, the magnitude of the
response was less than that observed with the wildtype S. typhimurium. The fact that the
sipA mutant is able to induce an aggregation response in erythrophore cells suggests that
the inability of the sipA/sopB mutant to induce a similar aggregation response in
erythrophore cells is due to lack of sopB. This is the first evidence in support of a role for
the SopB effector protein in pigment organelle aggregation in erythrophore cells. Even
though, it is clear the effector proteins secreted by T3SS-1 encoded by SPI-1 have a
certain degree of redundancy and they work in concert to facilitate S. typhimurium
invasion of the host cell, SopB appears to have a far more important role in inducing an
aggregation response in erythrophore cells than previously characterized in mammalian
host cells. SopB is an inositol phosphatase that contributes to extensive actin remodeling
in mammalian host cells by targeting host GEFs (guanine-nucleotide exchange factors) to
activate RhoG GTPase (138, 133). Additionally, SopB is involved in sealing the host
membrane in folding (139, 133).
Figure 12 illustrates a hypothetical model as to how S. typhimurium might impact
pigment organelle movement in erythrophore cells. Expression of hilD, in S.
typhimurium, is activated during late log growth phase. Fully expressed hilD, in turn,
activates expression of hilA. Expressed HilA activates expression of invF and prgHIJK,
SPI-1 operons. Alternatively, hilD can directly activate expression of the invF and
79 prgHIJK operons. Expressed InvF turns on the expression of genes responsible for
encoding the T3SS-1 effector proteins on SPI-1 or elsewhere. Activated prgHIJK operon
expresses the proteins that assemble in to the T3SS-1 needle-like apparatus. Presumably a
mutation in prgHIJK or hilD had a similar impact on erythrophore cell response because
both classes of mutations led to the failure to produce a needle like apparatus that could
deliver T3SS-1 effector proteins. Hypothetically, the T3SS-1 needle-like apparatus is
used to deliver the T3SS-1 effector proteins into the erythrophore cells whereby one or
several of the effector proteins directly impact pigment organelle movement. The
mutation in hilD had a more notable impact on pigment organelle movement, suggesting
that this function is crucial, presumably due to the genes regulated by HilD, and no other
regulatory protein can compensate for HilD function in S. typhimurium.
As illustrated in Figure 12, if the T3SS-1 needle-like apparatus has made contact
with the erythrophore cell, then effector proteins, such as SipA, SopB, SopE2, and SptP
might be injected into the erythrophore cell. If S. typhimurium effector proteins do gain
access to the erythrophore cell then how might they induce pigment organelle movement?
Although the results from this research effort did not suggest a direct role for SipA and
SopE2, given their function in leucocytes, these two effector proteins may be involved in
pigment organelle movement but other effector proteins may have similar function to
compensate for loss of SipA or SopE2 function. Additionally, S. typhimurium sptP
mutant exhibited an aggregation response in erythrophore cells with slightly higher
magnitude than the wildtype S. typhimurium. In leukocytes, SptP is secreted during the
early stage of pathogenesis after Salmonella enters the Salmonella containing vacuole
80 (SCV) and it reverses the activity of other effector proteins such as SopB, SopE, SopE2.
However, there is no evidence of SCV formation inside the erythrophore cell and the
secretion of SptP may require more time than the response that is observed in wildtype S.
typhimurium. This finding suggests that the internalization of S. typhimurium is not
required for inducing pigment organelle aggregation in erythrophore cells.
However,
results from this research effort suggest SopB has a role in triggering the movement of
the pigment organelles. SopB function may be mimicking function of proteins that are
involved in transporting pigment organelles along microtubules towards the perinuclear
region in erythrophore cell. Alternatively, SopB may affect the activity of a protein, such
as Rho G, that is involved in rearrangement of host cytoskeleton, resulting in pigment
organelle movement in the erythrophore cell. Results from this research effort suggest
that certain aspects of S. typhimurium’s pathogenic mechanism are involved in pigment
organelle movement in a neuron-like pigment cell, in this case an erythropore cell of the
chromatophore cell classification, isolated from the fish species, Betta splendens.
81 a. b. Figure 12. (a) A hypothetical model as to how S. typhimurium might enter an
erythrophore cell and the ensuing collaborative work of effector proteins of T3SS-1 to
induce pigment organelle movement. (b) Aggregated pigment organelles in the center of
an erythrophore cell.
82 Future Directions For This Research Effort
This study provides insight into how S. typhimurium induces pigment organelle
aggregation in B. splendens erythrophore cells. Previous studies have shown that S.
typhimurium requires a functional T3SS-1 needle-like apparatus for inducing an
aggregation response in B. splendens erythrophore cells. This study demonstrated that
effector protein, SopB, secreted by the T3SS-1 needle-like apparatus may also play a role
in pigment organelle aggregation in erythrophore cells. In addition, a major regulator of
SPI-1 expression, hilD, plays a crucial role in pigment organelle aggregation in
erythrophore cells. However, many factors involved in inducing a response in
erythrophore cells remain unclear. Further investigations of effector proteins such as
SipC and SipD will increase understanding and potentially reveal their involvement in
pigment aggregation of B. splendens erythrophore cells. Collectively, answering the why
and how erythrophore cells respond to S. typhimurium will result in better understanding
as to how to use this cell type as a biosensor for bacterial toxicity.
83 Chapter 6
Conclusion
This study describes Betta splendens erythrophore cells response to Salmonella
typhimurium, including an analysis of the bacterial mechanism(s) that may be involved in
this response. The outcome to this research effort is to increase understanding of the
biological basis of the erythrophore cell response to bacterial pathogens. Increased
understanding will lead to better utility of erythrophore cells as biosensors for bacterial
toxicity testing. Erythrophore cell-based biosensor is an emerging technology that has the
potential to respond to biological and environmental stimulants in a physiological manner
that can be measured and quantified. Cell based biosensors have the potential to
overcome the limitation of other molecular and immunology based methods that are not
function based.
This study tested the hypothesis that a S. typhimurium mutant, ST10, which no
longer induced a response in erythrophore cells, carried a mutation in either gene(s)
coding for the process regulating expression of the prgHIJI operon or the gene(s) coding
for the effector proteins that traveled through the PrgHIJK apparatus. Characterization of
ST10 mutant revealed that the EZ-Tn5™ transposome had interrupted the hilD gene.
Salmonella typhimurium mutant, ST10, containing an interrupted hilD gene was no
longer able to induce a response of pigment organelle aggregation in erythrophore cells.
The hilD wildtype gene, with partial Fur B box sequence, was cloned and the hilD
mutant, ST10, was complemented with this construct to demonstrate that the mutation is
84 linked to the resulting biological outcome of a mutant hilD. The result showed that
complemented ST10 strain induced an aggregation response in erythrophore cells similar
to the response induced by the wildtype S. typhimurium strain. However, the
erythrophore cell response induced by the complemented ST10 strain was delayed and
pigment organelle movement was reduced in magnitude as compared to that observed
with the wildtype S. typhimurium strain.
The second hypothesis tested in this study was that the effector proteins of T3SS
are the components of S. typhimurium’s invading mechanism directly involved in
inducing pigment organelles to undergo an aggregative response in erythrophore cells. To
test this hypothesis, the response of erythrophore cells to S. typhimurium strains
containing targeted, well characterized mutations in genes coding for T3SS effector
proteins SipA, SopB, SopE2 and SptP were analyzed. Mutations in either the sipA, sopE2
or sptP genes resulted in S. tyhpimurium mutants with a phenotype similar to the
wildtype S. typhimurium strain: all induced pigment organelle aggregation in
erythrophore cells. This resulted suggested that the SipA, SopE2 and SptP effector
proteins are not directly involved in the erythrophore response to S. typhimurium. In
contrast, S. typhimurium sopB and sipA/sopB mutants induced a response in erythrophore
cells that was different in magnitude and kinetics to that observed for the wildtype S.
typhimurium. Both mutants caused some aggregation (after 55 minutes of exposure) in
erythrophore cells; however, the magnitude of the response was lower than that of
observed by S. typhimurium wildtype. Presumably this altered response is due to a mutant
sopB function. This is the first evidence that S. typhimurium T3SS effector protein, SopB,
85 is directly involved in inducing a response, consisting of pigment organelle aggregation,
in B. splendens erythrophore cells.
86 Chapter 7
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109 Appendix
110 Biographical Information
Mohammad Farhad was born on October 10, 1987 in Kabul, Afghanistan to Haji
Gul Mohammad and Bibi Habiba. He was raised, until the age of six, in the middle of
civil war between the different Mujahideen groups following the withdrawal of the Soviet
army in 1989 from Afghanistan. The situation in Kabul worsened and his father’s life
was in risk because he was a Colonel in the Afghan National Army. His family migrated
to Quetta, Pakistan in 1995. His family suffered from extreme poverty in lawless regions
of Quetta. His father and older brother insisted on educating Farhad and his younger
siblings. He finished middle school, attended an English Language Center and worked in
a local pharmacy in Pakistan. He speaks five languages: Pashto (native language), Dari,
Farsi, Urdu and English.
After the U.S. Army invaded Afghanistan, his family returned to Kandahar,
Afghanistan in 2002. At the age of sixteen he was appointed as an instructor of computer
software and hardware in a newly established Computer Institute for one year. While still
attending High School, he worked for two years as an interpreter and translator for the
U.S. Army in Afghanistan to minimize the misunderstanding between Afghanis and U.S
Army. He graduated from Zahir Shahi High School in Kandahar in 2005. With the help
of his friend, Mike Manaton, he came to the United States in 2006 in pursuit of his higher
education. In June of 2008, he graduated from Portland Community College with an
Associate of Science degree with highest academic honors. He was awarded the PCC
Foundation Scholarship and the Dan and Kathy Moriarty scholarship for his academic
111 excellence. He received the first ever International Student Key Note Speaker Award. He
was also a member of Phi Theta Kappa Honor Society.
In 2008, he transferred to Oregon State University to pursue a Bachelor of
Science degree in Biology with a pre-medicine option. He was awarded an International
Cultural Service Program (ICSP) scholarship for educating people on the situation in
Afghanistan. He joined the Honors College at Oregon State University. As part of his
Honors College requirements he had to conduct research, write a thesis and defend it. He
conducted his research in Dr. Janine Trempy’s laboratory in the Department of
Microbiology as an undergraduate lab research assistant. He investigated diarrheal
disease in rural villages on the outskirt of Kabul. He completed his research and defended
his undergraduate thesis, titled: “Presence of Diarrhea and its Intensity due to Human
Behaviors in Deh Sabz, Kabul, Afghanistan.” He was awarded two scholarships from the
College of Science at Oregon State University: 1) Doc Gilfillan Scholarship and 2)
Science Scholar Scholarship. Mohammad Farhad graduated in 2010 from Oregon State
University with a Bachelors of Science degree in Biology with Honors. His compelling
life story, as well as his successes at Oregon State University, was one of four student
stories selected by Oregon State University’s President Ray to share at graduation
commencement.
In 2010, he was accepted into the Graduate Program in the Department of
Microbiology at Oregon State University and awarded a Graduate Research Assistantship
in Dr. Trempy’s laboratory to pursue his Masters of Science degree in Microbiology. In
addition to his research, he also served as a teaching assistant for the Department of
112 Microbiology’s lab courses. He defended his master’s thesis, “The Role of Salmonella
typhimurium Type Three Secretion System (T3SS) and Effector Proteins in Erythrophore
Cell Response”, on May 21st 2013, and graduated from OSU on Jun 15th 2013.
Mohammad Farhad was accepted into the Ph.D. Graduate Program in Molecular and
Cellular Biosciences at Oregon Health and Science University in Portland, OR. He will
begin his graduate studies for a Ph.D., September 2013.
Manuscripts in Preparation
1. Dierksen, K.P., Roach, H.B., Hutchison, J.R., Dukovcic, S., Farhad, M., Garcia, R.,
and Trempy, J.E. 2013. Biosensors employing chromatophore cells: a comparative
analysis of chromatophore cell types in the detection of food associated bacterial
pathogens. Manuscript in prep.
2. Dukovcic, S., Farhad, M., Dierksen, K.P., Hutchison, J.R., and Trempy, J. E. 2013.
The role of prgHIJI and hilD in the chromatophore cell response to Salmonella
typhimurium. Manuscript in prep.
3. Farhad, M., Dierksen, K.P., and Trempy, J.E. 2013. Salmonella typhimurium effector
proteins and their role in pigment organelle movement in chromatophore cells.
Manuscript in prep.