bioactive components from lactobacillus acidophilus and
TRANSCRIPT
Bioactive Components from Lactobacillus acidophilus and Lactobacillus
helveticus Fermented Milk Enhance Epithelial Membrane Integrity against
Salmonella enterica serovars Typhimurium Infection
by
Jingya Peng
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
in
Food Science
Guelph, Ontario, Canada
© Jingya Peng, May, 2014
ABSTRACT
BIOACTIVE COMPONENTS FROM LACTOBACILLUS ACIDOPHILUS AND
LACTOBACILLUS HELVETICUS FERMENTED MILK ENHANCE EPITHELIAL
MEMBRANE INTEGRITY AGAINST SALMONELLA ENTERICA SEROVARS
TYPHIMURIUM INFECTION
Jingya Peng Advisor:
University of Guelph, 2014 Professor Mansel W. Griffiths
Previous in vitro and in vivo studies showed that bioactive components produced by Lactobacillus
acidophilus (La-5) and Lactobacillus helveticus (LH-2) exerted protective effects against a variety of
enteric pathogens infection, including Salmonella. The purpose of this study is to determine the
mechanism of protective effect from La-5 and LH-2 against Salmonella Typhimurium infection on
epithelial cells.
Cell free spent medium (CFSM) were obtained after 48 h fermentation in whey protein-based medium
or milk for La-5 and LH-2, respectively. Human colonic carcinoma HT-29 cells were grown in
Transwell inserts for 40 days until they were polarized (TEER ~120 ohm×cm2). Cells were
pre-incubated with CFSMs 24 h prior to Salmonella infection and co-incubated during infection.
Lactate dehydrogenase (LDH) activity and TUNEL apoptotic assay were tested. Finally, an invasion
assay was carried out using chicken hepatoma LMH cells as an in vitro model for Salmonella
presence in poultry.
Results shown that Salmonella induced TEER loss was attenuated when pre-incubated and
co-incubated with non-toxic dose of CFSMs at 8th h post-infection (P <0.05). LDH was reduced up to
49.1% (La-5) and 46.8% (LH-2) (P <0.01). In terms of apoptosis, 75.8% (La-5) and 62.5% (LH-2)
less apoptotic cells were observed (P <0.01). The effectiveness of Salmonella invasion on LMH cells
was strain dependent. Less than one log10 cycle reduction was obtained when La-5 or LH-2 CFSM
was applied.
These data suggest that LH-2 and La-5 have antagonized function against Salmonella infection in
epithelial cells by enhancing epithelial membrane integrity and would bring further beneficial
evidence of consuming bioactives as functional supplements.
iii
ACKNOWLEDGEMENT
First and foremost, I would like to thank my supervisor, Prof. Mansel W. Griffiths, for his insightful
guidance and financial support. I thank him for giving me this chance and becoming someone whom I
can always rely on whenever I needed help throughout the course of my research. My sincerest
gratitude goes to my charming and intelligent committee members, Prof. Milena Corredig and Dr.
Angela Tellez, whose encouragements and suggestions always contributed to improving my work and
inspiring me in not only my research but also my life.
My appreciation extends to my fellow friends in the Canadian Research Institute for Food Safety and
Food Science department, especially to Rocio Morales, Sapana Sharma, Ruiqin Wu and Sandy Smith.
Thank you for all the encouragements, support and trust they provided for me. I would not arrive at
this stage of my life without all their help.
Above all, my greatest gratitude goes to my parents. I thank them for giving birth to me and being my
role models. I deeply appreciate their encouragement on challenging myself and chasing my dreams. I
thank my aunt, uncle and cousin in Toronto for their care throughout my study in Canada. Finally, I
thank my boyfriend for his unconditional love and support. I would not finish the Master degree on
time without the support from all of them.
Lastly, I thank the University of Guelph for endowing me with this precious journey. I will cherish
everything that I have learnt in Guelph. This moment is not a happy ending, but the beginning to a
new adventure, as my motto says, “live and learn”.
iv
TABLE OF CONTENTS
Abstract
Acknowledgements………………………………………………………...…………………………..iii
Table of contents…………………………………………………………………………………….....iv
List of tables……………………………………………………………………………………………vi
List of figures………………………………………………………………………………………….vii
List of abbreviations………………………………………………………………………………….viii
CHAPTER 1 .......................................................................................................................................... 1
Introduction ............................................................................................................................................. 1
Literature Review .................................................................................................................................... 2
1. Salmonella spp. ............................................................................................................................... 2
2. Probiotics and Their Fermented Products ....................................................................................... 3
3. Bioactive Peptides ........................................................................................................................... 8
Definition and Characteristics ......................................................................................................... 8
Bioactive Peptides Derived from Milk ........................................................................................... 8
4. Intestinal Epithelial Cells .............................................................................................................. 12
Epithelial Cells Polarization.......................................................................................................... 12
Trans-epitheliall Electrical Resistance .......................................................................................... 13
Epithelial Cells Immune Response to Salmonella Invasion ......................................................... 14
5. Apoptosis....................................................................................................................................... 15
Characteristics ............................................................................................................................... 15
Molecular Basics of Apoptosis ..................................................................................................... 18
Assays for Apoptosis ..................................................................................................................... 18
Flow Cytometry ............................................................................................................................ 19
Probiotics Protection against Pathogen-induced Apoptosis .......................................................... 20
Objectives ............................................................................................................................................. 23
CHAPTER 2: METHODOLOGY ..................................................................................................... 24
La-5 Cell Free Spent Medium (CFSM) Preparation ............................................................................. 24
LH-2 Cell Free Spent Medium (CFSM) Preparation ............................................................................ 25
Salmonella Strain and Growth Conditions ............................................................................................ 26
Cell Culture and Maintenance............................................................................................................... 27
Trypan Blue Exclusion Assay ............................................................................................................... 28
Confocal Scanning Laser Microscopy (CSLM) .................................................................................... 28
Trans-epithelial Electrical Resistance (TEER) ..................................................................................... 30
Epithelial monolayer Integrity Challenge Study ................................................................................... 31
Lactate Dehydrogenase (LDH) Cytotoxicity Assay .............................................................................. 32
Apoptosis TUNEL Assay ...................................................................................................................... 33
Sulforhodamine B (SRB) Assay ........................................................................................................... 34
Invasion Assay ...................................................................................................................................... 35
Statistical Analysis ................................................................................................................................ 37
CHAPTER 3: RESULT AND DISCUSSION ................................................................................... 38
Trypan Blue Exclusion Assay ............................................................................................................... 38
Confocal Scanning Laser Microscopy .................................................................................................. 40
v
Trans-epithelial Electrical Resistance (TEER) ..................................................................................... 43
Epithelial Barrier Integrity Study .......................................................................................................... 47
LDH Cytotoxicity Assay ....................................................................................................................... 54
Apoptosis Assay Using Flow Cytometry .............................................................................................. 58
SRB Assay ............................................................................................................................................ 66
Invasion Assay ...................................................................................................................................... 69
Conclusions ........................................................................................................................................... 71
CHAPTER 4 ........................................................................................................................................ 73
General Conclusions ............................................................................................................................. 73
Future Research .................................................................................................................................... 75
REFERENCE ...................................................................................................................................... 76
vi
LIST OF TABLES
TABLE PAGE
CHAPTER 1
Table 1.1 In vitro studies focus on pathogenic bacteria inhibitory function of probiotics………………….5
Table 1.2 In vivo studies focus on pathogenic bacteria inhibitory function of probiotics……………….....6
Table 1.3 Bioactive peptides identification within the fraction of cell-free supernatant from L. helveticus
LH-2 fermented milk using mass spectrometry and NCBI databas. ………………………………………11
Table 1.4 Bioactive peptides identification within the fraction of cell-free spent modified MRS medium
fermented with L. acidophilus La-5 using mass spectrometry and BLASTp………………………………11
CHAPTER 2
Table 2.1 Bacterial strains used in these studies …………………………………………………………..27
vii
LIST OF FIGURES
FIGURE PAGE
CHAPTER 1
Figure 1.1 Milk protein-derived bioactive peptides and their health promoting targets……………………9
Figure 1.2 Schematic representations of three forms of cell death. See context for detail description……17
Figure 1.3 Schematic representation of a flow cytometry….……………………………………………..22
CHAPTER 2
Figure 2.1 Catalytic function of the Lactate Dehydrogenase (LDH) enzyme……………………………..32
CHAPTER 3
Figure 3.1 La-5 and LH-2 toxic dose test on HT-29 cells proliferation, estimated by trypan blue exclusive
assay………………………………………………………………………………………………………..39
Figure 3.2 Confocal image of a single S. Typhimurium GFP cell .……………………………………….41
Figure 3.3 Merged confocal images of S. Typhimurium GFP invasion of HT-29 cells…………………...42
Figure 3.4 Determination of optimal HT-29 cell density in Transwell permeable inserts………………..45
Figure 3.5 Growth of HT-29 cells in Transwell permeable inserts………………………………………..46
Figure 3.6 Ratio of TEER of polarized HT-29 cell monolayers after exposure to Salmonella Typhimurium
to initial values.…………………………………………………………………………………………….50
Figure 3.7 Temperature dependent of TEER on polarized MDCK (Madin-Darby Canine Kidney) cells
grown on permeable inserts for 5 days ……………………………………………………………………54
Figure 3.8 Cytotoxicity (%) of HT-29 cells produced by S. Typhimurium at different times of exposure and
different MOI ……………………………………………………………………………………………..55
Figure 3.9 Evaluation of CFSMs incubation conditions………………………………………………….56
Figure 3.10 LDH % cytotoxicity of epithelial cells induced by Salmonella Typhimurium in the presence or
absence of CFSM treatment ………………………………………………………………………………57
Figure 3.11 Effect of CFSM on Salmonella Typhimurium induced apoptosis of polarized HT-29 cells...59
Figure 3.12 Apoptotic rate of polarized HT-29 cells and the protective effect of CFSM pre-incubation (24 h)
and co-incubation during exposure to Salmonella (1 h) (Flow cytometric TUNEL analysis)……………62
Figure 3.13 Cell viability by comparing efficiency of FITC (Y-axis)/PI (X-axis) dual stain signals……63
Figure 3.14 La-5 and LH-2 CFSM toxic dose test on LMH cells proliferation, estimated by SRB
colorimetric assay…………………………………………………………………………………………68
Figure 3.15 Invasion assay on LMH cell using Salmonella Typhimurium DT104 (SA2001-4368 &
SA2000-0406) isolated from chicken for 1 h infection with MOI of 10 …………………………………70
Figure 3.16 Invasion assay using LMH cells exposed to different infection conditions…………………70
viii
LIST OF ABBREVIATIONS
ANOVA
ATCC
CDC
CFSM
CFU
CSLM
EHEC
FACS
FAO
FITC
GFP
GI Tract
IEC
IFN-γ
IgG
IL-8
La-5
LAB
LDH
LH-2
NLR
MDCK
MOI
OD
PBS
PE
PI
S-IgA
SPI
SRB
T3SS
TCA
TEER
TJ
TLR
TNF-α
TSA
TSB
TUNEL
WHO
Analysis-of-Variance
American Type Culture Collection
Center for Disease Control
Cell Free Spent Medium
Colony-Forming Unit
Confocal Scanning Laser Microscopy
Enterohemorrhagic Escherichia coli
Fluorescence-Activated Cell Sorting
Food and Agriculture Organization
Fluorescein Isothiocyanate
Green Fluorescent Protein
Gastrointestinal Tract
Intestinal Epithelial Cell
Interferon Gamma
Immunoglobulin G
Interleukin 8
Lactobacillus acidophilus La-5
Lactic Acid Bacteria
Lactate Dehydrogenase
Lactobacillus helveticus LH-2
Nod-like Receptor
Madin-Darby Canine Kidney
Multiplicity of Infection
Optical Density
Phosphate-Buffered Saline
Phycoerythrin
Propidium Iodide
Secretory IgA
Salmonella Pathogenicity Island
Sulforhodamine B
Type Three Secretion System
Trichloroacetic Acid
Trans-epithelial Electrical Resistance
Tight Junction
Toll-like Receptors
Tumor necrosis Factor Alpha
Trypticase Soy Agar
Trypticase Soy Broth
Terminal eoxynucleotidyl transferase dUTP nick end labeling
World Health Organization
1
CHAPTER 1
Introduction
The public health effects and financial costs incurred following contamination of food with Salmonella
can be severe. In addition, the increase in antibiotic resistance of Salmonella has stressed the need to
find therapeutic alternatives. One such alternative may rest with bioactive peptides, especially those
produced following fermentation of milk by lactic acid bacteria (LAB). A variety of studies have
revealed the health promoting benefits of these peptides, including their ability to interfere with
virulence mechanisms of pathogens. Based on previous in vitro and in vivo observations made in our
laboratory, bioactive fractions from cell-free spent medium (CFSM) of L. acidophilus and L.
helveticus showed potent immunomodulating effects on the host and suppression of infectivity of
pathogens (Chin, 2002; Ding, Wang, & Griffiths, 2005; M. J. Medellin-Peña, 2007; Ng, 2000; Tellez
Garay, 2009). However, the mechanisms whereby CFSMs exert these protective functions on
epithelial cells are still not fully understood. We hypothesize that CFSMs enhance resistance of
epithelial cells against Salmonella infections by maintaining cell membrane integrity and constrain
Salmonella induced apoptosis. Herein, we propose to have a better comprehension of the mechanisms
behind these effects, which could support the use of bioactive components from LAB fermented milk
as supplements in functional food.
2
Literature Review
1. Salmonella spp.
Salmonella are one of the main causes of human food-borne illnesses. Digestion of Salmonella
contaminated food leads to two major disease patterns in humans: a systemic disease (typhoid fever)
and a self-limiting gastrointestinal illness, salmonellosis (Caron et al., 2006). Non-typhoidal
Salmonella are the strains mainly associated with gastrointestinal infections. Approximately 1.2 million
illnesses, 23,000 hospitalizations, and 450 deaths result from non-typhoidal Salmonella infections
every year in the United States, resulting in an estimated $365 million dollars spent in direct medical
costs annually (CDC, 2013). No significant decline of Salmonella infection has been found in more than
a decade (CDC, 2011), whereas the increasing resistance of Salmonella to clinical-used antibiotics
continues to be noticed since 1996. In 2013, CDC showed that around 5% non-typhoidal Salmonella
were resistant to five or more types of antibiotic (CDC, 2013).
The taxonomy and nomenclature of the genus Salmonella used to be a prevalent topic (Tindall,
Grimont, Garrity, & Euzeby, 2005). Over 2500 serotypes are found in the Salmonella genus, and
possibly all of them are pathogenic (Burkholder & Bhunia, 2009). In 2007, the World Health
Organization (WHO) defined that Salmonella consisted of only two species according to molecular
detection, S. enterica and S. bongori. There are 6 subspecies in S. enterica, namely S. enterica subsp.
enterica; S. enterica subsp. salamae; S. enterica subsp. arizonae; S. enterica subsp. diarizonae; S.
enterica subsp. houtenae and S. enterica subsp. indica. Specifically, Salmonella enterica serovar
Typhimurium (S. Typhimurium) is one of the most common species involved in human food-borne
illnesses, and often contaminates poultry, pork, beef and dairy products (Burkholder & Bhunia, 2009;
Gaggìa, Mattarelli, & Biavati, 2010). As a highly antibiotic-resistant strain, S. Typhimurium DT104
3
has attracted a great deal of public health attention (Burkholder & Bhunia, 2009; Wu, Carlson, &
Meyerholz, 2002). In addition to its clinical value, Salmonella is an interesting bacterial model to
examine for host-pathogen interaction since it can manipulate the functions of the host cells in order
to prolong its own survival. Its virulence functions are connected to Salmonella pathogenicity islands
(SPI1 and SPI2) and it possesses a needle-like Type Three Secretion System (T3SS) (Bayoumi &
Griffiths, 2010). Therefore, S. Typhimurium DT104 strains were selected in this study to test for in
vitro interference of epithelial cell functions.
2. Probiotics and Their Fermented Products
The Food and Agriculture Organization of the United Nations (FAO) and the World Health
Organization (WHO) defined probiotics as live microorganisms which, when administered in adequate
amounts, confer a health benefit on the host (FAO & WHO, 2001; Khani, Hosseini, Taheri, Nourani, &
Fooladi, 2012). Probiotics include bacteria, molds, and yeasts, among which lactic acid bacteria (LAB)
are one of the most intriguing groups due to their presence in yogurt, fermented milks and other
fermented foods. After Grigoroff isolated the first LAB in 1905 (Grigoroff, 1905), numerous studies
of LAB have been done to ensure LAB fermented foods have health benefits along with a GRAS
status (generally recognized as safe) (Divya, Varsha, Nampoothiri, Ismail, & Pandey, 2012). It is
reported in many reviews that benefits of consuming LAB and their bioactive components include: (1)
aiding in digestion and nutrient assimilation; (2) stimulating the immune system; (3) competing with
unfavorable pathogens and producing antimicrobial bioactive molecules; (4) preventing the risk of
certain cancers; (5) reducing the prevalence of allergy in susceptible individuals; (6) alleviating
symptoms of lactose intolerance and diarrhea; (7) lowering serum cholesterol concentrations and (h)
reducing blood pressure in hypertensive individuals (Divya et al., 2012; Khani et al., 2012; Masood,
4
Qadir, Shirazi, & Khan, 2011; Parvez, Malik, Kang, & Kim, 2006; Schrezenmeir & De Vrese, 2001).
Although it is well known that consuming LAB fermented foods exert positive effects on human and
animal health, possible mechanisms behind these effects still remain largely unknown. The ingestion of
LAB is considered as a promising alternative for antibiotic in order to deal with pathogens‟ increased
antibiotic resistance. Many in vivo and in vitro studies have focused on using specific LAB strains, with
or without their culture medium, which exert antagonistic effects against various types of pathogens.
Tables 1.1 and 1.2 summarize information on protection afforded to the host against specific pathogens
by probiotics for both in vivo and in vitro studies, respectively.
5
Table 1.1 In vitro studies focus on pathogenic bacteria inhibitory function of probiotics.
Probiotic Sample format Targeted
pathogen Findings Reference
Lactobacillus
plantarum 299v &
Lactobacillus
rhamnosus GG
Live microorganisms Escherichi coli Inhibited enteropathogenic E. coli adherence in vitro by
inducing intestinal mucin gene expression.
(Mack, Michail, Wei,
McDougall, & Hollingsworth,
1999)
Lactobacillus spp. Live microorganisms
Yersinia
enterocolitica
DSM4780
Affected the likelihood of Y. enterocolitica survival by
compromising urease functionality and cell viability.
(Lavermicocca, Valerio,
Lonigro, Di Leo, & Visconti,
2008)
Lactobacillus casei
strain Shirota
Live microorganism &
cell-free culture
supernatant
Helicobacter
pylori SS1
Inhibited H. pylori growth on solid agar as well as in liquid
medium with the presence of living L. casei strain Shirota. (Sgouras et al., 2004)
Lactobacillus casei
Rhamnosus Live microorganism
Escherichia coli
C25
Inhibited bacterial translocation of E. coli C25 in a
dose-dependent manner.
(Mattar, Drongowski, Coran,
& Harmon, 2001)
Lactobacillus casei
& Lactobacillus
acidophilus
Live microorganism Shigella sonnei Lactobacillus affected shigella growth rate. (Apella, Gonzalez, Demacias,
Romero, & Oliver, 1992)
Lactobacillus
acidophilus La5 cell-free spent medium
Escherichia coli
O157:H7
L. acidophilus La-5 secreted a molecule(s) that could block or
interfere with EHEC‟s virulence genes involved in
colonization.
(M. Medellin-Peña, Wang,
Johnson, Anand, & Griffiths,
2007; M. Medellin-Peña &
Griffiths, 2009)
Bifidobacterium
bifidum Cell-free spent medium
Salmonella
Typhimurium
Down-regulated S. Typhimurium reporter gene expression
driven by both hilA and ssrB at a dose dependent manner. (Bayoumi & Griffiths, 2010)
Bifidobacterium
bifidum
fraction from Cell-free
culture medium
Salmonella
Typhimurium &
Fraction #67 and #68 down-regulated S. Typhimurium and
E. coli reporter gene expression driven by hilA and ssrB, (Bayoumi & Griffiths, 2012)
6
Escherichia coli LEE1, respectively; reduced pathogens colonization on
eukaryotic cells; diminished survival and multiply capacities
of Salmonella within macrophages.
Lactobacilli spp.,
Bifidobacteria spp.,
Lactococcus lactis
& Stococcus
thermophilus
Cell-free extracts from
fermented milk
Campylobacter
jejuni
Cell-free extracts from ten probiotic bacteria inhibited
expression of the C. jejuni flaA s28 promoter, which was
independent of pH and lactic acid concentration. Two
non-probiotic lactic acid bacterial strains, Lactococcus lactis
and Stococcus thermophilus, were less inhibitory.
(Ding et al., 2005)
Lactobacillus
crispatus K313 and
K243
S-proteins
Salmonella
Braenderup
H9812
Inhibited S. Braenderup H9812 adherence to HT-29 cells;
attenuated the response of HT-29 infected with S.
braenderup H9812.
(Sun et al., 2012)
Lactobacillus
reuteri & Bacillus
licheniformis
Live microorganism
Salmonella
Typhimurium &
Salmonella
Choleraesuis
S. Typhimurium stimulated secretion of IL-8 was inhibited
basolaterally in the presence of B. licheniformis.
(Skjolaas, Burkey, Dritz, &
Minton, 2007)
Table 1.2 In vivo studies focus on pathogenic bacteria inhibitory function of probiotics.
Probiotic Sample format Targeted pathogen Findings Reference
Lactobacillus casei
strain Shirota
Live microorganism
& cell-free culture
supernatant
Helicobacter pylori SS1
Administration of L. casei strain Shirota reduced colonizing H.
pylori viable counts and the associated inflammation of the
gastric mucosa in the H. pylori SS1 murine infection model.
(Sgouras et al., 2004)
Lactobacillus
salivarius UCC118 Live microorganism Listeria monocytogenes
Produced bacteriocin Abp118 that could significantly protect
mice against infection with the invasive foodborne pathogen L.
monocytogenes.
(Corr et al., 2007)
7
Lactobacillus casei
GG Live microorganism Escherichia coli K1
Decreased the frequency of E. coli K1A translocation in a
neonatal rabbit model.
(Lee, Drongowski,
Coran, & Harmon, 2000)
Lactobacillus casei
& Latobacillus
acidophilus
Fermented milk
Listeria monocytogenes
& enteroinvasive
Escherichia coli
Higher survival rate, higher levels of anti-pathogen sera &
intestinal, less pathogen colonization of liver & spleen were
observed among LAB treated mice when challenged with
pathogens.
(Demacias, Romero,
Apella, Gonzalez, &
Oliver, 1993)
Lactobacillus casei Live microorganism Salmonella
Typhimurium
Oral administration of LAB increased the mucosal intestinal
immunity.
(Perdigon, Alvarez,
Demacias, Roux, &
Holgado, 1990)
Lactobacillus casei
& Lactobacillus
acidophilus
Ferment milk Salmonella
Typhimurium
Anti-salmonella protective immunity mainly mediated by the
mucosal tissue using L. casei + L. acidophilus mixture
fermented milk.
(Perdigon, Demacias,
Alvarez, Oliver, &
Holgado, 1990)
Lactobacillus
helveticus
A peptidic fraction
from fermented milk
Salmonella
Typhimurium
A peptide fraction composed of α-lactalbumin and β-casein
derived peptides played a dose-dependent role in protecting
mice against Salmonella translocation; mechanism involved
cell-mediated immune response and inference with virulence
gene expression.
(Tellez, Corredig,
Turner, Morales, &
Griffiths, 2011)
Lactobacillus
helveticus
cell-free fractions
from fermented milk
Bioluminescent
Salmonella Enteritidis
(lux CDABE)
Bioluminescence emitted by mice and the physical condition
of the mice indicated that animals fed with fermented milk or
fermented milk components prior to infection were less
susceptible to bacterial colonization and, subsequently,
bacteremia.
(Brovko et al., 2003)
Lactobacillus
acidophilus La5
cell-free spent
medium
enterohemorrhagic
Escherichia coli
(EHEC).
Cell-free spent medium fractions were able to down-regulate
several virulence genes of EHEC, including stxB2, qseA, luxS,
tir, ler, eaeA, and hlyB.
(Zeinhom et al., 2012)
8
3. Bioactive Peptides
Definition and Characteristics
Bioactive peptides are defined as specific protein fragments that have a positive impact on body
functions and conditions, ultimately benefiting health (Kitts & Weiler, 2003). Most bioactive peptides
are obtained from proteins of animal origin, such as milk, egg, gelatin, fish, as well as plant proteins
such as wheat gluten and soy (Elawadli, 2012). During digestion in the gastrointestinal tract, bioactive
peptides can be generated from inactive parental protein sequences through three ways of proteolysis:
(1) enzymatic hydrolysis by digestive enzymes, (2) fermentation with proteolytic starter cultures and
(3) proteolysis by enzyme derived from microorganisms or plants (Korhonen, 2009; Muro Urista,
Alvarez Fernandez, Riera Rodriguez, Arana Cuenca, & Tellez Jurado, 2011). A combination of the
above methods could also be used in the effective generation of short functional peptides (Korhonen,
2009). The size of the active peptides ranges from two to twenty amino acid residues. Their
bioactivities rely on the parental protein source and the composition of amino acid sequences they
have inherited. Many peptides are known to exhibit multi-functional properties (Meisel & FitzGerald,
2003).
Bioactive Peptides Derived from Milk
Bioactive peptides derived from milk protein have received increasing attention due to their positive
health-promoting effects on digestive, endocrine, cardiovascular, immune and nervous sytems
(Korhonen, 2009). Research done on peptide sequences have expanded our horizons allowing us to
have a better understanding of antimicrobial, antioxidative, antithrombotic, antihypertensive,
immunomodulatory and opioid activities that bioactive peptides possess (Muro Urista et al., 2011;
9
Silva & Malcata, 2005). Figure 1.1 exhibits the potential health benefits of various milk protein
derived bioactive peptides. Moreover, it is revealed that LAB have an ability to degrade milk protein
in order to fulfill their nutritional requirements for essential amino acids (Benkerroum, 2010).
Because of the many different sources of proteinases and their modes of action, bioactive peptides
from milk hydrolysed by lactic acid bacteria or their proteases represent a fundamental difference
from those generated by digestive proteinases (Benkerroum, 2010). As a result, there has been
growing scientific and commercial interest on the evaluation of milk fermentation with microbial
proteolysis on human health; specifically on reducing the risk of chronic diseases or enhancing natural
immune protection (Hartmann & Meisel, 2007).
Figure 1.1 Milk protein-derived bioactive peptides and their health promoting targets, adapted from Korhonen,
2009.
10
The wide range of nutritional, functional and biological benefits of milk proteins place them as the
most valuable source of bioactive peptides at present (Korhonen, 2009). The protein concentration of
bovine milk is about 32 g/L, 80% of which are caseins and 20% of which are whey proteins. LAB
prefer the substrate casein due to its porous structure and poor solubility but whey protein also
undergoes limited degradation (Griffiths & Tellez, 2013). Caseins can be divided into α-, β- and
κ-caseins (Muro Urista et al., 2011). The whey fraction contains α-lactalbumin, β-lactoglobulin and
other proteins, e.g., immunoglobulins, lactoferrin and serum albumin (Ebringer, Ferencik, &
Krajcovic, 2008; Haug, Hostmark, & Harstad, 2007).
Since the functionality of peptides is closely related to parental protein in milk, a great number of
peptide sequences with specific functionality in caseins and whey milk proteins have been identified
(Muro Urista et al., 2011). Previous studies on peptide isolation and purification have been conducted
in our laboratory. Tellez Garay (2009) found five putative bioactive peptides from a cell-free
supernatant of L. hevleticus LH-2 fermented milk. The fraction, which included these five peptides,
showed an immunomodulatory effect and antagonistic effect against Salmonella infection in both in
vitro and in vivo studies (Tellez Garay, 2009). Table 1.3 shows the five putative bioactive peptide
sequences within this fraction. Other related research carried out by Medellin-Peña (2007) found three
bioactive peptides in cell-free spent modified MRS medium (CFSM) fermented with L. acidophilus
La-5. The bioactive molecule(s) from the CFSM not only down-regulated different virulence genes in
enterohemorrhagic E. coli (EHEC) O157:H7 but also interfered with E. coli quorum sensing. Both in
vitro and in vivo studies indicated increased resistance against infection and colonization with EHEC
O157:H7 when biologically active La-5 CFSM fractions were applied (M. J. Medellin-Peña, 2007).
Table 1.4 shows the three bioactive peptide sequences from biological active La-5 CFSM fractions.
11
Furthermore, databases (BIOPEPE etc.) and programs (BLAST etc.) are available to simulate and
demonstrate proteolysis processes targeted at obtaining expected bioactive peptides from precursor
protein (Minkiewicz, Dziuba, Iwaniak, Dziuba, & Darewicz, 2008).
Table 1.3 Bioactive peptides identification within the fraction of cell-free supernatant from L. helveticus LH-2
fermented milk using mass spectrometry and NCBI database, adapted from Tellez Garay, 2009.
Peptide Protein Sequence assignment
HQPHQPLPPTVMFPPQ β –Casein 145–160
HQPHQPLPPT β –Casein 145–154
WMHQPHQPLPPT β –Casein 143–154
LYQEPVLGPVR β –Casein 192–202
LDQWLCEK α-Lactalbumin 115–122
Table 1.4 Bioactive peptides identification within the fraction of cell-free spent modified MRS medium
fermented with L. acidophilus La-5 using mass spectrometry and BLASTp, adapted from M. J. Medellin-Peña,
2007.
Peak sequence/sequence aligned BLASTp protein
YPVEPF/YPVEPF YP 194702 ncopullulanase
[L. acidophilus NCFM]
YPPGGP/YPPG YP 193877 ornithine decarboxylase chain A
[L. acidophilus NCFM]
NQPY/NQPY YP 193484 glutamine ABC transporter
[L. acidophilus NCFM]
12
4. Intestinal Epithelial Cells
Enterocytes in the gastrointestinal (GI) tract consist of a single monolayer of intestinal epithelial cells
(IECs) and this monolayer is the frontline in host defense. The IEC is the most important physical
barrier that separates the vast number of microbes in the intestinal lumen from the host tissue. IEC also
plays a role in innate immune defense since the GI tract has the greatest number of lymphoid organs in
the human body, which can interface with a myriad of internal and external stimuli (Ouwehand,
Salminen, & Isolauri, 2002). IEC helps monitor the luminal bacterial signals in the GI tract, interpreting
and transmitting this information to mucosal innate and adaptive immune cells in the lamina propria,
and collaborates with lymphoid tissue to initiate an immune response to certain stimuli (Goto & Ivaylo,
2013; Pinto et al., 2009).
Epithelial Cells Polarization
In vitro models using isolated epithelia are invaluable for observing intracellular activities in response
to external stimuli. The most common epithelial cell lines used in studies are small intestine isolated
carcinoma Caco-2 cells and large intestine isolated carcinoma HT-29 cells for their ability to better
mimic the natural intestinal mucosal barrier when they are polarized (Backert, Boehm, Wessler, &
Tegtmeyer, 2013). Numerous in vitro studies revealed that epithelial cell polarization improves
relevance of real human intestinal epithelia. Human IEC, together with in vitro polarized epithelial
cells are both endowed with physiological, and biochemical characteristics as well as structural
markers. One of the most distinct structural features is the tight junction (TJ). TJs form between
adjacent cells leading to apical and basolateral cell membrane formation. Additionally, a brush border
composed of microvilli can be observed on the apical cell surface. Each microvillus possesses a
bundle of actin filaments, which are cross-linked by numerous actin-bundling proteins like villin,
13
fimbrin and espin to form the microvilli structural core (Chalghoumi et al., 2009; Cohen, Ophir, & Ben
Shaul, 1999; Le Bivic, Hirn, & Reggio, 1988; Rodriguez-Boulan & Nelson, 1989).
Transwell permeable inserts with membrane filters in cell culture well plates have long been adopted
for cross-epithelial transport assessment (Hubatsch, Ragnarsson, & Artursson, 2007; Zemans et al.,
2011). The permeable insert is reported to be beneficial for epithelial cell polarization and
differentiation. Due to the similar procedure for polarization of HT-29 cells in the permeable inserts to
that observed during intestine embryonic development, HT-29 cells are utilized in epithelial
differentiation studies (Le Bivic et al., 1988). When cultured in glucose for a certain period of time,
HT-29 cells form very tight junctions and microvillus brush border at the apical surface, indicating cell
polarization (Fitzgerald, Omary, & Triadafilopoulos, 1997; Höner zu Bentrup et al., 2006).
Consequently, their resemblance to native IEC makes this model more representative than cells grown
in mono-culture. Even though mono-cultures of epithelial cells are still useful and continue to be used,
their restrictions, such as the inability to transport agents (from small molecules to immune cells)
across the epithelial monolayer, are concerned (Höner zu Bentrup et al., 2006).
Trans-epitheliall Electrical Resistance
The increase in Trans-epithelial Electrical Resistance (TEER) indicates the polarized status of
epithelial cells grown in Transwell permeable inserts. The measurement of TEER is a well-developed
approach to quantitatively monitor tight junction integrity and qualitatively observe monolayer health
(Grajek & Olejnik, 2004). The Millicell ERS (EMD Millipore, Billerica, MA, USA) is a device
designed to facilitate measurements of TEER to determine cultured epithelial monolayer integrity
directly in tissue culture wells. The Millicell ERS-2 uses alternating currents to eliminate adverse
effects on the cell membrane. It contains a silver electrode with a fixed pair of probes. These “chopstick”
14
like probes can measure the voltage deflection at 37°C in tissue culture medium (Balda et al., 1996).
TEER evaluates tight junction formation under a wide variety of experimental conditions (Ferrell et al.,
2010; Höner zu Bentrup et al., 2006). This model has provided invaluable insights into the intracellular
events that occur in response to pathogenic bacterial stimuli. Solano et al. (2001) used TEER
measurement as one of the methods to compare the virulence of different Salmonella Enteritidis
strains, since the more virulent strains produce a greater disruption of the epithelial cell monolayer
(Solano et al., 2001). In addition, probiotic bacteria have been reported to strengthen the intestinal
barrier function as well as limit pathogen induced TEER loss. L. plantarum MB452 (Anderson et al.,
2010), St. thermophilus, L. acidophilus (Resta-Lenert & Barrett, 2003) and L. amylophilus D14 (Yu,
Wang, & Yang, 2012) inhibited pathogen-induced cell junction loss and mucosal barrier damage.
Epithelial Cells Immune Response to Salmonella Invasion
The intestine contains a great deal of immunoglobulin producing cells, e.g., IgA, comprising the
largest immunological organ in the body (Tellez Garay, 2009). The epithelial cells located in the
gastrointestinal tract collaborate with intraepithelial lymphocytes by possessing Toll-like receptors
(TLRs) and Nod-like receptors (NLRs) on their surface to sense the existence of pathogens or
pathogen-associated molecular patterns (Magrone & Jirillo, 2013; Tlaskalova-Hogenova et al., 2002).
Furthermore, cytokines secretion is also involved in the immune responses to pathogen invasion.
Cytokines are secreted soluble polypeptide or glycoprotein which play diverse biological roles in
normal and pathological events, such as cell growth, differentiation, and immune response activation
(Steinke & Borish, 2006; Zanabria Eyzaguirre, 2013). They may affect other cytokines production and
action in order to achieve regulated immune response by balancing between provoking and
suppressive influences (Zanabria Eyzaguirre, 2013).
15
Cytokines play essential roles in apoptosis and inflammation on epithelial cells. Accordingly,
Salmonella infection in all organs stimulates IL-1 and TNF-α expression (Eckmann & Kagnoff, 2001).
In response to bacterial endotoxin, the first released cytokine is tumor necrosis factor TNF (Zanabria
Eyzaguirre, 2013). Moreover, TNF often triggers the initiation and development of apoptotic and
inflammatory processes (Benderska et al., 2012). Even though the detail of the cytokine‟ function still
remain to be elucidated, Eckmann & Kagnoff (2001) assumed that they may have indirect functions,
like overall endothelial adhesion molecules up-regulation in order to initiate macrophage functions.
IFN-γ is another most studied cytokine, which plays an important role in the host defense against
Salmonella, since production of IFN-γ is readily detectable in Salmonella infected mice (Eckmann &
Kagnoff, 2001). Kagaya et al., (1989) hypothesized that the most likely mechanism of operation of
IFN-γ is the ability to activate macrophages to kill Salmonella (Kagaya, Watanabe, & Fukazawa,
1989).
5. Apoptosis
Characteristics
Programmed cell death is essential in the hosts‟ normal development. Not only does it help with organs
differentiation, but it also eliminates useless and dangerous cells. It serves as a balance with cell
division to maintain the normal cell number over time (Raff, 1992). Salmonella manipulates the cell
death pathway of mammalian cells by inducing their apoptosis (Kim et al., 1998). It is hypothesized
that their ability to trigger apoptosis is a crucial step in the pathogenesis of Salmonella (Ashida et al.,
2011; Gobbato, Galdeano, & Perdigon, 2008; Knodler, Finlay, & Steele-Mortimer, 2005). By inducing
cell death, previously replicated Salmonella inside the host cells are released in the late apoptosis phase
by evading the immune response, hence more effectively re-infecting other cells, resulting in systemic
16
infection (Ashida et al., 2011; De Moreno De Leblanc et al., 2010; Knodler & Finlay, 2001). Yet this
mechanism is not fully proven whether apoptosis is a direct outcome of pathogen infection or is the
result of inflammatory mediators released by the host (Torchinsky, Garaude, & Blander, 2010). Indeed,
Kim et al. (1998) proposed that human colon epithelial cells generated inflammatory mediators in
response to bacterial invasion. Some of these mediators also could induce apoptosis (Kim et al.,
1998).
The term apoptosis was first introduced in 1972 by Kerr et al., who differentiated a naturally
programmed cell death from severe tissue injury necrosis (Kerr, Wyllie, & Currie, 1972). There are
three types of cell death involved in mammalian cells categorized by morphological criteria, namely
apoptosis, autophagy, and necrosis. Apoptotic cells exhibit alterations to their nuclear morphology,
including DNA fragmentation, chromatin condensation, membrane blebbing, overall cell shrinkage,
and formation of apoptotic bodies that contain nuclear or cytoplasmic material (Elmore, 2007;
Hans-Jurgen, 2008). Autophagy is characterized by an extensive accumulation of autophagosomes
(double-membrane vacuoles) followed by fusion with lysosomes, leading to the degeneration of
contents of autophagosomes. Cytoplasmic swelling is the characteristic of Necrosis, and the swelling
occurs until the intracellular contents are released through the ruptured plasma membrane resulting in
pro-inflammatory leakage (Hans-Jurgen, 2008). Figure 1.2 shows the different morphological
alterations in the three forms of cell death. Among the three types, apoptosis has been studied
extensively since it is a major form of removing unwanted and harmful cells with little inflammatory
response and little disturbance of tissue homeostasis in the host (Ashida et al., 2011; de LeBlanc,
Castillo, & Perdigon, 2010; Knodler & Finlay, 2001).
17
Figure 1.2 Schematic representations of three forms of cell death. See context for detail description, adapted
from Hans-Jurgen, 2008.
18
Molecular Basics of Apoptosis
The molecular mechanism of apoptosis is dependent on a family of cysteine proteases called caspases.
They mediate apoptosis with irreversible activation of proteins involved in DNA repair, DNA
replication, and RNA splicing (Santini, Rainaldi, & Indovina, 2000). Even though all members of the
caspase family have some overlapping amino acid sequences and possess similar structures,
individual caspases play different physiological roles in apoptosis or inflammatory responses (Fink &
Cookson, 2005). There are two major signaling routes to initiate caspase activation: the extrinsic death
receptor pathway and the intrinsic mitochondrial pathway (Ashida et al., 2011; Elmore, 2007;
Hans-Jurgen, 2008). Extracellular signals such as toxins (Popov et al., 2002), hormones, growth
factors, nitric oxide (Brune, 2003) and cytokines (Benderska et al., 2012), either penetrate through the
plasma membrane or transduce through membrane receptors to activate the executioners caspase-3
and -7 (Ashida et al., 2011). These signals may trigger (positively affect) or inhibit (negatively affect)
apoptotic activity in order to maintain tissue homeostasis, especially in the immune system (Ashida et
al., 2011; Hans-Jurgen, 2008; Levi et al., 2014). Intrinsic signaling pathways are mitochondrial
initiated events involved with a diverse array of non-receptor mediated stimuli, which can activate
caspase-9 production and directly aim at targets within the cells (Ashida et al., 2011). The “cross-talk”
between the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway leads to the
final execution phase of apoptosis (Elmore, 2007). In this phase, morphological and biochemical
alterations such as DNA fragmentation and membrane blebbing appear (Slee, Adrain, & Martin,
2001).
Assays for Apoptosis
Due to the distinct features of apoptotic cells, there are a variety of assays to determine apoptotic
19
activity activators, effectors and regulators and count the functional consequences of their actions.
These assays can be classified into five categories: (1) morphological alterations detected by
microscopy (Elmore, 2007; Fink & Cookson, 2005); (2) DNA fragmentation detected by DNA
laddering (Compton, 1992) and TUNEL (Fink & Cookson, 2005; Hans-Jurgen, 2008); (3) membrane
alterations detected by Annexin-V binding (van Engeland, Nieland, Ramaekers, Schutte, &
Reutelingsperger, 1998), LDH and impermeable DNA dyes (Elmore, 2007; Fink & Cookson, 2005;
Hans-Jurgen, 2008); (4) caspase activation detected by western blotting, PCR microarray (Elmore,
2007), colorimetric or fluorometric assays (Fink & Cookson, 2005; Hans-Jurgen, 2008); (5)
mitochondrial damage detected by ATP production and mitochondrial dyes (Elmore, 2007;
Hans-Jurgen, 2008)
Flow Cytometry
Amongst the assays previously described, the detection of DNA fragmentation is currently the most
frequently used method when studying apoptosis (Fink & Cookson, 2005) together with flow
cytometry, which can facilitate apoptosis detection. Flow cytometry, also referred to as
fluorescence-activated cell sorting (FACS), is laser-based biophysical method, which allows
multi-parameter measurements. It requires samples prepared in single cell format, to be measured in a
fluid stream, which will be spectrophotometrically measured by the laser light. The properties of the
designated single cell are acquired, including cell size (shown on forward-scattered light graph),
conformation of inner structure (shown on side-scattered light graph), and relative fluorescence
intensity (fluorescent intensity graph).
Fluidics, optics and electronics are the three main systems that make up the flow cytometer. The fluidics
20
system transports cells in single file through a narrow nozzle with arranged sheath fluid. A fluorescent
photon resonance vibrating mechanism helps separate the stream of cells into individual droplets. The
optics system consists of lasers at different wavelengths to illuminate cells in the same stream and
optical filters to channel designated light to the appropriate detection system. Last, but not least, the
electronics system converts detected light signals into a measurement that can be analyzed by computer
software. As a result, electronic signals can be interpreted after adjusting the sorting settings of the
software in order to obtain valuable information. Dong et al. (2008) concluded that flow cytometry was
a versatile method of detecting the apoptotic process, including morphological alteration and chromatin
shrinkage, activation of caspases, DNA condensation, etc. (Dong, Kleinberg, Davidson, & Risberg,
2008). Figure 1.3 provides a schematic representation of a flow cytometer (Jahan-Tigh, Ryan,
Obermoser, & Schwarzenberger, 2012).
Apoptotic cells share noticeable characteristics, which can be captured by flow cytometry with
appropriate fluorescent labels or dyes. From the data, it is able to differentiate the different phases of
cell death (Vermes, Haanen, & Reutelingsperger, 2000). In addition, flow cytometry allows several
measurements to be obtained on each cell simultaneously by applying different stains such as
fluorescein isothiocyanate (FITC), propidium iodide (PI), or phycoerythrin (PE) (Corver, Cornelisse,
& Fleuren, 1994). The stains‟ fluorescent intensity can be quantified using CellQuest™ software (BD
Bioscience, Mississauga, ON, Canada). In this study, the Apo-Direct™ Kit (BD Bioscience,
Mississauga, ON, Canada) was employed to assess DNA strand breakage using FITC/PI dual stains.
Probiotics Protection against Pathogen-induced Apoptosis
Although the detailed mechanisms of pathogen-induced apoptosis are still under evaluation, the
21
hypothesis that probiotic strains or bioactive components from LAB culture medium may interfere
with pathogen-induced apoptosis was investigated. Myllyluoma et al. (2008), performed a 1 h
pretreatment of H. pylori infected polarized Caco-2 cells in the presence of L. rhamnosus GG, L.
rhamnosus Lc705 and B. breve Bb99 cells and found that they reduced caspase-3 production
significantly at 24 h post-infection (Myllyluoma, Ahonen, Korpela, Vapaatalo, & Kankuri, 2008). An in
vitro study undertaken by Valdez et al. (2001) showed that the probiotic strains L. delbrueckii subsp.
bulgaricus and St. thermophilus down-regulated Salmonella induced apoptosis in macrophages. In this
case, the inhibitory effect did not seem to be related to an increase in the uptake of LAB, but more to
secretory IgA (S-IgA) production. The latter is an immunoglobulin known to be associated with
inhibition of pathogen internalization and toxin neutralization (Mantis & Forbes, 2010). In addition to
pro-inflammatory cytokine production, such as TNF-α, oxidant radicals (superoxide, hydrogen
peroxide and nitric oxide) released from macrophages stimulated by LAB may also partially explain the
protective capacity of LAB (Valdez, Rachid, Gobbato, & Perdigon, 2001). Further, an in vivo study by
Gobbato et al. (2008) found administration of L. delbrueckii subsp. bulgaricus and St. thermophilus
strains improved microbiocidal activity and down-regulated apoptosis produced by S. Typhimurium
infection, possibly through increasing cytokine IFN- γ, immunoglobulin IgA, and apoptosis related
protein Bcl-2 release (Gobbato et al., 2008).
There are a limited number of publications describing the putative mechanisms of the anti-apoptotic
effect on epithelial cells induced by cell free preparations of probiotics against pathogens. Li et al.
(2001) showed that Lactobacillus acidophilus S-layer protein inhibited caspase-3 production by
Caco-2 cells following Salmonella infection. In addition, the extracellular signal-regulated kinase 1 and
2 (ERK 1/2) signaling pathway plays a fundamental role in epithelial recovery after pathogen infection,
22
and the L. acidophilus S-layer protein activated the signaling pathway, reducing further apoptotic cell
damage (Li, Yin, Yu, & Yang, 2011). Yan et al. (2007), found Lactobacillus rhamnosus GG (LGG)
soluble factors p75 and p40 could regulate signaling pathways in order to prevent cytokine-induced
apoptosis in human and mouse intestinal epithelial cells. Soluble factors p75 and p40 not only
suppressed cytokine-induced apoptosis, but also promoted cell survival by stimulating Akt related
proapoptotic pathways (Yan et al., 2007).
Figure 1.3 Schematic representation of a flow cytometry (adapted from Jahan-Tigh et al., 2012). Samples are
prepared in single cell format and are labeled by fluorochrome-linked antibodies or stained with fluorescent
nuclear, cytoplasmic, or membrane dyes. The labeled/stained cells go through a beam of laser light with known
speed and position. A set of optical system is used to detect forward-scatter, side-scatter and fluorescence. Lastly,
an electronic system converts these signals into electronic signals on computer-based software.
23
Objectives
Bioactive peptides from LAB fermentations have shown potential as a therapeutic alternative of
clinic-dependent antibiotic, therefore, attracted attention both from academic research and related
industries. Based on previous in vitro and in vivo studies in our laboratory, cell-free spent medium
(CFSM) of L. acidophilus and L. helveticus showed potent immunomodulatory function on the host
and interference of pathogens virulence (Chin, 2002; Ding et al., 2005; M. J. Medellin-Peña, 2007; Ng,
2000; Tellez Garay, 2009). Our hypothesis that CFSMs reinforce epithelial cell membrane integrity
when invaded by Salmonella is tested in four individual steps.
1. Using Trypan Blue Exclusion, LDH Assay and SRB Assay to determine the non-toxic dose of
bioactive compounds from L. acidophilus or L. helveticus fermented milk (La-5 or LH-2) when
applied on epithelial cells;
2. Examine the protective effect on epithelial cell membrane integrity present in the bioactive
components against Salmonella infection through TEER Measurement and LDH Assay;
3. Employing TUNEL Assay to investigate down-regulation of Salmonella-induced apoptosis in
epithelial cells when La-5 and LH-2 are applied;
4. Evaluate Salmonella internalization interference of La-5 and LH-2 in an in vitro chicken
model by counting amount of invaded Salmonella.
24
CHAPTER 2: Methodology
La-5 Cell Free Spent Medium (CFSM) Preparation
Lactobacillus acidophilus La-5 strain was obtained from the culture collection of the Canadian
Research Institute for Food Safety (University of Guelph, Guelph, Canada), streaked on Lactobacillis
MRS Agar (BD Difco™, Mississauga, ON, Canada) and incubated under anaerobic conditions with a
BBL GasPak system (BD Bioscience, Mississauga, ON, Canada) at 37 °C for 48 h. Chemically defined
medium (CDM) was composed of 28 g whey protein isolate (Ergogenics Nutrition, Vancouver, BC,
Canada), and 10 ml of 0.25 g/ml sterile sucrose solution (Sigma, Markham, ON, Canada) in 500 ml
sterile distilled water. The CDM medium (500 ml) was inoculated with L. acidophilus La-5 (50 colonies
removed from an MRS agar plate cultured as described above) and incubated anaerobically with a BBL
GasPak system (BD Bioscience, Mississauga, ON, Canada) at 37°C for 48 h. Following growth,
bacterial cells were removed by centrifugation at 12,000 × g for 30 min at 4 °C (Avanti J-20 XPI,
Beckman Coulter, Canada). The supernatant was collected and sequentially filtered through a 0.7 μm
pore-size filter (EMD Millipore, Billerica, MA, USA), and then through a 0.45 μm pore-size filter
(Fisher Scientific, Mississauga, ON, Canada) in order to remove any bacterial cells present. The
cell-free preparation was frozen at -80°C (Thermo Fisher Scientific, Mississauga, ON, Canada),
followed by freeze drying (Unitop 600 SL, VirTis Co., Inc. Gardiner, NY, USA). The freeze dried
samples were stored at -80 °C (Thermo Fisher Scientific, Canada). The freeze-dried supernatant was
reconstituted with cell culture grade water (Water for Injection, WFI, Life technologies, Burlington, ON,
Canada) to 1/10 of its original volume before use. Portions of the reconstituted samples were also kept
at -20°C until analyzed. The protein concentration was measured spectrophotometrically using the
25
Protein A280 program in Nanodrop according to manufacturer‟s instruction (ND-1000, Thermo Fisher
Scientific, Mississauga, ON, Canada). The protein concentration of La-5 CFSM was 99.5 ± 0.4 mg/ml.
LH-2 Cell Free Spent Medium (CFSM) Preparation
Lactobacillus helveticus LH-2 strain was obtained from the culture collection of the Canadian Research
Institute for Food Safety (University of Guelph, Guelph, Canada), streaked on Lactobacillis MRS Agar
(BD Difco™, Mississauga, ON, Canada) and incubated under anaerobic conditions with a BBL GasPak
system (BD Bioscience, Mississauga, ON, Canada) at 37 °C for 48 h. Skim milk powder (Smucker's,
Markham, ON, Canada) was reconstituted 10% (w/w) with distilled water. Reconstituted skim milk was
heat treated at 95°C for 30 min and then cooled in the biosafety cabinet until it reached room
temperature. Isolated colonies of the L. helveticus strain were used to inoculate four replicates of 5 ml
sterilized reconstituted milk, which were then incubated anaerobically with a BBL GasPak system (BD
Bioscience, Mississauga, ON, Canada) at 37°C for 24 h, followed by aerobic incubation at the same
temperature for 26 h. The fermented milk samples were centrifuged at 16,000 × g for 10 min at 15 °C
(Avanti J-20 XPI, Beckman Coulter, Canada). The supernatant was filtered through a 0.2 μm pore-size
filter (EMD Millipore, Billerica, MA, USA) in order to remove any bacterial cells present. From 800 ml
of fermented milk, 500 ml of supernatant were obtained. The cell-free supernatant (10 ml) was
dispensed in 50 ml tubes and was frozen at -80°C (Thermo Fisher Scientific, Mississauga, ON, Canada),
followed by freeze drying (Unitop 600 SL, VirTis Co., Inc. Gardiner, NY, USA). The freeze dried
samples were stored at -80 °C (Thermo Fisher Scientific, Canada). The freeze-dried supernatant was
reconstituted with cell culture grade water (Water for Injection, WFI, Life technologies, Burlington, ON,
Canada) to 1/10 of its original volume before use. Portions of the reconstituted samples were also kept
26
at -20°C until analyzed. The protein concentration was measured spectrophotometrically using
Nanodrop (ND-1000, Thermo Fisher Scientific, Mississauga, ON, Canada) as described above. The
protein concentration of LH-2 CFSM was 37.1 ± 0.1 mg/ml.
Salmonella Strain and Growth Conditions
A human isolate (SA1997-0934) and two chicken isolates (SA2000-0406, SA2001-4368) of
Salmonella Typhimurium DT104 along with a GFP expressing mutant of Salmonella Typhimurium
were obtained from the culture collection of the Canadian Research Institute of Food Safety (University
of Guelph, Guelph, Canada). All strains were stored in 25% glycerol (v/v) (Fisher Scientific, Canada) at
-80°C, and transferred once to Trypticase Soy Agar (TSA, BD Bioscience, Mississauga, ON, Canada).
Following overnight growth at 37°C, a colony was removed aseptically from the plate and transferred
into 5 ml Trypticase Soy Broth (TSB, BD Bioscience, Mississauga, ON, Canada), which was then
incubated at 37°C with shaking (200 rpm; orbitary shaker incubator, New Brunswick Scientific, USA)
for 18~19 h. An aliquot (50 μl) of this culture was added into 5 ml of fresh TSB, and incubated at 37°C
with shaking for 4 h. By the end of 4 h incubation period, all S. Typhimurium strains were in the late
exponential phase and inoculum absorbance at OD600 was adjusted to 1.00. The bacteria count was
approximately 1 × 109 CFU (colony forming unit)/ml (= 9 log10 CFU/ml).
All the strains used in these experiments are listed in Table 2.1.
27
Table 2.1 Bacterial strains used in these studies
Species Strain No Source Gram stain Growth conditions
Salmonella Typhimurium
SA 1997-0934 CRIFS* Negative
TSB/TSA, 37°C SA 2000-0406 CRIFS
* Negative
SA 2001-4368 CRIFS* Negative
GFP CRIFS* Negative
Lactobacillus acidophilus La-5 CRIFS* Positive MRS, 37 °C
Lactobacillus helveticus LH-2 CRIFS* Positive MRS, 37 °C
* CRIFS: Canadian Research Institute for Food Safety, University of Guelph, ON, Canada
Cell Culture and Maintenance
The human colonic carcinoma cell line HT-29 was obtained from the culture collection of the Canadian
Research Institute of Food Safety (University of Guelph, Guelph, Canada). Cells were cultured in
Dulbecco‟s modified Eagle medium (DMEM, high glucose, Life technologies, Burlington, ON, Canada)
supplemented with 1% (v/v) 10, 000 U/mL penicillin–streptomycin (Life technologies, Burlington, ON,
Canada) and 10% heat-inactivated fetal bovine serum (HI FBS, Life technologies, Burlington, ON,
Canada). The HT-29 cells (20–40 passages) were kept in a humid atmosphere of 5% CO2 at 37°C in an
incubator (Forma™ Series II 3110 Water-Jacketed CO2 Incubators, Thermo Fisher Scientific,
Mississauga, ON, Canada), and grown as a monolayer in 25 or 75 cm2 flasks (Corning, NY, USA). The
medium was changed every two days and passed (80-90% confluence) using 0.25% trypsin-EDTA
reagent (Life technologies, Burlington, ON, Canada).
The chicken hepatoma cell line, LMH (ATCC CRL-2117) was purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA). Cells were cultured in Waymouth‟s MB 752/1 medium (Life
technologies, Burlington, ON, Canada) supplemented with 1% (v/v) 10, 000 U/mL penicillin–
streptomycin (Life technologies, Burlington, ON, Canada) and 10% fetal bovine serum (FBS, Life
technologies, Burlington, ON, Canada). LMH cells (6-23 passages) were kept in a humid atmosphere of
28
5% CO2 at 37°C, and grown as a monolayer in 0.1% gelatin (PCS-999-027, Cedar Lane Laboratories,
Burlington, Canada) coated 25 or 75 cm2 flasks (Corning, NY, USA). The medium was changed every
two days and was passed (80-90% confluence) using 0.25% trypsin-EDTA reagent (Life technologies,
Burlington, ON, Canada).
Trypan Blue Exclusion Assay
This assay based on the principle that viable cells have intact cell membranes which can resist trypan
blue dye penetration while dead cells do not. Hence live cells can be easily differentiated and counted
with a light microscope using trypan blue (Strober, 2001). Viable cells are clear, unstained, small, and
round under light microscopy, while dead cells are blue, stained, and swollen (Louis & Siegel, 2011) .
For this test, approximately 1 × 106 HT-29 cells/well were seeded in a 24-well plate (Fisher Scientific,
Canada) and left for twenty-four h at 37°C and 5% CO2. Cells were then stimulated with different
concentrations (v/v) of La-5 (0~12.5%) and LH-2 (0~10%) CFSM adjusted in cell culture medium and
left for twenty-four h at 37°C, in a CO2 (5 %) incubator. The cells were trypsinized using 250 μl of 0.25%
trypsin-EDTA reagent (Life technologies, Burlington, ON, Canada), and agitated pipetting with 750 μl
cell culture medium to make total volume of 1 ml cell suspension. The viable cells were counted by
adding 50 μl of the cell suspension in 450 μl of trypan blue dye (0.4% v/v, Life Technologies,
Burlington, ON, Canada). 50 μl of above cell suspension in trypan blue dye was loaded on
haemocytometer. The exclusion of trypan blue from cells were observed under 10× magnification light
microscopy (AE2000, Opti-Tech Scientific Inc., Scarborough, ON, Canada).
Confocal Scanning Laser Microscopy (CSLM)
The green fluorescent protein (GFP) from the jellyfish Aequorea victori can be used as an endogenous
29
fluorescent tag, which allows visualization of bacterial cells even when present intracellularly in host
cells (Ling, Wang, Xie, Lim, & Leung, 2000). The most outstanding advantage of using CSLM to study
Salmonella invasion is that bacterial internalization on epithelial cells can be observed in three
dimensions without physically disturbing the specimens (Takeuchi & Frank, 2001). The GFP protein
encoded on the plasmid inserted into Salmonella shares a common excitation and emission profile with
the commercially available GFP, but is optimized for an excitation wavelength of 488 nm (Auty et al.,
2005; Burnett, Chen, & Beuchat, 2000). For Leica CSLM, emitted light was collected through a 480 nm
dichroic mirror, a 520 nm long-pass filter, and a 680 nm short-pass filter (Takeuchi & Frank, 2001).
Leica confocal scanning laser microscopy (DMIRB Inverted Fluorescence Microscope, Leica
Microsystem, Concord, ON, Canada) was used to observe the intracellular populations of transformed S.
Typhimurium into the epithelial cell monolayer. In this study, 24 h prior to Salmonella infection, 5 × 106
HT-29 cells were seeded in 35mm glass bottom dishes (MatTek Corporation, Ashland, MA, USA). 2 ml
of a non-toxic concentration of CFSMs (1.5% of La-5 or 1% of LH-2) adjusted with antibiotics free cell
culture medium was added to designated dishes. The GFP labeled S. Typhimurium inoculum containing
5 × 107 CFU/ml (bacteria: cell MOI of 10) was added to dishes and incubated for 2 h at 37°C and 5%
CO2. A negative control was included, comprising HT-29 cells without exposure to bacteria and CFSM.
The positive control consisted of non-stimulated cells (not exposed to CFSM) infected with Salmonella
for 2 h. Test groups consisted of HT-29 cells stimulated with either La-5 or LH-2 CFSM and exposed to
Salmonella with the presence of CFSM for 2 h. After 2 h of infection, extracellular bacteria were
removed by extensive washing with PBS together with 1 ml of 350 μg/ml gentamicin sulfate (Sigma,
Markham, ON, Canada) treatment for 1 h at 37°C, in a CO2 (5 %) incubator. Medium was subsequently
changed to 1 ml of 200 μg/ml gentamicin for an additional 30 min. Following 2 washing steps with PBS,
30
1 ml of antibiotic-free cell culture medium was added in each dish. Then all the dishes were visualized
under CSLM (DMIRB Inverted Fluorescence Microscope, Leica Microsystem, Concord, ON, Canada)
according to manufacturer‟s instruction. Random fields were examined for bacterial invasion.
Trans-epithelial Electrical Resistance (TEER)
Millicell-ERS is designed to facilitate measurements of TEER of cultured epithelial monolayer
integrity directly in tissue culture wells. Millicell ERS-2 (EMD Millipore, Billerica, MA, USA) is a
device which uses alternating current to eliminate adverse effects on the cell membrane. It contains a
silver electrode with a fixed pair of probes, which can measure the voltage deflection at 37°C in tissue
culture medium (Balda et al., 1996). The change in TEER value is an indication of cell monolayer
confluence quantitatively and cell monolayer health qualitatively.
Transwell inserts (BD FalconTM
, Mississauga, ON, Canada) are permeable supports which create an
apical and a basolateral chamber in each well to allow epithelial and other cell types to be grown and
studied in a polarized state.
For TEER measurements, HT-29 cells at a concentration of 1 × 105 cells/ml were pipetted on top of
Transwell inserts, resulting in a seeding density of approximately 5×104 cells/insert. The medium was
changed every two days in both apical (500 μl) and basolateral (700 μl) chambers in each well. After 40
days, cells formed a fully polarized monolayer and reached the plateau TEER value of ~ 120 ohm × cm2.
TEER was measured with a Millicell ERS-2 apparatus (EMD Millipore, Billerica, MA, USA) by
immersing the shorter tip of electrode in the insert and the longer tip in the outer well at a 90° angle to
the plate insert. The shorter tip should not contact cells growing on the insert membrane and the longer
tip should just touch the bottom of the outer well. TEER values were obtained from blank inserts
31
(without cells) and test inserts (with cells) and was calculated to ohm × cm2. The final values were
obtained from triplicate inserts by subtracting average value of blank inserts from all samples and
multiplying by the area of the monolayer (Hasegawa et al., 1999).
Epithelial monolayer Integrity Challenge Study
In this study, we used polarized HT-29 cells which had been grown in Transwell inserts for 40 days with
~120 ohm × cm2
TEER value to investigate whether La-5 or LH-2 CFSM could prevent S.
Typhimurium from interfering with the HT-29 monolayer integrity. In each experiment, the monolayer
in all inserts was from the same passage number and stage of maturation. 24 h before Salmonella
infection, medium in both apical and basolateral chambers created by the Transwell insert in each well
was replaced with antibiotic-free cell culture medium or stimulated with 1.5% of La-5 or 1% of LH-2
CFSM adjusted in antibiotic-free cell culture medium. Subsequently, 1 × 107 CFU/ml S. Typhimurium
DT104 (SA1997-0934) inoculum (Multiplicity of Infection bacteria: cell = 10) was added to the apical
chamber of the inserts and left at 37°C, in a 5% CO2 incubator for 2 h. Gentamicin sulfate (Sigma,
Markham, ON, Canada) was used to eradicate extracellular Salmonella as described above. After
calibrating the Millicell ERS-2 using culture medium, the sterile probes were vertically immersed into
the apical or basolateral chamber at a 90° angle to the plate insert. The first measurement after the
addition of gentamicin was regarded as time t0. TEER readings were recorded at various time intervals
and expressed as the ratio of TEER at time t to the initial value at t0 for each series. This approach has
been utilized in the study of evaluating probiotic activity (Klingberg, Pedersen, Cencic, & Budde,
2005).
32
Lactate Dehydrogenase (LDH) Cytotoxicity Assay
LDH is a stable and soluble enzyme present in the cytoplasm of all mammalian cells. LDH is
impermeable in normal cells, but if the cell‟s plasma membrane is damaged then LDH is rapidly
released into the surrounding medium. In this research, LDH release caused by pathogenic bacteria
damage to the plasma membrane can be quantified as an accurate index of cell death or cytotoxicity.
The LDH levels in cell culture medium were determined using the LDH-Cytotoxicity Assay Kit II
(Abcam, Toronto, ON, Canada) according to the manufacturer‟s instructions. The assay utilizes an
enzymatic coupling reaction: LDH oxidized lactate to generate NADH, which then reacts with
cell-impermeable Tetrazolium Salt WST to generate yellow color (see Figure 2.1). The yellow color
formed can be detected spectrophotometrically at 450 nm and the intensity of the generated color is
proportional to the degree of cell lysis.
Figure 2.1 Catalytic function of the Lactate Dehydrogenase (LDH) enzyme.
Preliminary studies showed that the optimal cell density for LDH quantification was 1 × 105
cells/ml/well in 96-well plate. 24 h before Salmonella infection, 100 μl of HT-29 cells at a concentration
of 1 × 105 cells/ml were seeded into a 96-well plate, resulting in a seeding density of approximately 1 ×
104 cells/well. Simultaneously, 100 μl of non-toxic concentration of La-5 (1.5%) or LH-2 (1%) CFSM
(v/v) were adjusted using antibiotic-free cell culture medium and added in designated wells. Two wells
with cell culture medium only were included as background controls. The background value has to be
33
subtracted from all other values. The negative control consisted of supernatant from cells without
exposure to bacteria or CFSM. The positive control consisted of HT-29 cells infected with S.
Typhimurium DT104 (SA1997-0934; MOI of 10) in the presence of the appropriate CFSM for 2 h. All
supernatants were centrifuged (AllegraTM
21R Centrifuge, Beckman CoulterTM
, Mississauga, ON,
Canada) at 600 × g for 10 min to remove bacterial and eukaryotic cells. A 10 μl aliquot from each well
was dispensed into a new 96-well plate and reacted with 100 μl of WST substrate mix (within the kit).
The absorbance was measured at 450 nm using a Multilabel Counter (Wallac 1420 VictorTM
3V,
Perkin-Elmer Life Sciences, Woodbridge, ON, Canada). LDH cytotoxicity % was then determined as
shown.
Cytotoxicity (%) =(Test sample − Low Control)
(High Control − Low Control)× 100
Low Control: untreated cells
High Control: cells treated with 10 μl Cell Lysis Solution (within the kit)
Values of test groups were expressed as % cytotoxicity relative to the positive controls.
Apoptosis TUNEL Assay
Briefly, polarized HT-29 cells were seeded at 1 × 106
cells/well of a 24-well plate 2 days prior to S.
Typhimurium infection. 24 h later, designated wells were pre-incubated with 1.5% La-5 or 1% LH-2
CFSM in antibiotic-free cell culture medium. 1 × 107 CFU/ml S. Typhimurium DT104 (SA1997-0934;
MOI of 10) was added in designated well with or without the presence of appropriate concentration of
La-5 or LH-2. After 1 h, extracellular bacteria were removed by extensive washing with PBS and
addition of gentamicin incubation as described above. The cells were trypsinized using 250 μl of 0.25%
trypsin-EDTA reagent (Life technologies, Burlington, ON, Canada), and agitated pipetting with 750 μl
34
cell culture medium to make total volume of 1 ml cell suspension. After washing with PBS and
centrifuged at 500g, 5 min (AllegraTM
21R Centrifuge, Beckman CoulterTM
, Mississauga, ON, Canada)
several times, cells were fixed in 1 ml 1% paraformaldehyde (Sigma, Markham, ON, Canada) for 1 h on
ice, followed by PBS washing and suspending in cold 70% (v/v) ethanol and left at 4°C for at least 15 h
for cell membrane permeabilization. The Apo-Direct ™ Kit (BD Biosciences, Mississauga, ON,
Canada) was used in this research according to the manufacturer‟s instruction. Positive and negative
controls in this kit were run in parallel to the samples in order to define M1/M2 markers. 1 ml of BD
positive and negative control cells and all of 15 h frozen cells were both washed with 1 ml of BD wash
buffer (within the kit) and centrifuged at 500 g, 5 min (AllegraTM
21R Centrifuge, Beckman CoulterTM
,
Mississauga, ON, Canada) two times. Subsequently, cells were incubated with 50 μl DNA labeling
solution for 2 h. Then cells were washed with 1 ml of BD Rinse Buffer (within the kit) and centrifuged
at 500g, 5 min (AllegraTM
21R Centrifuge, Beckman CoulterTM
, Mississauga, ON, Canada) two times,
followed by adding 0.3 ml PI/RNase staining buffer. Based on DNA doublet discrimination and
exclusion of cell debris, cells were counted adjusting the sorting threshold. Afterwards, cells were
analyzed by flow cytometry (FACScan, BD, Mississauga, ON, Canada) with excitation at 488 nm.
Fluorescence emission by FITC was measured using a 530/30 band-pass filter while that of PI was
measured using a 585/42 band-pass filter. A total number of 5,000 cells were counted for each sample
and the percentage of FITC positive (apoptotic) cells was determined.
Sulforhodamine B (SRB) Assay
SRB is a bright-pink aminoxanthene dye. Under mild acidic conditions, it electrostatically binds to
basic amino-acid residues, while it dissociates under weakly basic conditions (Skehan et al., 1990). The
35
cell mass is correlated with the amount of dye extracted from cells. The SRB colorimetric assay is an
accurate and reproducible assay based on the sensitive linearity of quantitative staining of cellular
proteins with optical density (OD) between 560 and 580 nm wavelengths. This assay has been widely
used as an efficient and highly cost-effective method for screening drug toxicity on different types of
cell lines (Vichai & Kirtikara, 2006).
The SRB assay was tested on LMH cells in order to determine non-toxic doses of La-5 and LH-2 CFSM.
After seeding 3 × 103 cells/well in a 96-well plate and incubating for 24 h at 37°C in a 5% CO2 incubator,
cells were stimulated with different concentrations of La-5 (0~3%) or LH-2 (0~3%) CFSM, followed
by cells fixation with 50 μl of 50% (w/v) Trichloroacetic Acid (TCA, Sigma, Markham, ON, Canada).
After removing the TCA, cells were stained with 0.4% (w/v) SRB (Sigma, Markham, ON, Canada) in 1%
acetic acid (Glacial Acetic Acid, Fisher Scientific, Canada) for 30 min. Unbound dye was removed with
1% acetic acid, and the protein-bound dye was dissolved when 10 mM Tris-base
(Tris-hydroxymethyl-aminomethane, Sigma, Markham, ON, Canada) was added. The developed color
was measured spectrophotometrically at 570 nm using a microplate reader (Synergy H5, BioTek,
Winooski, VT, USA) and results expressed as percentage with respect to control wells (set as 100%)
grown under regular conditions.
Invasion Assay
The invasion assay was carried out on LMH cells in order to determine the protective ability of La-5 and
LH-2 CFSM to limit the invasion of S. Typhimurium in in vitro chicken model.
Invasion assay was assessed as described elsewhere (Bishop et al., 2008) with specific minor
modifications as follows. LMH cells were seeded at 2 × 106 cells/well in 0.1% gelatin pre-coated
36
12-well plate for 24 h at 37°C in a 5% CO2 incubator. Then 2 ml of a non-toxic concentration 1 % of
La-5 or 1% of LH-2 adjusted with antibiotics free cell culture medium was added to designated dishes
24 h prior to infection. Two chicken isolates of S. Typhimurium (SA2000-0406 or SA2001-4368)
containing 2 × 107 CFU/ml (bacteria: cell MOI of 10) were added to appropriate wells and incubated for
designated time in 37°C and 5% CO2 incubator. Negative controls were included, comprising LMH
cells without exposure to bacteria or CFSM. The positive control consisted of non-stimulated cells (not
exposed to CFSM) infected with Salmonella. Test groups consisted of LMH cells stimulated with either
La-5 or LH-2 CFSM and exposed to Salmonella with the presence of CFSM. At each time point,
extracellular bacteria were removed by extensive washing with PBS, followed by exposure to 350
μg/ml gentamicin sulfate (Sigma, Markham, ON, Canada) for 1 h, and subsequently 200 μg/ml
gentamicin for 30 min. Following 2 times of PBS washing, cells were lysed with 500 μl of 1% Triton
X-100 solution (Sigma, Markham, ON, Canada) in high-pure water (WFI water, Life Technologies,
Burlington, ON, Canada) for 2 min and suspended in 500 μl of PBS (Life Technologies, Burlington, ON,
Canada). Eckmann et al. (1993) stated that within 1 h after cell lysis, Triton would not affect bacterial
viability (Eckmann, Kagnoff, & Fierer, 1993). The number of released viable bacteria was determined
by serial dilution in PBS (Life technologies, Burlington, ON, Canada) and plating on TSA agar (BD
Bioscience, Mississauga, ON, Canada). After overnight incubation at 37°C, the numbers of bacterial
colony were enumerated. CFU calculation was shown in followed formula. Values of test groups were
expressed as % invasion relative to the positive controls.
CFU/ml =Number of Colonies
Dilution Factor × Volume (0.1 ml)
37
Statistical Analysis
Statistical analyses were performed using the Microsoft Excel 2010 analysis tool package. Unless
otherwise stated, all results are the average ± SD of two independent experiments with at least replicates.
Data from each experiment were analyzed using analysis of variance (ANOVA). Differences at P < 0.05
were considered to be statistically significant. Differences at P < 0.01 were considered to be statistically
very significant.
38
CHAPTER 3: Result and Discussion
Trypan Blue Exclusion Assay
In order to use tissue culture to determine the effect of CFSM on the infectivity of Salmonella, a
tolerable dose of CFSM for eukaryotic cells used in these studies needs to be established, since negative
effects (e.g. cell death) may be triggered when applying doses beyond the cells‟ threshold. Based on
previous in vivo studies using mice fed with a partially purified LH-2 CFSM fraction (F5), a high dose
of this fraction (0.08 μg/day) caused intolerance and the mice exhibited a lower survival rate after
Salmonella infection than the non-stimulated mice. However, a low dose of F5 (0.02 μg/day) protected
mice against Salmonella infection. One possible reason for negative effects in mice exposed to the high
dose of F5 might be perturbation of the immune system (Tellez Garay, 2009).
The trypan blue exclusion assay is based on the principle that viable cells have intact cell membranes
which can resist trypan blue dye penetration while dead cells do not. Hence live cells can be easily
differentiated and counted with a light microscope (Strober, 2001). This assay may show a relation
between the CFSM concentration and HT-29 cell proliferation. In the case of La-5, the CFSM showed
no significant effect (P ≥0.05) on cells at all concentrations (see Fig.3.1.A). Interestingly, HT-29 cells
showed highest viability in the presence of 1.6% La-5 CFSM. In the case of LH-2, only 1% showed no
effect (P ≥0.05) on cell viability (see Fig.3.1.B). Thus, 1.5% La-5 (protein concentration 1.5 mg/ml)
or 1% LH-2 (protein concentration 370 μg/ml) were chosen as a non-toxic dose on HT-29 cells and
used in subsequent experiments.
39
Figure 3.1 La-5 and LH-2 toxic dose test on HT-29 cells, estimated by trypan blue exclusive assay. A: La-5
treatment; B: LH-2 treatment. HT-29 cells were seeded and after 24 h the medium was replaced with new cell
culture medium containing different concentrations of La-5 CFSM or LH-2 CFSM and incubated for 24 h. Results
represent the means ± SD of two independent experiments performed in triplicate. Bars identified with ** are
significantly different from untreated cells (P < 0.01). NS means there is no significant difference compared to
untreated cells (P ≥0.05)
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
Via
ble
Ce
ll A
mo
ut/
ml
A NS
NS
NS NS NS NS
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
7.0E+05
10% LH-2 5% LH-2 2.5% LH-2 1% LH-2 untreatedcells
Via
ble
Ce
lls A
mo
un
t/m
l
B
**
**
**
NS
40
Confocal Scanning Laser Microscopy
In our preliminary studies, the effectiveness of gentamicin treatment in removing extracellular
Salmonella was assessed using confocal scanning laser microscopy (CSLM) and optical density
monitoring; we also evaluated effectiveness of Salmonella internalization through different infection
time using CSLM.
It is important to know whether the gentamicin treatment effectively removes all extracellular
Salmonella since S. Typhimurium, phage-type DT104 used in our experiments were reported to be
resistant to multiple antibiotics. In addition, some strains possess an enhanced virulence phenotype (Wu
et al., 2002). The inhibitory effect of different concentrations of gentamicin (from 50 μg/ml to 350
μg/ml) was tested on the growth of Salmonella Typhimurium DT104 (SA 1997-0934, SA 2000-0406 &
SA 2001-4368) by monitoring optical density (OD) at 600 nm in TSB for up to 40 h. The results showed
that the lowest tested concentration of gentamicin (50 μg/ml) inhibited the growth of all Salmonella
strains (data not shown). It was concluded that 1 h incubation in the presence of 350 μg/ml gentamicin
followed by 30 min incubation with 200 μg/ml gentamicin could effectively remove extracellular
bacteria. To confirm this, confocal scanning laser microscopy (CSLM) was used to visualize the
internalized and externalized Salmonella GFP strain.
By using CSLM it was possible to visualize Salmonella invasion status in three dimensions with
minimum disruption of the specimen. In Figure 3.2, since Salmonella cells are visible, and the cell
morphology is close to what has been reported in the literature (Collins & Kennedy, 1999). The cells
appeared rod-shaped and 2.0- 5.0 µm in size. Furthermore, the use of CSLM allowed the detection of
minimum infection time for S. Typhimurium to invade HT-29 cells. Different Salmonella infection time
41
courses (1 h, 2 h and 3 h) with MOI of 10 were investigated in our preliminary study. All tested infection
times showed internalized Salmonella. Figure 3.3 exhibits confocal images for 2 h infection with or
without La-5 or LH-2 CFSM treatment. In addition, by adjusting the Z-stacks of the scan field and
performing a 3D reconstruction using the Leica confocal software, intracellularly located bacterial cells
were distinguished from those remaining outside of the host cells. Rarely motile Salmonella cells were
observed in the medium. However, a very small portion of bacteria were attached to the external surface
of the host cell membrane. These attached but not internalized bacteria to some extent affect the
accuracy of internalized Salmonella count. However, 1 h incubation with 350 μg/ml gentamicin and 30
min incubation with 200 μg/ml gentamicin removed the majority of extracellular bacteria. Therefore,
this gentamicin treatment was used in the further studies.
Figure 3.2 Confocal image of a single S. Typhimurium GFP cell. Light channel + argon channel (set at GFP
specific parameters 488 nm) merged confocal image of invaded S. Typhimurium GFP in HT-29 cells after 2 h of
infection (MOI 10). Image was taken after remove extracellular Salmonella using gentamicin. A white arrow
indicates one single S. Typhimurium GFP cell. Scale bar: 2 µm.
42
Figure 3.3 Merged confocal images of S. Typhimurium GFP invasion of HT-29 cells. HT-29 cells were seeded and incubated for 24 h. Then the medium was replaced with new
medium only (A) or new medium containing 1.5% of La-5 CFSM (B) or 1% LH-2 CFSM (C) and incubated for 24 h. S.Typhimurium GFP (MOI 10) was added without any
CFSM (A) or with the presence of 1.5% La-5 (B) or with the presence of 1% LH-2 CFSM (C) and incubated for 2 h. Images were taken after using gentamicin to remove
extracellular Salmonella. Scale bar: 20 µm.
43
Trans-epithelial Electrical Resistance (TEER)
The permeable insert used in this study is reported to benefit epithelial cell polarization and
differentiation (Ferruzza, Rossi, Scarino, & Sambuy, 2012). Polarized cells improve physiological
relevance of cell culture studies as they possess tight junctions and microvilli composed brush border,
which are similar to those of human intestinal epithelial cells of the gastrointestinal tract (Chalghoumi
et al., 2009; Cohen et al., 1999; Le Bivic et al., 1988; Rodriguez-Boulan & Nelson, 1989). Therefore,
cell culture using permeable inserts provides a useful model that can mimic specific properties of the
human intestinal epithelium. When cultured in glucose for a certain period of time, HT-29 cells form
very tight junctions and microvillus brush border at the apical surface, indicating cell polarization
(Fitzgerald et al., 1997; Höner zu Bentrup et al., 2006). Taking all these benefits into consideration, the
following experiments were performed using polarized HT-29 cells.
Initial tests were carried out to determine the optimal HT-29 cell density necessary to seed in Transwell
permeable inserts. A 500 μl of four different cell concentrations (1×104 cells/ml, 6×10
4 cells/ml, 1×10
5
cells/ml and 2×105 cells/ml) was seeded in apical chamber of Transwell inserts. Medium in both apical
and basolateral chambers in each well was changed every two days. TEER values for each well were
measured by Millicell ERS-2 apparatus as described in Chapter 2. Consequently, recorded TEER value
was calculated to ohm × cm2. The final values were obtained from triplicate inserts of each cell density
by subtracting average value of blank inserts (without cell) and multiplying by the area of the
monolayer (0.33 cm2) (Hasegawa et al., 1999). As shown in Figure 3.4, it was found that 1×10
5 cells/ml
gave a more linear and stable response in TEER values with less fluctuation. Hence, a 1×105 cells/ml
level was used as optimal cell density for inoculation of Transwell inserts in subsequent experiments.
44
In order to know how long it took for cells to become polarized, the growth of HT-29 cells in the
Transwell permable inserts was determined. Similar with previous TEER measurement, we grew 1×105
cells/ml HT-29 cells and recorded the growth in TEER values. From the Figure 3.5, it can be seen that
before the 12th
day, the TEER value was lower than 20 ohm × cm2. Cells showed steady increase in
TEER values between the 12th day to the 40
th day. After the 40
th day, the TEER value was stable in the
range of 120~140 ohm × cm2. Since the increase in TEER value was an index of cell confluence, tight
junctions formation, and cell polarization establishment (Ghadimi, de Vrese, Heller, & Schrezenmeir,
2010), 120 ohm×cm2 TEER value on day 40 was considered as a mature status of cell polarization. Cells
with TEER value ≥ 120 ohm×cm2 were used in the following experiments.
45
Figure 3.4 Determination of optimal HT-29 cell density in Transwell permeable inserts. A 500 μl of four different cell concentrations (1×104 cells/ml, 6×10
4 cells/ml, 1×10
5
cells/ml and 2×105 cells/ml) was seeded in apical chamber of Transwell inserts. Medium in both apical and basolateral chambers in each well was changed every two days.
TEER values for each well were measured by Millicell ERS-2. Consequently, recorded TEER value was calculated to ohm × cm2. Results represent the means ± SD of two
independent experiments performed in triplicate.
-20
0
20
40
60
80
100
0 5 10 15 20 25 30 35
TEER
(o
hm
× c
m2)
Time (Day)
1×10^4
6×10^4
1×10^5
2×10^5
46
Figure 3.5 Growth of HT-29 cells in Transwell permeable inserts. 1×10
5 cells/ml HT-29 cells was seeded in the apical chambers of the inserts. Medium in both apical and
basolateral chambers in each well was changed every two days. TEER values for each well were measured by Millicell ERS-2. Consequently, recorded TEER value was
calculated to ohm × cm2. Results represent the means ± SD of two independent experiments performed in triplicate.
0
20
40
60
80
100
120
140
160
180
200
0 10 20 30 40 50 60 70
TEER
(o
hm
× c
m2)
Time (day)
47
Polarization of HT-29 cells occurs after a prolonged period of post-confluence culture. Instead of
waiting for 40 days to obtain polarized cells, other researchers have investigated using substances to
induce cell differentiation, including drugs, chemicals or by changing the energy source from glucose to
galactose in standard culture medium (Mitchell & Ball, 2004). Cohen et al. (1999) determined that 20 h
of exposure to the drug forskolin could induce up to 80% cell polarization among 21-day old HT-29
cells. Mocodazole and taxol, functioning as microtubule disrupters, were also discovered to affect brush
border formation in cells grown for 21 days. While another microtubule disrupting agent, colchicine,
could induce apical polarization of most HT-29 cells in only 7 days (Cohen et al., 1999). Fitzgerald et al.
(1997) revealed that HT-29 cells became polarized in 21 days when the extracellular environment was
adjusted to pH 5 (Fitzgerald et al., 1997). In addition, molecular engineering of HT-29 accelerates the
cell polarization period, as well as expands choices in cell line selection. Cloned HT-29 cell lines
exhibiting various cell forms in order to meet different research needs have been produced (Huet,
Sahuquillomerino, Coudrier, & Louvard, 1987; Mitchell & Ball, 2004). After treating cells with sodium
butyrate, the HT-29cl.19A cell line became differentiated and polarized, whereas HT-29cl.16E was
goblet-like and secretory (Mitchell & Ball, 2004). After replacing glucose as an energy source with
galactose, H29-18-C1 became absorptive, while HT29-18-N2 became mucus secreting (Huet et al.,
1987). In conclusion, the prolonged cell culture times used in this study could be replaced by other
methods to create polarized cells, which will shorten the experimental time.
Epithelial Barrier Integrity Study
Many investigators have demonstrated that pathogenic microorganisms have the ability to disrupt
epithelial cell integrity, resulting in abnormal effects on the host, e.g., diarrhea (Canil et al., 1993;
48
Guttman & Finlay, 2008; Solano et al., 2001). A polarized monolayer of epithelial cells form tight
junctions, while S. Typhimurium infection cause a rapid loss in monolayer integrity which can be
detected by TEER (Chalghoumi et al., 2009). TEER loss is correlated with Salmonella invasiveness,
since non-invasive Salmonella does not affect TEER values (Finlay & Falkow, 1990). In addition to
TEER loss, Salmonella can cause apical and basolateral cell depolarization, which is presumable the
reason for tight junction disruption (Finlay et al., 1988; Finlay & Falkow, 1990). When Salmonella
attach to the host cell surface, they use a Type Three Secretion System (T3SS) to inject signals which
assist in their internalization. The host cell membrane becomes ruffled to allow uptake of associated
bacteria (Guttman & Finlay, 2009; Solano et al., 2001).
Probiotics are reported to enhance epithelial barrier integrity and modify the host cell cytoskeleton
(Johnson-Henry, Hagen, Gordonpour, Tompkins, & Sherman, 2007; Resta-Lenert & Barrett, 2003).
Klingberg et al. (2005) concluded that measurement of TEER, where intestinal epithelial cells
monolayer grown in permeable inserts, could be used to evaluate probiotic activity. Hence, in this
epithelial barrier integrity study, the protection afforded by La-5 or LH-2 CFSM on cell integrity
following S. Tyhpimurium infection in polarized HT-29 cells was evaluated.
A preliminary study found that 24 h incubation with 1.5% La-5 CFSM or 1% LH-2 CFSM produced a
small but insignificant increase in TEER value of polarized HT-29 in the inserts (data not shown). In
accordance with previous findings, HT-29 inserts were used when TEER values were above 120 ohm ×
cm2. The initial TEER value in this study was 132 ± 10 ohm × cm
2. The first measurement at time t0
was conducted right after the gentamicin step used to reduce extracellular populations of Salmonella.
Following Salmonella infection, the TEER value at t0 fell broadly (60.4 ± 0.8%) compared to the
initial value. However, cells to which no Salmonella were added also showed a reduction in TEER of
49
43 ± 3%. Figure 3.6 shows the ratio of TEER values obtained at the time of analysis to that obtained
initially for all experimental groups. La-5 infected or LH-2 infected groups were pre-incubated with
1.5% La-5 CFSM or 1% LH-2 CFSM, respectively, and 107 CFU/ml S. Typhimurium (bacteria: cell
MOI of 10) were added. The positive control group comprised HT-29 cells, which were exposed to 107
CFU/ml S. Typhimurium (MOI of 10) only. The negative control group consisted of HT-29 cells to
which neither CFSM, nor Salmonella were added. There was an increase in TEER values for all
groups at t1 and t2 (the first 2 h post-infection). In Fig. 3.6, the negative control group maintained
stable TEER values after time t2. On the contrary, there was a gradual decline in TEER value for all
cells to which Salmonella was added. However, the reduction in TEER value was significantly
alleviated at time t8 (8 h post-infection) when La-5 (P <0.05) or LH-2 (P <0.01) CFSM was presented.
At t22 there was attenuation but insignificantly in TEER loss among La-5 or LH-2 infected groups
than positive control.
50
Figure 3.6 Ratio of TEER of polarized HT-29 cell monolayers after exposure to Salmonella Typhimurium to initial values. HT-29 cells were exposed to 107 CFU/ml S.
Typhimurium (MOI of 10) for 2 h. After 1, 2, 3, 4, 8 and 22 h of recovery in antibiotic-free cell culture medium, the changes in the TEER were measured using a Millicell ERS-2
apparatus. Result was expressed as the ratio of TEER at time t in relation to the time zero t0 (the first TEER measurement after gentamicin step) for each series. Results represent
the means ± SD of two independent experiments performed in triplicate. The dot identified with ** is significantly different (P < 0.01) compared to positive control group, and
of which identified with * is significant difference (P <0.05).
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
0 5 10 15 20 25
Rat
io o
f TE
ER (t/t 0
)
Time (h)
positive ctrl
La-5 infected
LH-2 infected
negative ctrl
**
*
51
The most vulnerable point to pathogen penetration is the paracellular space between cells. The normal
network of tight junction associated proteins is able to seal this pathway for microbial translocation
while allowing the transportation of electrolytes and other nutrients (Moorthy, Murali, & Devaraj,
2009). Probiotics have an advantageous impact on tight junction related proteins by increasing
expression of protein zonula occludens ZO-1 and preventing adverse alteration of protein occluding,
both of which benefit epithelial monolayer integrity (Yu et al., 2012). Klingberg et al. (2005) found a
time and dose dependent enhancement of monolayer integrity for polarized Caco-2 cells when probiotic
L. plantarum MF1298 and L. salivarius DC5 were presented. They concluded that the increase in
TEER value was correlated to the increase in the expression of ZO-1. Furthermore, they demonstrated
that L. monocytogenes-induced epithelial barrier malfunction was attenuated when cells were
pre-incubated with L. plantarum MF129 (Klingberg et al., 2005). Our finding that Lactobacillus CFSM
improved epithelial barrier integrity 8 h post-infection is in contrast with the Klingberg et al. work,
whereby they proposed that only live probiotics could exert the beneficial effects within 8 h since
heat-inactivated L. plantarum cells and L. plantarum cell-free supernatants from culture medium did
not increase TEER values (Klingberg et al., 2005). The reason for this difference could be the virulence
of the pathogen (Solano et al., 2001) or the probiotic strains applied. Yu et al. (2012) found that S.
Typhimurium induced more severe damage to cell integrity than E. coli (Yu et al., 2012). Interestingly,
even though Resta-Lenert & Barrett (2003) used Salmonella in their study, they still concluded that only
live probiotics improved epithelial barrier integrity since antibiotic killed, heat inactivated and spent
medium of Streptococcus thermophilus and Lactobacillus acidophilus failed to enhance TEER values.
This may be due to the amount of spent medium applied since no information was provided on the
concentrations (Resta-Lenert & Barrett, 2003). In another probiotic study, Yu et al. (2012) tested
52
expression of tight junction proteins, including ZO-1, claudin-1, and E-cadherin using a fluorescent dye.
The probiotic strain L. amylophilus D14 did not change the distribution of tight junction proteins, but it
was able to improve the expression of tight junction proteins and moderate their abnormal distribution
triggered by the presence of pathogenic E. coli or S. Typhimurium (Yu et al., 2012).
Probiotics are speculated to modulate Salmonella-induced cytokine-mediated alteration in paracellular
permeability, including interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) (Capaldo
& Nusrat, 2009). Epithelial cells respond to IFN-γ by restructuring actin, reducing tight junction protein
expression or displacing scaffolding protein ZO-1 (Blum et al., 1997; Youakim & Ahdieh, 1999). Even
though conflicting reports exist, TNF-α has been shown in several studies to directly impair tight
junction function associated with NF-κB pathway interruption (Ma et al., 2004; Ye, Ma, & Ma, 2006).
Furthermore, IFN-γ and TNF-α co-treatment exerts a synergistic effect, which increases barrier
sensitization and malfunction (Fish, Proujansky, & Reenstra, 1999; Wang et al., 2005). The CFSMs
used in this study were expected to perform cytokine adjustment, since Tellez Garay (2009) revealed
a dose dependent effect for IFN-γ and TNF-α modulation by a peptidic fraction of LH-2 CFSM (F5)
in vivo. She found that when the fraction was applied at a rate of 0.02 μg/mouse/day, the IFN-γ level
was significantly lower than in mice not given the fraction, but the fraction also induced TNF-α
production (Tellez Garay, 2009). Hence, the modulation of cytokines when epithelial cells are
stimulated with CFSMs may contribute to the epithelial barrier integrity.
Salmonella entry into the host cell induces membrane ruffling and rearrangement of actin filaments,
resulted in a reduction in TEER values. On the contrary, increase of TEER values were observed at
the first two h after Salmonella infection (see t1 & t2 in Fig. 3.6). Similar TEER increase trend was
also observed by Klingberg et al. (2005) when 107 CFU/cm
2 of Listeria monocytogenes was added in
53
polarized Caco-2 monolayer cells. However, the authors failed to give an explanation for this
phenomenon. The reason for the increase in TEER values at time t1 and t2 could be due to temperature
and homogeneity of culture medium. Matter and Balda (2003) discovered that the TEER value was
dependent on temperature using polarized Madin-Darby Canine Kidney (MDCK) cells. The first
measurement of TEER was carried out at 37°C. After 15 min, a second measurement was performed at
24°C and a final measurement after a further15 min was carried out at 4°C. They found that the higher
temperature the lower TEER value was. In order to get stable and representative values, TEER
measurements require a highly controlled ambient temperature (Matter & Balda, 2003). Figure 3.7
shows the effect of temperature on TEER value. In our experiment, the TEER value at t0 was obtained
after several rinses with PBS at room-temperature (24°C); whereas later TEER values were obtained at
37°C. Different temperature causes TEER value fluctuation. The other factor that may influence TEER
value is whether the cell culture medium is in homeostasis. Stable TEER result would be achieved if
cell culture medium are regulated and homogenized (Matter & Balda, 2003).
In conclusion, La-5 or LH-2 CFSM enhances the epithelial barrier integrity by protecting cells‟ tight
junctions. The measurement of TEER showed that CFSMs started to alleviate the TEER loss caused by
S. Typhimurium late after its infection (8 h post-infection).
54
Figure 3.7 Temperature dependent of TEER on polarized MDCK (Madin-Darby Canine Kidney) cells grown on
permeable inserts for 5 days. Data were obtained from four separate culture with 15 min time interval under
37°C, 24 °C and 4 °C conditions, adapted from Matter & Balda, 2003.
LDH Cytotoxicity Assay
The extracellular stimuli on plasma membrane damage can be quantified by measuring LDH
production. It is an accurate index of cell death or cytotoxicity, as well as a convenient method for the
evaluation of protective effect of CFSM on cell membrane integrity. First of all, we confirmed that 1.5%
La-5 or 1% LH-2 CFSM were not toxic for cells since the LDH production of cell incubated with CFSM
for 24 h was not significant different compared to untreated cells (data not shown). The optimal HT-29
cell seeding concentration was 1 × 105 cells/ml due to its rapid and reproducible detection of LDH.
Salmonella concentrations were evaluated to provide a Multiplicity of Infection (MOI, number of
bacteria: number of cells) of 10, 20, or 50 with 1, 2, or 3 h infection periods. The LDH percentage
cytotoxicity was calculated for each group, as shown in Figure 3.8. 1 h infection time was not adequate
for all MOI (data not shown). However, after 2 h of infection and with MOI of 10 the LDH cytotoxicity %
(22.3 ± 8.7%) was twice that observed for all the other test conditions. After 3 h exposure to
Salmonella (MOI of 50), destruction of HT-29 cells was nearly completed (98.6 ± 3.4%). Taking
previous confocal microscopy results into consideration that 2 h infection period allowed S.
55
Typhimurium to enter cells, the Salmonella Typhimurium infection condition with MOI of 10 for 2 h
was chosen for the LDH study.
Figure 3.8 Cytotoxicity (%) of HT-29 cells produced by S. Typhimurium at different times of exposure and
different MOI. 1 × 105 cells/ml HT-29 cells were seeded in a 96-well plate. LDH production was measured after
adding different MOI of S. Typhimurium and incubated for 2 or 3 h. Results represent the means ± SD of two
independent experiments performed in triplicate.
In addition, the effect of CFSM on Salmonella infection of HT-29 cells was investigated. Pre-exposure
to probiotic strains exerted an inhibitory effect on pathogen activities like adhesion, cytotoxicity and
invasion (Burkholder & Bhunia, 2009; Jankowska, Laubitz, Antushevich, Zabielski, & Grzesiuk, 2008;
Myllyluoma et al., 2008). Therefore, we compared the release of LDH from infected HT-29 cells when
they were pre-incubated with CFSM for 24 h prior to Salmonella infection or co-incubated with CFSM
during 2 h of Salmonella infection. From Figure 3.9, it is shown that co-incubation with CFSM had
almost triple LDH release than with which were co-incubated. The pre-incubation with La-5 CFSM
significantly reduced LDH production caused by Salmonella invasion than positive control. Therefore,
pre-incubation with CFSM for 24 h improved cell monolayer integrity. To strengthen the protective
22.3%
74.7%
12.7%
78.0%
12.5%
98.6%
0%
20%
40%
60%
80%
100%
120%
2 h 3 h
Cyt
oto
xici
ty %
S. Typhimurium infection time (h)
MOI10
MOI20
MOI50
56
effect of CFSM, in addition to pre-incubation, we also co-incubated non-toxic doses of CFSM with
HT-29 cells during exposure to Salmonella. Both pre-incubation and co-incubation with Salmonella
were applied in further studies.
Figure 3.9 Evaluation of CFSMs incubation conditions. 1 × 105 cells/ml polarized HT-29 cells were seeded in a
96-well plate. LDH production was measured after adding S. Typhimurium (SA1997-0934, MOI of 10) and
incubated for 2 h. Positive control represents S. Typhmurium infected cells. Negative control represents untreated
cells. Test groups were either pre-incubated with CFSM or co-incubated with CFSM. All the data were
normalized to positive control (positive control = 100% cytotoxicity). Results represent the means ± SD of two
independent experiments performed in triplicate. Bars identified with ** are very significantly different (P < 0.01)
compared to positive control group, and of which identified with * is significant different (P < 0.05). NS means
no significant difference compared to positive control (P ≥0.05)
As for the preliminary study, 100 μl of 1 × 105 HT-29 cells/ml cells was seeded in a 96-well plate. The
cells were pre-incubated with La-5 or LH-2 CFSM for 24 h and co-incubated with the CFSM when
exposed to Salmonella (MOI 10) for 2 h. Figure 3.10 shows the effect of CFSM on Salmonella infection
of epithelial cells. Compared with the positive control, very significant differences (P <0.01) were
found for both La-5 treated and LH-2 treated groups on normal HT-29 cells and polarized HT-29 cells.
100.0% 75.1%
62.9% 64.4%
192.8% 185.1%
0%
50%
100%
150%
200%
250%
La-5 LH-2 La-5 LH-2
positivecontrol
negativecontrol
pre-incubation co-incubation
Cyt
oto
cxic
ity
%
on polarized HT-29
** **
* NS
57
In the normal HT-29 cell line, cytotoxicity % (as measured by LDH release) in the La-5 treated group
and LH-2 treated group declined by 57 ± 1% and 58 ± 2%, respectively; whereas for the polarized
HT-29 cell line cytotoxicity % in the La-5 treated group and LH-2 treated group was reduced by 46 ± 3%
and 51 ± 2%, respectively. Interestingly, we found on both normal and polarized HT-29 cells, La-5
treated and LH-2 treated groups exhibited very significant reduction (P <0.01) in cytotoxicity
compared to the negative control. Besides protecting cells against S. Typhmurium infection, it is
speculated that CFSM could also benefit epithelial cells under normal growth conditions by reducing
the amount of necrotic cells, which would result in improvement of epithelial membrane integrity.
Figure 3.10 LDH cytotoxicity of epithelial cells induced by Salmonella Typhimurium in the presence or absence
of CFSM treatment. Positive control represents S. Typhmurium infected cells. Negative control represents
untreated cells. La-5 represents La-5 pre-incubation and co-incubation during S. Typhimurium infection. LH-2
represents LH-2 pre-incubation and co-incubation during S. Typhimurium infection. All the data were normalized
to positive control (positive control = 100% cytotoxicity). Results represent the means ± SD of two independent
experiments performed in four replications. Bars identified with ** are very significantly different (P < 0.01)
compared to positive control group.
LDH cytotoxicity evoked by pathogen invasion was alleviated by bioactive components from probiotic
strains. This result was in agreement with previous studies where the cytoprotective effect of
probiotics against a variety of pathogens or physiological stress have been reported. LDH liberation is
100.0% 100.0%
43.2% 53.2%
41.8% 50.9%
75.2% 68.8%
0%
20%
40%
60%
80%
100%
120%
on HT-29 on Polarized HT-29
Rel
ativ
e C
yto
tocx
icit
y %
positive control La-5 LH-2 negative control
** ** ** **
*
*
58
an indicator of cell membrane damage and cell viability. Hence, a reduction in the amount of LDH
released could demonstrate an advantagous impact of probiotics on the host. Burkholder & Bhunia
(2009) found that 1 h pre-incubation with L. rhamnosus GG (LGG) strain significantly attenuated S.
Typhimurium-induced cytotoxicity for both normal and thermally stressed (41°C, 1 h) Caco-2 cells.
Therefore, LGG effectively improved epithelial cell health and mucosal integrity during infection or
exposure to toxins (Burkholder & Bhunia, 2009). H. pylori-evoked cell membrane damage was very
significantly alleviated (P <0.001) by three probiotic strains, namely L. rhamnosus GG, L. rhamnosus
Lc705, P. freudenreichii subsp. shermanii Js (Myllyluoma et al., 2008). In conclusion, La-5 and LH-2
CFSM have potent ability to mitigate pathogen-induced cell damage.
Apoptosis Assay Using Flow Cytometry
To evaluate whether the beneficial effects of CFSM indicated by the LDH assay were reflected in a
reduction of Salmonella-induced apoptosis of HT-29 cells, flow cytometric TUNEL analysis of
apoptotic cells with FITC/PI dual stains was counted out.
The optimum Salmonella exposure time to cause cell apoptosis was determined. HT-29 cells were
pre-incubated with non-toxic doses of CFSM for 24 h, and this was followed by co-incubation in the
presence of CFSM and Salmonella (MOI 10). The selection of Salmonella infection period was based
on the results obtained using the LDH assay, as LDH release indicates the phase of late apoptosis/early
necrosis (Milovic et al., 2001). Infection periods of 1 h, 2 h or 3 h were then investigated for the
apoptosis assay. 1 h infection period caused up to 95% apoptosis on polarized HT-29 cells (data now
shown). Therefore, 1h infection time was chosen for the apoptosis assay.
DNA fragmentation is one of the most distinguishing features of apoptotic cells. When a DNA strand
59
breaks, the FITC stain labels the 3‟-hydroxyl (OH) end of double- and single-stranded DNA catalyzed
by the enzyme, terminal transferase (TdT; a template-independent addition). Apo-Direct™ Kit (BD
Bioscience, Mississauga, ON, Canada) was used to assess DNA strand breakage using FITC/PI dual
stains. Cell viability: vital cells, apoptosis and necrosis were discriminated by different staining. Cells
with the staining patterns FITC(-) and PI(-) were designated as vital cells, FITC(+) and PI(-) as
apoptotic cells, and FITC(+) and PI(+) as late apoptotic or necrotic cells (Punj et al., 2004). Figure 3.11
shows the effect of CFSM on Salmonella Typhimurium induced apoptosis of polarized HT-29 cells.
Cell diameter is reflected by forward-angle light scatter (FSC) and the conformation of inner cellular
structure is reflected by side-angle light scatter (SSC) (Vermes et al., 2000). Comparing light scattered
figures with different exogenous stimuli (A- D in figure 3.11), positive control (B), La-5 treatment
group (C) and LH-2 treatment group (D) resulted in a slight decrease of both FSC and SSC compared to
the negative control (A). Vermes et al. (2000) stated that during the initial phases of apoptosis, FSC
decreased while SSC increased or remained unchanged. After several h both FSC and SSC diminished;
indicating that cells underwent apoptosis (Vermes et al., 2000). Therefore, it was an evident that 1h
Salmonella infection (MOI 10) induced apoptosis of polarized HT-29 cells. To check the effects of
CFSM, FITC signals (Fig 3.11 E-H) and cell viability figures (Fig. 3.11 I-L) were examined.
Figure 3.11 E-H shows the intensity of FITC stain. The M1 and M2 regions in FL1-H reflected negative
FITC stain and positive FITC stain, respectively. The positive control (F) had its FITC signal peak in
the M2 region, indicating the greatest amount of apoptotic cells in this group compared to others. Even
though the peak of fluorescence intensity in graphs of the La-5 treatment group (G) and LH-2 treatment
group (H) was shifted more to the right side (higher FITC fluorescence intensity) than the negative
control (E), the major proportion of FITC signals in these groups were still in the M1 region.
60
A B C D
E F G H
Figure 3.11 Effect of CFSM on Salmonella Typhimurium induced apoptosis of polarized HT-29 cells. A-D showed Forward (FSC-H) and Side (SSC-H) light scattering graphs.
E-H showed the dot plot of FITC signals intensity. The first lane (A & E) represents negative control (untreated cells); the second lane (B & F) represents positive control (S.
Typhimurium infected cells); the third lane (C & G) represents La-5 treatment (La-5 pre-incubation and co-incubation during infection); the fourth lane (D & H) represents
LH-2 treatment (LH-2 pre-incubation and co-incubation during infection). Light scattering signals indicate morphological change of cells. FL1-H displays the number of FITC
positive stain cells. For statistical analysis see Figure 3.12.
61
The data shown in Figure 3.12 were obtained from the cell number ratio of M2/M1, indicating the
percentage of apoptotic cells. This analysis provided a better view of the effect of CFSM on Salmonella
induced apoptosis in polarized HT-29 cells. Compared with the positive control, very significant
differences (P <0.01) were found in cells treated with La-5 and LH-2. Apoptosis of La-5 treated or
LH-2 treated groups compared with positive control were dramatically reduced by 76 ± 2% and 62 ±
7%, respectively. Interestingly, we found La-5 stimulated or LH-2 stimulated groups exhibited a
slight reduction in apoptosis compared to the negative control. These findings further supported the
results obtained using the LDH assay and demonstrate that CFSM enhanced the health of epithelial
cells under normal growing conditions and protected them from Salmonella infection.
To further confirm the anti-apoptotic effect of CFSM, cell viability was determined by comparing
levels of FITC (FL1-H)/PI (FL2-A) dual stain signals (as shown in Figure 3.13).
As shown in Figure 3.13 FITC and PI dual stains differentiated cell viability. Cell population in the
lower left quadrant [FITC(-) and PI(-)] were designated as vital cells; cell populations in the upper left
quadrant [FITC(+) and PI(-)] were designated as apoptotic cells. The upper right quadrant represented
late apoptotic or necrotic cells, which was merely observed in all the tested groups. For the figures, the
positive control (B) consisted of cell population distributed mainly in the upper left quadrant, indicating
the presence of more apoptotic cells than other groups. Even though cells treated with La-5 or LH-2 was
distributed in the upper left quadrant than negative control, it was obvious that they were significantly
lower than the positive control. Cell viability, determined by comparing efficiency of the two stains,
suggested that CFSM treatment had a significant anti-apoptotic effect on Salmonella-induced apoptosis
of polarized HT-29 cells.
62
Figure 3.12 Apoptotic rate of polarized HT-29 cells and the protective effect of CFSM pre-incubation (24 h) and
co-incubation during exposure to Salmonella (1 h) (Flow cytometric TUNEL analysis). La-5 and LH-2 stimulated
cells represented cells only pre-incubated with La-5 and LH-2, respectively. La-5 and LH-2 groups represented
cells pre-incubated with CFSM and co-incubated with Salmonella during 1h infection. Negative control
represented untreated cells. Positive control represented cells infected with Salmonella for 1 h. Results were
obtained after analyzing M2/M1 populations in FL1-H graph (As shown in Fig. 3.10 E-H), displaying in means ±
SD of two independent experiments performed in replicate. Bars identified with ** are very significantly different
(P < 0.01) compared to positive control group.
0.8% 1.0% 1.7% 8.0%
21.3%
83.8%
0%
20%
40%
60%
80%
100%
120%
La-5stimulated
cells
LH-2stimulated
cells
negativecontrol
La-5infected
LH-2infected
positivecontrol
Ap
op
tosi
s R
ate
%
** **
63
A B C D E
Figure 3.13 Cell viability by comparing efficiency of FITC (Y-axis)/PI (X-axis) dual stain signals. A: negative control (untreated cells); B: positive control (S. Typhimurium
infected cells); C: La-5 treatment (La-5 pre-incubation and co-incubation during Salmonella infection); D: LH-2 treatment (LH-2 pre-incubation and co-incubation during
Salmonella infection). E: cartoon detailing elaboration of quadrant gates of figures A-D, adapted from Jahan-Tigh et al., 2012.
64
Many apoptotic studies revealed that pathogen induced apoptosis of epithelial cells was a prolonged
process. Conversely, our experiment found that S. Typhimurium with MOI of 10 caused up to 84 ± 15%
apoptosis of polarized HT-29 cells within 1 h. The differences could be due to using different strains,
different MOI, different treatment before fluorescence staining and, most likely, different cell models.
It is speculated that apoptosis is not easily induced in carcinoma derived cells. Kim et al. (1998) claimed
that it took 12-18 h for bacterial adhesion, invasion and replication, as well as expression of apoptotic
signals to initialize the appearance of apoptosis in HT-29 cells (Kim et al., 1998). Lundberge et al.
(1999) revealed that HeLa, Caco-2 and MDCK cells did not show apoptosis after Salmonella infection
(MOI 25, 30 min), while 80% of macrophages were not viable under the same conditions, elaborating
apoptosis induction from Salmonella infection was more rapid in macrophages than in epithelial cells
(Lundberg, Vinatzer, Berdnik, von Gabain, & Baccarini, 1999). A Salmonella-effector associated with
phosphoinositide phosphatase activity SigD, also named SopB, may account for the late appearance of
apoptosis in epithelial cells compare with macrophages (Knodler et al., 2005). Furthermore, Knodler
and Finlay (2001) compared the time for the occurrence of apoptosis among carcinoma HeLa cells and
a non-transformed rat intestinal epithelial cell line, IEC-6. They found much faster kinetics of
Salmonella induced apoptosis on IEC-6 cells than immortalized carcinoma HeLa cells (Knodler &
Finlay, 2001). This suggests that it is harder to show apoptosis in a carcinoma cell line. In our apoptosis
assay, the cell line we used was a polarized human colon carcinoma HT-29 cell line, which was grown
in Transwell inserts for up to 40 days. The biophysical features of these cells were closer to naïve
human intestine epithelial cells. It is speculated that cell polarization could exert a major role on the rate
of appearance of apoptosis.
Besides the diverse kinetics of using different strains, which may lead to variations in time to induce
65
apoptosis, another factor could be the amount of bacteria that were added. It is reported that the
apoptotic rate of epithelial cells increased with bacterial inoculum size (Kim et al., 1998). Cerquetti et al.
(2008) found more than 80% apoptosis caused by S. Enteritidis with MOI 10 in macrophage RAW
264.7 cells, whereas around 25% apoptosis and less than 10% apoptosis were produced by S. Enteritidis
with MOIs of 1 and 0.1, respectively (Cristina Cerquetti, Hovsepian, Sarnacki, & Goren, 2008).
Therefore, the amount of bacteria added to cells was a crucial factor to determine time to induce
apoptosis. Furthermore, how cells are treated before the fixation step of the assay, together with the
procedures for fluorescence staining may also affect the apoptosis timeline. After 1 h infection of S.
Dublin, Kim et al. (1998) left cells in the presence of gentamicin for a designated time before fixation.
As a result, they found apoptotic cells (cell nuclei stained with Hoechst dye 33258) did not always
appear in the presence of Salmonella (GFP expressing). Some apoptotic cells did not contain
GFP-labeled Salmonella inside. They hypothesized during apoptosis intracellular Salmonella cells were
either killed possibly by entry of gentamicin from culture medium and/or they were released from
apoptotic cells. Moreover, they found prostaglandin H synthase-2, which should inhibit apoptosis in
both bacteria-infected intestinal epithelial cells and non-infected cells, contractedly increased the
numbers of apoptotic cells in both infected and non-infected neighboring epithelial cells after more than
24 h post-infection (Kim et al., 1998). In our study, extracellular Salmonella cells were treated with
gentamicin for 1.5 h in total, followed by an instant fixation step. The exact mechanism of transmission
of apoptotic signals is yet to be elucidated. From the host perspective, an extended gentamicin step
would affect the amount of apoptotic cells to some extent. Instant fixation is crucial to determine the
total amount of cells undergoing Salmonella-induced apoptosis.
In recent years, there have been numerous publications indicating that probiotics suppress cellular
66
apoptosis following infection by a pathogen. Putative mechanisms involved in this suppression effect
have been investigated (as described in Chapter 1). Some of the cytokines, such as Interferon INF-γ
(Gobbato et al., 2008), Tumor Necrosis Factor TNF-α and Interleukin IL-8 (Hausmann, 2010) are
considered to play a central role for triggering apoptosis. Based on previous in vivo and in vitro studies,
La-5 and LH-2 CFSM showed an ability to modulate cytokines. Tellez (2009) revealed a dose
dependent modulation on cytokines such as IFN-γ and TNF-α in mice fed with a peptidic fraction of
LH-2 CFSM (Tellez Garay, 2009). Further study on dendritic cells revealed that levels of the
proinflammatory cytokine TNF-α increased significantly (P <0.0001) in the presence of LH-2 and
La-5 CFSM (1:10 dilution) compared to a negative control (PBS treated). However a 1:100 dilution of
LH-2 and La-5 CFSM, which is similar to the concentration used in the present study, resulted in a
small but insignificant increase in the production of TNF-α by dendritic cells (Elawadli, 2012). All
these results could lead to the conclusion that the modulation of cytokines when epithelial cells are
stimulated with CFSM may contribute to their anti-apoptotic capacity during Salmonella infection.
La-5 or LH-2 CFSMs showed a significant reduction of Salmonella-induced apoptosis on polarized
HT-29 cells line.
SRB Assay
As one of the most consumed meats, much attention has been focused on broiler chickens. Chicken
meat provides valuable nutrition with relative reasonable price. Unfortunately, it also offers the largest
and most vital reservoirs for Salmonella colonization. S. Typhimurium and S. Enteritidis are two main
pathogenic strains responsible for human Salmonellosis in the past few years. These two serovars have
the ability to asymptomatically colonize chickens (Chalghoumi et al., 2009). With the previous
67
beneficial results in human epithelial cells, we created an in vitro chicken model using LMH chicken
hepatoma cells to evaluate the protective efficiency of La-5 and LH-2 CFSM against S. Typhimurium
isolated from chicken.
SRB viability results (see Figure 3.14) show a correlation between the CFSM concentration and LMH
cell proliferation. For La-5 CFSM, cells treated with 1% (v/v) exhibited higher viability than untreated
cells. In terms of LH-2, cells treated with 0.5%, 1%, 1.5% or 3% of LH-2 CFSM showed no statistical
significant difference with untreated cells. Further LDH test confirmed that 1% of La-5 (protein
concentration 1.0 mg/ml) or 1% of LH-2 (370 μg/ml) was not toxic for LMH cell line (data not shown)
and was used in the following experiments.
68
Figure 3.14 La-5 and LH-2 CFSM toxic dose test on LMH cells proliferation, estimated by SRB colorimetric
assay. Cells were plated and after 24 h the medium was replaced with new medium containing different
concentrations of La-5 CFSM (A) or LH-2 CFSM (B) and incubated for 24 h. Results are normalized to untreated
cells (viability of untreated cells = 100%) and display as means ± SD of two independent experiments performed
in triplicate. Bars identified with ** are very significantly different from untreated cells (P < 0.01). NS means
there is no significant difference compared to untreated cells (P ≥0.05).
73.9%
110.9% 100.8% 100.0%
87.3% 100.0%
0%
20%
40%
60%
80%
100%
120%
0.5% La-5 1% La-5 1.5% La-5 2% La-5 3% La-5 untreatedcells
Via
bil
ity
%
A
**
NS NS NS
NS
99.1% 102.4% 102.8%
67.8%
101.9% 100.0%
0%
20%
40%
60%
80%
100%
120%
0.5% LH-2 1% LH-2 1.5% LH-2 2% LH-2 3% LH-2 untreatedcells
Via
bili
ty %
B
**
NS NS NS NS
69
Invasion Assay
Invasion by Salmonella results in bacteria internalization and systematic spread (Chalghoumi et al.,
2009). Bacterial pathogenicity is determined by the organism‟s propensity to colonize and invade the
host cells. Therefore, an assay was conducted to evaluate CFSMs regulation of Salmonella invasion in
the chicken model.
Salmonella infection depends on factors such as bacterial strain, duration of exposure to the pathogen,
and MOI. Initially, same incubation condition as apoptosis assay (MOI of 10, 1 h infection) was
assessed on LMH cell line infected with chicken isolated Salmonella Typhimurium DT104
(SA2001-4368 & SA2000-0406). The results are shown in Figure 3.15. Only LMH cells treated with
LH-2 CFSM showed a significant reduction and this was limited to the SA2000-0406 isolate. Hence, it
may be concluded that Salmonella internalization depends on the bacterial strain. Lin et al. (2008) also
found that levels of inhibition due to L. acidophilus LAP5 differed between strains S. Choleraesuis 2a
and 26a, and this was probably due to differences in their ability to adhere to and invade cells (Lin, Tsai,
Lin, Tsen, & Tsai, 2008).
Afterward, we assessed the regulation of LH-2 CFSM under different infection conditions (shown in
Fig. 3.16), e.g., 2 h infection with MOI of 20 using S. Typhimurium SA 2000-0406, or 1 h infection with
MOIs of 1 or 0.1 using S. Typhimurium SA2001-4368. Under these conditions, the ability of
Salmonella to invade cells was reduced, albeit not significantly, in the presence of LH-2 CFSM. This
led to the conclusion that a reduction of Salmonella internalization in host cells may be one of the
mechanisms enabling CFSM to execute an antagonistic effect against Salmonella infection, but it is not
the most important factor.
70
Figure 3.15 Invasion assay on LMH cell using Salmonella Typhimurium DT104 (SA2001-4368 & SA2000-0406)
isolated from chicken for 1 h infection with MOI of 10. Positive control: infected cells. La-5: cells pre-incubated
with La-5 CFSM and co-incubated Salmonella in the presence of La-5 CFSM. LH-2: cells pre-incubated with
LH-2 CFSM and co-incubated Salmonella in the presence of LH-2 CFSM. Results are normalized to positive
control (positive control = 100%) and display as means ± SD of two independent experiments performed in
triplicate. The bar identified with * is significantly different (P < 0.05) compared to positive control. NS means
there is no significant difference compared to positive control (P ≥0.05).
Figure 3.16 Invasion assay using LMH cells exposed to different infection conditions. Positive control: infected
cells. LH-2: cells pre-incubated with LH-2 CFSM and co-incubated Salmonella with the presence of LH-2 CFSM.
Results are normalized to positive control (positive control = 100%) and display as means ± SD of two
independent experiments performed in triplicate. NS means there is no significant difference compared to
positive control (P ≥0.05).
100.0% 100.0% 98.6% 98.9% 95.2% 94.6%
0%
20%
40%
60%
80%
100%
120%
SA2001-4368 SA2000-0406
Rel
ativ
e In
vasi
on
Rat
e %
positive control La-5 LH-2
* NS NS NS
100.0% 100.0% 100.0% 98.9% 97.9% 95.5%
0%
20%
40%
60%
80%
100%
120%
140%
MOI 20, 2 h MOI 1, 1 h MOI 0.1, 1 h
SA2000-0406 SA2001-4368
Rel
ativ
e In
vasi
on
Rat
e %
positive control LH-2
NS NS NS
71
The ability to prevent pathogen invasion depends on the specific probiotics and tested pathogens,
indicating a high variability. Lin et al. (2008) found both a culture of L. acidophilus LAP5 strain and its
spent culture supernatant had a strong antagonistic effect on S. Choleraesuis infection of Caco-2 cells,
of which live probiotic strain showed a stronger antagonistic effect than its spent culture supernatant
(Lin et al., 2008). Ingrassia et al. (2005) determined that E. coli invasion of differentiated and
undifferentiated intestinal epithelial cells was inhibited by pre-incubation (75% and 84%) or
co-incubation (43% and 62%) with L. casei. A better inhibitory effect on E. coli invasion was observed
when L. casei culture supernatant was added to the incubation medium together with its live cells, and
this effect was thought to be associated with the probiotic adhesion improvement to the cells when the
supernatant was present (Ingrassia, Leplingard, & Darfeuille-Michaud, 2005). Interestingly, mucus
pre-treated with probiotic strains such as Lactobacillus (Gueimonde, Jalonen, He, Hiramatsu, &
Salminen, 2006; Tuomola, Ouwehand, & Salminen, 1999) or Bifidobacterium (Collado, Gueimonde,
Hernandez, Sanz, & Salminen, 2005) have been reported to increase the adhesion of pathogenic E. coli,
L. monocytogenes and S. Typhimurium. The biological significance of these increases is unknown, but
Collado et al. (2007) thought, instead of decrease the pathogens‟ adhesive ability, the protective
function of these probiotic strains against pathogens might compete for the specific adhesion site(s) and
receptors or other factors (Collado, Meriluoto, & Salminen, 2007).
Conclusions
In summary, results demonstrate that bioactive components from L. acidophilus (La-5) and L.
helveticus (LH-2) fermented milk enhanced the epithelial membrane integrity and exerted antagonized
effect against Salmonella Typhimurium infection. First of all, non-toxic doses of CFSMs were tested.
72
Trypan blue exclusion and LDH results indicated that 1.5% La-5 or 1% LH-2 CFSM (v/v) was safe on
HT-29 cell line; whereas SRB and LDH results showed that 1% La-5 or 1% LH-2 CFSM (v/v) was safe
on LMH cell line. Afterward, when HT-29 cells were pre-incubated with non-toxic dose of CFSM and
co-incubated during Salmonella infection with the presence of CFSM, Salmonella triggered TEER loss
[8 h post-infection: La-5 (P < 0.05); LH-2 (P < 0.01)], LDH production [La-5 or LH-2 (P < 0.01)] and
apoptosis [La-5 or LH-2 (P < 0.01)] were significantly attenuated. Since Salmonella often colonizes
and contaminates chickens asymptomatically, chicken hepatoma LMH cell line together with
Salmonella isolates from chicken was utilized as in vitro poultry model. Then the evaluation of CFSMs
on Salmonella invasion was carried out under various infection conditions. Results showed CFSMs
disruption of Salmonella invasion was highly strain dependent. Therefore, a reduction of Salmonella
internalization in host cells may be served as one of the mechanisms, but not the most important factor,
endorsing CFSM to perform an antagonistic effect against Salmonella infection.
73
CHAPTER 4
General Conclusions
The main purpose of this study is to determine the mechanism whereby bioactive components from L.
acidophilus or L. helveticus fermented milk (La-5 or LH-2) protect epithelial cells from Salmonella
Typhimurium infection. Based on previous in vitro and in vivo studies related to milk fermented by
probiotic bacteria performed in our laboratory, it was found that the bioactive components derived
from the probiotic bacteria fermentation stimulate the immune system and exert an antagonistic effect
against various enteric infections (Chin, 2002; Elawadli, 2012; M. J. Medellin-Peña, 2007; Ng, 2000;
Tellez Garay, 2009). It was further hypothesized that the bioactive components from milk fermented
with two probiotic bacterial strains (L. acidophilus La-5 and L. helveticus LH-2) could inhibit
Salmonella infectivity through modulation of cell membrane integrity and inhibition of
Salmonella-induced apoptosis in epithelial cells. To test this hypothesis, the non-toxic dose of La-5 and
LH-2 cell free spent medium (CFSM) was determined for application to epithelial cells using the
Trypan Blue Exclusion assay, LDH and SRB colorimetric assays. The integrity of the epithelial
monolayer was assessed by measuring trans-epithelial electrical resistance (TEER), as well as release
of intracellular LDH by a colorimetric assay. Afterward, the inhibitory effect of CFSM on Salmonella
induced apoptosis in epithelial cells using the TUNEL assay was determined. Finally, the evaluation of
CFSMs on Salmonella invasion was carried out under various infection conditions.
The research was divided in three sections: (1) the preparation of CFSM, (2) an in vitro study using
human colon carcinoma HT-29 cells, as a human model, and (3) an in vitro study using chicken
hepatoma LMH cells, as a poultry model. L. acidophilus La-5 or L. helveticus LH-2 cell-free spent
74
medium (CFSM) were prepared from a 48-h fermentation either with whey protein-based medium for
La-5 or skim milk for LH-2. The CFSMs were filtered, lyophilized and concentrated (10×). Human
colon carcinoma HT-29 cells and chicken hepatoma LMH cells were used as the in vitro human
intestinal model and the chicken model, respectively. In the human model, HT-29 cells were grown in
Transwell permeable inserts for 40 days to allow the cells to become polarized, as polarized cells are
more physiologically and biochemically related to native human intestinal cells. Pre-incubation and
co-incubation with CFSM during Salmonella infection sensibilized and enhanced cell membrane
integrity. In TEER measurement, the presence of La-5 (P <0.05) or LH-2 (P <0.01) significantly
attenuated the S. Typhimurium-induced disruption of the epithelial barrier function at 8 h post-infection.
In addition, release of intracellular LDH, as an index of cell membrane damage induced by Salmonella,
was also attenuated (P <0.01) in the presence of both La-5 or LH-2. To further elaborate the mechanism
of the protective action on cells, apoptosis induced by Salmonella was investigated. When cells were
exposed to CFSM from La-5 or LH-2, significant (P <0.01) anti-apoptotic effects were observed.
Interestingly, without Salmonella infection, cell membrane integrity was also improved when cells
were exposed to La-5 or LH-2 CFSM.
The effects were less pronounced when chicken hepatoma LMH cells were used. Exposure of the LMH
cells to CFSMs of La-5 or LH-2 resulted in less than a log10 cycle reduction in the number of
internalized Salmonella cells. The impact varied depending on the Salmonella strain tested. Further
work using the chicken cell line needs to be done in order to elucidate whether CFSMs exert similar
protective mechanisms against Salmonella and whether this offers a promising approach to reduce
Salmonella carriage in poultry.
Overall, it can be concluded that the inhibitory function of CFSMs on Salmonella internalization may
75
not play a pivotal role in preventing salmonellosis, but the enhancement of cell membrane integrity and
anti-apoptotic effect on Salmonella-infected cells suggest that bioactive molecules produced by
probiotics hold promise as therapeutic and prophylactic agents to combat enteric infections.
Future Research
Encouraged by such in vitro results, future work could focus on the anti-microbial effects of CFSM
from various LAB strains and how medium composition affects the activity of the bioactives.
Moreover, the synergistic effects of diverse bioactive components from different LAB should be
investigated to determine if their combinatorial effects offer better protection to the intestine.
Furthermore, the stability of the bioactive components present in the CFSMs needs to be investigated.
Improvement could be achieved by using protective agents and other formulations e.g., encapsulation
material (Jankovic, Sybesma, Phothirath, Ananta, & Mercenier, 2010). Probiotic bacterial genes could
be modified to improve proteolytic activity during fermentation in order to acquire a greater array of
bioactive components. Along with the previous in vivo studies that revealed CFSMs had an
antagonistic effect against various enteric pathogens, in vivo anti-apoptotic activity of La-5 or LH-2
against S. Typhimurium infection as well as their active doses in laboratory animals or in clinical
trials need to be assessed. These findings will contribute to providing solid evidence to related
government departments, such as Health Canada, for the approval of using these CFSMs as
therapeutic supplements in functional foods.
76
REFERENCE
Anderson, R. C., Cookson, A. L., McNabb, W. C., Park, Z., McCann, M. J., Kelly, W. J., & Roy, N.
C. (2010). Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by
increasing the expression levels of genes involved in tight junction formation. Bmc Microbiology,
10, 316.
Apella, M. C., Gonzalez, S. N., Demacias, M. E. N., Romero, N., & Oliver, G. (1992). Invitro studies
on the inhibition of the growth of shigella-sonnei by lactobacillus-casei and lact-acidophilus.
Journal of Applied Bacteriology, 73(6), 480-483.
Ashida, H., Mimuro, H., Ogawa, M., Kobayashi, T., Sanada, T., Kim, M., & Sasakawa, C. (2011).
Host-pathogen interactions cell death and infection: A double-edged sword for host and
pathogen survival. Journal of Cell Biology, 195(6), 931-942.
Auty, M., Duffy, G., O'Beirne, D., McGovern, A., Gleeson, E., & Jordan, K. (2005). In situ
localization of escherichia coli O157 : H7 in food by confocal scanning laser microscopy.
Journal of Food Protection, 68(3), 482-486.
Backert, S., Boehm, M., Wessler, S., & Tegtmeyer, N. (2013). Transmigration route of campylobacter
jejuni across polarized intestinal epithelial cells: Paracellular, transcellular or both? Cell
Communication and Signaling, 11, 72.
Balda, M. S., Whitney, J. A., Flores, C., Gonzalez, S., Cereijido, M., & Matter, K. (1996). Functional
dissociation of paracellular permeability and transepithelial electrical resistance and disruption of
the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction
membrane protein. Journal of Cell Biology, 134(4), 1031-1049.
Bayoumi, M. A., & Griffiths, M. W. (2012). In vitro inhibition of expression of virulence genes
responsible for colonization and systemic spread of enteric pathogens using bifidobacterium
bifidum secreted molecules. International Journal of Food Microbiology, 156(3), 255-263.
Bayoumi, M. A., & Griffiths, M. W. (2010). Probiotics down-regulate genes in salmonella enterica
serovar typhimurium pathogenicity islands 1 and 2. Journal of Food Protection, 73(3), 452-460.
Benderska, N., Chakilam, S., Hugle, M., Ivanovska, J., Gandesiri, M., Schulze-Luehrmann, J., . . .
Schneider-Stock, R. (2012). Apoptosis signalling activated by TNF in the lower gastrointestinal
tract-review. Current Pharmaceutical Biotechnology, 13(11), 2248-2258.
Benkerroum, N. (2010). Antimicrobial peptides generated from milk proteins: A survey and prospects
for application in the food industry. A review. International Journal of Dairy Technology, 63(3),
320-338.
77
Bishop, A., House, D., Perkins, T., Baker, S., Kingsley, R. A., & Dougan, G. (2008). Interaction of
salmonella enterica serovar typhi with cultured epithelial cells: Roles of surface structures in
adhesion and invasion. Microbiology-(UK), 154(7), 1914-1926.
Blum, M. S., Toninelli, E., Anderson, J. M., Balda, M. S., Zhou, J. Y., O'Donnell, L., . . . Bender, J. R.
(1997). Cytoskeletal rearrangement mediates human microvascular endothelial tight junction
modulation by cytokines. American Journal of Physiology-Heart and Circulatory Physiology,
273(1), H286-H294.
Brovko, L. Y., Vandenende, C., Chu, B., Ng, K. Y., Brooks, A., & Griffiths, M. W. (2003). In vivo
assessment of effect of fermented milk diet on course of infection in mice with bioluminescent
salmonella. Journal of Food Protection, 66(11), 2160-2163.
Brune, B. (2003). Nitric oxide: NO apoptosis or turning it ON? Cell Death and Differentiation, 10(8),
864-869.
Burkholder, K. M., & Bhunia, A. K. (2009). Salmonella enterica serovar typhimurium adhesion and
cytotoxicity during epithelial cell stress is reduced by lactobacillus rhamnosus GG. Gut
Pathogens, 1, 14.
Burnett, S. L., Chen, J., & Beuchat, L. R. (2000). Attachment of escherichia coli O157: H7 to the
surfaces and internal structures of apples as detected by confocal scanning laser microscopy.
Applied and Environmental Microbiology, 66(11), 4679-4687.
Canil, C., Rosenshine, I., Ruschkowski, S., Donnenberg, M. S., Kaper, J. B., & Finlay, B. B. (1993).
Enteropathogenic escherichia-coli decreases the transepithelial electrical-resistance of polarized
epithelial monolayers. Infection and Immunity, 61(7), 2755-2762.
Capaldo, C. T., & Nusrat, A. (2009). Cytokine regulation of tight junctions. Biochimica Et Biophysica
Acta-Biomembranes, 1788(4), 864-871.
Caron, J., Lariviere, L., Nacache, M., Tam, M., Stevenson, M. M., McKerly, C., . . . Malo, D. (2006).
Influence of Slc11a1 on the outcome of salmonella enterica serovar enteritidis infection in mice
is associated with th polarization. Infection and Immunity, 74(5), 2787-2802.
CDC. (2011). Vital signs: Incidence and trends of infection with pathogens transmitted commonly
through food --- foodborne diseases active surveillance network, 10 U.S. sites, 1996—2010.
Atlanta, Georgia: U.S. Department of Health and Human Services, CDC.
CDC. (2013). Antibiotic resistance threats in the united states, 2013. Atlanta, Georgia: U.S.
Department of Health and Human Services, CDC.
Chalghoumi, R., Thewis, A., Beckers, Y., Marcq, C., Portetelle, D., & Schneider, Y. J. (2009).
Adhesion and growth inhibitory effect of chicken egg yolk antibody (IgY) on salmonella
78
enterica serovars enteritidis and typhimurium in vitro. Foodborne Pathogens and Disease, 6(5),
593-604.
Chin, N. M. L. (2002). In Griffiths M. W. (Ed.), Isolation and characterization of the
immunomodulating properties of milk peptides fermented by LB. helveticus (M.Sc. Thesis ed.).
Guelph, Ont.: University of Guelph.
Cohen, E., Ophir, I., & Ben Shaul, Y. (1999). Induced differentiation in HT29, a human colon
adenocarcinoma cell line. Journal of Cell Science, 112(16), 2657-2666.
Collado, M. C., Gueimonde, M., Hernandez, M., Sanz, Y., & Salminen, S. (2005). Adhesion of
selected bifidobacterium strains to human intestinal mucus and the role of adhesion in
enteropathogen exclusion. Journal of Food Protection, 68(12), 2672-2678.
Collado, M. C., Meriluoto, J., & Salminen, S. (2007). Role of commercial probiotic strains against
human pathogen adhesion to intestinal mucus. Letters in Applied Microbiology, 45(4), 454-460.
Collins, C. H., & Kennedy, D. A. (1999). Laboratory-acquired infections : History, incidence, causes,
and prevention (4th ed. ed.). Oxford ; Boston: Butterworth-Heinemann.
Compton, M. M. (1992). A biochemical hallmark of apoptosis - internucleosomal degradation of the
genome. Cancer and Metastasis Reviews, 11(2), 105-119.
Corr, S. C., Li, Y., Riedel, C. U., O'Toole, P., Hill, C., & Gahan, C. (2007). Bacteriocin production as
a mechanism for the antfinfective activity of lactobacillus salivarius UCC118. Proceedings of
the National Academy of Sciences of the United Statesof America, 104(18), 7617-7621.
Corver, W. E., Cornelisse, C. J., & Fleuren, G. J. (1994). Simultaneous measurement of 2 cellular
antigens and dna using fluorescein-isothiocyanate, R-phycoerythrin, and propidium iodide on a
standard facscan. Cytometry, 15(2), 117-128.
Cristina Cerquetti, M., Hovsepian, E., Sarnacki, S. H., & Goren, N. B. (2008). Salmonella enterica
serovar enteritidis dam mutant induces low NOS-2 and COX-2 expression in macrophages via
attenuation of MAPK and NF-kappa B pathways. Microbes and Infection, 10(14-15),
1431-1439.
de LeBlanc, A. M., Castillo, N. A., & Perdigon, G. (2010). Anti-infective mechanisms induced by a
probiotic lactobacillus strain against salmonella enterica serovar typhimurium infection.
International Journal of Food Microbiology, 138(3), 223-231.
De Moreno De Leblanc, A., Maldonado Galdeano, C., Dogi, C. A., Carmuega, E., Weill, R., &
Perdigon, G. (2010). Adjuvant effect of a probiotic fermented milk in the protection against
salmonella enteritidis serovar typhimurium infection: Mechanisms involved. International
Journal of Immunopathology and Pharmacology, 23(4), 1235-44.
79
Demacias, M. E. N., Romero, N. C., Apella, M. C., Gonzalez, S. N., & Oliver, G. (1993). Prevention
of infections produced by escherichia-coli and listeria-monocytogenes by feeding milk
fermented with lactobacilli. Journal of Food Protection, 56(5), 401-405.
Ding, W., Wang, H., & Griffiths, M. W. (2005). Probiotics down-regulate flaA sigma28 promoter in
campylobacter jejuni. Journal of Food Protection®, 68(11), 2295-2300.
Divya, J. B., Varsha, K. K., Nampoothiri, K. M., Ismail, B., & Pandey, A. (2012). Probiotic fermented
foods for health benefits. Engineering in Life Sciences, 12(4), 377-390.
Dong, H. P., Kleinberg, L., Davidson, B., & Risberg, B. (2008). Methods for simultaneous
measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow
cytometry. Nature Protocols, 3(6), 955-964.
Ebringer, L., Ferencik, M., & Krajcovic, J. (2008). Beneficial health effects of milk and fermented
dairy products--review. Folia Microbiologica, 53(5), 378-394.
Eckmann, L., Kagnoff, M. F., & Fierer, J. (1993). Epithelial cells secrete the chemokine interleukin-8
in response to bacterial entry. Infection and Immunity, 61(11), 4569-4574.
Eckmann, L., & Kagnoff, M. F. (2001). Cytokines in host defense against salmonella. Microbes and
Infection, 3(14-15), 1191-1200.
Elawadli, I. (2012). In Sharif S. (Ed.), Investigations into the effects of lactobacilli on murine
dendritic cells (M.Sc. Thesis ed.). Guelph, Ont: University of Guelph.
Elmore, S. (2007). Apoptosis: A review of programmed cell death. Toxicologic Pathology, 35(4),
495-516.
FAO, & WHO. (2001). Health and nutritional properties of probiotics in food including powder milk
with live lactic acid bacteria. Report of a joint FAO/ WHO expert consultation on evaluation of
health and nutritional properties of probiotics in food including powder milk with live lactic acid
bacteria. Retrieved March/30, 2014, from
http://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf
Ferrell, N., Desai, R. R., Fleischman, A. J., Roy, S., Humes, H. D., & Fissell, W. H. (2010). A
microfluidic bioreactor with integrated transepithelial electrical resistance ( TEER) measurement
electrodes for evaluation of renal epithelial cells. Biotechnology and Bioengineering, 107(4),
707-716.
Ferruzza, S., Rossi, C., Scarino, M. L., & Sambuy, Y. (2012). A protocol for differentiation of human
intestinal caco-2 cells in asymmetric serum-containing medium. Toxicology in Vitro, 26(8),
1252-1255.
Fink, S. L., & Cookson, B. T. (2005). Apoptosis, pyroptosis, and necrosis: Mechanistic description of
dead and dying eukaryotic cells. Infection and Immunity, 73(4), 1907-1916.
80
Finlay, B. B., & Falkow, S. (1990). Salmonella interactions with polarized human intestinal caco-2
epithelial-cells. Journal of Infectious Diseases, 162(5), 1096-1106.
Finlay, B. B., Starnbach, M. N., Francis, C. L., Stocker, B. A. D., Chatfield, S., Dougan, G., &
Falkow, S. (1988). Identification and characterization of tnphoa mutants of salmonella that are
unable to pass through a polarized mdck epithelial-cell monolayer. Molecular Microbiology,
2(6), 757-766.
Fish, S. M., Proujansky, R., & Reenstra, W. W. (1999). Synergistic effects of interferon gamma and
tumour necrosis factor alpha on T84 cell function. Gut, 45(2), 191-198.
Fitzgerald, R. C., Omary, M. B., & Triadafilopoulos, G. (1997). Acid modulation of HT29 cell growth
and differentiation - an in vitro model for barrett's esophagus. Journal of Cell Science, 110,
663-671.
Gaggìa, F., Mattarelli, P., & Biavati, B. (2010). Probiotics and prebiotics in animal feeding for safe
food production. International Journal of Food Microbiology, 141, 515-528.
Ghadimi, D., de Vrese, M., Heller, K. J., & Schrezenmeir, J. (2010). Effect of natural
commensal-origin DNA on toll-like receptor 9 (TLR9) signaling cascade, chemokine IL-8
expression, and barrier integritiy of polarized intestinal epithelial cells. Inflammatory Bowel
Diseases, 16(3), 410-427.
Gobbato, N., Galdeano, C. M., & Perdigon, G. (2008). Study of some of the mechanisms involved in
the prevention against salmonella enteritidis serovar typhimurium infection by lactic acid
bacteria. Food and Agricultural Immunology, 19(1), 11-23.
Goto, Y., & Ivaylo, I. (2013). Intestinal epithelial cells as mediators of the commensal–host immune
crosstalk. Immunology and Cell Biology, 91(3), 204-214.
Grajek, W., & Olejnik, A. (2004). Epithelial cell cultures in vitro as a model to study functional
properties of food. Polish Journal of Food and Nutrition Sciences, 13(SI 1), 5-24.
Griffiths, M. W., & Tellez, A. M. (2013). Lactobacillus helveticus: The proteolytic system. Frontiers
in Microbiology, 4, 30.
Grigoroff, S. (1905). E´tude sur un lait fermente comestible. le „„Kisselo-mleko‟‟ de bulgarie. Revue
Medical De La Suisse Romande, 25, 714-720.
Gueimonde, M., Jalonen, L., He, F., Hiramatsu, M., & Salminen, S. (2006). Adhesion and
competitive inhibition and displacement of human enteropathogens by selected lactobacilli.
Food Research International, 39(4), 467-471.
Guttman, J. A., & Finlay, B. B. (2008). Subcellular alterations that lead to diarrhea during bacterial
pathogenesis. Trends in Microbiology, 16(11), 535-542.
81
Guttman, J. A., & Finlay, B. B. (2009). Tight junctions as targets of infectious agents. Biochimica Et
Biophysica Acta-Biomembranes, 1788(4), 832-841.
Hans-Jurgen, R. (Ed.). (2008). Apoptosis, cytotoxicity and cell proliferation<br /> (Fourth Edition
ed.). Germany: Roche Diagnostics.
Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: From research to
food applications. Current Opinion in Biotechnology, 18(2), 163-169.
Hasegawa, H., Fujita, H., Katoh, H., Aoki, J., Nakamura, K., Ichikawa, A., & Negishi, M. (1999).
Opposite regulation of transepithelial electrical resistance and paracellular permeability by rho in
madin-darby canine kidney cells. Journal of Biological Chemistry, 274(30), 20982-20988.
Haug, A., Hostmark, A. T., & Harstad, O. M. (2007). Bovine milk in human nutrition - a review.
Lipids in Health and Disease, 6, 25.
Hausmann, M. (2010). How bacteria-induced apoptosis of intestinal epithelial cells contributes to
mucosal inflammation. International Journal of Inflammation, 2010, 574568-574568.
Höner zu Bentrup, K., Ramamurthy, R., Ott, C. M., Emami, K., Nelman-Gonzalez, M., Wilson, J.
W., . . . Nickerson, C. A. (2006). Three-dimensional organotypic models of human colonic
epithelium to study the early stages of enteric salmonellosis. Microbes and Infection, 8(7),
1813-1825.
Hubatsch, I., Ragnarsson, E., & Artursson, P. (2007). Determination of drug permeability and
prediction of drug absorption in caco-2 monolayers. NATURE PROTOCOLS, 2(9), 2111-2119.
Huet, C., Sahuquillomerino, C., Coudrier, E., & Louvard, D. (1987). Absorptive and mucus-secreting
subclones isolated from a multipotent intestinal-cell line (ht-29) provide new models for cell
polarity and terminal differentiation. Journal of Cell Biology, 105(1), 345-357.
Ingrassia, I., Leplingard, A., & Darfeuille-Michaud, A. (2005). Lactobacillus casei DN-114 001
inhibits the ability of adherent-invasive escherichia coli isolated from crohn's disease patients to
adhere to and to invade intestinal epithelial cells. Applied and Environmental Microbiology,
71(6), 2880-2887.
Jahan-Tigh, R. R., Ryan, C., Obermoser, G., & Schwarzenberger, K. (2012). Flow cytometry. Journal
of Investigative Dermatology, 132(10), 1-6.
Jankovic, I., Sybesma, W., Phothirath, P., Ananta, E., & Mercenier, A. (2010). Application of
probiotics in food products-challenges and new approaches. Current Opinion in Biotechnology,
21(2), 175-181.
Jankowska, A., Laubitz, D., Antushevich, H., Zabielski, R., & Grzesiuk, E. (2008). Competition of
lactobacillus paracasei with salmonella enterica for adhesion to caco-2 cells. Journal of
Biomedicine and Biotechnology, , 357964.
82
Johnson-Henry, K. C., Hagen, K. E., Gordonpour, M., Tompkins, T. A., & Sherman, P. M. (2007).
Surface-layer protein extracts from lactobacillus helveticus inhibit enterohaemorrhagic
escherichia coli O157 : H7 adhesion to epithelial cells. Cellular Microbiology, 9(2), 356-367.
Kagaya, K., Watanabe, K., & Fukazawa, Y. (1989). Capacity of recombinant gamma interferon to
activate macrophages for salmonella-killing activity. Infection and Immunity, 57(2), 609-615.
Kerr, J. F. R., Wyllie, A. H., & Currie, A. R. (1972). Apoptosis - basic biological phenomenon with
wide-ranging implications in tissue kinetics. British Journal of Cancer, 26(4), 239-257.
Khani, S., Hosseini, H. M., Taheri, M., Nourani, M. R., & Fooladi, A. A. I. (2012). Probiotics as an
alternative strategy for prevention and treatment of human diseases: A review. Inflammation &
Allergy Drug Targets, 11(2), 79-89.
Kim, J. M., Eckmann, L., Savidge, T. C., Lowe, D. C., Witthoft, T., & Kagnoff, M. F. (1998).
Apoptosis of human intestinal epithelial cells after bacterial invasion. Journal of Clinical
Investigation, 102(10), 1815-1823.
Kitts, D. D., & Weiler, K. (2003). Bioactive proteins and peptides from food sources. applications of
bioprocesses used in isolation and recovery. Current Pharmaceutical Design, 9(16), 1309-1323.
Klingberg, T. D., Pedersen, M. H., Cencic, A., & Budde, B. B. (2005). Application of measurements
of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic
activity. Applied and Environmental Microbiology, 71(11), 7528-7530.
Knodler, L. A., & Finlay, B. B. (2001). Salmonella and apoptosis: To live or let die? Microbes and
Infection, 3(14-15), 1321-1326.
Knodler, L. A., Finlay, B. B., & Steele-Mortimer, O. (2005). The salmonella effector protein SopB
protects epithelial cells from apoptosis by sustained activation of akt. Journal of Biological
Chemistry, 280(10), 9058-9064.
Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications. Journal of
Functional Foods, 1(2), 177-187.
Lavermicocca, P., Valerio, F., Lonigro, S. L., Di Leo, A., & Visconti, A. (2008). Antagonistic activity
of potential probiotic lactobacilli against the ureolytic pathogen yersinia enterocolitica. Current
Microbiology, 56(2), 175-181.
Le Bivic, A., Hirn, M., & Reggio, H. (1988). HT-29 cells are an in vitro model for the generation of
cell polarity in epithelia during embryonic differentiation. Proceedings of the National Academy
of Sciences of the United States of America, 85(1), 136-140.
Lee, D. J., Drongowski, R. A., Coran, A. G., & Harmon, C. M. (2000). Evaluation of probiotic
treatment in a neonatal animal model. Pediatric Surgery International, 16(4), 237-242.
83
Levi, C. A., Ejere, V. C., Asogwa, C. N., Iweh, P., Nwatu, K. U., & Levi, U. E. (2014). Apoptosis: Its
physiological implication and therapeutic possibilities . Journal of Pharmacy and Biological
Sciences, 9(1), 38-45.
Li, P., Yin, Y., Yu, Q., & Yang, Q. (2011). Lactobacillus acidophilus S-layer protein-mediated
inhibition of salmonella-induced apoptosis in caco-2 cells. Biochemical and Biophysical
Research Communications, 409(1), 142-147.
Lin, C., Tsai, H., Lin, P., Tsen, H., & Tsai, C. (2008). Lactobacillus acidophilus LAP5 able to inhibit
the salmonella choleraesuis invasion to the human caco-2 epithelial cell. Anaerobe, 14(5),
251-255.
Ling, S., Wang, X. H., Xie, L., Lim, T. M., & Leung, K. Y. (2000). Use of green fluorescent protein
( GFP) to study the invasion pathways of edwardsiella tarda in in vivo and in vitro fish models.
MICROBIOLOGY-SGM; Microbiology-(UK), 146(1), 7-19.
Louis, K. S., & Siegel, A. C. (2011). Cell viability analysis using trypan blue: Manual and automated
methods. Mammalian Cell Viability: Methods and Protocols, 740, 7-12.
Lundberg, U., Vinatzer, U., Berdnik, D., von Gabain, A., & Baccarini, M. (1999). Growth
phase-regulated induction of salmonella-induced macrophage apoptosis correlates with transient
expression of SPI-1 genes. Journal of Bacteriology, 181(11), 3433-3437.
Ma, T. Y., Iwamoto, G. K., Hoa, N. T., Akotia, V., Pedram, A., Boivin, M. A., & Said, H. M. (2004).
TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires
NF-kappa B activation. American Journal of Physiology-Gastrointestinal and Liver Physiology,
286(3), G367-G376.
Mack, D. R., Michail, S., Wei, S., McDougall, L., & Hollingsworth, M. A. (1999). Probiotics inhibit
enteropathogenic E- coli adherence in vitro by inducing intestinal mucin gene expression.
American Journal of Physiology-Gastrointestinal and Liver Physiology, 276(4), G941-G950.
Magrone, T., & Jirillo, E. (2013). The interplay between the gut immune system and microbiota in
health and disease: Nutraceutical intervention for restoring intestinal homeostasis. Current
Pharmaceutical Design, 19(7), 1329-1342.
Mantis, N. J., & Forbes, S. J. (2010). Secretory IgA: Arresting microbial pathogens at epithelial
borders. Immunological Investigations, 39(4-5), 383-406.
Masood, M. I., Qadir, M. I., Shirazi, J. H., & Khan, I. U. (2011). Beneficial effects of lactic acid
bacteria on human beings. Critical Reviews in Microbiology, 37(1), 91-98.
Mattar, A. F., Drongowski, R. A., Coran, A. G., & Harmon, C. M. (2001). Effect of probiotics on
enterocyte bacterial translocation in vitro. Pediatric Surgery International, 17(4), 265-268.
Matter, K., & Balda, M. S. (2003). Functional analysis of tight junctions. Methods, 30(3), 228-234.
84
Medellin-Peña, M., & Griffiths, M. W. (2009). Effect of molecules secreted by lactobacillus
acidophilus strain la-5 on escherichia coli O157: H7 colonization. Applied and Environmental
Microbiology, 75(4), 1165-1172.
Medellin-Peña, M., Wang, H., Johnson, R., Anand, S., & Griffiths, M. W. (2007). Probiotics affect
virulence-related gene expression in escherichia coli O157: H7. Applied and Environmental
Microbiology, 73(13), 4259-4267.
Medellin-Peña, M. J. (2007). In Griffiths M. W. (Ed.), Interference of virulence in escherichia coli
O157:H7 by molecule(s) secreted by probiotic lactic acid bacteria (Ph.D Thesis ed.). Guelph,
Ont.: University of Guelph.
Meisel, H., & FitzGerald, R. J. (2003). Biofunctional peptides from milk proteins: Mineral binding
and cytomodulatory effects. Current Pharmaceutical Design, 9(16), 1289-1295.
Milovic, V., Turchanowa, L., Khomutov, A. R., Khomutov, R. M., Caspary, W. F., & Stein, J. (2001).
Hydroxylamine-containing inhibitors of polyamine biosynthesis and impairment of colon cancer
cell growth. Biochemical Pharmacology, 61(2), 199-206.
Minkiewicz, P., Dziuba, J., Iwaniak, A., Dziuba, M., & Darewicz, M. (2008). BIOPEP database and
other programs for processing bioactive peptide sequences. Journal of AOAC International,
91(4), 965-980.
Mitchell, D. M., & Ball, J. M. (2004). Craracterization of a spontaneously polarizing HT-29 cell line,
HT-29/cl.f8. In Vitro Cellular & Developmental Biology-Animal, 40(10), 297-302.
Moorthy, G., Murali, M. R., & Devaraj, S. N. (2009). Lactobacilli facilitate maintenance of intestinal
membrane integrity during shigella dysenteriae 1 infection in rats. Nutrition, 25(3), 350-358.
Muro Urista, C., Alvarez Fernandez, R., Riera Rodriguez, F., Arana Cuenca, A., & Tellez Jurado, A.
(2011). Review: Production and functionality of active peptides from milk. Food Science and
Technology International, 17(4), 293-317.
Myllyluoma, E., Ahonen, A. -., Korpela, R., Vapaatalo, H., & Kankuri, E. (2008). Effects of
multispecies probiotic combination on helicobacter pylori infection in vitro. Clinical and
Vaccine Immunology, 15(9), 1472-1482.
Ng, K. (2000). In Griffiths M. W. (Ed.), Enhancement of macrophage cytokine production by cell-free
fractions of fermented milk (M.Sc. Thesis ed.). Guelph, Ont.: University of Guelph.
Ouwehand, A. C., Salminen, S., & Isolauri, E. (2002). Probiotics: An overview of beneficial effects.
Antonie Van Leeuwenhoek, 82(1-4), 279-289.
Parvez, S., Malik, K., Kang, S., & Kim, H. (2006). Probiotics and their fermented food products are
beneficial for health. Journal of Applied Microbiology, 100(6), 1171-1185.
85
Perdigon, G., Alvarez, S., Demacias, M. E. N., Roux, M. E., & Holgado, A. P. D. (1990). The
oral-administration of lactic-acid bacteria increase the mucosal intestinal immunity in response
to enteropathogens. Journal of Food Protection, 53(5), 404-410.
Perdigon, G., Demacias, M. E. N., Alvarez, S., Oliver, G., & Holgado, A. A. P. D. (1990). Prevention
of gastrointestinal infection using immunobiological methods with milk fermented with
lactobacillus-casei and lactobacillus-acidophilus. Journal of Dairy Research, 57(2), 255-264.
Pinto, M., Gomez, M. R., Seifert, S., Watzl, B., Holzapfel, W. H., & Franz, C. (2009). Lactobacilli
stimulate the innate immune response and modulate the TLR expression of HT29 intestinal
epithelial cells in vitro. International Journal of Food Microbiology, 133(1-2), 86-93.
Popov, S. G., Villasmil, R., Bernardi, J., Grene, E., Cardwell, J., Wu, A., . . . Alibek, K. (2002).
Lethal toxin of bacillus anthracis causes apoptosis of macrophages. Biochemical and Biophysical
Research Communications, 293(1), 349-355.
Punj, V., Bhattacharyya, S., Saint-Dic, D., Vasu, C., Cunningham, E. A., Graves, J., . . . Das Gupta, T.
(2004). Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast
cancer. Oncogene, 23(13), 2367-2378.
Raff, M. (1992). Social controls on cell-survival and cell-death. Nature, 356(6368), 397-400.
Resta-Lenert, S., & Barrett, K. E. (2003). Live probiotics protect intestinal epithelial cells from the
effects of infection with enteroinvasive escherichia coli (EIEC). Gut, 52(7), 988-997.
Rodriguez-Boulan, E., & Nelson, W. J. (1989). Morphogenesis of the polarized epithelial cell
phenotype. Science (New York, N.Y.), 245(4919), 718-725.
Santini, M. T., Rainaldi, G., & Indovina, P. L. (2000). Apoptosis, cell adhesion and the extracellular
matrix in the three-dimensional growth of multicellular tumor spheroids. Critical Reviews in
Oncology and Hematology, 36(2), 75-87.
Schrezenmeir, J., & De Vrese, M. (2001). Probiotics, prebiotics, and synbiotics--approaching a
definition. American Journal of Clinical Nutrition, 73(2), 361S-364S.
Sgouras, D., M., Sgouras, D., Maragkoudakis, P., Petraki, K., Martinez-Gonzalez, B., . . . Mentis, A.
(2004). In vitro and in vivo inhibition of helicobacter pylori by lactobacillus casei strain shirota.
Applied and Environmental Microbiology, 70(1), 518-526.
Silva, S. V., & Malcata, F. X. (2005). Caseins as source of bioactive peptides. International Dairy
Journal, 15(1), 1-15.
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., . . . Boyd, M. R. (1990).
New colorimetric cytotoxicity assay for anticancer-drug screening. Journal of the National
Cancer Institute, 82(13), 1107-1112.
86
Skjolaas, K. A., Burkey, T. E., Dritz, S. S., & Minton, J. E. (2007). Effects of salmonella enterica
serovar typhimurium, or serovar choleraesuis, lactobacillus reuteri and bacillus licheniformis on
chemokine and cytokine expression in the swine jejunal epithelial cell line, IPEC-J2. Veterinary
Immunology and Immunopathology, 115(3), 299-308.
Slee, E. A., Adrain, C., & Martin, S. J. (2001). Executioner caspase-3,-6, and-7 perform distinct,
non-redundant roles during the demolition phase of apoptosis. Journal of Biological Chemistry,
276(10), 7320-7326.
Solano, C., Sesma, B., Alvarez, M., Urdaneta, E., Garcia-Ros, D., Calvo, A., & Gamazo, C. (2001).
Virulent strains of salmonella enteritidis disrupt the epithelial barrier of caco-2 and HEp-2 cells.
Archives of Microbiology, 175(1), 46-51.
Steinke, J. W., & Borish, L. (2006). Cytokines and chemokines. Journal of Allergy and Clinical
Immunology, 117(2), S441-S445.
Strober, W. (2001). Trypan blue exclusion test of cell viability. Current Protocols in Immunology /
Edited by John E.Coligan ...[Et Al.], Appendix 3, 3B-Appendix 3B.
Sun, Z. L., Huang, L. H., Kong, J., Hu, S. M., Zhang, X. W., & Kong, W. T. (2012). In vitro
evaluation of lactobacillus crispatus K313 and K243: High-adhesion activity and
anti-inflammatory effect on salmonella braenderup infected intestinal epithelial cell. Veterinary
Microbiology, 159(1-2), 212-220.
Takeuchi, K., & Frank, J. F. (2001). Confocal microscopy and microbial viability detection for food
research. Journal of Food Protection, 64(12), 2088-2102.
Tellez Garay, A. M. (2009). In Griffiths M. W. (Ed.), Characterization of immuno active peptides
present in cell free preparations obtained from milk fermented by lactobacillus helveticus (Ph.D.
Thesis ed.). Guelph, Ont.: University of Guelph.
Tellez, A., Corredig, M., Turner, P. V., Morales, R., & Griffiths, M. W. (2011). A peptidic fraction
from milk fermented with Lactobacillus helveticus protects mice against Salmonella infection.
International Dairy Journal, 21(9), 607-614.
Tindall, B. J., Grimont, P. A. D., Garrity, G. M., & Euzeby, J. P. (2005). Nomenclature and taxonomy
of the genus salmonella. International Journal of Systematic and Evolutionary Microbiology, 55,
521-524.
Tlaskalova-Hogenova, H., Tuckova, L., Lodinova-Zadnikova, R., Stepankova, R., Cukrowska, B.,
Funda, D. P., . . . Sanchez, D. (2002). Mucosal immunity: Its role in defense and allergy.
International Archives of Allergy and Immunology, 128(2), 77-89.
Torchinsky, M. B., Garaude, J., & Blander, J. M. (2010). Infection and apoptosis as a combined
inflammatory trigger. Current Opinion in Immunology, 22(1), 55-62.
87
Tuomola, E. M., Ouwehand, A. C., & Salminen, S. J. (1999). The effect of probiotic bacteria on the
adhesion of pathogens to human intestinal mucus. FEMS Immunology and Medical
Microbiology, 26(2), 137-142.
Valdez, J. C., Rachid, M., Gobbato, N., & Perdigon, G. (2001). Lactic acid bacteria induce apoptosis
inhibition in salmonella typhimurium infected macrophages. Food and Agricultural Immunology,
13(3), 189-197.
van Engeland, M., Nieland, L. J. W., Ramaekers, F. C. S., Schutte, B., & Reutelingsperger, C. P. M.
(1998). Annexin V-affinity assay: A review on an apoptosis detection system based on
phosphatidylserine exposure. Cytometry, 31(1), 1-9.
Vermes, I., Haanen, C., & Reutelingsperger, C. (2000). Flow cytometry of apoptotic cell death.
Journal of Immunological Methods, 243(1-2), 167-190.
Wang, F. J., Graham, W. V., Wang, Y. M., Witkowski, E. D., Schwarz, B. T., & Turner, J. R. (2005).
Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial
barrier dysfunction by up-regulating myosin light chain kinase expression. American Journal of
Pathology, 166(2), 409-419.
Wu, M. T., Carlson, S. A., & Meyerholz, D. K. (2002). Cytopathic effects observed upon expression
of a repressed collagenase gene present in salmonella and related pathogens: Mimicry of a
cytotoxin from multiple antibiotic-resistant salmonella enterica serotype typhimurium phagetype
DT104. Microbial Pathogenesis, 33(6), 279-287.
Yan, F., Cao, H., Cover, T. L., Whitehead, R., Washington, M. K., & Polk, D. B. (2007). Soluble
proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth.
Gastroenterology, 132(2), 562-575.
Ye, D. M., Ma, I., & Ma, T. Y. (2006). Molecular mechanism of tumor necrosis factor-alpha
modulation of intestinal epithelial tight junction barrier. American Journal of
Physiology-Gastrointestinal and Liver Physiology, 290(3), G496-G504.
Youakim, A., & Ahdieh, M. (1999). Interferon-gamma decreases barrier function in T84 cells by
reducing ZO-1 levels and disrupting apical actin. American Journal of
Physiology-Gastrointestinal and Liver Physiology, 276(5), G1279-G1288.
Yu, Q., Wang, Z., & Yang, Q. (2012). Lactobacillus amylophilus D14 protects tight junction from
enteropathogenic bacteria damage in caco-2 cells. Journal of Dairy Science, 95(10), 5580-5587.
Zanabria Eyzaguirre, R. (2013). In Corredig M. (Ed.), Anticarcinogenic and immunomodulatory
properties of the milk fat globule membrane (Ph.D Thesis ed.). Guelph, Ont.: University of
Guelph.
88
Zeinhom, M., T., Zeinhom, M., Tellez, A. M., Delcenserie, V., El-Kholy, A., . . . Griffiths, M. W.
(2012). Yogurt containing bioactive molecules produced by lactobacillus acidophilus la-5 exerts
a protective effect against enterohemorrhagic escherichia coli in mice. Journal of Food
Protection, 75(10), 1796-1805.
Zemans, R. L., Briones, N., Campbell, M., McClendon, J., Young, S. K., Suzuki, T., . . . Downey, G.
P. (2011). Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin
signaling. Proceedings of the National Academy of Sciences of the United States of America,
108(38), 15990-15995.