Microbiology (2015), 161, 229243

Review

DOI 10.1099/mic.0.082362-0

The antibacterial properties of isothiocyanates

Virginie Dufour,1 Martin Stahl2 and Christine Baysse1

Correspondence
Virginie Dufour
virginie.dufour.pro@gmail.com

Received 16 July 2014

Accepted 4 November 2014

Equipe EA1254, Microbiologie Risques Infectieux, University of Rennes 1, F-35042 Rennes

cedex, France

Division of Gastroenterology, BCs Childrens Hospital, Child and Family Research Institute and
University of British Columbia, Vancouver, BC, Canada

Isothiocyanates (ITCs) are natural plant products generated by the enzymic hydrolysis of
glucosinolates found in Brassicaceae vegetables. These natural sulfur compounds and their
dithiocarbamate conjugates have been previously evaluated for their anti-cancerous properties.
Their antimicrobial properties have been previously studied as well, mainly for food preservation
and plant pathogen control. Recently, several revelations concerning the mode of action of ITCs in
prokaryotes have emerged. This review addresses these new studies and proposes a model to
summarize the current knowledge and hypotheses for the antibacterial effect of ITCs and whether
they may provide the basis for the design of novel antibiotics.

Introduction

Sources of ITCs

The interest in natural plant products as antimicrobials is

supported by the necessity to reduce the use of conventional antibiotics in food preservation and to overcome the
emergence of antibiotic resistance in bacterial pathogens.
Isothiocyanates (ITCs; RN5C5S) are the products of the
reaction of plant glucosinolates with myrosinase, an
enzyme released by the disruption of plant tissues. This
myrosinaseglucosinolate system is present in plants of the
family Brassicaceae, such as cauliflower, broccoli, mustard
and cabbage. ITCs are volatile substances that display
an inhibitory effect on a variety of pathogenic microorganisms at low concentrations, making them promising
antimicrobial candidates. The use of allyl ITC (AITC) is
already approved in Japan for food preservation, provided
it has a natural plant source (Nadarajah et al., 2005), but
little information exists about the antibacterial effects of
the other ITCs. ITCs can also be found in food with added
condiments made from Brassicaceae vegetables, usually
mustard, horseradish or wasabi (Delaquis & Sholberg,
1997). Additionally, the properties of ITCs as antibacterial,
anti-fungal and anticancer compounds have increased their
interest as food supplements (for reviews, see Singh &
Singh, 2012; Zhang, 2012). This review intends to
summarize the current knowledge about the cellular targets
of ITCs, with particular focus on bacterial targets, the
potential bacterial resistance to ITCs and the related
research perspectives.

Sulfur metabolism in plants is a source of natural products

able to affect human health. Amongst these products, the
ITCs are strongly electrophilic and are used to modify both
thiols and amines. ITCs are known to have a role in
protecting plants against both pest and microbial infection
(Bending & Lincoln, 2000; Brown et al., 1997; Fahey et al.,
2001). Glucosinolates themselves are not known to be
toxic; however, the products of their degradation by the
myrosinase (thioglucosidase) enzyme, which cleaves the
sulfurglucose bond, creates toxic ITCs. Myrosinase was
discovered in the fungus Aspergillus sydowi (Ohtsuru et al.,
1969). The first bacterial myrosinase enzyme was found in
Enterobacter cloacae (Tani et al., 1974a, b). It has also been
found in bacteria associated with mammal gut microflora
(Fahey et al., 2012) and in crucivorous aphids (MacGibbon
& Beuzenberg, 1978). In plants, myrosinase is known to
be a cytosolic enzyme; however, the cellular organization
of the myrosinaseglucosinolate system remains unclear.
Myrosinase and glucosinolate are kept separate to prevent
the production of toxic compounds in an intact plant. In
Arabidopsis thaliana, high glucosinolate concentrations are
found in specialized, sulfur-rich cells located between the
phloem and the endoderm. Myrosinase is located in the
adjacent phloem parenchyma (Koroleva et al., 2010). Upon
disruption of the tissues by chopping or chewing, glucosinolate and myrosinase meet and the degradation process
starts. Thioester glucosinolates are converted to reactive
ITCs, as well as thiocyanates, nitrites, epithionitriles and
oxazolidine 2-thione (Dinkova-Kostova & Kostov, 2012;
Fahey et al., 2001) (Fig. 1).

Abbreviations: BSH, bacillithiol; ER, erucin; GSH, glutathione; GST,

glutathione S-transferase; HSL, homoserine lactone; ITC, isothiocyanate;
AITC, allyl ITC; BITC, benzyl ITC; PEITC, 2-phenylethyl ITC; SFN,
sulforaphane.

082362 G 2015 The Authors

Printed in Great Britain

The family Brassicaceae, which includes more than 350

genera and 3000 species, is able to synthesize glucosinolates.
There are over 120 known glucosinolates (Agerbirk & Olsen,
229

V. Dufour, M. Stahl and C. Baysse

CH2OH
O

CH2OH
O
HO

HO
S

HO
Glucosinolate

OH

SO3 + H O
2

H
C

(3) H2C
S

(6) H2C

(5)
C
H

SH
N

C
H2

C
H2

(1)

OH

Myrosinase

(4)

OH

HO

(2)

SO3

H
C

CH3

NH

Isothiocyanate
R N C S

C
Plant tissue
Gut

+ R-SH
R

NH
C

Consuming host
Bacteria

R
S

S
Dithiocarbamate

+ R-NH2
NH
C

NH
Thiourea

+ H2O
NH2
+
O C S
R

(7)

+ H2O
CO2 + H2S

Fig. 1. Degradation of glucosinolates by myrosinase and reaction of ITC with thiol and amine groups. Myrosinases catalyse the
hydrolysis of glucosinolates to give unstable aglucones and glucose (1). The products that are formed vary depending on the
glucosinolate side chain R and on the reaction conditions. The products are thiohydroxamate-O-sulfonate (2), epithionitrile (3),
thiocyanate (4), nitrile (5), oxazolidine-2-thione (6), and ITC. ITC reacts with thiol groups to form labile dithiocarbamate and with
amine groups to form stable thiourea. ITC reacts with water to form CO2, H2S, and amine (7).

2012), which are both stable and water soluble. The sulfurlinked b-D-glucopyranose structure is invariant (Fig. 1),
whereas side chains differ since they are derived from different
amino acids (Table 1). These differences confer on the
resulting ITC rather different biological properties due to
their specific side chain structure. These variations depend on
the speed at which ITCs penetrate the cell, and the persistence and accumulation of each ITC (Ye & Zhang, 2001;
Zhang & Talalay, 1998). Table 1 presents the major ITCs
that are cited in this review and the originating glucosinolates (Fahey et al., 2001; Nugon-Baudon & Rabot,
1994). Glucosinolate side chains contain a variety of chemical structures. The most intensively studied glucosinolates
are the aliphatic, aromatic, heterocyclic and methylthioalkyl
variants, mostly found in Brassica. Many ITCs, such as the amethylsulfonylbutyl ITC, named erysolin, have also been
chemically synthesized in vitro for several decades (Kjaer &
Conti, 1954).
230

Chemistry of ITCs
The reactivity of ITCs can lead to random modifications of
proteins in vivo, which can disturb biochemical processes.
ITCs readily attack thiols and amines, but ITC activity is
mainly linked to their reactivity with sulfhydryl groups.
The carbon atom of the ITC group (N5C5S) is highly
electrophilic and reacts with amines, thiols and hydroxyls
to generate thiourea, thiocarbamates and carbamates
respectively (Fig. 1). ITCs react with a-amino groups in
N-terminal residues and/or amino groups of lysine through
alkylation at a rate at least 1000 times less than that of
thiocarbamation (Podhradsky et al., 1979). Cysteine, when
highly conserved in proteins, plays a crucial role in protein
structure and regulatory function owing to the ability of
thiol groups to stabilize protein structure with covalent
disulfide bonds, to coordinate metal ions, and to confer
redox properties. Therefore, ITC-thiol conjugates may have
pleiotropic effects. For example, ITCs are known to inhibit
Microbiology 161

The antibacterial properties of isothiocyanates

Table 1. Structures of some glucosinolates and names of glucosinolates and related ITC
Glucosinolate
Side chain R*
CH3CH3-CH2CH3-CH2-CH(CH3)CH25CH-CH2CH3-S-(CH2)4CH3-SO-(CH2)4CH3-SO-(CH2)5C6H5-CH2C6H5-(CH2)2-

Side chain name

Trivial name

Isothiocyanate

Methyl
Ethyl
1-Methylpropyl
Prop-2-enyl
4-Methylthiobutyl
3-Methylsulfinylpropyl
3-Methylsulfinylbutyl
Benzyl
2-Phenylethyl

Glucocapparin
Glucolepidiin
Glucocochlearin
Sinigrin
Glucoerucin
Glucoiberin
Glucoraphanin
Glucopaeolin
Gluconasturtiin

Methyl ITC
Ethyl ITC
Methylpropyl ITC
Allyl ITC
Erucin
Iberin
Sulforaphane
Benzyl ITC
Phenylethyl ITC

*for the general structure of glucosinolates, see Fig. 2.

ATP binding sites of P-ATPase (Breier & Ziegelhoffer,

2000) in mammals and also in bacteria (Escherichia coli) via
cysteine residue attack. ITC-thiol adducts are spontaneously generated but their formation is also catalysed
by specialized enzymes (glutathione S-transferases, GST)
when the target is the small thiol glutathione (GSH, c-Lglutamyl-L-cysteinyl-L-glycine). The reactivity of specific
amines and thiols in target proteins also depends on their
pKa values, which are affected by surrounding amino acid
residues (Podhradsky et al., 1979; Snyder et al., 1981). Not
all cysteine residues are reactive toward electrophiles. The
pKa value, itself affected by electrostatic interactions with
nearby residues, directs the protonation state of cysteine
and thus its reactivity toward ITCs. The hydrophobicity of
the environment and steric hindrance can also modulate
the reactivity of nucleophile residues (Nagahara et al.,
2009). Since thiol and amino groups react in their
dissociated form, higher pH values favour the interactions
with ITC (Drobnica & Sturdk, 1979).
The conjugation of ITCs with thiols is reversible (Jiao et al.,
1996). The decomposition rates of ITC conjugates seem to
influence their efficacy, as demonstrated by their activity
towards the cytochrome P450 enzyme (Conaway et al.,
2001). Although the thiocarbamyl linkage is unstable, the
thiol-ITC product, a dithiocarbamate, can react with adjacent amines to yield a more stable thiourea (Nakamura
et al., 2009). Dithiocarbamate can also react with water
without forming a covalent adduct (Hanschen et al., 2012)
(Fig. 2). This feature of ITC to spontaneously bind thiols or
OH
HO
HO

O
OH

S
R

Fig. 2. Structure of glucosinolates.

http://mic.sgmjournals.org

O S O
O
N

amine groups has already been explored in order to create

molecular research tools such as an ITC-fluorophore (fluorescein ITC) that react with thiol groups of proteins or
amino groups of oligonucleotides for labelling experiments.
Phenyl ITC and its derivatives have already been employed
for protein sequencing via the Edman reaction, owing to
their reactivity with the N-terminal amine (Jin et al., 1986).
Conjugation with GSH is a major metabolic route of ITC
accumulation and detoxification in both rodents and
humans. GSH is the most abundant non-protein thiol in
many organisms, being present in almost all eukaryotes but
only in a small number of prokaryotes, such as Cyanobacteria, Proteobacteria and a few Gram-positive strains
(Fahey & Sundquist, 1991; Newton et al., 1996). The thiol
group gives GSH its biological activity, while the c-linkage
between glutamic acid and cysteine prevents the action of
most proteases. Only the peptidase c-glutamyl transpeptidase (GGT) is known to be able to hydrolyse GSH. GGTs are
widely distributed from bacteria to mammals (Chikhi et al.,
1999; Tate & Meister, 1981). Small thiols such as GSH are
antioxidants and scavengers of reactive electrophiles; ITCs
may impair various detoxification processes by sequestering
these small thiols. ITCs in humans are excreted as S-(Nacetyl)cysteine conjugates in urine (Jiao et al., 1994). These
mercapturates are the degradation products of ITC-GSH
conjugates via the mercapturic acid pathway. GST enzymes
are involved in the first step of this process (Kolm et al.,
1995; Meyer et al., 1995). GSTs catalyse the conjugation of
GSH to the electrophilic centres of potentially harmful
compounds, including ITCs. The resulting conjugates are
more water soluble and can be further metabolized and
excreted. Mammalian GSTs are grouped in eight classes
based on primary structure similarities. Prokaryotic GSTs
are less well characterized (Sheehan et al., 2001).
Antibacterial properties of ITCs
MIC against various pathogens
ITCs exhibit biocidal activities against various bacterial
pathogens. In 1995, Brabban and Edwards reported that
231

V. Dufour, M. Stahl and C. Baysse

only the hydrolysis products of the glucosinolate sinigrin

displayed an effect upon the growth of micro-organisms
(Brabban & Edwards, 1995). Several years later, the volatile
ITC components of two Brassicaceae plant extracts were
identified for their activity against yeasts, bacteria and
fungi (Hashem & Saleh, 1999). There is now ample evidence for the antimicrobial properties of ITCs (Aires et al.,
2009a, b), but most reports of the suppression of bacterial
growth by ITCs are still limited to MIC determination.
However, MIC values are not a biological constant and are
greatly influenced by the method used. Therefore the
published MIC values of ITCs (some examples are
displayed in Table 2) cannot be compared since they were
not measured under well standardized conditions. There is
no general rule that arises concerning the efficacy of ITCs
toward various bacteria; however, the aromatic and/or
hydroxy ITCs seem to consistently display a higher antimicrobial activity than aliphatic ones between studies
(Table 2). The effect is dose-dependent, and both bacteriostatic and bactericidal effects have been described. More
importantly, the inhibitory concentrations of active ITCs
are in ranges equivalent to or below those of conventional
antibiotics (Aires et al., 2009b). Some ITCs display a synergy
with conventional antibiotics: it was demonstrated that
2-(4-hydroxyphenyl)ethyl ITC displayed antimicrobial synergism with aminoglycosides such as streptomycin, against
E. coli and Staphylococcus aureus grown in glucose-containing medium (Tajima et al., 2001). However, small changes in
the concentrations of both ITC and streptomycin affect
their combined action from synergism to suppression of
antimicrobial activity. The mechanism of synergism and
suppression remains unclear (Tajima et al., 2003). AITC was
effective at reducing the MIC of erythromycin against
Streptococcus pyogenes (Palaniappan & Holley, 2010), and
both AITC and PEITC displayed a synergy with streptomycin against Gram-negative bacteria such as E. coli and
Pseudomonas aeruginosa (Saavedra et al., 2010). PEITC was
tested against 11 isolates of E. coli with extended blactamases, in combination with the aminoglycoside gentamicin. A significant additive effect between PEITC and
gentamicin was observed for most of the isolates (Freitas
et al., 2013). Therefore, not only do ITCs display an
antimicrobial activity, but also they may work synergistically
with antibiotics, potentially allowing for reduced doses
achieving the same potency, but with reduced chance for the
development of bacterial resistance.
Food preservation and control of soil-borne disease

Antimicrobial food packaging aims to reduce, inhibit or

retard the growth of micro-organisms on food products.
AITC from mustard seeds is used both as a flavour
compound and as an antimicrobial. AITC is responsible for
the pungent taste of mustard, horseradish and wasabi and
it is sometimes added to some prepared vegetable meals to
enhance the flavour. This pungent volatile compound has
been used as a natural food preservative since the late 20th
century in Japan (Delaquis & Mazza, 1995; Nadarajah et al.,
232

2005). AITC vapour has been found to be more effective

than liquid AITC (Shin et al., 2010), but its strong odour
can limit its use in food systems. The use of AITC as a
flavouring substance has been evaluated by the Joint FAO/
WHO Expert Committee on Food Additives (JECFA) and
by the EFSA (European Food Safety Authority) Panel on
Food Additives, Flavourings, Processing Aids and Materials
in Contact with Food (AFC). This report concluded that
there were no safety concerns from AITC consumption at
the estimated levels of intake (EFSA, 2010). Moreover,
evaluations have been conducted of the use of 4hydroxybenzyl ITC, which is found in white mustard
essential oil (WMEO), derived from white mustard seeds
(Sinapis alba L.), and is obtained by the hydrolysis of the
glucosinolate sinalbin by myrosinase. WMEO was found to
reduce the number of Salmonella recovered from inoculated frozen vegetables and chicken particulates in a dosedependent manner (David et al., 2013).
Methyl ITC has proven its ability to reduce soil-borne disease
in agriculture systems. However, similar to other fumigants
such as methyl bromide, it is toxic and may leave chemical
residues in products. The use of glucosinolates, the precursors to ITC, was investigated in search of alternative
processes (Rosa & Rodriguez, 1999). The role of glucosinolates and their breakdown products in the selection of the
natural rhizosphere community was evaluated in Arabidopsis
thaliana (Bressan et al., 2009) using a transgenic Ara.
thaliana that overexpressed p-hydroxybenzyl glucosinolate
(gene from sorghum). The metabolites can be released via
root exudation, as shown in Brassica rapus (Choesin &
Boerner, 1991) and in mustard (Schreiner & Koide, 1993).
The authors hypothesized that glucosinolates and their
hydrolysis products diffused from the roots into the rhizosphere, where they are degraded by extracellular myrosinases
as described by Borek et al. (1996). In the rhizosphere of the
transgenic plant, the proteobacteria and fungal community
were significantly affected, with significant differences
detected with the heavy DNA profiles for a-Proteobacteria,
c-Proteobacteria and fungal communities in rhizospheric soil
(Bressan et al., 2009). Modification of the glucosinolate
content of the plant could be an alternative to the use of
pesticides, although no ITCs were experimentally detected
because of their low concentrations and rapid turnover.
Arabidopsis mutants with a reduced aliphatic glucosinolate
content [reduction of 60-fold of 4-methylsulfinylbutyl
glucosinolate, the precursor of sulforaphane (SFN)] showed
an increased susceptibility to Fusarium oxysporum, whereas
the resistance toward other pathogens, such as Alternaria
brassicicola, Erwinia carotovora, Pseudomonas syringae and
Botrytis cinerea, was unchanged compared with isogenic WT
plants (Tierens et al., 2001).
Mode of action of ITCs on prokaryotic cells
Effect on membranes
The antimicrobial mechanisms of ITCs are not well understood. The ability of ITCs to bind thiol groups of proteins
Microbiology 161

The antibacterial properties of isothiocyanates

or thiol peptides such as GSH was largely demonstrated in

vitro, whereas the physiological responses of bacteria to
ITC challenges have been poorly investigated.
Lin and collaborators have compared the responses of E.
coli O157 : H7, Salmonella Montevideo and Listeria monocytogenes to AITC with those to conventional antibiotics
with known targets, such as polymixin B, streptomycin and
penicillin, which affect the cell membrane, ribosomes and
the cell wall, respectively. AITC displayed bactericidal
activity at all growth stages, and when used as a vapour,
seemed to provoke the leakage of cellular metabolites,
similar to the effect of polymixin B (Lin et al., 2000).
However, the exact mechanism behind these effects was not
found. The hypothesis that AITC damages the cell wall was
countered by further studies of Ahn and collaborators, who
found that when L. monocytogenes was treated with AITC,
neither leakage of ATP nor damage to the cell wall was
observed (Ahn et al., 2001). Nevertheless, the treatment
induced altered internal structures as observed by electron
microscopy. In a similar way, in the presence of essential
oil from Salvadora persica, which contains 98 % benzyl ITC
(BITC), or pure BITC, small protrusions were observed by
electron microscopy in Aggregatibacter actinomycetemcomitans HK1519 after only 2 min exposure. These protrusions increased with the incubation time until a loss of
membrane integrity occurred (Sofrata et al., 2011). The
authors hypothesized that BITC, having both lipophilic
and electrophilic properties, might penetrate through the
outer bacterial membrane and hamper the ability of the
bacterium to maintain its membrane potential, similar to
the effect observed with cationic peptides (Sofrata et al., 2011).
Inhibition of enzymic or regulatory (quorum sensing,
QS) activities

AITC was reported to inhibit specific enzymic activity in E.

coli, interfering with the function of both thioredoxin
reductase and acetate kinase (Luciano & Holley, 2009). The
enzymic inhibition was monitored in vitro, using purified
enzymes. In Helicobacter pylori (a gastric pathogen), SFN
inhibits urease activity, which generates ammonia and thus
neutralizes gastric acidity and promotes inflammation
(Fahey et al., 2013). Recently, a number of sulfur-containing compounds have been employed as a new class of QS
inhibitors, including ajoene from garlic extract (with
disulfide and sulfinyl groups) (Jakobsen et al., 2012b)
and sulfur-containing molecules that were found to inhibit
the AI-2 QS system of Vibrio harveyi (Li et al., 2008; Peng
et al., 2009). Similarly, ITCs have also been found to
display QS-inhibiting activity.
The first whole transcriptomic assay on a bacterium with
ITC was carried out by Jakobsen and collaborators. In this
study, 69 common food products and plant extracts were
analysed for their ability to inhibit the QS system of Ps.
aeruginosa. Ps. aeruginosa produces two N-acylhomoserine
lactones (N-acyl-HSLs) as QS signalling molecules: 3-oxoC12-HSL, recognized by the receptor LasR, and C4-HSL,
http://mic.sgmjournals.org

the ligand of the receptor RhlR (Jakobsen et al., 2012a).

The QS regulons of both RhlR and LasR have been
identified (Jimenez et al., 2012). Amongst the 69 products
tested, horseradish (Armoracia rusticana) extract displayed
the better inhibitory activity, based on the repression of
both the lasB-gfp and rhlA-gfp transcriptional fusions.
Horseradish belongs to the family Brassicaceae and the
main component of horseradish oil is allyl ITC (AITC)
(7894.4 %) (Park et al., 2006; Yu et al., 2001). A recently
improved HPLC method identified AITC and PEITC as the
two main horseradish ITCs, whereas iberin and SFN were
at a concentration below the detection limit. Iberin and
SFN were, however, detected in both cauliflower and
cabbage (Wilson et al., 2012). Iberin (3-methylsulfinylpropyl ITC) is an analogue of SFN, differing only in a shorter
lateral chain (Table 1). Both ITCs possess a sulfinyl lateral
group. Interestingly, a sulfinyl lateral group is also present
in the QS-inhibiting compound ajoene. Despite being a
minor ITC, iberin was purified and identified as the QSinhibiting compound within the different horseradish
extract fractions tested by HPLC. Pure iberin was used to
confirm the QS-inhibiting activity and to perform the
transcriptomic analysis at several subinhibitory concentrations that did impact growth or cell viability. The expression of 64 Ps. aeruginosa genes was downregulated by
sublethal concentrations of pure iberin, including 49 QSregulated genes. These data confirmed an antagonist interaction between the QS system and iberin in Ps. aeruginosa,
although not all QS-regulated genes were affected at the
transcriptional level. It is interesting to note that the
subinhibitory concentrations of iberin did not decrease the
expression of the las reporter, whereas the rhl-lux reporter
transcription was decreased.
SFN and its deoxy analogue erucin (ER) were also tested
for their ability to inhibit the QS system in Ps. aeruginosa
(Ganin et al., 2013). ER is a precursor of SFN but does not
contain the sulfinyl group found in SFN, iberin and ajoene
(Table 1). The ITC ER is formed both from the enzymic
hydrolysis of glucoerucin (Table 1), a glucosinolate found
at high levels in rocket species of the family Brassicaceae
(Eruca sativa), and from the in vivo reduction of SFN
(Kassahun et al., 1997), derived from broccoli and the action
of myrosinase on glucoraphanin from Brassica oleracea. ER
possesses anticancer properties by the modulation of phase
I, II and III detoxification, the induction of apoptosis and
cell cycle arrest, the induction of oxidative stress and the
regulation of androgen receptor pathways (for review see
Melchini & Traka, 2010). The QS-inhibiting ability of ER
and SFN was assayed using a reporter strain of Ps. aeruginosa
containing luminescent transcriptional fusions activated
by either 3-oxo-C12-HSL or C4-HSL, the two signalling
molecules of Ps. aeruginosa, via their binding to their specific
intracellular receptors, LasR and RhlR respectively. ER and
SFN efficiently competed with 3-oxo-C12-HSL to inhibit in a
concentration-dependent manner the activation of LasRspecific reporter fusions, whereas no antagonism of C4-HSL
upon the RhlR specific reporter system was observed. The
233

ITC
Phenethyl
(extracted from
seed of Sinapis
alba L., white
mustard), acetyl,
allyl, benzoyl,
benzyl, butyl,
ethyl, methyl
Allyl, benzyl,
2-phenylethyl,
SFN

3-Butenyl,
4-pentenyl,
2-phenylethyl,
benzyl

Essential oil of
Salvadora persica
root containing
73.8 % of benzyl
ITC

Bacteria
Intestinal bacteria: Bifidobacterium
bifidum ATCC 29521, Bif. breve ATCC
15700, Bif. longum ATCC 15707,
Clostridium difficile ATCC 9689, Cl.
perfringens ATCC 13124, E. coli ATCC
11775, Lactobacillus acidophilus ATCC
4356, Lb. casei ATCC 27216

Methods
Paper disc agar diffusion method:
0.55 mg ITC per disc; EG agar
medium

Microbiology 161

Human faecal material isolates:

Paper disc agar diffusion
Enterococcus faecalis, Staphylococcus
method: 15 ml of 0.015, 0.15,
0.75 or 3 mmol ITC per disc;
aureus, Staph. saprophyticus,
MuellerHinton agar
Acinetobacter baumanii, Citrobacter
freundii, Enterobacter asburiae, Eb.
cloacae, Eb hormaechei, E. coli (two
strains), Hafnia alvei, Klebsiella oxytoca,
K. pneumoniae, Morganella morganii,
Proteus mirabilis, Pseudomonas
aeruginosa, Salmonella typhi,
Stenotrophomonas maltophilia
Bacillus cereus, Bac. subtilis, Listeria
Paper disc agar diffusion
monocytogenes, Staph. aureus, Aeromonas
method: 1 ml per disc ITC at
hydrophila, Ps. aeruginosa, Salmonella
0.052 ml ml21; 2-layer plate
choleraesuis, Salmn. enterica, Serratia
nutrient agar
marcescens, Shigella sonnei, Vibrio
parahaemolyticus
Oral periodontal Gram2 pathogens:
Viable colony counting:
Aggregibacter actinomycetemcomitans
bacteria incubated 90 min with
HK 1519, Porphyromonas gingivalis
0.11 % (v/v) essential oil in
ATCC 33277
species-specific medium (BHI,
Oral Gram+ pathogen: Streptococcus
MRS, TSB or LB); spreading
mutans (CCUG 27624)
onto same agar medium
Other: Salmn. enterica sv. Typhimurium
ATCC 14028, Ps. aeruginosa PAO1,
Haemophilus influenzae ATCC 49247,
E. coli MC4100, E. coli K-12 LPS-mutant
strain D21f2, Lb. acidophilus NCTC 1723,
Streptococcus pyogenes, Staph. aureus
8325-4, Lactobacillus fermentum DSM
20052, Ec. faecalis ATCC 29212, Ec.
faecium

Calculation

Main result

Reference

Diameter inhibition zone

Phenethyl, benzyl, benzoyl

ITC only inhibited harmful Cl.
difficile, Cl. perfringens, E. coli

Kim & Lee

(2009)

Diameter inhibition zone;

percentage of relative inhibition
zone diameter with control
antibiotics

SFN, BITC: highest antimicrobial

activities against Gram+ and
Gram2; PEITC: high inhibitory
activity against Gram+

Aires et al.
(2009b)

Diameter inhibition zone

2-Phenylethyl and benzyl ITC

(aromatic): higher antibacterial
activities than 3-butenyl and
4-pentenyl ITCs (aliphatic)

Jang et al.
(2010)

C.f.u. determination

BITC-rich essential oil of Salva.

persica: dose-dependent high
antibacterial activity against
Gram2 bacteria; no or limited
growth inhibition for Gram+
bacteria

Sofrata
et al.
(2011)

V. Dufour, M. Stahl and C. Baysse

234

Table 2. Antimicrobial activity of ITCs against various bacteria

http://mic.sgmjournals.org

Table 2. cont.
ITC
SFN

b-Phenylethyl

Several synthetic
hydroxyphenyl
ITCs

Allyl, benzyl

Methods

Calculation

Main result

MIC 0.068 mg ml21 (mean 2.5 mg

Three reference strains (26695, J99, ATCC Agar dilution method: NCCLS*
MIC defined as lowest concn each
43504), 45 clinical isolates of Helicobacter procedures
compound that resulted in no
ml21, median 2 mg ml21); MIC90D
pylori
visible growth after 3 days incubaTime-to-kill assay: Brucella broth,
4 mg ml21; for strain LBN20
u
incubation with SFN at 0.25, 0.5, 1, tion at 37 C under microaerophilic 1 : 1000-fold reduction within 24 h
exposure to 56MIC SFN
56MIC, 0, 2, 4, 6, 8, 24 h at 37 uC, conditions; bactericidal activity
defined as .1000-fold reduction
microaerophilic conditions; c.f.u.
in viable c.f.u.
determination
Diameter inhibition zone; MIC
Strong activity (i.z.d .20 mm)
Food-poisoning bacterial strains: V.
Paper disc agar diffusion method:
defined as lowest concn at which
against V. parahaemolyticus and
parahaemolyticus KCCM 11965, Salmn.
30 ml per disc ITC at 100, 200,
growth of strains cannot be
Staph. aureus; clear activity (i.z.
1000, 2000 mg ml21; 2-layer plate
choleraesuis KCCM 40050, Staph. aureus
detected by measurement of
1015 mm) against Bac. cereus at
nutrient agar; liquid culture in
KCCM 40935, Bac. cereus KCCM 41034
absorbance
1000 mg l21; MIC 500 mg ml21 for
nutrient broth plus ITC; OD600
at 12 h intervals for 72 h
V. parahaemolyticus; MIC
.2000 mg ml21 for other strains
Staph. aureus IFO12732; E. coli C
Twofold agar dilution method: ITC MIC, minimum concn at which no 2-(4-hydroxyphenyl)ethyl ITC:
colony observed; MBC, minimal
higher antimicrobial activity, MIC
in nutrient agar at 1250 mg ml21;
bacteria streaked onto plates and
concn at which no regrowth
15.6 mg ml21, MBC 62.5 and
31.3 mg ml21 for Staph. aureus
incubated 37 uC 48 h; plates rinsed observed
and E. coli, respectively
and washing transferred into
nutrient agar; incubated 37 uC 48h
Campylobacter jejuni reference strains
Agar dilution method: 10-fold serial MIC defined as lowest concn that MIC AITC 50200 mg ml21, MIC
BITC 2.55 mg ml21; identical
NCTC 11168 and 81-17 plus 24 Ca.
dilutions ITC in MuellerHinton
inhibited any visible growth of a
5
5
MIC and MBC values for
jejuni strains isolated from chicken faeces, agar; liquid culture in Mueller
10 5610 c.f.u. spot after 48 h
both ITCs
human infections (blood, faeces),
Hinton broth plus ITC; final concns at 37 uC in microaerobic
conditions; MBC defined as
contaminated processed meats
10, 5, 2.5, 1.25 mg ml21 for AITC,
1.25, 0.625, 0.312, 0.156 mg ml21
lowest concn ITC that kills 99.9 %
for BITC; OD600 before and after
bacteria (i.e. 3 log reduction)
18 h microaerobic incubation
after 18 h incubation
37 uC; c.f.u. determination
Gram+: E. coli ATCC 11775, Eb. cloacae
Paper disc method: soybeancasein Diameter growth inhibition zone
Gram+ bacteria more affected,
ATCC 13047, Salmn. typhimurium ATCC
digest agar, 2.57.5 mmol plate21
especially Bac. cereus and Staph.
13311, Proteus vulgaris ATCC 6380.
aureus
Gram2: Staph. aureus S-6, Staph. epidermidis
ATCC 14990, Bac. subtilis ATCC 6633, Bac.
cereus var. mycoides ATCC 11778

*NCCLS, National Committee for Clinical Laboratory Standards.

DMIC90, MIC at which growth of 90 % of strains is inhibited.
di.z., Diameter of inhibition zone.

Reference
Fahey et al.
(2002)

Hong &
Kim
(2008)

Tajima
et al.
(1998)

Dufour
et al.
(2012)

Uda et al.
(1993)

235

The antibacterial properties of isothiocyanates

4-Methylthio-3butenyl ITC

Bacteria

V. Dufour, M. Stahl and C. Baysse

authors postulated that ER and SFN were inactivating 3oxo-C12-HSL binding to LasR by the formation of a complex with the nucleophilic cysteine residue of the binding
pocket. However, the authors failed to isolate an ITC-LasR
dithiocarbamate conjugate. Nevertheless, in this case, they
ruled out the implication of the sulfoxide moiety in the QSinhibitory activity, although the QS-inhibiting ability of
SFN and ER on the Las QS system showed slight differences
in efficacy (Ganin et al., 2013). A recent study has confirmed
the QS-inhibitory activity of three ITCs (AITC, PEITC and
BITC), using the CviIR QS system in Chromobacterium
violaceum (Borges et al., 2014). Each ITC reduced violacein
(a QS-induced pigment) production in Ch. violaceum but at
growth-inhibitory levels, with BITC being the most potent
inhibitor.
Effect of ITCs on respiratory enzymes

The study carried out by Jakobsen et al. found that ITC

molecules altered physiological pathways other than just
the QS system in bacteria. At sublethal concentrations in
Ps. aeruginosa, iberin activated the expression of the
respiratory genes cyoA and cyoB, encoding the cytochrome
O ubiquinol oxidase (bo3 oxidase), whereas the gene
PA4133, encoding an uncharacterized subunit I of a putative cytochrome c oxidase cbb3 type, was downregulated
(Jakobsen et al., 2012a). Ps. aeruginosa possesses five
described terminal oxidases for aerobic respiration, with
the cbb3 type being O2 high-affinity oxidases (including
CIO, cbb3-1 and cbb3-2 oxidases). They can support microaerobic growth with as little as 2 % oxygen and are
probably involved in energy conservation under low O2
concentrations (Kawakami et al., 2010). ITCs seem to affect
normal O2 respiration while activating alternative respiratory pathways to maintain energy levels. The effect of
ITCs on respiration was previously described in yeast by
Kojima & Ogawa (1971), for which oxygen uptake was
diminished by several ITCs (AITC, methyl ITC, PITC and
PEITC), probably through the inhibition of cytochrome c
oxidase. However, this study was performed with concentrations of ITCs up to 2006MIC. Genes encoding several
oxidoreductases of unknown function were also upregulated by iberin in Ps. aeruginosa, suggesting that iberin
perturbed the redox status of the cells (Jakobsen et al.,
2012a). Recently, bacterial biosensors were used to study
the antibacterial mode of action of plant metabolites,
including AITC and four terpenes (carvacrol, cinnamaldehyde, citral and thymol). E. coli HB101, containing the
luxCDABE genes on a multi-copy plasmid, was used as
biosensor for cellular metabolism, since light production
reflected the levels of respiration and intracellular NADPH
and ATP. Light intensity reduction should be proportional
to the degree of toxicity. Acinetobacter baylyi, with chromosomally integrated recA-luxCDABE transcriptional fusion,
served as biosensor for DNA-damaging agents. Amongst the
five selected plant metabolites, AITC displayed the greatest
biocidal activity and the greatest metabolic toxicity.
However, AITC did not induce recA expression, indicating
236

that it was not acting by compromising genome integrity

(Chan et al., 2013).
Induction of heat-shock and oxidative stress responses

In Ps. aeruginosa, groEL and dnaK, encoding heat-shock

proteins (Hsp), were induced by iberin (Jakobsen et al.,
2012a). Both genes encode folding chaperones that rely on
ATP-driven conformational changes to mediate refolding
or unfolding of their protein targets (Lund, 2001).
However, in this study, the expression levels of groES,
dnaJ and grpE, encoding the associated factors of these two
chaperones, were not significantly changed in iberintreated Ps. aeruginosa cells. Overexpression of GroEL/
GroES or DnaK/DnaJ prevented protein aggregation in E.
coli (Gragerov et al., 1992). Interestingly, similar differential expression of heat-shock proteins was observed in
ITC-treated eukaryotic cells. In Caco-2, HeLa and rat colon
carcinoma cells, SFN induced a heat-shock response,
mainly resulting in an overexpression of Hsp27, a small
heat-shock protein, usually induced by a variety of stresses,
and displaying several functions such as redox homeostasis
and chaperone activity. The overexpression of Hsp27
by SFN was mediated by activation of the heat-shock
transcription factor Hsf1, but the activation mechanism
remains unknown (Gan et al., 2010). How ITC impacts on
the heat-shock response is variable according to the cell
lines and experimental conditions used. In pancreatic
cancer cells, SFN blocked the interaction of Hsp90 with its
co-chaperone P50 (Li et al., 2011) and in breast cancer cell
lines, SFN downregulated the expression of Hsf1 and of
several Hsps, inducing a cascade that led to increased
apoptosis (Sarkar et al., 2012). Although there is a link
between ITCs and the heat-shock response, the effect seems
to depend on the conditions used.
We have recently performed a whole transcriptomic
analysis of Campylobacter jejuni treated with subinhibitory
concentrations of BITC (Dufour et al., 2013). We had
previously determined the MIC and bactericidal action of
BITC on several Ca. jejuni strains, showing that BITC was
the most efficient ITC at killing Ca. jejuni, relative to AITC
and ethyl ITC (Dufour et al., 2012). From this transcriptomic study, it clearly appeared that BITC elicited a heatshock-like response in Ca. jejuni, with the genes clpB, dnaK,
grpE, groEL, groES, cbpA and hrcA all being upregulated in
response to BITC exposure. ClpB functions to solubilize
and reactivate aggregated proteins via an ATP-driven
process, coupled with the action of DnaK-DnaJ-GrpE
activity. CbpA is a DnaJ homologue and a DnaK cochaperone (Chae et al., 2004). HrcA is a transcriptional
repressor that can fold into an active conformation only in
non-heat-shock-treated cells since it requires the assistance
of the chaperones GroEL/ES to fold properly. Indeed, in
cells undergoing heat shock, the available GroEL/ES
chaperone levels are decreased by the accumulation
of unfolded proteins (Holmes et al., 2010; Lund, 2001).
All of these upregulated heat-shock-related proteins are
Microbiology 161

The antibacterial properties of isothiocyanates

ATP-dependent. It is interesting to note that this

upregulation is correlated with an increase in ATP content
in BITC-treated Ca. jejuni cells, along with an overexpression of genes from the TCA cycle and respiratory
pathways such as nitrate reductase, lactate oxidase,
hydrogenases and fumarate reductase (Dufour et al.,
2013). In E. coli, an aerobic organism for which O2 is the
preferential electron acceptor, treatment with 2-(4-hydroxyphenyl)ethyl ITC decreased the cellular content of ATP
(Tajima et al., 1998). The two O2-dependent respiratory
chains of E. coli may be sensitive to ITC while, in the
microaerophile Ca. jejuni, the upregulation of O2-independent pathways for fumarate and nitrate respiration may
overcome the inhibition of O2 respiration by ITCs. We
have also demonstrated that subinhibitory concentrations
of BITC reduced O2 consumption by Ca. jejuni and
promoted protein aggregation in treated Ca. jejuni cells
(Dufour et al., 2013). This effect may contribute to the
antimicrobial action of ITC in bacterial cells (Fig. 3). BITC
also induces an oxidative stress response in Ca. jejuni, with
the upregulation of genes encoding superoxide dismutase
(SodB), rubrerythrin-like proteins (Rrc) (Yamasaki et al.,
2004), and a sulfite oxidase involved in nitrogen-reactive
species detoxification (Cj0379c) (Hitchcock et al., 2010).

These data confirmed that ITC disrupted the redox homeostasis of the cells, as previously described in eukaryotic cells.
Induction of a stringent response

Nowicki et al. (2014) have reported the efficacy of PEITC

in inhibiting the growth of enterohaemorrhagic E. coli
(EHEC) as well as Shiga toxin production. The authors
correlated these effects to the induction of a stringent
response. PEITC provoked a strong inhibition of RNA
synthesis, except in relA and relA spoT mutants deficient in
ppGpp synthase and therefore unable to induce a stringent
response. Moreover, PEITC caused an accumulation of
ppGpp and its precursor, pppGpp. The authors proposed
that the induction of the stringent response was due to the
depletion of cellular amino acids, through their interaction
with PEITC. Indeed, supplementation of glycine or
arginine in the growth medium, thereby increasing the
pool of free amino acids at inhibitory concentrations of
ITC, was able to restore growth of bacterial cultures treated
with PEITC and increased the MIC of PEITC for E. coli.
This MIC-increasing effect was also observed by supplementing lysine, methionine, phenylalanine, serine and
threonine (Nowicki et al., 2014).

Degradation
Efflux
pumps

GGT?
sax genes?
Other?

ITC
R

Thiol
S +

SH

Conjugation
GST
Spontaneous
Other?

Thiourea

S
R

N
H

N
H

Lys

S
R

N
H

SH

S
Redox unbalance
ROS
Oxidative stress response

Protein aggregation
Misfolding
Heat-shock response
Stringent response

N
H

Dithiocarbamate
S

Cys

Enzyme and regulator inhibition

Impact on: respiration, ATP
synthesis, metabolism, quorum
sensing, etc.

Fig. 3. Hypothetical model for the antimicrobial effects of ITC and resistance mechanisms. ITCs accumulate in bacteria as
GSH, small thiol-like BSH or thioredoxin-dithiocarbamate conjugates and attack the active centre of enzymes by binding to thiol
or amine groups, thus impacting on enzymic activities such as respiration, metabolism and the transcription of genes.
Conjugation of ITCs with the thiol groups of proteins also promotes the aggregation of misfolded proteins and activation of
heat-shock responses. Conjugation with amino acids may promote a stringent response by the depletion of free amino acids.
Conjugation of ITCs with thioredoxin and with small thiols may affect cell redox homeostasis. Conjugation of ITCs with GSH (via
GST enzyme), BSH or thioredoxin may be a first step in the detoxification of ITCs. Other resistance mechanisms such as the
product of sax gene homologues or efflux systems have been suggested. SH represents thioredoxin or small thiols such as
GSH or BSH.
http://mic.sgmjournals.org

237

V. Dufour, M. Stahl and C. Baysse

Mechanisms of resistance to ITCs

Resistance to SFN was investigated in the plant pathogen
Ps. syringae by Fan et al. (2011). They identified a group of
genes (the sax genes, for Survival in Arabidopsis eXtract)
responsible for the resistance of virulent host-associated Ps.
syringae to SFN produced by Arabidopsis. When introduced into E. coli, the first three genes of the operon,
saxABC, conferred the ability to overcome the plant defences
and therefore to grow on Arabidopsis extracts. How the sax
genes mediate resistance of virulent Ps. syringae is unclear, as
are the mechanisms of homologous genes known to be
present in various pathogens of ITC-producing plants. The
sax operon is composed of seven genes, amongst which only
three are required for ITC resistance. The product of saxA is
related to class B b-lactamase, although it does not confer
resistance to conventional b-lactam antibiotics. SaxB is an
isochorismatase, and SaxC is a transcriptional regulator of
the AraC/XylS family. Only the absence of saxA and/or saxC
totally abolished the bacterial resistance to ITC-containing
extracts. saxA-like genes are found only in the genome of
pathogenic strains of Ps. syringae. Further investigations are
required to understand the implication of a b-lactamase-like
enzyme in the resistance to ITC. Recently, Chan and
collaborators showed that AITC was not mutagenic for E.
coli, since it did not activate the DNA repair gene recA. As a
result, the authors postulated that the risk of evolving
antimicrobial resistance to AITC by mutagenesis was reduced
(Chan et al., 2013). A transcriptomic analysis of the response
of Ps. aeruginosa to iberin revealed some potential resistance
mechanisms. Amongst the 51 upregulated genes, the authors
pointed out the genes mexEF-oprN, encoding a well
characterized efflux pump in Ps. aeruginosa. Originally
identified as being linked to fluoroquinolone resistance,
MexEF-OprN mediates resistance to a variety of antimicrobials, including trimethoprim and chloramphenicol (Kohler
et al., 1997). Recently, it was demonstrated that MexEF-OprN
was induced by nitrosative stress, while not being directly
involved in nitric oxide resistance (Fetar et al., 2011).
Interestingly, the genes are under the control of the regulator
MexT (LysR-type regulator), which was recently found to be a
redox-responsive regulator controlling disulfide stress provoked by diamides, which deplete cellular GSH in a fashion
similar to ITC (Fargier et al., 2012). The authors postulated
that this efflux pump may be involved in the efflux of
electrophilic compounds; therefore it is tempting to postulate
that MexEF-OprN may also be involved in the efflux of iberin.
In E. coli O157 : H7, the complete deletion of the twocomponent system BaeSR increased sensitivity to AITC via
a currently undefined mechanism (Cordeiro et al., 2014).
BaeSR has been previously linked to the ability of E. coli
to resist exposure to Acacia mearnsii (black wattle) tannin
extract (Zoetendal et al., 2008), as well as to the induction
of the multidrug resistance cluster mdtABCD (Baranova &
Nikaido, 2002).
In a further study on the effects of ITC on Ps. aeruginosa, a
gene encoding a GST was identified as being upregulated
238

by iberin (Jakobsen et al., 2012a). The genome of Ps.

aeruginosa PAO1 encodes 10 putative GSTs (www.
pseudomonas.com), meaning that, while the upregulation
of only one GST gene by iberin suggests that Ps. aeruginosa
does use the GST-GSH detoxification system for iberin,
these GSTs have specific substrates, of which only one may
apply to iberin exposure. Additionally, GST has also been
reported as a detoxification enzyme for ITC in the cyanobacterium Synechococcus elongatus PCC 6301 (Wiktelius &
Stenberg, 2007). As mentioned previously, GSTs are also
part of a common route of ITC detoxification in eukaryotes (Kolm et al., 1995), and expression of GST was found
to be induced by BITC in the pathogenic fungus Alt.
brassicicola (Sellam et al., 2006). GSH is not, however,
present in all bacterial species. In low-GC Gram-positive
bacteria, such as Bacillus and Staphylococcus, bacillithiol
(BSH), a glycoside formed between L-cysteinyl-D-glucosamine and malic acid, is the major low molecular mass thiol,
along with coenzyme A and cysteine (Helmann, 2011). The
thiol group of BSH functions as a nucleophile, reacting with
electrophilic molecules. A recent study has demonstrated the
implication of BSH in the detoxification of toxins and thiolreactive compounds such as rifamycin in Staph. aureus, via
the formation of BSH conjugates and the generation of
mercapturic acid (Newton et al., 2012). So far, the
detoxification processes for electrophilic molecules in
bacteria lacking both BSH and GSH are unknown.
Given that several bacteria, such as Ca. jejuni, possess
neither GST nor GSH biosynthesis pathways, how do these
bacteria mediate the detoxification of ITC conjugates? The
first step in the process needs to be a thiol exchange
mechanism to separate the dithiocarbamate conjugates, as
demonstrated for the GSH/GST system. The conjugation of
electrophilic ITC with GSH is a means to decrease
the intracellular levels of both free ITC and the proteinITC conjugates. Interestingly, the transcriptomic analysis of
Ca. jejuni exposed to BITC showed the upregulation of the
thioredoxin gene trxA (Dufour et al., 2013). The thioredoxin/thioredoxin reductase systems are often redundant
with the GSH/glutaredoxin/glutathione reductase system or
can replace it (Toledano et al., 2007). These systems are
responsible for maintaining a reducing environment and for
the regulation of thiol-based enzymic activity. All thioredoxins have a three-dimensional structure comprising a
central core of four b-strands, surrounded by three
a-helices (Martin, 1995). The active site contains two
redox-active cysteines. The pKa of the N-terminal cysteine
is lower than the pKa of a free cysteine, which enables
thioredoxin to attack the disulfide in proteins as a nucleophile (Holmgren, 1985; Roos et al., 2009). Because of their
low redox potential (2270 to 2330 mV in E. coli) (Aslund
et al., 1997; Krause & Holmgren, 1991), thioredoxins are
efficient thiol-disulfide reductants. In bacteria, thioredoxins
serve as enzyme reductants, hydrogen donors to ribonucleotide reductases (DNA synthesis) and phosphoadenosine phosphosulfate reductases (sulfur assimilation), and
methionine sulfoxide reductases (protein repair). They
Microbiology 161

The antibacterial properties of isothiocyanates

provide reducing equivalents to peroxiredoxins, involved in

peroxide detoxification (for review see Zeller & Klug, 2006).
So far, their role in the resistance to ITC via their possible
thiol exchange ability, the reduction of dithiocarbamate
conjugates or ITC depletion has not been experimentally
evaluated. However, it is interesting to notice that, in
eukaryotic cells, treatment with ITCs often resulted in overexpression of thioredoxin reductase or thioredoxin-encoding
genes, suggesting that these proteins have a role in the defence
against ITCs. In human colon carcinoma Caco-2 cells, iberin
triggered the overexpression of the thioredoxin reductase-1
gene (Jakubikova et al., 2006), and a similar result was
obtained in human hepatoma HepG2 cells cultured with SFN
(Zhang et al., 2003). Along with the thioredoxin reductase
genes, SFN also induced the expression of thioredoxin in both
HepG2 and Caco-2 cells (Bacon et al., 2007). In mice, both
intraperitoneal and oral SFN induced increased production of
thioredoxin protein in retinal tissues, reducing light damage
to retinol (Tanito et al., 2005). Thioredoxin may have an
essential role in maintaining reduced thiol groups as a means
to scavenge electrophilic ITC. In eukaryotic cells treated with
14
C-radiolabelled PEITC or SFN, only a small number of
proteins were found to contain radioactive ITC conjugates
(Mi et al., 2008, 2011), suggesting that either ITC binding in
vivo was selective or, as previously reported, ITC conjugates
dissociate to reach an equilibrium with free ITCs. The
proportion and identification of prokaryotic ITC target
proteins in vivo have not as of yet been investigated.
Conclusion and perspectives
Fig. 3 is a hypothetical model of the different ITC activities
in prokaryotes based on experiments carried out in
different species: (1) ITCs may accumulate in bacteria as
GSH, small thiols like BSH or thioredoxin dithiocarbamate
conjugates, and attack the active centre of enzymes (such as
receptors of QS signalling molecules), or affect protein
conformation via exchange processes (2) and binding to
thiol or amine groups. These interactions impact specialized enzymic activities, respiration, metabolism and
transcription of genes. Moreover, conjugation of ITCs
with the thiol groups of proteins may promote the
aggregation of misfolded proteins and thus the activation
of heat-shock responses, while the conjugation with amino
acids may induce a stringent response (3). The binding of
ITCs by thioredoxin, and of small thiols such as GSH or
BSH, may affect oxidative stress responses by impairing
peroxiredoxin regeneration (reduction) and cell redox
homeostasis (4). The conjugation of ITCs to GSH (via the
enzyme GST), BSH or thioredoxin may be a first step in the
detoxification of ITCs. The product of the sax gene
homologues, found in SFN-resistant Ps. syringae (Fan
et al., 2011), may also be involved in ITC detoxification
(5). Efflux systems (such as MexEF-OprN of Ps. aeruginosa) may promote the extrusion of ITCs from the cell (6).
These hypotheses are based mainly on gene expression
assays and are awaiting experimental confirmation.
http://mic.sgmjournals.org

The use of ITCs as antimicrobials is often impaired by their

high volatility and strong odour. In the absence of myrosinase, such as when plants are cooked and myrosinase is
heat-inactivated, animals can efficiently convert glucosinolates to ITCs through the action of myrosinase derived
from the microbiota of the gastrointestinal tract (Fahey
et al., 2012). In this case, the dietary intake of Brassicaceae
vegetables is sufficient to obtain beneficial effects on health.
However, for the purpose of treating food for preservation
or to fight against infectious diseases, processes including a
combination of glucosinolates and myrosinase are not as
easy to implement. Several packaging systems are being
evaluated to reduce the detrimental effect of ITC on food
appearance and taste. So far there is no report of the
antimicrobial effect of ITC conjugates; however, in eukaryotic cells, GSH-ITC and cysteine-ITC dithiocarbamates,
formed from AITC and BITC, displayed a similar biological
activity to their parent ITCs in inhibiting the growth of
epithelial cancer cells. Other studies demonstrated that ITC
conjugates could act as prodrugs, with similar efficacy to the
accumulated ITCs in cells treated with GS-ITC conjugates. This result demonstrates that dithiocarbamates are
potent ITC donors in eukaryotic cells (Zhang, 2000). Since
GSH-ITC and other ITC conjugates are reversible complexes
(Hwang & Jeffery, 2005), they may be able to release free ITC
inside the cells, and thus not deplete endogenous GSH or
other thiols. However, the inhibitory efficacy of these
conjugates may vary significantly compared with free ITC.
Indeed, some bacteria overcome the inhibitory effect of free
ITC via their conjugation to GSH, but an excess of GSH-ITC
conjugates may be a sufficient source of ITC for endogenous
thiol-exchange processes. Conaway et al. (2005) have
demonstrated that thiol conjugates of ITCs in an aqueous
medium exist in equilibrium with the free form, and that
free ITCs, rather than conjugates, exert enzyme-inhibitory
activity (tested in vitro on cytochrome P450). They have also
shown that N-acetyl-cysteine conjugates are more stable
than GSH and cysteine conjugates and postulated that
increasing the number of alkyl carbons should increase the
stability of ITC-thiol conjugates. Further investigations will
be required to determine the effect of thiol conjugates and
related chemical factors on bacteria.
In summary, ITCs are natural plant products with interesting
antimicrobial and anticancerous properties. Further studies
are needed to better understand their mode of action and
reveal potential synergetic activity with antibiotics.
Acknowledgements
This work was supported by an ARED doctoral training grant from
Region Bretagne to V. D. and by a doctoral mobility grant from VAS
Graduate School also to V. D.

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