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TitleEffects of anethole on the growth and virulence expression of toxigenic
Vibrio cholerae and its therapeutic effects in animal models
Author(s) MD. SHAMIM HASAN ZAHID
Editor(s)
Citation
Issue Date 2015
URL http://hdl.handle.net/10466/14458
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大阪府立大学博士（獣医学）学位論文
Effects of anethole on the growth and virulence expression of toxigenic Vibrio cholerae and its therapeutic effects in
animal models
(アネトールがコレラ菌の増殖及び病原因子の発現に及ぼす影響
とその動物モデルでの治療効果)
MD. SHAMIM HASAN ZAHID
2015 年
CONTENTS
GENERAL INTRODUCTION………………………………………………………………1 CHAPTER 1. Anethole suppresses the growth and virulence potential of toxigenic Vibrio cholerae
1.1. INTRODUCTION…………………………………………………………. ………….12
1.2. MATERIALS AND METHODS………………………………………........................14
1.3. RESULTS……………………………………………………………………...……….19
1.4. DISCUSSION…………………………………………………………………………..22
TABLES AND FIGURES………………………………………………………….......26
CHAPTER 2. Anethole might affect virulence regulatory cascade in Vibrio cholerae via cyclic AMP (cAMP)-cAMP receptor protein (CRP) signaling system
2.1. INTRODUCTION…………………………………………………………. …………..33
2.2. MATERIALS AND METHODS……………………………………….........................34
2.3. RESULTS……………………………………………………………………...………..39
2.4. DISCUSSION……………………………………………………………………….......42
TABLES AND FIGURES…………………………………………………………........47
CHAPTER 3. Effects of anethole on the pathogenesis of Vibrio cholerae in animal models
3.1. INTRODUCTION…………………………………………………………. …………..57
3.2. MATERIALS AND METHODS………………………………………..........................58
3.3. RESULTS……………………………………………………………………...………..61
3.4. DISCUSSION…………………………………………………………………………...63
TABLES AND FIGURES…………………………………………………………........66
GENERAL DISCUSSION………………………………………………………………….70
ACKNOWLEDGMENTS………………………………………………………....................75
REFERENCES…………………………………………………………………….................77
1
GENERAL INTRODUCTION
Antimicrobial agents have been playing a pivotal role in controlling bacterial infections
since their discovery. However, the current status of global emergence and spread of
multidrug resistant (MDR) bacteria has caused loss of their effectiveness against many
bacterial pathogens (Bax et al., 2000). Like other bacterial pathogens, multidrug resistance in
toxigenic Vibrio cholerae, the causative agent of cholera pandemics is a growing concern.
Multidrug resistance against nalidixic acid, furazolidone, trimethoprim, sulfamethoxazole and
tetracycline in V. cholerae is spreading widely (Kitaoka et al., 2011). Moreover,
azithromycin resistant V. cholerae has been already reported, and resistance to ciprofloxacin
is emerging, indicating a very few effective antimicrobial options for future treatment of
cholera (Quilici et al., 2010; Tran et al., 2012).
V. cholerae is a Gram-negative, curved rod shaped facultative anaerobic and highly
motile bacterium due to having a single polar flagellum (Fig I). In 1883, a German scientist
Robert Koch identified V. cholerae in the stools of cholera patients in Egypt, and in 1884,
this organism was first isolated in pure culture by himself. Actually, cholera toxin (CT,
encoded by the ctxAB genes) is the major virulence factor in toxigenic V. cholerae and is
mostly responsible for the profuse watery diarrhea (Fig. IIA), leading to severe dehydration
(Fig. IIB) (Harris et al., 2012; Sanchez and Holmgren, 2011). Although till date, >200
different ‘O’ serogroups of V. cholerae have already been documented; only O1 and O139
are responsible for cholera outbreaks (Ramamurthy et al., 2003). Serogroups other than O1
and O139 are collectively known as non-O1/-O139, and associated with occasional cases of
diarrhea and extra-intestinal infections (Chatterjee et al., 2009). V. cholerae strains belonging
to O1 serogroup are further subdivided into two biotypes, El Tor and classical. The Two
biotypes of V. cholerae O1 differ in certain phenotypic and genetic characteristics (Table I).
The disease cholera is a global problem, affecting 45-60 countries during the present
decade, mostly in tropical Africa followed by Asia, America and Europe (Fig. III). According
2
to World Health Organization reports (WHO, 2014), in 2013, 47 countries reported a total of
129,064 cholera cases including 2,102 deaths. In 2012, a total of 245,393 cholera cases
including 3,034 deaths were reported worldwide, whereas during 2011 there was an increased
mortality and morbidity with a total of 589,854 cases notified in 58 countries, including 7,816
deaths. (Fig. IV). Moreover, due to limitations in surveillance systems and fear of trade and
travel sanctions worldwide, most of the cholera cases remain unreported to WHO.
Cholera is an ancient disease, and so far, seven pandemics have been recorded since the
first pandemic began in 1817 (Faruque et al., 1998). Among V. cholerae O1 serogroup, the
O1 El Tor biotype is responsible for the ongoing seventh pandemic of cholera, which
originated in Indonesia in 1961, is the most extensive in terms of geographic spread and
duration. On the other hand, the sixth and presumably the earlier pandemics were caused by
the O1 classical biotype strains, which is now possibly extinct from the environment
(Siddique et al., 2010). V. cholerae strains belonging to O139 serogroup emerged in 1992 and
caused severe outbreaks, particularly in India and Bangladesh (Cholera working group,
ICDDR,B, 1993; Ramamurthy et al., 1993) (Fig. V). According to some previous studies, the
strains belonging to O139 serogroup has evolved from the O1 El Tor strains with few genetic
modifications (Faruque et al., 1997; Sharma et al., 1997; Yamasaki et al., 1997). On the other
hand, recently emerged V. cholerae O1 El Tor hybrid strains (possess some attributes of
classical biotype including ctxB gene allele) also categorized as El Tor variant strains produce
more CT and cause more severe diarrhea than prototype El Tor (Faruque et al., 2007; Ghosh-
Banerjee et al., 2010). The detail phenotypic and genetic characteristics of El Tor variant
strains are illustrated in Table I. Analysis of the strains from recent devastating cholera
outbreak in Haiti reveals that MDR O1 El Tor variant strains are now ruling the cholera
world (Sjölund-Karlsson et al., 2011; Son et al., 2011).
The disease cholera is caused by toxigenic V. cholerae mediated secretion of CT. CT
belongs to the AB5 toxin superfamily, and consists of six separate subunits: a single ‘A’
3
subunit (29 kDa) encoded by the ctxA gene is surrounded by five copies of B subunit (11 kDa
each) encoded by the ctxB gene. The pentameric ring-like structure of B subunits assembles
with the A subunit in a non-covalent fashion. The A subunit consists of two fragments (A1
[22 kDa] and A2 [5 kDa]) which are linked to each other via a disulphide bond (Sixma et al.,
1991; Zhang et al., 1995). Interestingly, the B subunit of V. cholerae O1 classical biotype
differs from that of O1 El Tor biotype in two amino acid residues: histidine instead of
tyrosine at position 39, and threonine instead of isoleucine at position 68 (Popovic et al.,
1994).
Along with CT, by using another virulence factor toxin-coregulated pilus (TCP, encoded
by the tcpA gene), V. cholerae causes diarrheal disease to human host. In order to cause
diarrhea V. cholerae has to attach to the intestinal epithelium, where it colonizes and secretes
CT. The colonization process is aided by TCP (Herrington et al., 1988). Although the
virulence regulon in toxigenic V. cholerae was recognized as the ToxR regulon, ToxT is the
direct transcriptional activator of the genes encoding CT and TCP. Indeed, activation of toxT
occurs via synergistic coupling of two membrane-localized heterodimers ToxR/ToxS and
TcpP/TcpH (Matson et al., 2007; Higgins and DiRita, 1994; Hase and Mekalanos, 1998). On
the other hand, histone-like nucleoid structuring (HNS, encoded by the hns gene) protein is a
basal repressor of the genes encoding CT, TCP and ToxT under non-permissive conditions of
osmolarity, temperature, pH, oxygen concentration etc. (Nye et al., 2000; Krishnan et al.,
2004) (Fig. VI).
After successful passage through the acid barrier of the stomach, V. cholerae colonizes in
the human intestine and secrets CT. Then, the B subunit ring binds to GM1 ganglioside on the
surface of host intestinal mucosal cells followed by internalization of the entire CT complex
and splitting of A subunit in to A1 and A2 fragments. Then, A1 fragment catalyses ADP
ribosylation, which causes constitutive activation of adenylate cyclase and subsequently
increase the level of cyclic AMP (cAMP) in the host cell (Cassel and Selinger, 1977). This
4
results in rapid efflux of huge amount of electrolytes (Cl¯, Na+ , K+, HCO3
-) and water from
toxin-damaged mucosal cells, and leads to diarrhea and vomiting (Zhang et al., 1995; DiRita,
2001). If this condition remains uncured, death is the ultimate result. A schematic diagram of
the CT activation mechanism as describe above is shown in Fig. VII.
Due to huge fluid and electrolyte loss from the body, cholera is generally treated with
rehydration of fluids and electrolytes (Sack et al., 2004). Antimicrobial agents are also
usually given to patients with severe cholera as a second line of treatment to kill the pathogen.
But effectiveness of antimicrobial agents can be hampered due to the emergence of
antimicrobial resistant V. cholerae strains (Sack et al., 2001). Uptake of foreign genetic
material via horizontal gene transfer (e.g., via conjugative plasmid, SXT elements etc.) can
facilitate the emergence of MDR V. cholerae strains (Mazel et al., 1998; Mwansa et al.,
2007; Kitaoka et al., 2011). Like other bacteria, role of active efflux pumps (e.g., MDR
pump) has also related to antimicrobial resistance in V. cholerae (Jane et al., 1998). In the
past two decades, a large number of epidemic V. cholerae strains in cholera endemic
countries have gained resistance to several antimicrobial agents including tetracycline,
ampicillin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, trimethoprim,
gentamicin, etc. (Das et al., 2008).
It is generally expected that bioactive compounds as antimicrobial agents of plant origin
are effective against infectious diseases and simultaneously subside many of the side effects
often shown by the synthetic antimicrobials. Since ancient times, natural products from
medicinal plants, such as spices, herbs, etc., have been used to treat various diseases
including enteric infections (Low Dog, 2006). So, natural products of plant origin could be a
reservoir to explore new antimicrobial agents against toxigenic MDR V. cholerae. Much
effort and attention have already been paid to search effective antimicrobial agents from
natural sources against V. cholerae. Previous reports have demonstrated that extracts from
Japanese green tea (Camellia sinensis), ‘neem’ (Azadirachta indica), ‘elephant garlic’ and
5
Vitex negundo leaf could effectively inhibit V. cholerae growth (Toda et al., 1992; Thakurta
et al., 2007; Rattanachaikunsopon and Phumkhachorn, 2009; Kamruzzman et al., 2013).
Alternatively, use of natural compounds as anti-virulence drugs, that suppresses virulence
of bacterial pathogen could be a novel therapeutic approach (Marra, 2004) to combat diseases
caused by MDR toxigenic V. cholerae. As CT is the major virulence factor in toxigenic V.
cholerae, much attention has been paid to search suitable anti-virulence drug candidates
against CT. Previous studies demonstrated that bile can repress ctxA and tcpA transcriptions
in V. cholerae in a ToxT-independent manner (Chatterjee et al., 2007). A synthetic
compound virstatin also showed inhibition of CT but in a ToxT-dependent manner (Hung et
al., 2005). In another recent study synthetic compound toxtazin B has been found to affect
ToxT by inhibiting tcpP transcription, but mechanisms behind tcpP inhibition is still obscure
(Anthouard and DiRita, 2013).
However, there is still very limited information regarding the effect of natural products on
virulence expression regulation in V. cholerae. In our previous study, we showed that sub-
bactericidal concentration of extracts of some spices, such as, red chili, sweet fennel and star
anise seed can effectively inhibit CT production in V. cholerae (Yamasaki et al., 2011). One
of the active ingredients in red chilli is capsaicin. We have recently reported a mechanism of
virulence gene suppression in V. cholerae by capsaicin. Capsaicin drastically suppressed CT
in vitro in a toxT-dependent manner by upregulating hns transcription (Chatterjee et al.,
2010), but failed to show such activity in vivo.
Trans-anethole [(1-methoxy 4-propenyl benzene) Fig. VIII] is the major active
component (80-90%) of the essential oil derived from sweet fennel and star anise seeds
(Sandberg and Corrigan, 2001). Antimicrobial activities of anethole (trans-anethole) against
bacteria, yeast and fungi have already been reported (De et al., 2002). Anethole has both
bacteriostatic and bactericidal effects against Salmonella enterica (Kubo and Fujita, 2001).
6
But, the effects of anethole on the growth or virulence expression of V. cholerae has not yet
been evaluated.
In this study, we have evaluated anethole as a potential antimicrobial and anti-virulence
drug candidate against MDR toxigenic V. cholerae. Furthermore, the possible molecular
mechanisms behind anethole-mediated virulence gene inhibition in V. cholerae were also
investigated. In chapter 1, the in vitro inhibitory effects of anethole on the growth and
virulence factors production of MDR-toxigenic V. cholerae have been evaluated. In chapter 2,
the molecular mechanism behind anethole-mediated in vitro virulence suppression in V.
cholerae is investigated. Finally, in chapter 3, the potential therapeutic benefits of anethole
were evaluated in animal models of V. cholerae infection.
Tabl
e I.
Cha
ract
eris
tics o
f Vib
rio
chol
erae
O1
clas
sica
l, E
l Tor
and
El T
or v
aria
nt s
trai
ns
Bio
type
Tr
ait
clas
sica
l El
Tor
El
Tor
var
iant
B
iolo
gica
l
Su
scep
tibili
ty to
pol
ymyx
in B
(50
U)
+ -
-
Voge
s-pr
oska
uer t
est
- +
+
Hae
mol
ysin
-
+ +/
-
Chi
cken
cel
l agg
lutin
atio
n -
+ +
Ly
sis b
y cl
assi
cal I
V p
hage
+
- -
Ly
sis b
y El
Tor
pha
ge V
-
+ +
Gen
etic
# ct
xB g
enot
ype
clas
sica
l E
l Tor
cl
assi
cal
tc
pA a
llele
cl
assi
cal
El T
or
El T
or
# Cla
ssic
al ty
pe C
T-B
pos
sess
es h
istid
ine
and
thre
onin
e at
pos
ition
s 39
and
68, r
espe
ctiv
ely,
whi
le E
l Tor
type
CT-
B p
osse
sses
tyro
sine
and
iso
leuc
ine,
resp
ectiv
ely
in th
ose
posit
ions
(Ray
chou
dhur
i et a
l., 2
008)
.
Fig. I. Photograph of curved rod shape Vibrio cholerae with single polar flagellum. Magnification approximately X 10,000 (Waldor and Raychowdhuri, 2000). Thebacterium is the causative agent of cholera epidemics, affecting millions of people every year.
(A) Rice water stool (B) Severe dehydration
Fig. II. Two hallmark symptoms of cholera patients: excretion of rice water stool (A), which caasues severe dehydration (B).
8
Fig. III: A global picture of cholera affected countries during 2004-10 (Source: WHO, http://www.who.int/gho/epidemic_diseases/cholera/en/index.html).
Fig. IV: World-wide cholera cases reported to WHO during 1989-2013. Each year 45-60 countries are affected with this disease (Source: WHO, http://www.who.int/gho/epidemic_diseases/cholera/cholera_005.jpg).
9
Fig. V. Key evolutionary events in cholera epidemiology since 1817. All the pandemics were caused by V. cholerae O1. The sixth and presumably the earlier pandemics were caused by the classical biotype, while the ongoing seventh pandemic is caused by the El Tor biotype strains. Recently, the atypical El Tor designated as El Tor variant is the predominant cause of cholera epidemics. (Safa et al., 2010).
Fig. VI. Schematic representation of the regulatory cascade of virulence expression in V. cholerae. Generally, ctxAB and tcpA transcriptions are directly regulated by the ToxTprotein, and activation of toxT depends on the synergistic coupling of toxR/toxS and tcpP/tcpH (Matson et al., 2007; Higgins et al., 1994; Hase et al., 1998). On the other hand, activation of toxR/toxS, as well as tcpP/tcpH genes are influenced by certain environmental stimuli (Skorupski and Taylor, 1997). A global prokaryotic gene regulator, histone-like nucleoid structuring protein (H-NS) is a basal repressor of toxT, ctxAB and tcpA genes in unfavorable conditions (Nye et al., 2000). In all cases, arrows indicate positive regulation while T-bar denotes negative or inhibitory effects.
10
Fig. VII. Mode of action of cholera toxin (CT). After colonize in epithelial cells, V. cholerae produces CT (composed of one A subunit and five B subunits). B subunits binds to GM1 ganglioside receptor resulting in conformational changes of holotoxin and A subunit enters the cell. Then, dissociation of A subunit into A1 and A2 fragments occurs by reduction. A1 fragment causes continuous activation of adenylate cyclase and increases intracellular cAMP level. Therefore, the secretion of H2O, Na+, K+, Cl-, and HCO3- into the lumen of the small intestine occurs. Finally, huge loss of fliuds with nutrients cause severe dehydration (diarrhea) (http://www.slideshare.net/dranjayvet/food-borne-bacterial-toxins).
11
Epithelial cellmembrane
Fig. VIII. Chemical structure of trans-anethole (1-methoxy 4-propenyl benzene). Trans-anethole is the naturally occurring isomer of anethole, and usually recognized as anethole. It is the major component (80-90%) of the essential oil derived from sweet fennel and star anise seeds (Sandberg and Corrigan, 2001).
12
Chapter 1: Anethole suppresses the growth and virulence potential of toxigenic Vibrio
cholerae
1.1. INTRODUCTION
Like other bacterial infectious diseases, antimicrobial agents are generally accepted as a
key therapeutic measure to treat cholera. However, despite the use of traditional antimicrobial
agents and oral rehydration therapy, cholera still remains a major public health concern,
particularly in the developing countries (WHO, 2014). Moreover, the majority of the wild
type V. cholerae strains in the cholera endemic regions have gained resistance to commonly
used antimicrobial agents (Das et al., 2008). Due to the emergence of multidrug resistant
(MDR) V. cholerae, sometimes it is very difficult to find proper antimicrobial agents to treat
cholera patients (Mwansa et al., 2007). Hence, development of either new antimicrobial
agents or alternative approaches to antimicrobials (such as searching anti-virulence drugs
which disarm the bacterial pathogen by eliminating its virulence potential rather than killing
the organisms) from cheap sources are badly needed to combat MDR V. cholerae.
Since ancient times, natural products from medicinal plants, such as, spices, herbs, etc.,
have been identified as effective against diarrheal diseases (Low Dog, 2006). More
importantly, spices from different medicinal plants and their constituents are considered to be
safe because of their traditional use without any documented harmful effects to hosts (Smid
and Gorris, 1999). According to previous reports, spices like cinnamon, cardamom, clove,
turmeric, different peppers, red chili, ginger, garlic, etc., have many kinds of medicinal
properties including antimicrobial activity against infectious pathogens (Low Dog, 2006; Ahn
et al., 1990; Oh et al., 1996). Moreover, spices are cheap and easily available. So, spices and
their constituents could be a reservoir to explore either new antimicrobial or anti-virulence
agents against toxigenic MDR V. cholerae.
13
During the second half of the 20th century, researchers are encouraged to search natural
products from medicinal plants (such as herbs, spices, etc.) that can be used in large scale to
reduce enteric infections including V. cholerae-mediated diarrhea. It has been reported that
extracts from Japanese green tea (Camellia sinensis), ‘neem’ (Azadirachta indica), ‘elephant
garlic’ and Vitex negundo leaf could effectively inhibit V. cholerae growth in vitro (Toda et
al., 1992; Thakurta et al., 2007; Rattanachaikunsopon and Phumkhachorn, 2009;
Kamruzzman et al., 2013). In contrast, targeting repression of bacterial virulence factors
rather than killing by natural compounds would be advantageous, such as preserving the host-
indigenous microflora, and can be expected to impose less selective pressure on the
development of antimicrobial resistance (Clatworthy et al, 2007). But, still there is very
limited information regarding the influence of natural compounds on virulence expression
regulation in V. cholerae.
As cholera toxin (CT) is the major virulence factor, researchers are now focusing to
search suitable anti-virulence drug candidates against CT. Previous studies demonstrated that
synthetic compounds virstatin and toxtazin B have anti-virulence properties against CT
(Hung et al., 2005; Anthouard and DiRita, 2013). However, there is still very limited
information regarding the effect of natural compounds on virulence expression inhibition in V.
cholerae. According to previous report, bile can repress CT and TCP expression in V.
cholerae (Chatterjee et al., 2007). Recently, we have also shown that sub-bactericidal
concentration of extracts of some spices, such as, red chili, sweet fennel and star anise seed
can effectively inhibit CT production in V. cholerae (Yamasaki et al., 2011). We have
already reported that capsaicin, the major component of of red chili, could suppress virulence
potential of toxigenic V. cholerae in vitro (Chatterjee et al., 2010).
As potential inhibition of virulence expression was observed in toxigenic V. cholerae by
sub-bactericidal concentration of extracts of sweet fennel and star anise seeds, it would be
very useful if we could identify the active compounds exerting such effects. As an approach
14
of searching active compounds in extracts of sweet fennel and star anise seeds, we targeted
first trans-anethole which accounts for 80-90% of the essential oil derived from sweet fennel
and star anise seeds (Sandberg and Corrigan, 2001).
Besides having various medicinal properties, anethole (trans-anethole) and its natural
reservoir sweet fennel seeds are generally used as a food additive or as a spice component.
Although antimicrobial activities of anethole (trans-anethole) against some bacteria, yeast
and fungi are well established (De et al., 2002; Kubo et al., 2008), still there is no report
regarding its effects on the growth or virulence factors production in V. cholerae. In chapter 1,
the in vitro inhibitory effects of anethole on the growth and virulence factors production of
MDR-toxigenic V. cholerae has been evaluated.
1.2. MATERIALS AND METHODS
1.2.1. Vibrio cholerae strains and growth conditions
A total of 22 toxigenic V. cholerae strains of different isolation origin and belonging to
various serogroups and biotypes were selected randomly from the strain collection of the
Laboratory of International Prevention of epidemics, Osaka Prefecture University. The
relevant characteristics of different toxigenic V. cholerae strains used in this study are listed
in Table 1-1. All V. cholerae strains irrespective of their serogroups were recovered from -
80°C glycerol stock on Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar (Eiken, Tokyo,
Japan) plates. Then, single yellow colony from TCBS agar plates was picked up and
inoculated in either AKI-medium [0.5% NaCl, 0.4% Yeast extract, 1.5% Bactopeptone and
0.3% NaHCO3 (pH 7.4)] at 37°C for El Tor/O139 strains (Iwanaga et al., 1986) or in Luria–
Bertani (LB) broth (pH 6.6, Difco, KS, USA) at 30°C for classical or non-O1/non-O139
strains for optimum growth. The following culture conditions were adjusted according to the
purpose of the study.
15
1.2.2. DNA template preparation and ctxB genotyping
For PCR analysis of the tested V. cholerae strains, DNA template was prepared by boiling
method from the overnight cultures as described previously (Hoshino et al., 1998). In brief,
single yellow colony from TCBS agar plate was inoculated into 3.0 ml LB broth and
incubated at 37˚C overnight with shaking (180 rpm). The culture was diluted 10 times with
TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), boiled for 10 min followed by instant
cooling on ice. After centrifugation at 8,900 g for 3 min, the supernatant was used as DNA
template and stored at -30˚C for future use. To determine the ctxB gene alleles (classical or El
Tor) of the tested strains, a mismatch amplification mutation PCR assay (MAMA-PCR) was
carried out as described previously (Morita et al., 2008). The reaction was performed in a
Gene Amp PCR system 9700 (Applied Biosystems Inc., CA, USA).
1.2.3. Antimicrobial susceptibility testing of the analyzed strains
Antimicrobial resistance pattern of the tested strains was determined by disc diffusion
method according to guidelines provided by the Clinical and Laboratory Standards Institute
(CLSI, 2011). In brief, single colony of V. cholerae from thiosulfate-citrate-bile salts-sucrose
(TCBS) agar plate was grown in 3 ml of Mueller Hinton broth (Difco, KS, USA) at 37C
until the turbidity reached to 0.5 McFarland standards. By using sterile cotton swabs, an
evenly distributed bacterial lawn was prepared on Mueller Hinton agar plates. Then, the
antimicrobial discs were softly placed on each bacterial lawn. The inhibition zone of each
antimicrobial agent was analyzed after overnight incubation at 37C. The inhibition zone was
determined as resistant or susceptible based on those of bacterial cells belonging to the family
Enterobacteriaceae as guidelines of CLSI, 2011. Escherichia coli strain ATCC 25922 was
used as an internal control. Antimicrobials (all from Becton Dickinson) used are as follows:
sulfamethoxazole/trimethoprim (SXT) (1.25/23.75 µg), ampicillin (10 µg), chloramphenicol
(30 µg), tetracycline (30 µg), norfloxacin (10 µg), nalidixic acid (30 µg), streptomycin (10
16
µg), kanamycin (30 µg), doxycycline (30 µg), azythromycin (15 µg), gentamycin (10 µg),
furazolidone (50 µg) and ciprofloxacin (5 µg).
1.2.4. Culture conditions to analyze the effect of anethole on V. cholerae growth
Based upon the serogroups, single colony of V. cholerae was inoculated either in AKI
medium or in LB broth from the TCBS agar plate as described earlier. After 12 hr of growth,
optical density (OD) at 600 nm (OD600) was adjusted to 1.0 and diluted 100-fold with fresh
AKI medium [~107 colony-forming unit (CFU)/ml] and incubated both in the presence and
absence of trans-anethole [1-methoxy 4-propenyl benzene (Nacalai Tesque, Kyoto, Japan;
purity 98%)]. The culture condition was maintained according to Iwanaga et al. (1986), with
slight modifications. Briefly, cultures with or without anethole (trans-anethole) were kept
under stationary condition for an initial 4 hr and then shifted to a shaking condition at 180
rpm for another 4 hr, unless otherwise needed. As anethole was dissolved and diluted in
methanol (MeOH), the final concentrations of methanol were always kept ≤ 0.5% in cultures.
Appropriate dilutions of the culture samples were made with phosphate-buffered saline (PBS,
pH 7.0) and spread on LB agar to see the bacterial viability as CFU/ml.
1.2.5. Determination of MIC and MBC of anethole
The minimum inhibitory concentration (MIC) of anethole against tested V. cholerae
strains was determined by broth macrodilution methods as described previously (Nihei et al.,
2004) with some modifications. Briefly, cultures (~107 CFU/ml) without or with different
concentrations of anethole were co-cultured in one ml of AKI medium according to our
desired culture conditions. Then, the MIC was determined as the lowest concentration of
anethole, in which no growth of V. cholerae was observed at OD600, by using a
spectrophotometer.
The minimum bactericidal concentration (MBC) of anethole was determined by complete
(~100%) killing of V. cholerae cells compared to that of untreated control. The MBC of
17
anethole was confirmed by re-inoculating the broth cultures showing no visible bacterial
growth onto the LB agar plates following overnight incubation at 37C.
1.2.6. Time-kill studies
To examine the effect of anethole on killing of V. cholerae strains in more detail, time-
kill studies were performed. Aliquots of the cultures with (MBC) or without anethole were
withdrawn at desired time-points, and bacterial viability was checked by spreading
appropriate dilutions of samples onto LB-agar plates. Time–kill studies also demonstrate the
least required time to exert bactericidal effect by anethole.
1.2.7. Quantification of CT production by bead-ELISA
For quantification of CT production by V. cholerae strains, culture condition was
maintained as described earlier. Bacterial cultures both in the presence (50 μg/ml) and
absence of anethole were kept under stationary condition for an initial 4 hr and then shifted to
a shaking condition at 180 rpm for another 4 hr at 37C, unless otherwise needed. A cell-free
supernatant (CFS) was prepared by centrifugation of a bacterial culture at 12,000 g for 10
min, followed by filtration through a 0.22- µm filter (Iwaki, Tokyo, Japan). Appropriate
dilutions of the CFS of the culture samples were made in PBS (pH 7.0). Dilutions of purified
CT of known concentrations were used to estimate the amount of CT in CFS by a bead-
ELISA as described previously (Oku et al., 1988). Before CFS preparation, bacterial viability
from each culture was tested by spreading the PBS-diluted culture on LB-agar. As anethole
was dissolved and diluted in 99.9% methanol, methanol (≤ 1%) alone was also added in a
control assay to determine its effects on bacterial growth and CT production.
1.2.8. Western blot analysis of TcpA
To analyze the effect of anethole on TcpA expression, OD600 of V. cholerae cells grown
under appropriate conditions were adjusted to 5.0 in a buffer containing 100 mM Tris-HCl
(pH 8.0), 100 mM sucrose, and 0.2 mM EDTA. Samples were quickly frozen in liquid
nitrogen, thawed in cold water and subjected to DNase I treatment (5 µl of 1.U/µl) for 15 min
18
at room temperature. Samples were then treated with protease inhibitor cocktail (Sigma-
Aldrich) and whole-cell lystaes were prepared by using a sonicator (Astrason W-385
ultrasonic processor) with 5 to 7 pulses of 30 sec each. Proteins from equal volume of cell
extracts of all samples were separated by SDS-PAGE using 15% (wt/vol) polyacrylamide
gels, transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) in a Trans-blot
apparatus (Bio-Rad), and probed with rabbit anti-TcpA antibody (1:3,000 dilution) followed
by HRP-conjugated anti-rabbit IgG (GE Healthcare). Expression of TcpA was then visualized
by using Amersham ECL western blotting detection reagents, according to the product
guideline. The signal intensity of specific protein bands were determined with the ImageJ
software (http://imagej.nih.gov/ij/) and normalized to that of wild type without anethole
samples.
1.2.9. Dose-dependent effects of anethole on CT and TCP expression
A representative V. cholerae O1 El Tor variant strain CRC41 (Please see Table 1-1) was
cultured without (0.5% MeOH) or with different sub-bactericidal concentrations (12.5, 25.0,
50.0 and 100 µg/ml) of anethole under appropriate conditions and CT production was
measured by bead-ELISA as described above. Simultaneously, western blot analysis of TcpA
was carried out by using the same bacterial cultures.
1.2.10. Time-course effect of anethole on CT production
To analyze the time-course effect of anethole on CT production, V. cholerae strain
CRC41 was cultured with an initial 4 hr stationary followed by various length of shaking
conditions both in the absence (0.5% MeOH) and presence of 50 μg/ml anethole. At desired
time points, CT production was measured from the CFS as described earlier.
1.2.11. Statistical analysis
All the experiments were done in triplicate and mean values with standard deviation (SD)
were calculated. Student’s two-sample t-test was performed using the Microsoft Excel
19
program to analyze the significant differences. A P-value of <0.05 was considered as
significant.
1.3. RESULTS
1.3.1. ctxB genotyping and antimicrobial resistance profile of the tested strains
Among 22 different ctx-positive V. cholerae strains of various serogroups and biotypes,
the ctxB gene alleles of twelve O1 El Tor, one O139 (CRC142) and two O1 classical (569B
and O395) strains were confirmed to be of classical type by MAMA-PCR, while another five
O1 El Tor, one O139 (SG24) and one non-O1/non-O139 (CRC127) strains were determined
to be of El Tor type (Table 1-1). Antimicrobial resistance pattern of the tested strains is also
presented in Table 1-1. Surprisingly, most of the O1 El Tor variant strains (10 out of 12)
which are the current epidemic strains, showed resistance to the commonly used multiple
antimicrobial agents. On the other hand, strains belonging to other serogroups including
prototype O1 El Tor strains did not show resistance to multiple antimicrobial agents.
1.3.2. Determination of MIC and MBC of anethole
We tested the effect of anethole on the growth of 22 different toxigenic V. cholerae
strains by co-culturing with different concentrations of anethole. Methanol (0.5%) alone was
also added in a control assay as it had no detectable effect on V. cholerae growth (data not
shown). As shown in Table 1-2, ≤ 100 µg/ml anethole did not show significant growth
inhibitory effect on the analyzed V. cholerae strains compared to those of anethole untreated
controls. But, 150 µg/ml anethole showed potent antibacterial activity against all of the tested
strains. Moreover, no visible growth (determined at OD600) was observed in the analyzed
strains after co-cultured with ≥ 200 µg/ml anethole.
We further investigated whether the effect of anethole on the growth of V. cholerae is
bacteriostatic or bactericidal. To test this, we re-examined the cultures of each of the
representative O1 El Tor (P130), O1 El Tor variant (CRC41), O139 (SG24), O1 classical and
20
non-O1/-O139 (CRC127) strains both in the absence and presence of different concentrations
of anethole by inoculating on the LB agar plate and compared the cell viability as CFU/ml.
Due to some limitations in spreading bacterial cultures onto agar plate, we were unable to
detect the viable cells below 10 CFU/ml. As shown in Fig. 1-1, a significant growth
inhibition (p < 0.01) of the tested strains was observed in presence of 150 µg/ml anethole
compared to the anethole untreated controls. Moreover, no viable bacteria were detected after
co-culturing with 200 µg/ml anethole in case of the tested strains. We found that 200 µg/ml
anethole is bactericidal at least in case of the tested representative strains.
1.3.3. Anethole exerts rapid bactericidal effect
We tested the time-dependent killing effect of anethole on the strains P130 (O1 El Tor)
and CRC41 (O1 El Tor variant) under our experimental conditions. Initially, we observed
that 200 µg/ml anethole potentially killed the tested strains within one hr of incubation at
37C (data not shown), as no CFU was detected following incubation of those cultures on the
LB agar plates. Then, to monitor the anethole-mediated bactericidal effects in more detail, we
shorten the incubation time (within 60 min) and analyzed the bactericidal effect of anethole
against the strain CRC41. As shown in Fig. 1-2, anethole caused rapid death of V. cholerae
cells. Anethole killed 100% of the incubated V. cholerae cells within 20 min, as no CFU was
detected following incubation of the cultures onto the LB agar plates.
1.3.4. Inhibition of CT production in V. cholerae by anethole
Since 100 μg/ml anethole did not affect the growth of a total of 22 different ctx-positive V.
cholerae strains (Table 1-2), those V. cholerae strains were also subjected to analyze the CT
production level both in the presence and absence of anethole. CT in the culture supernatant
fluid was estimated by bead-ELISA. The effects of anethole (50 μg/ml) on CT production in
the tested strains are presented in Fig. 1-3. Addition of anethole inhibited CT production in
all strains irrespective of the serogroups or biotypes, although there were apparent variation
in the degree of CT production and inhibition by anethole for different strains. Anethole-
21
mediated CT inhibition among O1 El Tor, O1 El Tor variant, O139, O1 classical and non-
O1/O139 strains were 68-99%, 80-98%, 73-96%, 59-60% and 43%, respectively. Although
only two classical biotype O1 strains were analyzed, anethole-mediated CT inhibition was
relatively less in them compared to that of El Tor biotype strains.
1.3.5. Dose dependent effect of anethole on CT production
Dose dependent effect of anethole on CT production was analyzed in a representative
MDR and high CT-producing O1 El Tor variant strain CRC41 (Fig. 1-3 & Table 1-1). We
found that although CT production was inhibited in the presence of anethole (≤100 μg/ml) in
a dose dependent manner, bacterial viability was not affected. As shown in Fig. 1-4A, 50 and
100 μg/ml anethole could suppress 85% and 95% of CT, respectively, compared to anethole-
free control culture. Furthermore, these concentrations had no significant effect on CRC41
growth (Fig. 1-4A). Taken together, these results indicated that suppression of CT production
in CRC41 by anethole was not due to bacterial growth inhibition.
1.3.6. Dose dependent effect of anethole on TCP expression
Since CT expression is coordinately regulated with the expression of TCP (Taylor et al.,
1987) in toxigenic V. cholerae, expression of TCP was also examined. For this purpose,
western blot analysis was carried out with the same CRC41 cultures, used to see the dose-
dependent effect of anethole on CT inhibition. As expected, reduction of TcpA (the major
subunit of TCP) expression was observed (Fig. 1-4B) and well correlated with CT inhibition
by anethole (Fig. 1-4A). TcpA expression was reduced ~89% (determined with the ImageJ
software; see Methods section) in presence of 50 μg/ml anethole, compared to the anethole-
free culture. Thus, anethole inhibited the expression of both of the two major virulence
factors of toxigenic V. cholerae in a dose dependent manner.
1.3.7. Anethole inhibits CT production irrespective of the time and culture conditions in
AKI medium
22
Previous studies suggested that initial stationary condition plays a crucial role in the
initiation of CT production by El Tor biotype strains in AKI medium (0.3% NaHCO3) and an
enhanced production of CT was noticed following 4 hr stationary culture (Iwanaga et al.,
1986; Abuaita and Withey, 2009). To analyze the trend of anethole-mediated CT inhibition in
the strain CRC41, a time-course assay of CT production with initial 4 hr stationary and
followed by various length of shaking conditions was conducted. We found that CT
production (366 ng/ml) in the absence of anethole peaked at 2 hr of shaking culture following
initial stationary condition, and a high amount of CT (212 ng/ml) was induced at the end of 4
hr stationary phase (Fig. 1-5). Further extension of stationary phase (up to 8 hr) did not
increase the amount of CT production (202 ng/ml) compared to that of 4 hr stationary phase.
However, addition of anethole inhibited CT production under these conditions until 24 hr of
incubation.
1.4. DISCUSSION
During the second half of the 20th century, it was generally accepted that antimicrobial
agents are effective to treat bacterial infections. However, multidrug resistant (MDR)
pathogenic bacteria have been spreading widely, and traditional antimicrobial agents are no
longer effective against them (Bax et al., 2000). So, researchers have been encouraged to
investigate alternatives of antimicrobial drugs, such as searching natural compounds with
inhibitory effect on virulence rather than killing the whole organism (Clatworthy et al., 2007).
It is believed that purified compounds from medicinal plant could exert better antimicrobial
effect than whole extracts. The present study is the first report which demonstrates that
anethole, purified from natural sources is a potent inhibitor of growth of MDR toxigenic V.
cholerae. Moreover, the present study also demonstrates that sub-bactericidal concentration
of anethole drastically suppressed the virulence expression in toxigenic V. cholerae.
23
Recent cholera outbreaks caused by the O1 El Tor variant strains become more severe in
terms of generating symptoms than past, might be due to the co-existence of MDR and higher
cholera toxin production phenomena in them than prototype El Tor (Sjölund-Karlsson et al.,
2011; Son et al., 2011; Ghosh-Banerjee et al., 2010). In this study, most of the O1 El Tor
variant strains have recently been emerged, simultaneously acquired MDR phenomena, and
produced higher amount of CT compared to the prototype El Tor strains (Table 1-1 & Fig. 1-
3). If these trends continue, in near future, to treat infections mediated by MDR toxigenic V.
cholerae O1 El Tor variant strains will be very difficult unless discovery of novel
antimicrobials or alternative approaches to traditional antimicrobials.
We tested the effects of anethole on the growth and virulence expression of V. cholerae
strains in AKI medium [0.3% NaHCO3 (pH 7.4)] which resembles the environment of human
small intestine. Moreover, this medium is specially considered for maximum virulence
induction by 7th cholera pandemic causing O1 El Tor strains (predominant strains in our
study, 17 out of 22), when cultures were kept for an initial 4 hr stationary followed by a
shaking growth phase (Iwanaga et al., 1986; Abuaita and Withey, 2009). So, by considering
the environment of host small intestine during early phase of V. cholerae infection, its closely
mimic in vitro virulence inducing conditions in AKI medium was used to analyze the effects
of anethole on the growth and virulence production of diverse toxigenic V. cholerae strains.
In this study, MBC of anethole was evaluated as 200 µg/ml against all of the tested V.
cholerae strains belonging to various serogroups and biotypes, and also demonstrate the
potentiality of the anethole as an antimicrobial agent (Table 1-2 & Fig. 1-1). Moreover, rapid-
killing of V. cholerae cells by MBC of anethole (Fig. 1-2) demonstrates the efficacy of
anethole as an antimicrobial drug. Although we did not studied detail mechanisms of
bactericidal effect of anethole, microscopic observations revealed morphological changes in
anethole-treated cells compared to the curved-rod shape of untreated V. cholerae cells (Fig.
1-6).
24
Since ≤ 100 µg/ml anethole did not show significant growth inhibitory effect on the V.
cholerae strains tested in this study (Table 1-2), we analyzed whether these sub-bactericidal
concentrations of anethole have effect on virulence production by V. cholerae or not. As
shown in Fig. 1-3, anethole potentially inhibited CT production in toxigenic V. cholerae,
irrespective of their serogroups and biotypes. Although O1 classical biotype strains analyzed
in this study was only two in number, anethole caused relatively less CT inhibition in them
compared to El Tor biotype strains. It is reported that CT production is differently regulated
among the two biotypes of the V. cholerae strains belonging to O1 serogroup in response to
certain environmental stimuli (Murley et al., 2000). So, we hypothesize that the effectiveness
of anethole as inhibitor of CT production could varied in these two biotypes, but further
studies are needed to reach in conclusion. Based upon the previous reports (Chatterjee et al.,
2010) and anethole-mediated in vitro CT inhibition data in our present study (Fig. 1-3), a
high CT-producing O1 El Tor variant strain CRC41 was selected to analyze the effects of
anethole in detail on virulence inhibition in V. cholerae. In the present study, it was found
that sub-bactericidal concentrations of anethole have dose dependent inhibitory effects on not
only for CT but also for TcpA expression by the strain CRC41 (Fig. 1-4). As TCP is the
major colonization factor, it is speculated that anethole might have inhibitory effect on V.
cholerae colonization in vivo. Furthermore, we did not observe any reduction of CT quantity,
when we incubated a known concentration of purified CT with either anethole (50 μg/ml) or
its solvent methanol (0.5%) at our experimental set up (data not shown). So, we denied a
possibility that anethole or its solvent methanol directly acts on CT to cause alteration of its
immunological property under our experimental conditions, and also indicated that anethole
might affect virulence regulatory cascade to inhibit CT and TcpA expression in V. cholerae.
V. cholerae cells exposed to very low level of oxygen at stationary growth condition at
37C in AKI medium (0.3% NaHCO3) might resemble the environment of host small
intestine during the course of infection. In a time-course study, irrespective of the time
25
duration (until 24 hr) and culture conditions in AKI medium anethole was capable of
inhibiting CT production in the strain CRC41 (Fig. 1-5). Although CT production by the
strain CRC41 in absence of anethole was found to be maximum at 2 hr shaking following
initial stationary condition, this condition differs from what happens during the course of V.
cholerae infection in natural system. So, CT inhibition regulation studies by anethole at
stationary culture in AKI medium (0.3% NaHCO3) might have significant impact on better
understanding of the virulence expression of V. cholerae during early phase of infection in
human small intestine.
CONCLUSIONS
Anethole showed potential antibacterial activity against MDR toxigenic V. cholerae.
Sub-bactericidal concentration of anethole potentially inhibited CT production in
toxigenic V. cholerae.
Inhibition of CT production by anethole was more drastic in El Tor compared to
classical biotype, among V. cholerae O1 strains analyzed in this study.
Anethole showed dose dependent inhibitory effect on not only for CT but also for
TCP expression by V. cholerae.
In vitro inhibition of TCP indicated the possible in vivo colonization defect of V.
cholerae by anethole.
Anethole inhibited CT production, irrespective of the culture conditions in AKI
medium.
None, not resistant to the tested antimicrobials; SM, streptomycin; SXT, sulfamethoxazole/trimethoprim; NA, nalidixic acid; FR, furazolidone; TE, tetracycline
Lab. CollectionNANon-O1/-O139, ctxB genotype: El TorCRC12722
India, 1964SMO1 Clasical, ctxB genotype: ClassicalO39521
India, 1948NoneO1 Clasical, ctxB genotype: Classical569B20
India, 2000NAO139, ctxB genotype: ClassicalCRC14219
India, 1992SMO139, ctxB genotype: El TorSG2418
Bangladesh, 2006SM, SXT, NA, FR, TEO1 El Tor variant, ctxB genotype: Classical268426917
Bangladesh, 2006SM, SXT, NA, FRO1 El Tor variant, ctxB genotype: Classical268071316
India, 2005SM, SXT, NAO1 El Tor variant, ctxB genotype: Classical5'/200515
India, 2005SM, SXT, NAO1 El Tor variant, ctxB genotype: Classical2'/200514
India, 2005SM, SXT, NAO1 El Tor variant, ctxB genotype: Classical1'/200513
Mozambique, 2004SM, SXT, NA, FRO1 El Tor variant, ctxB genotype: ClassicalB3312
India, 2000SM, SXT, NA, FRO1 El Tor variant, ctxB genotype: ClassicalCRC8711
India, 2000SM, SXT, NA, FRO1 El Tor variant, ctxB genotype: ClassicalCRC4110
India, 2000SM, SXT, NA, FRO1 El Tor variant, ctxB genotype: ClassicalCRC279
India, 1994SM, SXT, NA, FRO1 El Tor variant, ctxB genotype: ClassicalCO5338
Bangladesh, 1993SMO1 El Tor variant, ctxB genotype: ClassicalAI-0917
India, 1992SMO1 El Tor variant, ctxB genotype: ClassicalVC3016
India, 1993NoneO1 El Tor, ctxB genotype: El TorVC1905
Peru, 1991NoneO1 El Tor, ctxB genotype: El TorP1304
India, 1980NoneO1 El Tor, ctxB genotype: El TorNICED-33
India, 1970NoneO1 El Tor, ctxB genotype: El TorNICED-102
India, 1970NoneO1 El Tor, ctxB genotype: El TorNICED-11
Isolation Yearresistance profilebiotypeIDNo.
Origin, Antimicrobial Serogroup/strain Serial
Table 1-1. The relevant characteristics of V. cholerae strains used in this study
26
000.41±0.11.55±0.111.64±0.071.78±0.07CRC12722
0000.80±0.021.06±0.051.24±0.06O39521
0001.04±0.071.22±0.061.29±0.08569B20
000.70±0.132.10±0.172.31±0.102.39±0.08CRC14219
000.37±0.131.39±0.061.65±0.081.90±0.08SG2418
000.72±0.082.23±0062.55±0.082.68±0.09268426917
000.57±0.122.24±0.142.53±0.052.62±0.09268071316
000.46±0.051.68±0.061.85±0.101.86±0.115'/200515
000.19±0.041.34±0.071.64±0.071.74±0.072'/200514
000.48±0.121.41±0.081.66±0.041.77±0.061'/200513
000.62±0.061.84±0.071.87±0.102.08±0.06B3312
000.14±0.071.70±0.081.97±0.072.12±0.06CRC8711
000.75±0.122.32±0.112.47±0.072.59±0.08CRC4110
000.61±0.072.18±0.062.29±0.062.46±0.06CRC279
000.52±0.062.14±0.062.50±0.102.60±0.05CO5338
000.47±0.142.12±0.112.40±0.092.47±0.08AI-0917
000.36±0.101.25±0.041.35±0.041.57±0.07VC3016
000.38±0.051.80±0.101.94±0.062.15±0.05VC1905
000.48±0.091.68±0.051.76±0.071.86±0.11P1304
000.11±0.021.30±0.071.37±0.061.38±0.06NICED-33
000.67±0.141.57±0.091.74±0.051.80±0.09NICED-102
000.47±0.091.73±0.051.85±0.131.83±0.11NICED-11
300200150100500IDNo.
OD600 at different concentration of anethole (µg/ml)strain Serial
Table 1-2. Effect of anethole on the growth of different toxigenic V. cholerae strains
OD600, Optical density at 600nm; In all cases, values represent the mean (OD600) ± SD of three independent bacterial cultures at respective anethole concentration
27
Fig. 1-1. Recovery of V. cholerae cells after incubating with different concentrations of anethole. V. cholerae cells belonging to various serogroups and biotypes were incubated with different concentrations of anethole in AKI medium at 37C under initial 4 hr stationary followed by shaking. x-axis indicates the strain ID. ‘ND’ indicates no viable CFU was recovered after spreading of the 100 µl of bacterial cultures onto the agar plates followed by overnight incubation at 37oC. Values represent averages ± SD of three independent experiments. By using two-sample t-test, two asterisks (**) represent p < 0.01 as compared with the anethole-free culture.
Fig. 1-2. Time-killing effect of anethole on V. cholerae. O1 El Tor variant strain CRC41 was incubated with 200 µg/ml of anethole for 60 min. Viable bacteria were counted by inoculating cultures from each of the desired time points onto the LB agar plates. ≤10 CFU/ml indicates the below detection limit of the recovered bacteria. Data represented as the mean ± SD of three independent experiments.
28
Fig.
1-3
. Effe
ct o
f ane
thol
eon
CT
prod
uctio
n in
V. c
hole
rae.
Ane
thol
e(5
0 μg
/ml)
dras
tical
ly in
hibi
ted
CT
prod
uctio
n in
var
ious
se
rogr
oups
and
biot
ypes
of
V. c
hole
rae.
Ope
n an
d cl
osed
bar
s in
dica
te C
T pr
oduc
tion
leve
l as
ng/m
l with
out a
nd w
ith a
neth
ole,
re
spec
tivel
y. N
umer
ical
val
ues
in th
e x-
axis
repr
esen
t the
stra
in id
entit
y (s
ee T
able
1-1
). (+
/-) b
elow
the
stra
in n
umbe
r in
the
x-ax
isre
pres
ents
the
pres
ence
or
abse
nce
of s
peci
fic c
txB
alle
le a
nd s
erog
roup
s/bi
otyp
es a
re d
escr
ibed
bel
ow th
e re
spec
tive
ctxB
alle
le.
Val
ues r
epre
sent
the
aver
ages
±SD
of t
hree
inde
pend
ent e
xper
imen
ts.
29
Fig.
1-4
. Dos
e-de
pend
ent e
ffect
s of
ane
thol
eon
CT
and
TCP
inhi
bitio
n in
V. c
hole
rae
O1
El T
orva
riant
stra
in C
RC
41. (
A)
Effe
ct o
f an
etho
leon
CT
prod
uctio
n an
d ba
cter
ial
viab
ility
pre
sent
ed i
n th
e pr
imar
y an
d se
cond
ary
y-ax
is,
resp
ectiv
ely.
x-a
xis
indi
cate
s th
e co
ncen
tratio
ns o
f ane
thol
eus
ed in
thes
e as
says
. Dat
a ar
e pr
esen
ted
as th
e av
erag
es ±
SD o
f thr
ee in
depe
nden
t obs
erva
tions
. By
usin
g tw
o sa
mpl
e t-t
est,
a sin
gle
aste
risk
(*) r
epre
sent
s p
<0.0
5 an
d tw
o as
teris
ks (*
*) re
pres
ents
p <
0.0
1 as
com
pare
d w
ith th
e an
etho
le-f
ree
cultu
re.
(B)
Dos
e-de
pend
ent
effe
ct o
f an
etho
leon
Tcp
Aex
pres
sion.
Thr
ee
inde
pend
ent
expe
rimen
ts w
ere
cond
ucte
d an
d a
repr
esen
tativ
e w
este
rn b
lot
imag
e is
show
n he
re. T
he b
and
signa
lint
ensit
ies
(sho
wn
belo
w t
he i
mag
e) o
f th
e im
age
of w
este
rn b
lots
w
ere
quan
tifie
d by
Im
ageJ
softw
are
(http
://im
agej
.nih
.gov
/ij/)
and
norm
aliz
ed to
that
of w
ild-t
ype
with
out a
neth
ole
sam
ple
(arb
itrar
ily
take
n as
100
%)
30
Fig.
1-5
. A t
ime-
cour
se e
ffect
of
anet
hole
on C
T pr
oduc
tion
in V
. cho
lera
est
rain
CR
C41
. CT
was
est
imat
ed f
rom
the
CFS
of
initi
al s
tatio
nary
and
follo
wed
by
diffe
rent
leng
th o
f sha
king
con
ditio
ns, b
oth
in th
e pr
esen
ce (
50 μ
g/m
l) an
d ab
senc
e of
ane
thol
e.
Ope
n an
d cl
osed
bar
s in
dica
te C
T pr
oduc
tion
leve
l as
ng/m
l with
out a
nd w
ith a
neth
ole,
res
pect
ivel
y. R
esul
ts r
epre
sent
ed a
s th
e m
ean
±SD
of t
hree
inde
pend
ent e
xper
imen
ts.
31
(A)
(B)
Fig. 1-6. Microscopic observations of V. cholerae O1 El Tor variant strain CRC41: Untreated (A) and treated with 200 µg/ml of anethole (B). Microscopic observations were performed after 30 mins of incubation at 37oC. In untreated cells, 0.5% methanol was used as a control to anaalyze its effect on the morphology of V. cholerae cells. Magnification approximately X 1000.
32
33
Chapter 2: Anethole might affect virulence regulatory cascade in Vibrio cholerae via
cyclic AMP (cAMP)-cAMP receptor protein (CRP) signaling system
2.1. INTRODUCTION
As cholera toxin (CT) is the major virulence factor in toxigenic Vibrio cholerae, much
research and attention have gone into understanding how its expression is regulated. CT
production mechanisms in V. cholerae are extremely intricate and depend on the activation of
ToxT by synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH (Matson et al.,
2007; Higgins and DiRita, 1994; Hase and Mekalanos, 1998). Moreover, CT expression is
differently regulated even among the two biotypes (classical and El Tor) of the V. cholerae
O1 strains, in response to certain environmental stimuli (Murley et al., 2000). It was found
that over production of TcpP overcomes the requirement for ToxR in activating toxT, but
over production of ToxR was unable to overcome the requirement for TcpP (Krukonis et al.,
2000). This suggests that TcpP is more directly responsible for transcriptional activation of
toxT and that ToxR/ToxS plays an indirect role. TcpH protects the periplasmic domain of
TcpP from proteolytic cleavage and thus maintain the cellular level of TcpP (Beck et al.,
2004). Transcription of tcpP/tcpH is activated by transcriptional activators AphA and AphB.
On the other hand, cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) complex is the
down regulator of tcpP/tcpH (Kovacikova and Skorupski, 2001). Apart from activation,
histone-like nucleoid structuring protein (H-NS) has been shown as a direct repressor of the
transcriptions of toxT, ctxAB and tcpA (Nye et al., 2000).
In chapter 1, it was found that sub-bactericidal concentration of anethole is a potent
inhibitor of CT and TCP expression in V. cholerae. In a previous study, synthetic compound
virstatin (4-N-[1,8-naphthalimide]-n-butyric acid) showed the same effects by affecting ToxT
post transcriptionally (Hung et al., 2005). Another recent study showed that synthetic
compound toxtazin B affected ToxT by inhibiting tcpP transcription, but mechanisms behind
34
tcpP inhibition were not studied well (Anthouard and DiRita, 2013). Apart from synthetic
compounds, natural compounds like extracts of red bayberry could also inhibit the CT
production in V. cholerae by repressing the virulence gene transcription (Zhong et al., 2008).
Bile was also able to repress ctxA and tcpA transcriptions in a ToxT independent manner and
through the activation of hns (Chatterjee et al., 2007). Recently, our group also reported that
capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide), the major component of extract of red
chili also inhibits ctxA and tcpA transcriptions by upregulating hns transcription, but in a
ToxT dependent fashion (Chatterjee et al., 2010). So, different bioactive compounds could
exert different mechanisms to inhibit virulence expression in V. cholerae.
In previous chapter, we did not observe any direct effect of anethole on purified CT to
cause alteration of its immunological property, suggesting that anethole caused virulence
repression in V. cholerae by affecting virulence regulatory cascade. In this chapter, we
attempted to resolve the underlying molecular mechanisms behind anethole-mediated in vitro
virulence suppression in V. cholerae O1 El Tor variant strain, via quantitative reverse
transcription real time-PCR (qRT-PCR) assay and western blot analyses of the expression of
virulence/virulence regulatory genes.
2.2. MATERIALS AND METHODS
2.2.1. Bacterial strains, plasmids and growth conditions
A description of different bacterial strains, the relevant characteristics of specific gene
mutant strains and properties of plasmids used in this section are listed in Table 2-1. Among
V. cholerae strains, a high CT-producing O1 El Tor variant strain CRC41 was used
throughout the in vitro mechanisms study of CT production inhibition by anethole. AKI-
medium [0.3% NaHCO3 (pH 7.4)] at 37C was used as optimal growth and virulence
production by the strain CRC41, unless otherwise mentioned. Escherichia coli DH5α λpir
and SM10 λpir were used for cloning and conjugation study, respectively. Antimicrobials
35
were used at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml;
nalidixic acid, 30 μg/ml, and gentamycin, 20 μg/ml
2.2.2. DNA manipulations
Restriction and DNA modification enzymes were purchased from TaKaRa Bio Inc.
(Shiga, Japan). As the genomic sequence of V. cholerae O1 El Tor variant strain CRC41 is
currently unknown, we used the relatively close published sequence of V. cholerae O1 El Tor
strain N16961 [accession no. AE003852 (Heidelberg et al., 2000)] for designing primers and
probes, with the help of Primer Express Software Version 3.0 (Applied Biosystem Inc., CA,
USA). Each gene probe was labeled with 6-FAM (6-carboxyfluorescein) as a reporter dye
and TAMRA as quencher dye at the 5´and 3´ ends, respectively. All the PCR amplicons and
cloned products from CRC41 were sequence-verified by using ABI PRISM 3100-Avant
genetic analyzer (Applied Biosystem Inc.). The nucleotide sequences were aligned and
analyzed by using a Laser-gene DNASTAR (Madison, WI) software package.
2.2.3. RNA extraction and qRT-PCR
Total RNA was extracted from V. cholerae cells grown under appropriate conditions by
using TRIzol reagent (Invitrogen), according to the product guidelines. Isolated RNA was
treated with RNase-free DNase I (1 U/μg, amplification grade; Invitrogen) to avoid genomic
DNA contamination. The reverse transcription was performed with 1 μg of RNA, according
to the instruction of quick RNA-cDNA kit (Applied Biosystems Inc.). The obtained cDNA
was diluted 1:4 with nuclease-free water, and 4 μl was used for qRT-PCR assay. The qRT-
PCR assay was carried out by following the TaqMan probe method with each gene-specific
primers and TaqMan Gene Expression master mix (Applied Biosystems Inc.). PCR
conditions were 50ºC for 2 min, 95ºC for 10 min and 40 cycles, each having 95ºC for 15 sec
and 60ºC for 1 min in an ABI PRISM 7500 sequence detection system (Applied Biosystems
Inc.). Genomic DNA and DNase-treated RNA that had not been reverse transcribed were
used as positive and negative controls, respectively. The housekeeping gene recA was used as
36
an internal control. The relative transcription of each gene in comparison with the internal
control was analyzed as described previously (Hagihara et al., 2004). Primers and probes
used for qRT-PCR analysis are listed in Table 2-2.
2.2.4. Recombinant strains and plasmids construction
The virulence regulatory cyaA and crp deletion mutants were constructed in the strain
CRC41 by following in-frame deletion mutagenesis as described previously (Warrens et al.,
1997). Briefly, deletion in each gene mentioned earlier was generated by an overlapped
fusion PCR using the primers listed in Table 2-3. Then, the cyaA and crp genes with desired
deletions were cloned into the MCS of the suicide vector pWM91 to construct the plasmids
pΔcyaA and pΔcrp, respectively. The resulting plasmids were then electroporated into E. coli
strain DH5α λpir for maintaining and subsequently to the SM10 λpir to mobilize into the V.
cholerae strain CRC41 via conjugation. Resolution of the cointegration was done by sucrose-
encounter selection. Recombination and the loss of the wild-type allele were confirmed by
PCR using primers flanking the deletions.
pCyaA and pCRP were constructed in pBR322 vector to analyze the complemention
effect of the proteins in respective V. cholerae mutants. To construct pCyaA, a 2.65-kb
fragment that includes entire cyaA gene (VC0122) with 126-bp upstream of putative
promoter region, was amplified and cloned in to the compatible sites of pBR322. By
following the same procedure entire crp gene (VC2614) including 183-bp of putative
promoter region was cloned into pBR322 to construct pCRP. To express recombinat TcpP
(rTcpP), plasmid pTcpPH was constructed in pET-28-a(+) vector. The entire tcpPH
(VC0826-0827) genes were cloned in to the compatible sites of pET-28-a(+) vector. The
nucleotide sequences used to construct plasmids are presented in Table 2-3.
2.2.5. Rabbit immunization for anti-TcpP Immunoglobulin G (IgG)
Antiserum against purified rTcpP was basically prepared as described previously with
some modifications (Yutsudo et al., 1987). In brief, 100 µg of purified rTcpP in phosphate
37
buffered saline [PBS (pH 7.0)] was emulsified with an equal volume of Freund’s complete
adjuvant (Difco Laboratories, Detroit MI, USA). The emulsion was administered
intramuscularly into an adult New Zealand white male rabbit (~2 kg). Subsequently,
additional 6 doses of 100 µg rTcpP were given in similar fashion at 7 days interval to
previous dose. The serum antibody titer was regularly monitored by western blotting with
known concentrations of rTcpP. At the end, the rabbits were anesthetized by intramuscular
injection of 45 mg kg-1 ketamine (Ketalar, Daiichi Sankyo Co., Ltd.) and 5 mg kg-1 xylazine
(Selactar, Bayer Healthcare) and whole blood was collected from the rabbit and the serum
was separated and stored at -80˚C for further use.
2.2.6. Western blot analysis of TcpP
To analyze the effect of anethole on TcpP expression, OD600 of V. cholerae cells grown
under appropriate conditions were adjusted to 5.0 in 100 mM Tris-HCl buffer (pH 8.0)
containing 100 mM sucrose, and 0.2 mM EDTA. Samples were quickly frozen in liquid
nitrogen, thawed in cold water and subjected to DNase I treatment (5 µl of 1 U/µl) for 15 min
at room temperature. Samples were then treated with protease inhibitor cocktail (Sigma-
Aldrich) and whole-cell lystaes were prepared by using a sonicator (Astrason W-385
ultrasonic processor) with 5 to 7 pulses of 30 sec each. Proteins from equal volume of cell
extracts of all samples were separated by SDS-PAGE using 15% (wt/vol) polyacrylamide
gels, transferred to a polyvinylidene difluoride (PVDF) membrane in a Trans-blot apparatus,
and probed with rabbit anti-TcpP antibody (1:500 dilution) followed by HRP-conjugated
anti-rabbit IgG (GE Healthcare). Expression of TcpP was then visualized by using Amersham
ECL western blotting detection reagents, according to the product guideline. The signal
intensity of specific protein bands was determined with the ImageJ software
(http://imagej.nih.gov/ij/) and normalized to that of wild type without anethole samples.
Initially, rabbit anti-TcpP antibody was kindly provided by Dr. V. J. DiRita. Later, we also
38
developed anti-TcpP antibody in adult New Zealand white male rabbit (~2 kg) as described
earlier.
2.2.7. Construction of promoter-lacZ fusion plasmids
lacZ transcriptional fusions with promoter sequences of tcpPH, cyaA, crp, hapR and aphA
were constructed into a promoter-less lacZ reporter gene (encodes β-galactosidase)
containing gentamycin resistant vector pHRP309 as described previously (Parales and
Harwood, 1993). The PCR-amplified promoter regions with ~200-300 bp, immediately
upstream of the start codons of the genes mentioned earlier were ligated into the XbaI-EcoRI
sites of pHRP309 and electroporated into an E. coli strain JM109 (lacZ -). Plasmids pPro
tcpPH, pPro cyaA, pPro crp, pPro hapR and pPro aphA (Table 2-1) were constructed for the
promoter regions of tcpPH, cyaA, crp, hapR and aphA genes, respectively. The desired
sequences of promoter regions of fusion plasmids were then verified by sequencing. The
primers used for amplification of the promoter regions are presented in Table 2-3.
2.2.8. β-Galactosidase assay
β-Galactosidase assays were performed as describe by Miller (1972) with little
modifications. Briefly, OD600 of overnight cultures were adjusted to 1.0. Subsequently,
cultures were 50-fold diluted with M9 medium (250 mM Na2HPO4 7H2O, 100 mM KH2PO4,
40 mM NaCl, 100 mM NH4Cl, pH 7.0) (Parales and Harwood, 1993) supplemented with
0.4% glucose and incubated both in the presence and absence of anethole at 37o C for 12 hr.
One ml portions of the cultures were then harvested and washed with 1 ml of Z-buffer (60
mM Na2HPO4, 35 mM NaH2PO4, 10 mM KCl, 2 mM MgSO4, pH 7.0). Then, cells were
lysed by adding 100 μl chloroform and 50 μl of 0.1% SDS (sodium dodecyl sulfate),
followed by resuspension. The cell lysates were incubated at room temperature for 5 min.
After that, different dilutions of cell lysates (50 μl) were placed into 96-well microtiter plates
and 20 μl o-nitrophenyl-β-D-galactopyranoside solution (4 mg/ml) were added to each well.
The reaction was stopped by adding 1M Na2CO3 (150 μl) and color intensities were measured
39
at OD420 and OD550. The duration of color development was marked and β-galactosidase
activity (in Miller unit) was calculated as described previously (Miller, 1972).
2.3. RESULTS
2.3.1. Anethole suppresses the virulence regulatory cascade of V. cholerae by down
regulating tcpPH transcriptions
To determine at which point of the virulence regulatory cascade anethole affects CT and
TCP expression, transcriptional level of various regulatory genes, including toxT, toxR, toxS,
tcpP, tcpH and hns were analyzed via qRT-PCR analysis. The relative expression of each
gene was normalized with that of housekeeping gene recA, which was used as an internal
control. Based upon our initial experiments showing the trend of inhibition of CT expression
(Fig. 1-5), a culture condition with 4 hr stationary which resembles the environment of host
small intestine and followed by 2 hr of shaking (at which point CT production by CRC41 was
found maximum) were chosen for assaying the transcription of the various virulence genes as
well as the regulatory genes, in the presence of 50 μg/ml anethole.
As shown in Fig. 2-1A, in presence of anethole at initial stationary culture ctxA gene
transcription was repressed ~10 fold (p <0.01). Transcription of major colonization factor
tcpA was also repressed ~ 60 fold (p <0.01). At this stage, transcriptions of other virulence
regulatory genes were also repressed by anethole, at various extents: toxT (~13 fold; p <0.01),
tcpP (2.3 fold; p <0.01) and tcpH (2.8 fold; p <0.01). On the other hand, transcription of toxR,
toxS and hns were not affected significantly by anethole. Under shaking condition (Fig. 2-1B),
the transcriptions of all the analyzed genes showed the same trend as observed under
stationary condition. At shaking stage, along with ctxA (~15 fold, p < 0.01) and tcpA (~90
fold, p < 0.01), transcriptions of toxT (~30 fold; p < 0.01), tcpP (~3 fold; p < 0.01) and tcpH
(~4 fold; p < 0.01) were inhibited drastically by anethole compared to those of untreated
controls. As observed under stationary condition, the transcriptions of toxR, toxS and hns
40
were not affected significantly by anethole. As expected, the transcription of housekeeping
gene recA was not affected in presence of anethole compared to the untreated controls at
either culture conditions (data not shown).
2.3.2. Suppression of TcpP expression by anethole
The validity of the qRT-PCR data was further verified by analyzing the expression of
TcpP both in the presence and absence of anethole (50 μg/ml) from initial stationary and
followed by shaking culture conditions. The relative signal intensities of TcpP by western
blot (Fig. 2-2) correlated well with the observation of tcpP transcription by qRT-PCR (Fig. 2-
1). The relative intensities of TcpP expression in the anethole treated cells were 34%
(determined with the ImageJ software; see Methods section) and 38% at 4 hr stationary and
followed by 2 hr shaking conditions, respectively, compared to those of anethole untreated
controls.
2.3.3. Anethole might affect cAMP-CRP signaling system to suppress tcpP/tcpH
Since there are also upstream regulatory genes for tcpPH, the effect of anethole on the
transcriptions of probable upstream regulators were also examined. tcpP/tcpH are
overlapping operons and positively regulated by the membrane-located transcription factor
AphA/AphB in V. cholerae (Kovacikova and Skorupski, 1999). The cAMP-CRP complex
has overlapping binding sites with AphA and AphB in tcpPH promoter and can negatively
regulate the expression of tcpPH (Skorupski and Taylor, 1997; Kovacikova and Skorupski,
2001), whereas the quorum sensing regulator HapR has a negative effect on AphA (Lin et al.,
2007). Therefore, we also analyzed the transcriptions of possible regulators of tcpPH,
including cyaA, crp, hapR, aphA and aphB in the presence of anethole.
As shown in Fig. 2-3A, after 4 h stationary condition the relative transcription of cyaA
(1.5 fold; p <0.05), crp (2.4 fold; p <0.01) and hapR (1.8 fold; p <0.05) increased but aphA
(2.8 fold; p <0.01) decreased significantly in presence of 50 μg/ml anethole compared to
those of cultures without anethole. Although we observed certain variation in expression of
41
these genes under stationary and shaking condition (Fig. 2-3B), the transcription of crp
remained consistently elevated in presence of anethole irrespective of the culture conditions.
Taken altogether, a hypothesis can be raised that anethole might initiate inhibition of tcpPH
transcriptions as well as CT through cAMP-CRP complex-mediated signal.
2.3.4. Anethole might influence the crp-promoter activity
To see whether anethole have any effect on the promoter of tcpPH or its regulatory genes,
promoter activity assay was performed in an E. coli system as described in the Methods
section. Based upon the transcriptional analysis (Figs. 2-1 & 2-3); tcpPH, cyaA, crp, hapR,
and aphA were considered for promoter activity testing both in the absence and presence of
anethole (50 μg/ml). To reduce the background lacZ expression, the promoter-lacZ fusion
plasmids were transformed into a lacZ deficient E. coli strain, such as JM109 (Yanisch-
Perron et al., 1985). Then the β-galactosidase expression by the cloned promoters of desired
genes was measured both in the presence and absence of anethole. This assay procedure was
carried out in M9 medium supplemented with 0.4% glucose, to minimize the effect of other
compound than anethole on the promoters of desired genes. As the results shown in Fig. 2-4,
no other promoter activity was significantly changed except for crp, in presence of anethole
compared to the anethole-free cultures. So, anethole might have upregulatory effect on crp
promoter.
2.3.5. Anethole causes growth inhibition of ΔcyaA and Δcrp mutants compared to wild
type V. cholerae.
To further investigate the role of cAMP-CRP signaling system in anethole-mediated
suppression of CT expression, we constructed ΔcyaA and Δcrp isogenic mutants of the strain
CRC41, respectively. These mutants were apparently slow growing with increased doubling
times in AKI medium at 37ºC as well as a phenotypic growth defect on TCBS agar compared
to the wild-type strain. Complementation of the mutations by transformation with
recombinant plasmids carrying the cloned cyaA and crp genes (pCyaA and pCRP
42
respectively) also restored the growth rate to the level of the wild type strain (Fig. 2-5). But
we failed to analyze the effect of anethole on CT production in these mutants, since they
showed significant growth inhibition at 50 μg/ml anethole compared to anethole-free culture
(Fig. 2-6).
2.4. DISCUSSION
Due to the emergence of multidrug-resistant (MDR) Vibrio cholerae strains and declining
performance of traditional antimicrobial agents against them, approaches to identify suitable
anti-virulence drugs have already become an alternative research area (Chatterjee et al., 2007;
Hung et al., 2005; Anthouard and DiRita, 2013; Yamasaki et al., 2011; Chatterjee et al.,
2010). In those studies, it has been demonstrated that different compounds inhibited virulence
expression in V. cholerae by exerting different mechanisms. In the present study, we have
also proposed a novel mechanism of virulence expression inhibition in MDR V. cholerae by
natural compound anethole. We have studied the molecular mechanisms behind anethole-
mediated virulence suppression in most toxigenic and current epidemic causing O1 El Tor
biotype variant strain of V. cholerae.
Oxygen limiting condition, a factor similar to the host intestinal environment promoted V.
cholerae virulence genes expression (Marrero et al., 2009). When V. cholerae cells were
exposed to very low level of oxygen at static growth conditions at 37ºC in AKI medium
( 0.3% NaHCO3-), all of which might resemble the environment of host small intestine during
the course of infection. It has been shown that HCO3- could stimulate CT synthesis by O1 El
Tor strains in AKI medium, when grown statically. But it enhances neither toxT transcription
nor ToxT expression compared to the HCO3- -free culture (Meddrano et al., 1999). Indeed,
HCO3- enhances ToxT activity post-translationally to produce high amount of CT by O1 El
Tor strains at static condition in AKI medium (Abuaita and Withey, 2009). In our present
study, inhibition of transcription of ctxA along with tcpA and toxT (Fig. 2-1), suggested that
43
anethole affects the virulence regulatory cascade prior to toxT. These results also ruled out
the possibility of interfering anethole with the activity of HCO3- in AKI medium, as HCO3
-
enhanced ToxT activity post-translationally. In a recent study, our research group found that
another natural compound capsaicin causes drastic inhibition of ctxA along with tcpA and
toxT by upregulating hns transcription (Chatterjee et al., 2010). In this study, we did not find
any significant enhancement of hns transcription in presence of anethole. Even when the
same strain and culture conditions used to see capsaicin-mediated virulence regulatory gene
transcriptions were applied, anethole was unable to upregulate hns transcription (data not
shown).
It is well established that ToxR is essential for activation of toxT in V. cholerae. But only
ToxR is not sufficient for its activation, and it is unable to successfully activate toxT (Higgins
and DiRita, 1994). On the other hand, overproduction of TcpP obviates the requirement of
ToxR and can alone activate the toxT promoter (Krukonis et al., 2000). In our present study,
we observed a drastic repression of tcpPH resulting in inhibition of CT, despite any
significant changes in toxR/toxS transcripts level in presence of anethole (Fig. 2-1), also
suggesting a toxR-independent but tcpP-dependent inhibition of toxT. Moreover, low level
detection of TcpP (Fig. 2-2) in anethole treated cells compared to the untreated control
further confirmed our observations.
Transcription analyses of the upstream regulatory genes of tcpPH (Fig. 2-3A) raised a
hypothesis that anethole might initiate inhibition of tcpPH transcriptions as well as CT by
affecting quorum sensing regulatory genes via cAMP-CRP complex-mediated signal.
Promoter activity assay (Fig. 2-4) of the tcpPH regulatory genes also supported the idea that
anethole might have upregulatory effect on crp transcription. Apart from generating quorum
sensing signal, cAMP-CRP complex could directly inhibit tcpPH promoter activity by
binding to the competitive site for AphA and AphB (Kovacikova and Skorupski, 2001).
However, at shaking stage (Fig. 2-3B) although significant elevation of crp was observed,
44
transcription of other tcpPH regulatory genes were not significantly affected by anethole.
This suggested that cAMP-CRP complex mediated activation of quorum sensing pathway
might be terminated at this stage. It has been reported that although initial expression of ToxT
is dependent on the activity of ToxR and TcpP, once produced, ToxT itself is able to maintain
its expression by activating distal tcpA promoter (auto-regulatory loop) leading to
transcription of toxT (Yu and DiRita, 1999; Abuaita and Withey, 2011). Taken together, it
can be hypothesized that in anethole exposed cells, activation of cAMP-CRP signaling
system leads to a very low level production of TcpP, which might fail to activate toxT
transcription initially and subsequently prevent the activation of auto-regulatory loop of toxT
transcription. Thus, due to the failure of activation of auto-regulatory loop of toxT
transcription at initial stationary phase, further activation of toxT transcription (Fig. 2-1B)
might not occur, and thus contribute in anethole-mediated suppression of CT production at
aerobic growth phase. As ΔcyaA and Δcrp isogenic mutants of the strain CRC41 showed
significant growth inhibition at 50 μg/ml anethole compared to anethole-free culture (Fig. 2-
6), we failed to evaluate the effect of anethole on CT production in these mutants.
Based upon the findings in this study, we propose a model showing the hypothetical
mechanisms of how anethole-mediated signal affect the general toxR regulon of CT
expression (Fig. 2-7). In this scenario, anethole initiates CT production inhibition by
activating crp at stationary phase grown V. cholerae cells. In response to crp, transient
activation of cyaA occurs. Then, cAMP-CRP complex could exert dual inhibitory effect on
tcpPH promoter either binding directly or by activating quorum-sensing regulatory genes.
Thus, very low level production of TcpP inhibits initiation of toxT transcription directly and
subsequently prevents the activation of virulence factors production mechanisms in V.
cholerae at later growth phase.
Although we showed evidences in favor of our hypothesis that anethole might initiate CT
production inhibition in V. cholerae by activating cAMP-CRP signaling system, contribution
45
of other factors along with cAMP-CRP complex mediated signal in anethole-mediated
virulence suppression could not be excluded. Recently, it has been reported that
extracytoplasmic stress response can induce integral membrane zinc metalloprotease RseP
(formerly known as YaeL protease) in V. cholerae, which causes degradation of TcpP
(Matson and DiRita, 2005; Chatterjee et al., 2013). It is possible that anethole could induce
extracytoplasmic stress response and thereby induce the expression of some proteases, such
as RseP or major serine proteases DegS in V. cholerae. But we failed to find any significant
differences of rseP and degS in their transcription level between anethole-treated and
untreated cells at our experimental set up (Fig. 2-8). Due to anethole-mediated tcpH
suppression, proteolytic cleavage of TcpP could be another possibility, as TcpH protects the
periplasmic domain of TcpP from proteolytic cleavage (Beck et al., 2004). It has been also
shown that at non-permissive conditions, a protein named PepA partially inhibits tcpP
transcription in V. cholerae (Behari et al., 2001). tcpP promoter can also be negatively
regulated by PhoB, which binds to a distinct site from both the AphA and AphB binding sites
(Pratt et al., 2010). Moreover, oxidative modification of AphB could repress virulence
expression in V. cholerae by affecting tcpP transcription (Liu et al., 2011). So, involvement
of synergistic activation of any of the mentioned pathways along with the cAMP-CRP
signaling system in anethole-mediated virulence suppression in V. cholerae could not be
excluded, unless tested.
CONCLUSIONS
Anethole affected virulence regulatory cascade prior to toxT, to inhibit CT and TCP
production in V. cholerae.
Anethole suppressed the virulence factors production (CT and TCP) in V. cholerae by
down regulating TcpP expression at the transcriptional level.
46
Anethole-mediated activation of cAMP-CRP signaling system might contribute to
inhibit tcpPH transcriptions as well as virulence factors production in V. cholerae.
47
Table 2-1. Properties of bacterial strains and plasmids used in this study Strains or plasmids Relevant characteristics Source or reference V. cholerae strains
CRC41 O1 El Tor variant, ctxB genotype: Classical India, 2000 ΔcyaA-CRC41 Derivative of El Tor variant strain CRC41 carrying
deletion of cyaA (VC0122) This study
Δcrp-CRC41 Derivative of El Tor variant strain CRC41 carrying deletion of crp (VC2614)
This study
E.coli strains DH5α λpir supE44 DlacU169 (/80 lacZDM15) hsdR17 recA1 endA1
gyrA96 thi-1 relA1 ( λ pirR6K) Miller et al., 1988
SM10 λpir thi-1, thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Mu, Kmr, (λ pirR6K)
Miller et al., 1988
JM109 recA1 endA1 gyrA96 thi-1 hsdR17 (rK– mK+)supE44 relA1 Δ(lac-proAB) [F´ traD36 proAB lacIqZΔM15].
Yanisch-Perron et al., 1985
Plasmids pWM91 oriR6K plasmid vector used for sacB-mediated allelic
exchange, Ampr Metcalf et al., 1996
pΔcyaA pWM91::ΔVC0122, Ampr This study pΔcrp pWM91::ΔVC2614, Ampr This study
pCyaA pBR322 carrying cyaA gene including putative promoter
region, Ampr, Tetr This study
pCRP pBR322 carrying crp gene including putative promoter
region, Ampr, Tetr This study pTcpPH pET-28-a(+) carrying entire tcpPH gene, Kmr This study pTcpA pET-28-a(+) carrying entire tcpA gene, Kmr This study
pHRP309 IncQ, lacZ transcriptional fusion vector for promoter cloning; Gmr
Parales et al., 1993
pPro tcpPH pHRP309 containing promoter (342-bp) of tcpPH gene;Gmr This study pPro cyaA pHRP309 containing promoter (195-bp) of cyaA gene; Gmr This study pPro crp pHRP309 containing promoter (277-bp) of crp gene; Gmr This study
pPro hapR pHRP309 containing promoter (242-bp) of hapR gene; Gmr This study
pPro aphA pHRP309 containing promoter (366-bp) of aphA gene; Gmr This study
48
Table 2-2. Primers and probes used for qRT-PCR analysis Primer*/probe*† Sequence (5'-3')
ctxA-rt-F GGA GGG AAG AGC CGT GGA T ctxA-rt-P CAT CAT GCA CCG CCG GGT TG ctxA-rt-R CAT CGA TGA TCT TGG AGC ATT C tcpA-rt-F GGG ATA TGT TTC CAT TTA TCA ACG T tcpA-rt-P TGC TTT CGC TGC TGT CGC TGA TCT T tcpA-rt-R GCG ACA CTC GTT TCG AAA TCA toxT-rt-F TGA TGA TCT TGA TGC TAT GGA GAA A toxT-rt-P TAC GCG TAA TTG GCG TTG GGC AG toxT-rt-R TCA TCC GAT TCG TTC TTA ATT CAC toxR-rt-F GCT TTC GCG AGC CAT CTC T toxR-rt-P CTT CAA CCG TTT CCA CTC GGG CG toxR-rt-R CGA AAC GCG GTT ACC AAT TG toxS-rt-F TGC CAT TAG GCA GAT ATT TCA CA toxS-rt-P TGA CGT CTA CCC GAC TGA GTG GCC C toxS-rt-R GCA ACC GCC CGG CTA T tcpP-rt-F TGG TAC ACC AAG CAT AAT ACA GAC TAA G tcpP-rt-P TAC TCT GTG AAT ATC ATC CTG CCC CCT GTC tcpP-rt-R AGG CCA AAG TGC TTT AAT TAT TTG A tcpH-rt-F GCC GTG ATT ACA ATG TGT TGA GTA T tcpH-rt-P TCA ACT CGG CAA AGG TTG TTT TCT CGC tcpH-rt-R TCA GCC GTT AGC AGC TTG TAA G hns-rt-F TCG ACC TCG AAG CGC TTA TT hns-rt-P CTG CGC TAT CAG GCG AAA CTA AAA CGA AA hns-rt-R GGT GCA CGT TTG CCT TTT G
cyaA-rt-F CAC ACT GCT CAA CCC ACA AAT T cyaA-rt-P CCC CAG ACC TGC ATG AGC CCG cyaA-rt-R CCA GCA CAA ACC TCA ATA AAA CTT AA crp-rt-F GAT GCG CCT TTC AGG TCA A crp-rt-P TCG TCG TCT GCA AGT GAC CAG CCA crp-rt-R CGC AAG GTC GCC AAC TTT
hapR-rt-F GCG CAA TCT CGG CAA TAT CT hapR-rt-P CAC CAC GAC CAA TGC CGC GTT TA hapR-rt-R AAA TCG CGT TGG AAG TGT TTG aphA-rt-F GCA GCA ACG TTT AGA GCG TTT aphA-rt-P CGT CGT AAT TTA CTG GTT CGC CAA GCA aphA-rt-R TCG TCC GCC CAT TGA ATC aphB-rt-F GCA TGA GCG TAA TGC CTA AAC C aphB-rt-P TCT GAA CAT GCG CTG CGA ACA ACA aphB-rt-R TTC AAG CCA GCG CAC TGA rseP-rt-F CGG GAA TCG CAC CAA AAG rseP-rt-P CGC AGA ATG GCC GCA AAA CTA TCG rseP-rt-R CGA CTC AAA TAC ACC AAA TTG CA degS-rt-F GCT ACC GGA CGT TCA TCC A degS-rt-P CGC TGA TGG TCG CCA AGC CTT TAT T degS-rt-R CAT TGA TTG CGG CAT CAG TTT recA-rt-F CAA TTT GGT AAA GGC TCC ATC AT recA-rt-P CTT AGG CGA CAA CCG CGC recA-rt-R CCG GTC GAA ATG GTT TCT ACA
* All the primers and probes were designed using PRIMER EXPRESS software version 3.0 (Applied Biosystems Inc.). † FAM was used as a 5'-reporter dye and TAMRA as a 3'quencher dye to design each TaqMan probe. F, forward primer; P, probe; R, reverse primer.
49
Table 2-3. Primer sequences used for mutant construction, recombinant protein expression and cloning of promoter regions Primer* Sequence (5'-3') For mutant construction
cyaA-FO GACCGGAACATCTTTCATTG cyaA-5O GATCGGGATCCATAAGCCTTTACCGCCCACT cyaA-5I AGAGACGACGCGTGCCAGTCCTGCAAGTTTGCTTCCCTG cyaA-3O GATCGGCGGCCGCCGAGAGCCGACAACAAAAAC cyaA-3I GACTGGCACGCGTCGTCTCTAGCTCAAGCCCAGTTTTTG
cyaA-RO TATACAGTGGCCCAGTTTGC crp-FO ATTCCATCGTCCGTTCAATG crp-5O GATCGCTCGAGATTCCAACGCTGGATGAGAG crp-5I AGAGACGACGCGTGCCAGTCAGTGTTGGATCGGTTTGAGG crp-3O GATCGGGATCCTGCAAGCGATTGTTGAAAAG crp-3I GACTGGCACGCGTCGTCTCTCGGTTCGTGCTTTCAAAGAT
crp-RO CATTTTGAACATCCCGATCC For recombinant protein expression
TcpA-pr-F GATCGGGATCCATGCAATTATTAAAACAGC TcpA-pr-R GATCGCTCGAGTTAACTGTTACCAAAAGC TcpPH-pr-F GATCGGGATCCATGGGGTATGTCCGCGTG TcpPH-pr-R GATCGCTCGAGCTAAAAATCGCTTTGAC
For complementation of protein in mutant strain CyaA-PBR-comp F GATCGAAGCTTGTGATGTGCTTCCAAGAGC CyaA-PBR-comp R GATCGGGATCCTTAGGCATTGACCACTTG CRP-PBR-comp F GATCGAAGCTTGCAAATTGGACTACTGACACGA CRP-PBR-comp R GATCGGGATCC TTAGCGAGTGCCGTAAACC
For promoter cloning tcpPH-pro-F GATCGTCTAGACAAACGTAAGGGGCAAAATG tcpPH-pro-R GATCGGAATTCTGCATTCATTCCACCAAAGG cyaA-pro-F GATCGTCTAGAGGCAATTTGCAACAACACTG cyaA-pro-R GATCGGAATTCCTGCAAGTTTGCTTCCCTG crp-pro-F GATCGTCTAGAATCCGACCAGTGATGAATGC crp-pro-R GATCGGAATTCAGTGTTGGATCGGTTTGAGG
hapR-pro-F GATCGTCTAGAGAGTTCGAGGGCGTTTTTC hapR-pro-R GATCGGAATTCCCCTACTCATCCTCGCTTTG aphA-pro-F GATCGTCTAGACACAACTTTGTGGCCTTTTG aphA-pro-R GATCGGAATTCTCGCGTGTGCTAAGAACAG
* All the primers and probes were designed using PRIMER EXPRESS software version 3.0 (Applied Biosystems Inc.). F, forward primer; R, reverse primer; O, outer primer; I, inner primer. Underlines below the nucleotide sequences indicate restriction enzyme sites BamHI, MluI, NotI, XhoI, HindIII, XbaI and EcoRI.
Fig.
2-1
. Effe
ct o
f an
etho
leon
the
tra
nscr
iptio
ns o
f vi
rule
nce
regu
lato
ry g
enes
in
V. c
hole
rae
O1
El T
orva
riant
stra
in C
RC
41.
qRT-
PCR
ass
ay o
f the
gen
es b
elon
ging
to v
irule
nce
regu
lato
ry c
asca
dew
as p
erfo
rmed
with
V. c
hole
rae
cells
cul
ture
d (A
)at
4 h
st
atio
nary
and
(B)f
ollo
wed
by
2h s
haki
ng c
ondi
tion,
bot
h in
the
pres
ence
(50
μg/
ml)
and
abse
nce
(0.5
% M
eOH
) of a
neth
ole.
‘C’
indi
cate
s th
e co
ntro
l val
ue o
f ea
ch t
arge
t ge
ne t
rans
crip
tion
with
out
anet
hole
(arb
itrar
ily t
aken
as
1). D
ata
are
pres
ente
d as
the
av
erag
e ±
SD o
f th
ree
inde
pend
ent
expe
rimen
ts.
By
usin
g tw
o-sa
mpl
e t-t
est,
a do
uble
ast
erisk
(**
) re
pres
ents
p <
0.01
as
com
pare
d w
ith a
neth
ole
untre
ated
con
trol.
50
Relative arbitrary units Relative arbitrary units
Cct
xAtc
pAto
xTto
xRto
xStc
pPtc
pHhn
s
Cct
xAtc
pAto
xTto
xRto
xStc
pPtc
pHhn
s
Fig.
2-2
. Det
ectio
n of
Tcp
Pby
Wes
tern
blo
tting
. Lan
es a
and
b in
dica
te T
cpP
leve
l in
the
pres
ence
(50 μg
/ml)
and
abse
nce
(0.5
%
MeO
H) o
f ane
thol
e, re
spec
tivel
y. L
ane
c in
dica
tes
the
reco
mbi
nant
His 6
-Tcp
P, w
hich
was
use
d as
a p
ositi
ve c
ontro
l for
det
ectio
n of
Tcp
P. In
left
pane
l (la
nes a
1 &
b1)
, pro
tein
s w
ere
obta
ined
from
initi
al 4
hr o
f sta
tiona
ry c
ultu
re a
nd ri
ght p
anel
(lan
es a
2 &
b2)
,pr
otei
ns w
ere
from
initi
al st
atio
nary
follo
wed
by
2 hr
sha
king
cul
ture
. The
rela
tive
band
sig
nal i
nten
sitie
s (s
how
n be
low
the
imag
e)
of t
he i
mag
e of
wes
tern
blo
t w
as q
uant
ified
by
Imag
eJso
ftwar
e (h
ttp://
imag
ej.n
ih.g
ov/ij
/) an
d no
rmal
ized
to
that
of
wild
-type
w
ithou
t ane
thol
esa
mpl
e (a
rbitr
arily
take
n as
100
%).
51
a1
b
1
c
a
2
b2
Rel
ativ
eIn
tens
ity (%
)
α-Tc
pP
Fig.
2-3
. Ef
fect
s of
ane
thol
eon
the
tra
nscr
iptio
n of
tcp
PHre
gula
tory
gen
es i
n V.
cho
lera
eO
1 El
Tor
varia
nt s
train
CR
C41
. R
elat
ive
trans
crip
tiona
l lev
el o
f th
e tc
pPH
regu
lato
ry g
enes
wer
e ex
amin
ed b
oth
in t
he p
rese
nce
(50 μg
/ml)
and
abse
nce
(0.5
%
MeO
H)
of a
neth
ole,
with
V.
chol
erae
cells
cul
ture
d (A
)at
4 h
r st
atio
nary
and
(B
)fo
llow
ed b
y 2
hr s
haki
ng c
ondi
tions
. ‘C
’in
dica
tes
the
cont
rol v
alue
of
each
tar
get
gene
tra
nscr
iptio
n w
ithou
t an
etho
le(a
rbitr
arily
tak
en a
s 1)
. Dat
a ar
e pr
esen
ted
as t
he
aver
age
±SD
of t
hree
inde
pend
ent e
xper
imen
ts. B
y us
ing
two-
sam
ple
t-tes
t, a
singl
e as
teris
k (*
) re
pres
ents
p <
0.05
and
a (
**)
repr
esen
ts p
<0.
01 a
s com
pare
d w
ith a
neth
ole
untre
ated
con
trol.
52
C
cyaA
crp
hapR
aphA
aphB
C
cyaA
crp
hapR
aphA
aphB
A B
Fig.
2-4
. Pro
mot
er a
ctiv
ity o
f tcp
PHal
ong
with
its
upst
ream
reg
ulat
ors,
both
in th
e ab
senc
e an
d pr
esen
ce o
f ane
thol
e(5
0 μg
/ml).
Pr
omot
er a
ctiv
ity (
lacZ
expr
essio
n) i
s de
note
d as
Mill
er u
nit
in t
he y
-axi
s. ‘C
’in
dica
tes
the
cont
rol
lacZ
expr
essio
nva
lue
of
prom
oter
less
lacZ
(-) v
ecto
r pH
RP3
09.D
ata
are
pres
ente
d as
the
aver
ages
±SD
of t
hree
inde
pend
ent e
xper
imen
ts. B
y us
ing
two
sam
ple
t-tes
t, a
two
aste
risks
(**)
repr
esen
ts p
< 0
.01
as c
ompa
red
with
the
anet
hole
-fre
e cu
lture
.
53
0
1000
2000
3000
4000
5000
Miller Units**
Ane
(-)An
e(+
)
C
tcpP
Hcr
pcy
aAha
pRap
hA
Fig. 2-5. Effects of complementation of CRP and CyaA in respective mutants of V. cholerae O1 El Tor variant strain CRC41. Growth of each mutant strain was compared with that of wild type strain CRC41. x-axis indicates the culture conditions used to analyze the bacterial growth. Primary y-axis indicates the optical density of the culture at 600nm and secondary y-axis indicates the bacterial count (cfu/ml) at desired time point.
Fig. 2-6. Effects of anethole (50 µg/ml) on the growth of Δcrp and ΔcyaA mutants of V. cholerae O1 El Tor variant strain CRC41. x-axis indicates the culture conditions used to analyze the samples. Primary y-axis indicates the OD value of the culture at 600nm and secondary y-axis indicates the bacterial count at desired time point.
54
Fig.
2-7
. The
hyp
othe
tical
regu
lato
ry c
asca
de o
f CT
prod
uctio
n in
hibi
tion
in V
. cho
lera
eby
ane
thol
e. In
all
case
s, ar
row
indi
cate
s po
sitiv
e re
gula
tion
whi
le b
ar d
enot
es n
egat
ive
or in
hibi
tory
eff
ects
. Arr
ow b
esid
es th
e ge
nes
nam
e re
pres
ent s
igni
fican
t inc
reas
e or
de
crea
se o
f tra
nscr
iptio
n in
pre
senc
e of
ane
thol
e. T
hick
arr
ows r
epre
sent
the
anet
hole
-med
iate
d ef
fect
on
tcpP
Hsu
ppre
ssio
n.
55
Fig.
2-8
. Effe
cts
of a
neth
ole
on th
e tra
nscr
iptio
ns o
f zin
c m
etal
lopr
otea
seen
codi
ng r
seP
and
maj
or s
erin
e pr
otea
se e
ncod
ing
degS
inV.
cho
lera
eO
1 El
Tor
varia
nt s
train
CR
C41
. Rel
ativ
e tra
nscr
iptio
nal l
evel
of t
he tc
pPH
regu
lato
ry g
enes
was
exa
min
ed b
oth
in
the
pres
ence
(50
μg/
ml)
and
abse
nce
(0.5
% M
eOH
) of
ane
thol
e, w
ith V
. cho
lera
ece
lls c
ultu
red
(A)
at 4
h s
tatio
nary
and
(B
)fo
llow
ed b
y 2
h sh
akin
g co
nditi
ons.
‘C’
indi
cate
s th
e co
ntro
l val
ue o
f eac
h ta
rget
gen
e tra
nscr
iptio
n w
ithou
t ane
thol
e(a
rbitr
arily
ta
ken
as 1
). D
ata
are
pres
ente
d as
the
aver
age
±SD
of t
hree
inde
pend
ent e
xper
imen
ts.
56
57
Chapter 3: Effects of anethole on the pathogenesis of Vibrio cholerae in animal models
3.1. INTRODUCTION
The results obtained in chapters 1 and 2, suggested that anethole has effects both on the
growth and toxin production against MDR toxigenic Vibrio cholerae in vitro. At relatively
higher concentration (≥ 150 µg/ml) anethole could act as an antibacterial agent. On the other
hand, sub-bactericidal concentrations of anethole (≤ 100 µg/ml) drastically suppressed the
virulence factors production in V. cholerae. However, the expression of a large number of V.
cholerae virulence regulatory genes for successful colonization and CT production in the
human small intestine might differs from those of in vitro conditions (Peterson, 2002). Henec,
to establish anethole as a potential anti-virulence drug candidate against toxigenic MDR V.
cholerae, evaluation of its therapeutic benefits in animal models is worthwhile.
Ligated rabbit ileal loop (RIL) assay is one of the most extensively used assays to
determine the CT-mediated enterotoxic potency of toxigenic V. cholerae (De and Chatterjee,
1953). In our previous study, we found that capsaicin, the major component of red chili
caused drastic inhibition of CT production by toxigenic V. cholerae in vitro, but failed to
show similar activity in vivo (Chatterjee et al., 2010). In a recent study, it has been reported
that 6-gingerol, an active component of ginger could inhibit CT-mediated fluid accumulation
in ligated RIL by binding to CT, hindering its interaction with the GM1 receptor present on
the intestinal epithelial cells (Saha et al., 2013). Previous studies have also demonstrated that
few plant polyphenols can suppress CT activity by inhibiting the fluid accumulation in rabbit
ileal loop or by repressing binding of CT to the Vero and CHO cells (Oi et al., 2002;
Morinaga et al., 2005). However, those studies performed with the purified CT but not with
live toxigenic V. cholerae. In chapter 2, we found that anethole inhibited virulence expression
by affecting virulence regulatory cascade in vitro. So, in this section we attempted to evaluate
58
whether anethole is capable to show the similar effects or not in ligated RIL, when cocultured
with live toxigenic V. cholerae cells.
Although clinical manifestation of the disease cholera is mostly due to CT, prior to secret
CT V. cholerae needs to attach and colonize in the human small intestine. The colonization
process in the human upper small intestine is aided by toxin-coregulated pilus (TCP)
(Herrington et al., 1988). As the in vitro expression of TcpA, which is the major colonization
factor was coordinately repressed with CT by anethole (Fig. 1-4), it is speculated that
anethole might have effect on V. cholerae colonization. Therefore, in chapter 3 we also
evaluated whether anethole have any effect on V. cholerae colonization in mice model.
3.2. MATERIALS AND METHODS
3.2.1. Rabbit ileal loop (RIL) assay
In vivo effect of anethole on cholera toxin (CT) production was analyzed by a rabbit ileal
loop (RIL) assay using 7-week-old New Zealand white male rabbits (ca 1.8 kg) as described
previously (De and Chatterjee, 1953). Briefly, overnight fasted rabbits were anesthetized by
intramuscular injection of 45 mg/kg ketamine (Ketalar; Daiichi Sankyo Co., Ltd.) and 5
mg/kg xylazine (Selactar; Bayer Healthcare). Laparotomy was performed in the anesthetized
animals from the lower liver margin and 8 loops (~8 cm long) with a 3 cm inter loop were
ligated. Exponential phase growth culture of O1 El Tor variant strain (CRC41) was washed
twice with phosphate buffered saline (PBS, pH 7.0) by centrifugation at 4,000 g for 5 min
and then the cells were re-suspended in PBS. Ligated segments of RILs were then inoculated
with fresh CRC41 culture both in the presence and absence of anethole. Loops were then
placed back in the peritoneal cavity. After 6 hr incubation, the animals were killed by
injecting 200 mg/kg pentobarbital (Nembutal; Dainippon Sumitomo Pharma Co., Ltd., Osaka,
Japan). Fluid accumulation (FA), recovered bacterial number (CFU) and CT production (ng)
of intestinal fluids of each ligated loop were then analyzed. All animal experiments were
59
performed according to the Guidelines for Animal Experimentation of Osaka Prefecture
University and approved by the Animal Experiment Committee of Osaka Prefecture
University.
3.2.2. Assessment of FA ratio, recovered CFU and CT amount in intestinal fluids
FA ratio of intestinal fluids of each ligated loop is determined as ml of fluid per cm of the
loop. Recovered bacterial number (CFU) in the ileal fluid was counted by plating on
thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates followed by overnight incubation at
37°C. Total recovered bacterial number (total CFU) were determined from the total volume
of the fluid obtained after 6 hr of incubation. RILs which showed no fluid accumulation were
also washed internally with PBS and the washings were plated on TCBS agar plates to
determine CFU. For estimation of CT, at first intestinal fluids were subjected to
centrifugation at 4,000 g for 5 min to settle down both the bacteria and intestinal debris
followed by filtration through a 0.22- µm filter (Iwaki, Tokyo, Japan) to get CFS. Appropriate
dilutions of the CFS of the intestinal fluids were made with PBS (pH 7.0). Total CT amount
in the CFS of the intestinal fluids were then estimated by a bead-ELISA as described earlier.
3.2.3. Optimization of bacterial inoculum for fluid accumulation in RIL
To optimize bacterial inoculum size for significant fluid accumulation in ligated RIL, we
inoculated different doses (106 -109 CFU/loop) of exponential phase grown CRC41 culture
and incubated for 6 hr. In no.1 loop, relatively high dose (109 CFU) of CRC41 was inoculated
by considering as a ‘positive control’ for fluid accumulation. On the other hand, loop no. 8
was inoculated with only PBS as a ‘negative control’ for fluid accumulation. Loop no. 2, 4
and 6 were inoculated with 108, 107 and 106 CFU, respectively. To see whether anethole has
any effect on inhibition of fluid, a relatively high amount of anethole (10 mg) was inoculated
in loop no. 3, 5 and 7, along with bacterial inoculum of 108, 107 and 106 CFU, respectively.
60
3.2.4. Assessment of dose-dependent effect of anethole on fluid accumulation in RIL
To analyze the in vivo dose-dependent effect of anethole on fluid accumulation, ligated
segments of rabbit ileal loops were inoculated with 108 CFU/loop of CRC41, both in the
presence and absence of anethole. Among the ligated 8 loops, the first loop was injected with
only PBS and the last loop was injected with 108 CFU as negative and positive controls of
fluid accumulation, respectively. The intermediate 6 loops were inoculated with 108 CFU of
CRC41 with various concentration of anethole ranging from 0.8 mg to 10 mg/loop.
3.2.5. Assessment of the effect of anethole on V. cholerae colonization in mice model
Mouse inoculations of V. cholerae strain CRC41 were performed according to the
previous authors with some modifications (Oliver et al., 2007). In brief, 3-week-old specific
pathogen-free (SPF) BALB/c mice were acclimatized with foods and water for 2 days. Then,
mice were given water containing 3 mg/ml streptomycin for 2 days to ablate intestinal flora.
Twenty to twenty-four hours, prior to inoculation, food was removed from cages to empty the
stomach. Acidic environment in the mice stomach are neutralized by oral inoculation of 100
µl of 8.5% (w/v) NaHCO3. Before inoculation of V. cholerae, one group of mice (n=4)
received 500 µg of anethole dissolved in 100 µl PBS (pH 7.0). On the other hand, control
group of mice (n=4) received 100 µl of PBS containing 1% MeOH. Then, both groups of
mice were challenged with oral inoculation of 109 CFU of the strain CRC41. After
inoculation, mice were kept in microisolator cages with free access to food and sterile water
with 300 µg /ml streptomycin. Three hours post-inoculation, anethole-treated mice groups
received another dose of anethole (500 µg). Following 3 days, anethole-treated mice received
2 doses of anethole (500 µg) per day. After that, single dose of anethole (500 µg) was given
daily. On the other hand, anethole-untreated control mice were inoculated with PBS
containing 1% MeOH, as same schedule followed for anethole-treated mice. After
inoculation of infectious doses of the strain CRC41, colonization (CFU/gm of feces) was
enumerated at desired time points. Approximately 100 mg of feces were collected from each
61
mouse, and dissolved in 1 ml of cold PBS (pH 7.0). After that appropriate dilution were made
in PBS and each diluent was spread on TCBS agar. Bacterial viability (CFU/ml) was then
checked after overnight incubation at 37°C. All animal experiments were performed
according to the Guidelines for Animal Experimentation of Osaka Prefecture University and
approved by the Animal Experiment Committee of Osaka Prefecture University.
3.3. RESULTS
3.3.1. Optimization of bacterial inoculums for significant amount of fluid accumulation
in rabbit intestine
After incubation for 6 hr, the overall picture of different ligated loops inoculated with
different doses (106 -109 CFU/loop) of exponential phase grown CRC41 culture is shown in
Fig. 3-1. Fluid accumulation (FA) ratios were 0.90, 0.50 and 0.25 in the loops inoculated with
109, 108 and 107 CFU of CRC41, respectively. On the other hand, in loop 6 (inoculated with
106 CFU) significant amount of fluid was not accumulated. Moreover, we found that a
relatively high amount of anethole (10 mg) was able to suppress those amounts of fluid
accumulation, when inoculated with 108 and 107 CFU (loops 3 and 5, respectively). Based
upon these results, to analyze the dose dependent effects of anethole, we considered 108 CFU
of CRC41 as ‘positive control’ for fluid accumulation in ligated rabbit intestine within 6 hr of
incubation.
3.3.2. Anethole inhibits fluid accumulation in rabbit intestine caused by toxigenic V.
cholerae in a dose dependent manner
To test whether anethole can effectively suppress expression of CT in RIL, we inoculated
different doses of anethole along with 108 CFU of toxigenic V. cholerae strain CRC41. In
addition, as described in Methods section the first loop (loop 1) was injected with only PBS
and the last loop (loop 8) was injected with 108 CFU as negative and positive controls of fluid
accumulation, respectively. As anethole was dissolved and diluted in methanol (MeOH), we
62
evaluated the effect of ≤1% MeOH on fluid accumulation or growth of the strain CRC41 in
RIL. We observed that ≤1% MeOH have no significant effect on fluid accumulation or
growth inhibition of CRC41 in RIL (data not shown). So, 1% MeOH was also added into the
positive control loop (loop 8) for fluid accumulation.
After 6 hr incubation, the overall picture of different ligated loops inoculated with 108
CFU both in the absence and presence of different concentrations of anethole is shown in Fig.
3-2. The Effects of anethole on fluid accumulation, colonization and CT production by
CRC41 in RILs are summarized in Table 3-1. The FA ratio for loop inoculated with 108 CFU,
as a‘positive control’ was 0.67. No fluid was accumulated in the loop inoculated with only
PBS. Detectable amount of fluid was not observed from the loops inoculated with 108 CFU
with anethole ranging from 10 ~ 0.625 mg. On the other hand, loops inoculated with 108 CFU
with anethole ranging from 0.325 ~0.078 mg, fluid accumulation and CT production were
inhibited in a dose-dependent manner of anethole (Fig. 3-2). As shown in Table 3-1, the FA
ratios were 0.17, 0.27 and 0.53 in presence of 0.325, 0.156 and 0.078 mg of anethole,
respectively. CT production was also inhibited in a dose dependent manner of anethole in
those loops. Moreover, in comparison to CT production and fluid accumulation, the
recovered bacteria (1.3x109 CFU) from the loop inoculated with 0.156 mg of anethole (loop
3), were not significantly reduced compared to the anethole-free ‘positive control’ loop for
fluid accumulation. Although significant amount of CT was not detected, viable bacteria
(>108 CFU) capable of producing CT were recovered from the loops inoculated with ≤ 0.625
mg anethole, indicating that reduced fluid accumulation was not completely due to the
bacterial growth inhibition.
3.3.3. Anethole facilitates the clearance of V. cholerae shedding in mice intestine
To optimize the inoculum dose, various dosages (106 -109 CFU) of the strain CRC41 were
inoculated into 3-week-old SPF BALB/c mice and found that 109 CFU was able to colonize
(data not shown). Then, to evaluate the effect of anethole on V. cholerae colonization in mice,
63
we orally inoculated 109 CFU of CRC41 by considering the facts that host intestinal flora and
acidic environment in the stomach are the major physical barrier to V. cholerae infection.
After inoculation of infectious dose of the strain CRC41, colonization (CFU/gm of feces) was
enumerated at desired time points both from the anethole treated and untreated control mice.
As shown in Fig. 3-3, intestinal colonization of the strain CRC41 was significantly decreased
in anethole treated mice throughout the experimental period compared to untreated controls.
At day 10, we observed almost 2 log reduction of CRC41 colonization in anethole treated
mice compared to that of anethole untreated mice. Moreover, it was found that V. cholerae
cleared from the anethole-treated mice earlier compared to control mice.
3.4. DISCUSSION
Since the major biological function of CT is to promote the loss of fluid in small intestine,
we used the ligated rabbit ileal loop (RIL) assay to determine whether anethole can
effectively suppress CT-mediated fluid accumulation in vivo. Previously, some chemical
compounds/natural products showed reduced fluid accumulation in ligated RIL by directly
interfering with the activity of purified CT (Saha et al., 2013; Oi et al., 2002; Morinaga et al.,
2005). In the present study, we co-cultured anethole with live toxigenic V. cholerae O1 El
Tor variant strain CRC41 in ligated RIL, to analyze whether anethole can suppress CT
expression in them in vivo.
In this study, relatively short incubation period (6 hr) in RIL was considered because of
the possibility of absorption or degradation of anethole in intestine. As shown in Fig. 3-2 and
Table 3-1, there was marked reduction in fluid accumulation when various sub-lethal doses of
anethole were administered together with 108 CFU of a toxigenic strain CRC41 as compared
to loop in which bacteria were inoculated without anethole. Thus, CT production as indicated
by fluid accumulation was inhibited by anethole in a dose-dependent manner under in vivo
conditions. As TCP is the major colonization factor and its expression was also repressed by
64
anethole (Fig. 1-4B), some extent of colonization defect of CRC41 in ligated RIL by anethole
is expected. But, we observed that although recovered bacteria from the loop inoculated with
0.156 mg of anethole was not drastically varied, total CT production was suppressed ~10
times compared to the anethole-free ‘positive control’ loop for fluid accumulation (Table 3-1),
indicating that inhibition of fluid accumulation or CT production were not completely due to
the bacterial growth inhibition as demonstrated by in vitro experiments.
As the in vitro expression of TCP was coordinately repressed with CT by anethole, it is
speculated that anethole might have effect on the reduction of V. cholerae colonization in
vivo. We verified the in vivo effect of anethole on TCP suppression by a colonization assay in
mice model of V. cholerae infections. We found that oral feeding of anethole (500 µg) before
inoculation of infectious dose of the strain CRC41 and subsequent daily ingestion of anethole
might reduce colonization of CRC41 in the intestine of adult BALB/c mice compared to
those of untreated controls (Fig. 3-3)
Thus, suppression of CT-mediated fluid accumulation in RIL and reduced V. cholerae
colonization in the mice model by anethole demonstrated that anethole could be a potential
anti-virulence/preventive drug candidate against toxigenic V. cholerae-mediated diarrhea.
Previous studies suggested that anti-virulence drugs could be used either alone or in
combination with antimicrobials to increase the clinical value of these drugs (Cegelski et al.,
2008; Paul and Leibovici, 2009). The in vivo beneficial effects of anethole on the
pathogenesis of V. cholerae in animal models evaluated in the current studies clearly
demonstrated the potentiality of anethole as an anti-virulence/preventive drug candidate
against V. cholerae-mediated diseases, such as cholera. Although further studies are needed,
we believe that anethole could be a potential anti-virulence drug candidate against toxigenic
V. cholerae-mediated diarrhea, which remains a significant public health concern in the
developing countries despite the use of traditional antimicrobial agents and oral rehydration
therapy.
65
CONCLUSIONS
Anethole inhibited cholera toxin-mediated fluid accumulation caused by toxigenic V.
cholerae in rabbit intestine.
Anethole inhibited cholera toxin-mediated fluid accumulation in rabbit intestine in a
dose dependent manner.
Inhibition of toxigenic V. cholerae-mediated secretion of cholera toxin or fluid
accumulation in ligated rabbit intestine by anethole was not due to the bacterial
growth inhibition.
Anethole facilitated the clearance of V. cholerae shedding in SPF BALB/c mice
intestine.
330.
3 (±
28)
(2.6
±0.
67)X
109
0.66
(±0.
11)
108
+ 0
#8
Not
det
(9.2
±2.
90)X
107
ND
108
+ 10
7
Not
det
(1.1
±0.
12)X
108
ND
108
+ 2.
5 6
2.7
(±0.
6)(1
.4 ±
0.07
)X10
80.
03 (±
0.03
)10
8+
0.62
55
9.3
(±0.
6)(2
.9 ±
2.00
)X10
80.
17 (±
0.02
)10
8+
0.31
24
35.7
(±7.
5)(1
.3 ±
0.05
)X10
90.
27 (±
0.03
)10
8+
0.15
6 3
180.
7 (±
6.5)
(1.8
±0.
17)X
109
0.53
(±0.
09)
108
+ 0.
078
2
Not
det
<10
ND
PBS
1
(ng)
(Tot
al C
FU)
( flu
id in
mL
/leng
th in
cm
)(c
fu*
+ an
e**
in m
g)
Tot
al C
T p
rodu
ctio
ndR
ecov
ered
bac
teri
acFl
uid
accu
mul
atio
n ra
tiob
Inoc
ulum
aL
oop
Tab
le 3
-1. E
ffec
ts o
f ane
thol
eon
flui
d ac
cum
ulat
ion,
col
oniz
atio
n an
d C
T p
rodu
ctio
n by
V. c
hole
rae
O1
El
Tor
vari
ant s
trai
n C
RC
41in
rab
bit i
leal
loop
s (R
ILs)
a* c
olon
y fo
rmin
g un
it, **
anet
hole
, # (1%
MeO
H);
b N
D, N
ot d
eter
min
ed (s
igni
fican
t am
ount
of f
luid
was
not
acc
umul
ated
);
c <1
0, n
o cf
uw
as d
etec
ted
in 1
00 µ
l of P
BS
was
hing
sam
ples
; dN
ot d
et, N
ot d
etec
ted
(sig
nific
ant a
mou
nt o
f CT
was
not
det
ecte
d in
flui
ds/w
ashi
ngs)
. In
all c
ases
, val
ues p
rese
nted
as t
he m
ean
with
±SD
of t
hree
inde
pend
ent r
abbi
t exp
erim
ents
66
Fig.
3-1
. Dos
e-de
pend
ent e
ffect
s of V
. cho
lera
eO
1 El
Tor
varia
nt st
rain
CR
C41
on
fluid
acc
umul
atio
n in
RIL
sin
the
pres
ence
or
abse
nce
of a
neth
ole.
Fre
sh C
RC
41 c
ultu
res w
ere
inoc
ulat
ed a
nd in
cuba
ted
for 6
h in
liga
ted
RIL
. Loo
ps n
o. 3
, 5 a
nd 7
repr
esen
t the
ef
fect
of 1
0 m
g of
ane
thol
eon
flui
d ac
cum
ulat
ion
by 1
08 , 10
7 an
d 10
6C
FU o
f CR
C41
, res
pect
ivel
y.
* Col
ony
form
ing
unit,
**A
neth
ole,
C1%
MeO
H, FA
Flui
d ac
cum
ulat
ion,
ND
Not
det
erm
ined
67
ND
PBS
8
ND
106
+ 10
7
ND
106
+ 0c
6
ND
107
+ 10
5
0.25
107
+ 0c
4
ND
108
+ 10
3
0.50
108
+ 0c
2
0.90
109
+ 0c
1
FA ra
tioIn
ocul
umC
FU* +
Ane
** (m
g)Lo
op
No.
Fig.
3-2
. Dos
e-de
pend
ent e
ffect
of a
neth
ole
on to
xige
nic
V. c
hole
rae
(CR
C41
)-m
edia
ted
fluid
acc
umul
atio
n in
rabb
it ile
allo
ops (
RIL
).
Flui
d ac
cum
ulat
ion
(F/A
) rat
io, b
acte
rial c
olon
izat
ion
and
CT
prod
uctio
n of
eac
h lo
op a
re su
mm
ariz
ed in
Tab
le 3
-1.
* Col
ony
form
ing
unit,
**A
neth
ole,
C1%
MeO
H
68
108
+ 0c
8
108
+ 10
7
108
+ 2.
56
108
+ 0.
625
5
108
+ 0.
312
4
108
+ 0.
156
3
108
+ 0.
078
2
PBS
1
Inoc
ulum
CFU
* + A
ne**
(mg)
Loop
N
o.
Fig.
3-3
. Effe
ct o
f an
etho
leon
the
col
oniz
atio
n of
V. c
hole
rae
in m
ice
inte
stin
e. ‘
A’
and
‘C’
deno
te t
he a
neth
ole
treat
ed a
nd
untre
ated
mic
e, r
espe
ctiv
ely.
V. c
hole
rae
O1
El T
orva
riant
stra
in C
RC
41 (
109
CFU
) w
as in
ocul
ated
in b
oth
grou
p of
mic
e. x
-ax
isin
dica
tes
the
desir
ed ti
me
poin
ts to
ana
lyze
col
oniz
atio
n. y
-axi
sre
pres
ents
the
colo
niza
tion
of C
RC
41, i
n te
rms
of c
olon
y fo
rmin
g un
its (C
FU)/g
of m
ice
fece
s.
69
2
4
6
8
1
0
12
14
16
18
20
2
2
24
26
28
CFU/g of feces
Day
s af
ter i
nocu
latio
n
♦C
1C
2C
3C
4
A1
A2
A3
A4
▲■ ●
◊ □ Δ ○
70
GENERAL DISCUSSION
Emergence and spread of multidrug resistant (MDR) pathogenic bacteria have been
increasingly recognized as one of the most important global issues. Particularly, the
emergence of MDR accompanying with higher cholera toxin (CT) producing varieties of
Vibrio cholerae is the ongoing concern to treat the disease cholera. V. cholerae O1 El Tor
strains, the causative agent of the ongoing 7th cholera pandemic is undergoing frequent
genetic changes to attain their fitness in the ecosystem. As a consequence, highly toxigenic
multidrug resistant (MDR) O1 El Tor variant strains (possess classical type ctxB gene allele)
have been emerged. Hence, either development of new antimicrobial agents or alternative
approaches, such as targeting virulence factors than killing the whole organism are needed to
combat them.
It is generally accepted that natural compounds of plant origin have fewer side effects
compared to synthetic. Moreover, natural products of medicinal plants, such as spices, herbs,
etc., have been used to treat diarrheal diseases including cholera since ancient times. So,
researchers have been encouraged to search suitable antimicrobial drug components from
medicinal plants against V. cholerae. As a consequence, some natural compounds having
antibacterial activity against V. cholerae have already been identified. But use of any kind of
antimicrobial agents targeting bacterial viability can be expected to impose selective pressure
on the development of antimicrobial resistance. So, search for natural compounds having
inhibitory effect on the virulence factors production of V. cholerae is important, particularly
in view of growing multidrug resistance among them. However, only few studies have been
conducted focusing the effect of natural compounds on the virulence expression of V.
cholerae. Among the identified anti-virulence drug candidates, some showed in vitro
inhibition of virulence factors production by V. cholerae, but failed to show similar effects in
vivo. Moreover, very few information are available regarding the precise mechanisms of their
anti-virulence actions.
71
Optimum amount of spices in daily use have no documented side effects. Moreover,
spices are cheap and easily available. For these reasons,, we targeted spice components to
explore new antibacterial or anti-virulence drugs against toxigenic V. cholerae. Here, we
have evaluated that trans-anethole (anethole), purified from natural compound, is a potent
inhibitor of both the growth and virulence expression of toxigenic V. cholerae, belonging to
various serogroups and biotypes. Furthermore, we propose a novel molecular mechanism
behind anethole-mediated in vitro virulence suppression in MDR toxigenic V. cholerae via
bead-ELISA, quantitative reverse transcription real time-PCR (qRT-PCR) and western blot
analyses of the expression of virulence/virulence regulatory genes. Finally, the potential
therapeutic benefits of anethole were evaluated in rabbit and mice models of V. cholerae
infection. The findings of this study, have evaluated anethole as a potential anti-
virulence/antimicrobial drug candidate against toxigenic V. cholerae-mediated infections.
We found that ≤100 µg/ml of anethole did not have any detectable effect on the growth of
various toxigenic V. cholerae strains. On the other hand, ≥ 150 µg/ml anethole showed
significant growth inhibitory effect and ≥ 200 µg/ml exerted complete bactericidal effect
within 20 minutes against the tested strains. Moreover, rapid-killing of V. cholerae cells
demonstrates the efficacy of anethole as an antimicrobial drug. Although detail data are not
shown in this study, we found that ≥ 150 µg/ml anethole is bactericidal against two virulent
Vibrio parahaemolyticus strains, also supports the idea that anethole might have broad-
spectrum antibacterial activity. Because of having fewer side effects to hosts, anethole is
confirmed as “GRAS” (Generally Recognised as Safe) by the FDA (Food and Drug
Administration) and FEMA (Flavor Extract Manufactures Association) in the USA.
Correlating these with the findings in this study, it is suggested that anethole could be used as
future alternatives to control V. cholerae contamination in foods.
We found that sub-bactericidal concentration of anethole (50 µg/ml) inhibited CT
production in the tested V. cholerae strains irrespective of their serogroups or biotypes.
72
Moreover, anethole showed dose-dependent inhibitory effect on both CT and TCP expression,
when MDR V. cholerae O1 El Tor variant strain was co-cultured with various sub-lethal
doses of anethole. Although O1 classical biotype strains analyzed in this study was only two
in number, anethole causes relatively less CT inhibition in them compared to El Tor biotype
strains. It is noted that tcpP transcription is differently regulated in the two biotypes of O1
(classical and El Tor) to certain environmental stimuli and later we also found that anethole
might inhibit virulence expression in V. cholerae by affecting tcpP transcription. Correlating
anethole-mediated suppression of tcpP transcription with the natural differences in the
regulation of tcpP, we hypothesize the effectiveness of anethole as inhibitor of CT production
could be varied in these two biotypes.
To explore anethole-mediated virulence suppression mechanisms, we found that anethole
affects the transcription of toxT in virulence regulatory cascade of V. cholerae, by down
regulating TcpP expression at the transcriptional level. On the other hand, the transcription of
another important virulence regulatory factor toxR was not affected significantly by anethole.
These observations also supported the previous findings that TcpP is more directly
responsible for transcriptional activation of toxT and ToxR plays an indirect role. Among the
tcpPH regulatory genes, although we observed certain variation in expression of these genes
under stationary and shaking conditions, the transcription of crp remained consistently
elevated in presence of anethole irrespective of the culture conditions. Additionally, among
the tcpPH regulatory genes, significant enhancement of crp promoter activity in presence of
anethole also suggested that anethole might have upregulatory effect on the crp transcription.
Moreover, transcriptional analyses of the upstream regulatory genes of tcpPH at stationary
phase grown culture, raised the hypothesis that anethole might initiate inhibition of tcpPH
transcription as well as CT by affecting quorum sensing regulatory genes via cAMP-CRP
complex-mediated signal. As cAMP-CRP complex mediated signaling system is also well
conserved in other enteropathogens, in future, it might be possible to observe cAMP-CRP
73
signaling system mediated regulation of virulence expression in them by anethole. However,
we observed differential transcriptions of tcpPH regulatory genes at shaking condition
compared to those under stationary codition in presence of anethole. So, contribution of other
factors along with cAMP-CRP complex mediated signal in anethole-mediated virulence
suppression at shaking phase grown culture, could not be fully excluded.
The present study demonstrated that anethole is capable of inhibiting CT-mediated fluid
accumulation in ligated loops of rabbit, when co-cultured with infectious doses of toxigenic V.
cholerae. Moreover, our observations suggest that anethole inhibited fluid accumulation in
RIL without exerting significant effect on bacterial viability. Suppression of CT-mediated
fluid accumulation in RIL by anethole demonstrated its potentiality as an anti-virulence drug
candidate against toxigenic V. cholerae-mediated diarrhea. As CT production is not essential
for V. cholerae survival, the use of anethole as anti-CT drug might contribute a milder
evolutionary pressure on the development of resistance. On the other hand, the specificity of
the effect of anethole as anti-CT might preserve the bacteria that inhabit the normal flora.
Moreover, lack of CT production is expected to limit the amplification and spread of
toxigenic V. cholerae.
Since ancient times, different kinds of spices and their constituents have been used to
treat diarrheal diseases. Moreover, especially in the Indian subcontinent where cholera is
endemic from ancient times, people usually take sweet fennel seeds (natural reservoir of
anethole) after meal as a gastrointestinal refreshener. The scientific reasons behind that is still
a mystery. The present study, demonstrating the inhibitory effect of anethole both on the
growth and virulence expression of V. cholerae could be an initiative to solve that mystery.
In summary, dual beneficiary effects of natural compound anethole have been evaluated
against the pathogen V. cholerae. We found that ≥150 µg/ml anethole potentially inhibited
the growth and sub-bactericidal concentrations (≤100 µg/ml) showed potential anti-virulence
activities against toxigenic V. cholerae. We have given evidences that anethole inhibited CT
74
and TCP expression by affecting TcpP at transcriptional level, remaining toxR transcription
being unaffected. Here we propose a mechanism that anethole might suppress TcpP in V.
cholerae by activating cAMP-CRP complex mediated signal, which is also well conserved in
other bacterial pathogens. So, it might be possible to observe cAMP-CRP signaling system
mediated virulence expression regulation in them by anethole. Finally, suppression of CT-
mediated fluid accumulation in RIL and reduced V. cholerae colonization in the mice model
by anethole demonstrated its potentiality as a future therapeutic candidate against MDR
toxigenic V. cholerae. Although further studies are needed, we believe that daily intake of
sweet fennel seeds containing anethole could be a cheap and alternative approach to prevent
enteric infections including cholera.
75
Acknowledgments
Four years is not a short period in one’s life. I really enjoyed this period very much in the
Laboratory of International Prevention of Epidemics, Osaka Prefecture University (OPU). So,
I would like to express my sincere regards and appreciation for all those, who have
contributed both professionally and emotionally in making this dissertation to its current
shape.
First of all, I would like to express gratitude and respect from the core of my heart to my
supervisor Prof. Shinji Yamasaki of the Laboratory of International Prevention of
Epidemics, OPU, for his adept guidance, constructive criticism, fruitful suggestions and
enthusiastic supports throughout my study and in completion of this dissertation.
The author is grateful to Prof. Masami Miyake, Laboratory of Veterinary Public Health,
and Prof. Masafumi Mukamoto, Laboratory of Veterinary Epidemiology, OPU for critically
reviewing this dissertation and providing valuable scientific comments and suggestions.
Special thanks to Prof. Toshio Inaba, Dean, Life and Environmental Sciences, OPU for
his kind support to carry out the present study in this faculty and Assoc. Prof. Yoshihiro
Ohnishi, Laboratory of International Prevention of Epidemics, OPU for his moral supports
and encouragements.
I want to express my sincere gratitude to Asst. Professor Dr. Atsushi Hinoneya,
Laboratory of International Prevention of Epidemics, OPU for his all-out supports,
suggestions and careful observations while performing laboratory experiments. His kindness
was not limited inside the Laboratory, but also extended to my family life. I am also grateful
to Dr. Masahiro Asakura, Fuso Pharmaceutical Industries Ltd., Osaka. I learned many
aspects of research, especially Bioinformatics from him. I am very lucky to get Asst.
Professor Dr. Sharda Prasad Awasthi in the Laboratory of International Prevention of
Epidemics, OPU throughout my study. He helped me a lot in Laboratory works, especially in
completion of a part of this study.
I was very fortunate to get Dr. Nityananda Chowdhury and Dr. Sucharit Basu Neogi,
in my early life in the Laboratory of International Prevention of Epidemics. They taught
many aspects of research and helped to adapt in the Laboratory of International Prevention of
Epidemics. I am grateful to Dr. Shruti Chattergee, whose initial findings inspired me to
continue this work.
I express my heartfelt gratitude to all of my colleagues of the Laboratory of International
Prevention of Epidemics. Without their friendly support and inspiration the work would have
been impossible. I am really grateful to Dr. Ayaka Shima, who helped me a lot to acclimatize
76
with daily life during my early days in Japan. Special thanks to Noritomo Yasuda, Noritoshi
Hatanaka and Kentaro Okuno to take care of my daily life in Japan, both inside and outside of
the Laboratory. Moreover, I would like to mention the kind help of Dr. Kenichi Okazaki, Dr.
Sachi Shiramaru, Dr. Lutful Kabir, Dr. Suleiman M. Saidi, Mr. Kazumasa Kamei, Mr. Rabee
Alhossiny, Ms. Srinuan Somroop, Mr. Sikander Sheikh, Ms. Hoang Hoai Phuong, Ms.
Azimun Nahar, Ms. Yiming Li, Mr. Jayedul Hassan, Mr. Goutham Belagula Manjunath, Mr.
Hiroki Kawabata, Ms. Midori Morita, Ms. Sayaka Nishikawa, Ms. Yoku Niwa, Mr. Hidetoshi
Ichimura, Ms. Yuko Hiratou, Ms. Mao Okujima, Mr. N. Arai, Mr. Y. Saburi, Ms. Y. Matsui
and all other undergraduate students whom I met during my study in this Laboratory.
I am very much grateful to Dr. Shah M. Faruque, Scientist and Head, Centre for Food
and Water Borne Diseases, International Centre for Diarrhoeal Disease Research, Bangladesh
(ICDDR,B) for his guidance, valuable scientific comments and critically reviewing a part of
the thesis. I am grateful to Dr. Rupak Bhadra from Indian Institute of Chemical Biology,
India for reviewing a part of the thesis. I am also grateful to Dr. Victor J. DiRita, Prof. of
department of Microbiology and Immunology, University of Michigan for his generous gift
of rabbit antiserum.
I am greatly indebted to the Ministry of Education, Culture, Sports, Science and
Technology of Japan for the great support and financial assistance under Monbukagakusho
Scholarship Scheme for PhD program.
I could not express my gratitude in this pen and paper format to my mother Most.
Anzumanara Begum for providing emotional support and continuous inspiration throughout
this study. I am also grateful to my brothers, sisters, fater-in-law, mother-in-law and other
relatives in the country for constant encouragement throughout my stay in Japan.
I would not have been able to survive without my wife Most. Nazmuna Rahman Jui’s
unweaving love and support, who sacrificed her personal happiness or demands for the well
being of my study. She never complained, when I had to work late and brought peace during
my down times. She was my trusted listener and consultant during all ups and downs
throughout the study. The innocent smiles of my daughter Shoha Tasnuva Zahidy provided
me enormous mental peace to carry out my daily research works. I don’t have words to
express thanks to my father, who had a strong desire that one day his son will be able to get
PhD degree. But unfortunately, I lost him forever during my studies in Japan. This study is
dedicated to my beloved father.
77
REFERENCES
Abuaita BH and Withey JH (2009) Bicarbonate induces Vibrio cholerae virulence gene
expression by enhancing ToxT activity. Infect Immun 77: 4111–4120.
Abuaita BH and Withey JH (2011) Termination of Vibrio cholerae virulence gene
expression is mediated by proteolysis of the major virulence activator, ToxT. Mol Microbiol
81: 1640–1653.
Ahn YJ, Kim MJ, Yamamoto T, Fujisawa T and Mitsuoka T (1990) Selective growth
responses of human intestinal bacteria to Araliaceae plant extracts. Microb Ecol Health Dis 3:
169-175.
Anthouard R and DiRita VJ (2013) Small-Molecule Inhibitors of toxT Expression in Vibrio
cholerae. MBio 4: e00403-13.
Bax R, Mullan N and Verhoef J (2000) The millennium bugs--the need for and
development of new antibacterials. Int J Antimicrob Agents 16: 51-59.
Beck NA, Krukonis ES and DiRita VJ (2004) TcpH influences virulence gene expression
in Vibrio cholerae by inhibiting degradation of the transcription activator TcpP. J Bacteriol
186: 8309–8316.
Behari J, Stagon L and Calderwood SB (2001) pepA, a gene mediating pH regulation of
virulence genes in Vibrio cholerae. J Bacteriol 183: 178–188.
Cassel D and Selinger Z (1977) Mechanism of adenylate cyclase activation by cholera
toxin: Inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci USA 74: 3307-
3311.
Cegelski L, Marshall GR, Eldridge GR and Hultgren SJ (2008) The biology and future
prospects of antivirulence therapies. Nat Rev Microbiol 6: 17–27.
Chatterjee A, Dutta PK and Chowdhury R (2007) Effect of fatty acids and cholesterol
present in bile on the expression of virulence factors & motility of Vibrio cholerae. Infect
Immun 5: 1946–1953.
78
Chatterjee E and Chowdhury R (2013) Reduced virulence of the Vibrio cholerae fadD
mutant is due to the induction of the extracytoplasmic stress response. Infect Immun 81:
3935-3941.
Chatterjee S, Asakura M, Chowdhury N, Neogi SB, Sugimoto N, Haldar S, Awasthi SP,
Hinenoya A, Aoki S and Yamasaki S (2010) Capsaicin, a potential inhibitor of cholera
toxin production in Vibrio cholerae. FEMS Microbiol Lett 306: 54-60.
Chatterjee S, Ghosh K, Raychoudhuri A, Chowdhury G, Bhattacharya MK,
Mukhopadhyay AK, Ramamurthy T, Bhattacharya SK, Klose KE and Nandy RK
(2009) Incidence, virulence factors, and clonality among clinical strains of non-O1, non-
O139 Vibrio cholerae isolates from hospitalized diarrheal patients in Kolkata, India. J Clin
Microbiol 47: 1087–1095.
Cholera Working Group, International Centre for Diarrhoeal Disease Research,
Bangladesh (1993) Large epidemic of cholera-like disease in Bangladesh caused by Vibrio
cholerae O139 synonym Bengal. Lancet 342: 387-390.
Clatworthy AE, Pierson E and Hung DT (2007) Targeting virulence: a new paradigm for
antimicrobial therapy. Nat Chem Biol 3: 541-548.
Clinical and Laboratory Standards Institute (CLSI) (2011) Performance Standards for
Antimicrobial Susceptibility Testing. 21st Informational supplement.
Colmer JA, Fralick JA and Hamood AN (1998) Isolation and characterization of a putative
multidrug resistance pump from Vibrio cholerae. Mol Microbiol 27: 63-72.
Das S, Saha R and Kaur IR (2008) Trend of antibiotic resistance of Vibrio cholerae strains
from East Delhi. Ind J Med Res 127: 478-482.
De M, De AK, Sen P and Banerjee AB (2002) Antimicrobial properties of star anise
(Illicium verum Hook f). Phytother Res 16: 94-95.
De SN and Chatterjee DN (1953) An experimental study of the mechanism of action of
Vibrio cholerae on the intestinal mucous membrane. J Pathol bacteriol 66: 559-562.
79
DiRita VJ (2001) Molecular basis of Vibrio cholerae pathogenesis. Principles of bacterial
pathogenesis (Groisman, E. A., ed.), pp 457-508.
Faruque SM, Ahmed KM, Alim ARA, Qadri F, Siddique AK and Albert MJ (1997)
Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V.
cholerae O139 Bengal in Bangladesh. J Clin Microbiol 35: 624-630.
Faruque SM, Albert MJ and Mekalanos JJ (1998) Epidemiology, genetics, and ecology of
toxigenic Vibrio cholerae. Microbiol Mol Biol Rev 62: 1301-1314.
Faruque SM, Tam VC, Chowdhury N, Diraphat P, Dziejman M, Heidelberg JF,
Clemens JD, Mekalanos JJ and Nair GB (2007) Genomic Analysis of the Mozambique
Strain of Vibrio cholerae O1 reveals the origin of El Tor strains carrying classical CTX
prophage. Proc Natl Acad Sci USA 104: 5151-5156.
Ghosh-Banerjee J, Senoh M, Takahashi T, Hamabata T, Barman S, Koley H,
Mukhopadhyay AK, Ramamurthy T, Chatterjee S, Asakura M, Yamasaki S, Nair GB
and Takeda Y (2010) Cholera toxin production by the El Tor variant of Vibrio cholerae O1
compared to prototype El Tor and classical biotypes. J Clin Microbiol 48: 4283-4286.
Hagihara K, Nishikawa T, Isobe T, Song J, Sugamata Y and Yoshizaki K (2004) IL-6
plays a critical role in the synergistic induction of human serum amyloid (SAA) gene when
stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time
quantitative RT-PCR assay system. Biochem Biophys Res Commun 314: 363-369.
Harris JB, LaRocque RC, Qadri F, Ryan ET and Calderwood SB (2012) cholera. Lancet
379: 2466-2476.
Hase CC and Mekalanos JJ (1998) TcpP protein is a positive regulator of virulence gene
expression in Vibrio cholerae. Proc Natl Acad Sci USA 95: 730–734.
Heidelberg JF, Eisen JA, Nelson WC, Clyaton RA, Gwinn ML, Dodson RJ, Haft DH,
Hickey EK, Peterson JD and Umayam L et al. (2000) DNA sequence of both
chromosomes of the cholera pathogen Vibrio cholerae. Nature 406: 477-483.
80
Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Tylor RK and Levine MM (1988)
Toxin, toxin coregulated pili and ToxR regulon are essential for Vibrio cholerae pathogenesis
in humans. J Exp Med 168:1487-1492.
Higgins DE and DiRita VJ (1994) Transcriptional control of toxT, a regulatory gene in the
ToxR regulon of Vibrio cholerae. Mol Microbiol 14: 17–29.
Hoshino K, Yamasaki S, Mukhopadhyay AK, Chakraborty S, Basu A, Bhattacharya SK,
Nair GB, Shimada T and Takeda Y (1998) Development and evaluation of a multiplex
PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. FEMS Immunol
Med Microbiol 20: 201-207.
Hung DT, Shakhnovich EA, Pierson E and Mekalanos JJ (2005) Small-molecule inhibitor
of Vibrio cholerae virulence and intestinal colonization. Science 310: 670–674.
Iwanaga M, Yamamoto K, Higa N, Ichinose Y, Nakasone N and Tanabe M (1986)
Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor.
Microbiol Immunol 30: 1075–1083.
Jane A, Colmer JA, Fralick and Hamood AN (1998) Isolation and characterization of a
putative multidrug resistance pump from Vibrio cholerae. Mol Microbiol 27: 63-72.
Kamruzzman M, Bari SM and Faruque SM (2013) In vitro and in vivo bactericidal
activity of Vitex negundo leaf extract against diverse multidrug resistant enteric bacterial
pathogens. Asian Pac J Trop Med 6: 352-359.
Kitaoka M, Miyata ST, Unterweger D and Pukatzki S (2011) Antibiotic resistance
mechanisms of Vibrio cholerae. J Med Microbiol 60: 397–407.
Kovacikova G and Skorupski K (1999) A Vibrio cholerae LysR homolog, AphB,
cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence
cascade. J Bacteriol 181: 4250–4256.
81
Kovacikova G and Skorupski K (2001) Overlapping binding sites for the virulence gene
regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter. Mol
Microbiol 41: 393–407.
Krishnan HH, Ghosh A, Paul K and Chowdhury R (2004) Effect of anaerobiosis on
expression of virulence factors in Vibrio cholerae. Infect Immun 72: 3961-3967.
Krukonis ES, Yu RR and DiRita VJ (2000) The Vibrio cholerae ToxR/TcpP/ToxT
virulence cascade: distinct roles for two membrane-localized transcriptional activators on a
single promoter. Mol Microbiol 38: 67–84.
Kubo I and Fujita K (2001) Naturally occurring anti-Salmonella agents. J Agric Food Chem
49: 5750–5754.
Kubo I, Fujita K and Nihei K (2008) Antimicrobial activity of anethole and related
compounds from aniseed. J Sci Food Agric 88: 242-247.
Lin W, Kovacikova G and Skorupski K (2007) The quorum sensing regulator HapR
downregulates the expression of the virulence gene transcription factor AphA in Vibrio
cholerae by antagonizing Lrp- and VpsR-mediated activation. Mol Microbiol 64: 953–967.
Liu Z, Yang M, Peterfreund GL, Tsou AM, Selamoglu N, Daldal F, Zhong Z, Kan B
and Zhu J (2011) Vibrio cholerae anaerobic induction of virulence gene expression is
controlled by thiol-based switches of virulence regulator AphB. Proc Natl Acad Sci USA
108: 810-815.
Low Dog T (2006) A reason to season: the therapeutic benefits of spices and culinary herbs.
Explore (NY) 2: 446-449.
Marra A (2004) Can virulence factors be viable antibacterial targets? Expert Rev Anti Infect
Ther 2: 61-72.
Marrero K, Sánchez A, Rodríguez-Ulloa A, González LJ, Castellanos-Serra L, Paz-
Lago D, Campos J, Rodríguez BL, Suzarte E, Ledón T, Padrón G and Fando R (2009)
82
Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio
pathogenicity island in Vibrio cholerae El Tor. Res Microbiol 160: 48–56.
Matson JS and DiRita VJ (2005) Degradation of the membrane-localized virulence
activator TcpP by the YaeL protease in Vibrio cholerae. Proc Natl Acad Sci USA 102:
16403-16408.
Matson JS, Withey JH and DiRita VJ (2007) Regulatory networks controlling Vibrio
cholerae virulence gene expression. Infect Immun 75: 5542–5549.
Mazel D, Dychinco B, Webb VA and Davies J (1998) A distinctive class of integron in the
Vibrio cholerae genome. Science 280: 605-608.
Meddrano AI, DiRita VJ, Castillo G and Sanchez J (1999) Transient transcriptional
activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture
conditions. Infect Immun 67: 2178–2183.
Metcalf WW, Jiang W, Daniels LL, Kim SK, Haldimann A and Wanner BL. (1996)
Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning,
mutagenesis, and allele replacement in bacteria. Plasmid 35:1-13.
Miller JH (1972) Assay of β-galactosidase, Experiments in molecular genetics. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY. p. 352–355
Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of
insertion mutations: osmoregulation of outer membrane proteins and virulence determinants
in Vibrio cholerae requires toxR. J Bacteriol 170: 2575–2583.
Morinaga N, Iwamura Y, Yahiro K, Tagashira M, Moss J and Noda M (2005).
Differential activities of plant polyphenols on the binding and internalization of cholera toxin
in vero cells. J Biol Chem 280:23303-23309.
Morita M, Ohnishi M, Arakawa E, Bhuiyan NA, Nusrin S, Alam M, Siddique AK,
Qadri F, Izumiya H, Nair GB and Watanabe H (2008) Development and validation of a
83
mismatch amplification mutation PCR assay to monitor the dissemination of an emerging
variant of Vibrio cholerae O1 biotype El Tor. Microbiol Immunol 52: 314–317.
Murley YM, Behari J, Griffin R and Calderwood SB (2000) Classical and El Tor biotypes
of Vibrio cholerae differ in timing of transcription of tcpPH during growth in inducing
conditions. Infect Immun 68: 3010–3014.
Mwansa JC, Mwaba J, Lukwesa C, Bhuiyan NA, Ansaruzzaman M, Ramamurthy T,
Alam M and Nair GB (2007) Multiply antibiotic-resistant Vibrio cholerae O1 biotype El
Tor strains emerge during cholera outbreaks in Zambia. Epidemiol Infect 135: 847-853.
Nihei K, Nihei A and Kubo I (2004) Molecular design of multifunctional food additives:
antioxidative antifungal agents. J Agric Food Chem 52: 5011–5020.
Nye MB, Pfau JD, Skorupski K and Taylor RK (2000) Vibrio cholerae H-NS silences
virulence gene expression at multiple steps in the ToxR regulatory cascade. J Bacteriol 182:
4295-4303.
Oh JW, Ahn YJ and Kim HY (1996) Medicinal compounds from forest resources for the
development of new forest income crops; Ministry of Agriculture and Forestry: Seoul, Korea.
Oi H, Matsuura D, Miyake M, Ueno M, Takai I, Yamamoto T, Kubo M, Moss J and
Noda M (2002). Identification in traditional herbal medications and confirmation by
synthesis of factors that inhibit cholera toxin-induced fluid accumulation. Proc Natl Acad Sci
USA 99: 3042-3046.
Oku Y, Uesaka Y, Hirayama T, Takeda Y (1988) Development of a highly sensitive bead-
ELISA to detect bacterial protein toxins. Microbiol Immunol 32: 807-816.
Olivier V, Salzman NH and Satchell KJ (2007) Prolonged colonization of mice by Vibrio
cholerae El Tor O1 depends on accessory toxins. Infect Immun 75: 5043–5051.
Parales RE and Harwood CS (1993) Construction and use of a new broad-host-range lacZ
transcriptional fusion vector, pHRP309, for gram (-)ve bacteria. Gene 133: 23–30.
84
Paul M and Leibovici L (2009) Combination antimicrobial treatment versus monotherapy:
the contribution of meta-analyses. Infect Dis Clin North Am 23(2): 277–293.
Peterson KM (2002) Expression of Vibrio cholerae virulence genes in response to
environmental signals. Curr Issues Intest Microbiol 3: 29-38.
Popovic T, Fields PI and Olsvik O (1994) Detection of cholera toxin genes. In: Wachsmuth
IK, Blake PA, Olsvik O, editors. Vibrio cholerae and cholera: molecular to global
perspectives. Washington: American Society for Microbiology, pp 41-52.
Pratt JT, Ismail AM and Camilli A (2010) PhoB regulates both environmental and
virulence gene expression in Vibrio cholerae. Mol Microbiol 77: 1595–1605.
Quilici ML, Massenet D, Gake B, Bwalki B and Olson DM (2010) Vibrio cholerae O1
Variant with Reduced Susceptibility to Ciprofloxacin, Western Africa. Emerg Infect Dis 16:
1804-1805.
Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, Takeda T,
Karasawa T, Kurazano H and Pal A (1993) Emergence of novel strain of Vibrio cholerae
with epidemic potential in southern and eastern India. Lancet 341: 703-704.
Ramamurthy T, Yamasaki S, Takeda Y and Nair GB (2003) Vibrio cholerae O139
Bengal: Odyssey of a fortuitous variant. Microbes Infect 5: 329–344.
Rattanachaikunsopon P and Phumkhachorn P (2009) Antimicrobial activity of elephant
garlic oil against Vibrio cholerae in vitro and in a food model. Biosci Biotechnol Biochem
73: 1623-1627.
Raychoudhuri A, Mukhopadhayay AK, Ramamurthy T, Nandy RK, Takeda Y and
Nair GB (2008) Biotyping of Vibrio cholerae O1: Time to redefine the scheme. Ind J Med
Res 128:695-698.
85
Sack DA, Lyke C, McLaughlin C and Suwanvanichkij V (2001) Antimicrobial resistance
in shigellosis, cholera and campylobacteriosis. In: WHO report (Background document for
the WHO Strategy for Containment of Antimicrobial Resistance). Geneva: WHO, pp 1-51.
Sack DA, Sack RB, Nair GB and Siddique AK (2004) Cholera. Lancet 363: 223-233.
Safa A, Nair GB and Kong RY (2010) Evolution of new variants of Vibrio cholerae O1.
Trends Microbiol 18: 46-54.
Saha P, Das B, Griffin R and Chaudhuri K (2013) Role of 6-gingerol in reduction of
cholera toxin activity in vitro and in vivo. Antimicrob Agents Chemother 57: 4373–4380.
Sanchez J and Holmgren J (2011) Cholera toxin—a foe & a friend. Indian J Med Res 133:
153–163.
Sandberg F and Corrigan D (2001) Natural remedies: Their origins and uses. 1st ed., pp.
124, Taylor & Francis, London.
Sharma C, Nair GB, Mukhopadhyay AK, Bhattacharya SK, Ghosh RK and Ghosh A
(1997) Molecular characterization of V. cholerae O1 biotype El Tor strains isolated between
1992 and 1995 in Calcutta, India: evidence for the emergence of a new clone of the El Tor
biotype. J Infect Dis 175: 1134-1141.
Siddique AK, Nair GB, Alam M, Sack DA, Huq A, Nizam A, Longini IMJr, Qadri F,
Faruque SM, Colwell RR, Ahmed S, Iqbal A, Bhuiyan NA and Sack RB (2010) El Tor
cholera with severe disease: a new threat to Asia and beyond. Epidemiol Infect 138: 347-352.
Sixma TK, Pronk SE, Kalk KH, Wartna ES, van Zanten BA, Witholt B and Hol WG
(1991) Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature
351: 371-377.
Sjölund-Karlsson M, Reimer A, Folster JP, Walker M, Dahourou GA, Batra DG,
Martin I, Joyce K, Parsons MB, Boncy J, Whichard JM and Gilmour MW (2011) Drug-
86
resistance mechanisms in Vibrio cholerae O1 outbreak strain, Haiti, 2010. Emerg Infect Dis
17: 2151-2154.
Skorupski K and Taylor RK (1997) Cyclic AMP and its receptor protein negatively
regulate the coordinate expression of cholera toxin and toxin-coregulated pilus in Vibrio
cholerae. Proc Natl Acad Sci USA 94: 265–270.
Skorupski K and Taylor RK (1997) Control of the ToxR virulence regulon in Vibrio
cholerae by environmental stimuli. Mol Microbiol 25:1003-1009.
Smid EJ and Gorris LGM (1999) Natural antimicrobials for food preservation. In: Rahman,
M.S. (Ed.), Handbook of Food Preservation. Marcel Dekker, New York, pp 285-308.
Son MS, Megli CJ, Kovacikova G, Qadri F and Taylor RK (2011) Characterization of
Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti,
including a molecular genetic analysis of virulence genes. J Clin Microbiol 49: 3739–3749.
Taylor RK, Miller VL, Furlong DB and Mekalanos JJ (1987) Use of phoA gene fusions to
identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad
Sci USA 84: 2833–2837
Thakurta P, Bhowmi P, Mukherjee S, Hajra TK, Patra A and Bag PK (2007)
Antibacterial, antisecretory and antihemorrhagic activity of Azadirachta indica used to treat
cholera and diarrhea in India. J Ethnopharmacol 111: 607-612.
Toda M, Okubo S, Ikigai H, Suzuki T, Suzuki Y, Hara Y and Shimamura T (1992) The
protective activity of tea catechins against experimental infection by Vibrio cholerae O1.
Microbiol Immunol 36: 999-1001.
Tran HD, Alam M, Trung NV, Kinh NV, Nguyen HH, Pham VC, Ansaruzzaman M,
Rashed SM, Bhuiyan NA, Dao TT, Endtz HP and Wertheim HF (2012) Multi-drug
resistant Vibrio cholerae O1 variant El Tor isolated in northern Vietnam between 2007 and
2010. J Med Microbiol 61: 431–437.
87
Waldor MK and Raychoudhuri D (2000) Treasure trove for cholera research. Nature 406:
469-470.
Warrens AN, Jones MD and Lechler RI (1997) Splicing by overlap extension by PCR
using asymmetric amplification: an improved technique for the generation of hybrid proteins
of immunological interest. Gene 186: 29-35.
WHO (2014) Cholera, 2013. Wkly Epidemiol Rec 89: 345-356.
Yamasak S, Nair GB, Bhattacharya SK, Yamamoto S, Kurazono H and Takeda Y
(1997) Cryptic appearance of a new clone of Vibrio cholerae O1 biotype El Tor in Calcutta,
India. Microbiol Immunol 41: 1-6.
Yamasaki S, Asakura M, Neogi SB, Hinenoya A, Iwaoka E and Aoki S (2011) Inhibition
of virulence potential of Vibrio cholerae by natural compounds. Indian J Med Res 133: 232–
239.
Yanisch-Perron C, Vieira J and Messing J (1985) Improved M13 phage cloning vectors
and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–
119.
Yu RR and DiRita VJ (1999) Analysis of an autoregulatory loop controlling ToxT, cholera
toxin, and toxin coregulated pilus production in Vibrio cholerae. J Bacteriol 181: 2584–2592.
Yutsudo T, Nakabayashi N, Hirayama T and Takeda Y. (1987) Purification and some
properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated
to shiga toxin. Microb Pathog 3: 21-30.
Zhang RG, Scott DL, Westbrook ML, Nance S, Spangler BD, Shipley GG and
Westbrook EM (1995) The three-dimensional crystal structure of cholera toxin. J Mol Biol
251: 563-573.
Zhong Z, Yu Z and Zhu J (2008) Red bayberry extract inhibits growth and virulence gene
expression of the human pathogen Vibrio cholerae. J Antimicrob Chemother 61: 753-754.
