Effects of iron and oxygen species scavengers onListeria spp. chemiluminescence

Philippe Andre,* Sandra Bilger, Pauline Remy, Sebastien Bettinger,and Dominique J.-M. Vidon

Universit�ee Louis Pasteur, U.F.R. des Sciences Pharmaceutiques, Laboratoire de Bact�eeriologie et Cryptogamie, EA 3429 and INSERM U392,

74 route du Rhin, B.P. 24, F-67401, Illkirch Cedex, France

Received 2 April 2003

Abstract

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is

amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth

curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is

tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as

well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the

CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL,

whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide

radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Listeria monocytogenes; Listeria innocua; Chemiluminescence; Iron; Tiron; Reactive oxygen species

Listeria are Gram-positive, regular rod-shaped bac-

teria which are widely distributed in the environment.

Listeria monocytogenes is pathogenic to humans andcauses severe infections in humans and animals. The

nonpathogenic species Listeria innocua is frequently

found associated with L. monocytogenes and serves as an

indicator for the possible presence of the pathogen.

Roth and Kaeberle [1] first showed that L. monocyto-

genes produces weak CL correlated with the presence of

superoxide anion, H2O2, and carbonate ion. We have

shown that addition of luminol 10-fold increases the CLproduced by L. monocytogenes [2]. We have used this

property to develop a simple CL-based method for rapid

enumeration of Listeria spp. microcolonies in raw milk

[3]. Iron is essential to bacterial survival and it is well

known that Listeria requires iron to support growth [4–

6]. This metal is also used by pathogenic bacteria as a

signal molecule for the regulation of virulence gene ex-

pression [7]. It has been shown that expression of hly,

which encodes the listeriolysin toxin, is induced in iron

limited medium [8]. More recently, B€oockmann et al. [9]reported that in vitro, specific attachment of PrfA to its

promoter is strongly inhibited by iron. Moreover, this

metal enters as a cofactor in the composition of a

number of cellular enzymes such as catalase and per-

oxidases, which are implicated in the metabolism of re-

active oxygen species (ROS). Chemically, iron(II)

complexes can act as one-electron reducing agents and

iron(III) complexes as one-electron oxidizing agents.Furthermore, iron complexes can react with molecular

oxygen or with its reduced species, such as superoxide

and hydrogen peroxide, leading to highly reactive

compounds [10]. The fundamental mechanism of CL is

not entirely elucidated, but it is closely related to the

release in the medium of superoxide radical, hydroxyl

radical, and singlet oxygen. Luminol is the most com-

monly used CL probe for measuring O�2 concentration

in biological systems [11]. It has been used to detect

the ROS production by different bacteria such as

Biochemical and Biophysical Research Communications 304 (2003) 807–811

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BBRC

* Corresponding author. Fax: +33-3-88-67-92-42.

E-mail address: andre@pharma.u-strasbg.fr (P. Andre).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00671-5

Helicobacter pylori [12] or Staphylococcus aureus [13].Iron being an essential element for Listeria spp. growth,

the aim of the present paper was to investigate the role

of iron and to precise the type of reactive oxygen species

implicated in the CL signal emitted by L. monocytogenes

and L. innocua.

Materials and methods

Bacterial strains, culture conditions, and reagents. Listeria mono-

cytogenes B38 (serovar 4b) and L. innocua H16 (serovar 6a) were

isolated from cheese and frozen corn, respectively. Cultures were

grown in brain heart infusion (BHI) (Bio-Rad) broth (pH: 7.2) at

37 �C. The inoculum was prepared by suspending overnight cultures on

trypticase soy agar (BioMerieux) at 37 �C in distilled water to an OD620

of 0.156. One ml of that suspension corresponding to 1–3� 108 CFU

was added to 10ml of BHI. Charcoal pretreatment was achieved by

addition of 0.2% activated charcoal (Sigma) to BHI broth. Charcoal-

BHI was agitated for 2 h at room temperature, then centrifuged 15min

to 3500g, and the supernatant was filtered through 0.45lm pore size

filters (Dutscher). Iron-free BHI was obtained by mixing BHI medium

with 2.5% Chelex-100 (Bio-Rad) for 24 h under agitation (200 rpm) at

room temperature. The resin was removed by decantation and filtra-

tion of supernatant. Reagents were added at indicated concentrations

in growth medium. Luminol, sodium hydrogen carbonate, tryptophan,

mannitol, tiron, and citrate ferric (17.4% iron) were provided by Sig-

ma–Aldrich. Desferal (deferoxamine B) was purchased from Novartis.

Chemiluminescence and bacterial growth measurements. CL was

measured at room temperature (+22 to +25 �C) with a Lumac M-2500

Biocounter (Perstorp Analytical), using polystyrene cuvettes. Growing

culture 0.1ml samples were added to cuvettes containing 0.1ml of

sodium carbonate 1.0M. Luminol 10�4 M (0.1ml) was added just be-

fore measuring the luminescence over 10-s periods. CL is expressed in

relative light unit (RLU). Bacterial growth was monitored by mea-

suring the absorbance at OD620 of vortexed cultures at +37 �C with a

digital photometer (Dr lange).

Results and discussion

As shown in Fig. 1, there is a close relationship be-tween the growth curve and the emitted CL, in both L.

monocytogenes and L. innocua. The maximum CL was

obtained just before the entry of bacterial growth into

the stationary phase. This maximum CL was dependent

on bacterial growth level and was followed by a rapid

and dramatical drop in luminescence. No significant

difference between the CL emitted by L. monocytogenes

and L. innocua was observed. This CL was also observedfor other different strains of L. monocytogenes and L.

innocua (data not shown). We have investigated the role

of iron in the production of luminescence. At the con-

centrations used, ferric citrate (3 g/L) and tiron (5mM),

a Fe(III) chelator, did not modify the growth curve

profiles of the two bacteria (data not shown). In the

presence of ferric citrate (Fig. 2A), the CL increased

strongly from 5:2� 104 RLU to 1:5� 105 RLU and4:7� 104 RLU to 1:4� 105 RLU for L. monocytogenes

and L. innocua, respectively, while addition of tiron

5.0mM (Fig. 2B) and charcoal pretreatment of BHI

(Fig. 2C), which has been assumed to adsorb iron fromthe medium [14], completely inhibited the CL of L.

monocytogenes and L. innocua. In the same condition,

deferoxamine B 5.0mM a strong iron chelator, inhibited

at 80% the CL of the two bacteria (data not shown). In

iron-free BHI both L. innocua and L. monocytogenes are

unable to grow and did not emit CL (data not shown).

These results show that iron is likely implicated in the

CL emitted by both bacteria. But which type of ROS areimplicated in this phenomenon? The nature of ROS

produced by metabolic systems can be determined by

using antioxidants or compounds that are able to

scavenge the oxyradical produced. Different types of

antioxidants were used as scavengers for superoxide

radicals, hydroxyl radicals, and singlet oxygen:tiron

[15,16], mannitol [17], and tryptophan [18], respectively.

Addition of mannitol (0.1, 1.0, and 10mM) or trypto-phan (0.01, 0.1, and 1.0mM) did not affect both kinetic

growth curves (data not shown) and the CL signal

emitted by the two bacteria (Fig. 3). On the other hand,

addition of tiron (0.05, 0.5, and 5.0mM) inhibited in a

dose-dependent manner the CL of L. monocytogenes

and L. innocua (Fig. 3) with a maximum of 60%

inhibition in the presence of 5.0mM tiron, without

Fig. 1. (A) Growth of L. monocytogenes (�) and L. innocua (j) in

BHI; (B) kinetics of chemiluminescence of L. monocytogenes (�) and

L. innocua (j). The results are means of triplicate experiments, n ¼ 4,

�SE.

808 P. Andre et al. / Biochemical and Biophysical Research Communications 304 (2003) 807–811

modifying the growth curve of the bacteria. These re-

sults show that superoxide radicals were clearly involved

in the CL signal. We have previously shown that L.

monocytogenes possesses a ferric-reductase activity [19],

which releases Fe(II). Ferrous ligand in the presence of

oxidizing agent leads to superoxide radicals as depicted

in the equation

FeðIIÞ þO2 ! FeðIIIÞ þO��2

Complexation of superoxide radicals and iron by tironinhibited the CL of both bacteria species, while addition

of ferric citrate, which is reduced in ferrous ligand by

Listeria ferric reductase activity, results in an increase in

bacterial superoxide production and CL enhancing. We

have also examined whether CL signal is correlated with

bacterial virulence modulation. In this way, it has been

shown by different authors that fermentable carbohy-

drates cause a strong repression of virulence genes in L.

monocytogenes; for example cellobiose reduces PrfA-dependent virulence gene expression [20,21]. In an op-

posite manner, exposure to high temperature (+42 to

+44 �C) increases the expression of a set of genes among

A B

C

Fig. 2. Effects of ferric citrate 3 g/L (A), tiron 5mM (B) and charcoal-BHI pretreatment (C) on L. monocytogenes (open symbols) and L. innocua

(closed symbols) chemiluminescence. Symbols: �, L. monocytogenes; s, L. monocytogenes+ ferric citrate (3 g/L) or tiron (5mM) or charcoal-BHI

pretreatment; j, L. innocua; d, L. innocua+ ferric citrate (3 g/L) or tiron (5mM) or charcoal-BHI pretreatment. The results are means of triplicate

experiments, n ¼ 3, �SE.

Fig. 3. Effects of tryptophan, mannitol, and tiron on chemiluminescence of L. monocytogenes (open columns) and L. innocua (black columns)

chemiluminescence. L. monocytogenes and L. innocua controls correspond to 100%.

P. Andre et al. / Biochemical and Biophysical Research Communications 304 (2003) 807–811 809

which hly [22], via induction of prfA. As shown in Fig.

4B, the CL signal emitted by L. monocytogenes is not

modified by the presence of cellobiose (25mM), while L.

innocua CL is twofold increased from 3:2� 104 to6:5� 104 RLU. At +42 �C, the growth curve is delayed

compared to +37 �C (Fig. 4C) and the CL (Fig. 4D) is

decreased as expected by the lower OD620 obtained at

+42 �C compared to OD620 reached at +37 �C. These

results suggest that modification of virulence genes ex-

pression is not correlated with the CL of L. monocytog-

enes. Roth and Kaeberle have shown that superoxide

dismutase (SOD) and catalase negatively affected thisphenomenon. Subsequently, it has been shown that SOD

[23] and catalase [24] appear to have only a minor im-

plication in Listeria virulence. In conclusion, L. mono-

cytogenes and L. innocua produce a transient strong peak

of CL, amplified by luminol, at the end of the exponen-

tial growth phase. This CL is correlated with the pro-

duction of superoxide radicals, which are generated by

iron oxidation. Furthermore, this effect does not seem tobe related to virulence genes expression.

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