International Journal of Food Microbiology 167 (2013) 202207

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Comparative proteomic analysis of Listeria monocytogenes exposed to

enterocin AS-48 in planktonic and sessile states
Natacha Caballero Gmez a, Hikmate Abriouel a, Said Ennahar b, Antonio Glvez a,
a
b

rea de Microbiologa, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jan, 23071 Jan, Spain
Equipe de Chimie Analytique des Molcules Bio-Actives, UMR 7178, IPHC-DSA, Universit de Strasbourg, CNRS, 67400 Illkirch-Graffenstaden, France

a r t i c l e

i n f o

Article history:
Received 9 April 2013
Received in revised form 25 July 2013
Accepted 23 August 2013
Available online 31 August 2013
Keywords:
Enterocin AS-48
Listeria monocytogenes
Comparative proteomics
Biolm

a b s t r a c t
Enterocin AS-48 is a cyclic peptide of great interest for application in food preservation and sanitation. In the present study, the proteome response of Listeria monocytogenes to puried enterocin AS-48 was studied under two
different conditions: planktonic cells and sessile cells grown on polystyrene plates. Ten different proteins were
differentially expressed in planktonic L. monocytogenes cells treated with 0.1 g/ml enterocin AS-48 compared
to the untreated controls. Overexpressed proteins were related to stress response (DnaK) or carbohydrate transport and metabolism, while underexpressed and unexpressed proteins were related to metabolism (such as
glyceraldehyde-3-phosphate dehydrogenase, pyruvate oxidase, glutamate dehydrogenase or glutamate decarboxylase) or stress (GroEL). In the sessile state, L. monocytogenes cells tolerated up to 10 g/ml bacteriocin, and
the treated biolm cells overexpressed a set of 11 proteins, some of which could be related to stress response
(DnaK, GroEL), protein synthesis and carbohydrate metabolism, while glyceraldehyde-3-phosphate dehydrogenase was the only unexpressed protein. Some of the overexpressed proteins (such as elongation factor Tu and
GroEL) could also be implicated in cell adhesion. These results suggest different cell responses of L. monocytogenes
to enterocin AS-48 in the planktonic and in the sessile state, including stress response and cell metabolism proteins. While in the planktonic state the bacterium may tend to compensate for the cytoplasmic cell permeability
changes induced by AS-48 by reinforcing carbohydrate transport and metabolism, sessile cells seem to respond
by shifting carbohydrate metabolism and reinforcing protein synthesis. Stress response proteins also seem to be
important in the response to AS-48, but the stress response seems to be different in planktonic and in sessile cells.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Listeria monocytogenes (McLauchlin et al., 2004) is a psychrotrophic
foodborne Gram-positive bacterium which is widely found in the environment such as plants, soil, animals, water, dirt, dust and silage.
Although listeriosis is a relatively rare disease compared to other
foodborne illnesses, it is considered to have the second-highest case fatality rate (20.5%) and the highest hospitalization rate (90%) among
foodborne illnesses (Lianou and Sofos, 2007; EFSA, 2010; Scallan et al.,
2011). The high capacity of L. monocytogenes to survive in the presence
of several environmental stressors such as heat, cold, salt, high osmolarity
and acidic conditions (Gandhi and Chikindas, 2007), its ubiquitous
character and its capacity to form biolms on biotic or abiotic surfaces including stainless steel, rubber, glass and polypropylene (Lundn et al.,
2000; Latorre et al., 2010) are main factors for L. monocytogenes to be continuously introduced into the processing environment and crosscontaminate food contact surfaces, equipment, oors, drains and other
Corresponding author at: rea de Microbiologa, Departamento de Ciencias de la
Salud, Facultad de Ciencias Experimentales, Edif. B3, Universidad de Jan, Campus Las
Lagunillas s/n, 23071-Jan, Spain. Tel.: +34 953 212160; fax: +34 953 212943.
E-mail address: agalvez@ujaen.es (A. Glvez).
0168-1605/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2013.08.019

locations. To reduce the incidence of listeriosis, various new strategies

have been proposed including natural antimicrobials as alternative or
natural sanitizers instead of chemicals, e.g. essential oils (Desai et al.,
2012), nisin (Guerra et al., 2005) and reuterin alone or in combination
with nisin (El-Ziney and Jakobsen, 2009).
Enterocin AS-48 is a cyclic antimicrobial peptide produced by Enterococcus faecalis and Enterococcus faecium strains (Maqueda et al.,
2004; Abriouel et al., 2010). This bacteriocin exhibits a broad antimicrobial spectrum including several foodborne pathogens both in liquid
cultures and in foods (Abriouel et al., 2010). Planktonic cells of
L. monocytogenes have shown to be highly sensitive to the enterocin
AS-48 in culture media (Mendoza et al., 1999), while biolms of
L. monocytogenes CECT 4032 formed on polystyrene microtiter plates required higher bacteriocin concentrations (Caballero Gmez et al., 2012).
The degree of biolm sensitivity to AS-48 may depend on several parameters such as surface composition and structure, bacteriocin concentration, target bacteria, environmental conditions and the presence of
other antimicrobials. In this context, data obtained by Caballero
Gmez et al. (2012) showed that the combination of AS-48 with different biocides remarkably increased the inactivation of L. monocytogenes
biolms formed on polystyrene microtiter plates and that previous conditioning of plates with enterocin solutions decreased adherence and

N. Caballero Gmez et al. / International Journal of Food Microbiology 167 (2013) 202207

biolm formation. However, no data are available about the response of

L. monocytogenes to AS-48 at the molecular level to elucidate the mechanisms adopted by this bacterium under stress conditions. Protein expression is in continuous change through different stages of the
cellular cycle but also in response to external actions. So, the differences
between a normal condition and a stress condition are translated by
differences in protein expression, which is one of the main tools
used by bacteria to interact with their environment. Several studies
have explored the complex phenotypic and genotypic responses of
L. monocytogenes exposed to different bacteriocins (Palmer et al.,
2009; Tadesse et al., 2009, 2011). However, no proteomic analysis was
done with L. monocytogenes exposed to the cyclic enterocin AS-48. The
purpose of this study was to investigate differences in the proteome of
L. monocytogenes CECT 4032 when exposed to AS-48 under different
cellular states (planktonic and sessile).
2. Materials and methods
2.1. Bacterial strains and growth conditions
L. monocytogenes CECT 4032 (serovar 4b) was obtained from the
Spanish Type Culture Collection (CECT) and was stored as frozen stocks
at 20 C in BHI broth containing 20% glycerol. For routine use,
L. monocytogenes CECT 4032 was propagated at 37 C on tryptone soy
broth (TSB; Scharlab, Barcelona, Spain) or in tryptone soy agar (TSA)
slants and stored at 4 C. For preparation of inocula, L. monocytogenes
CECT 4032 was grown for 15 h in TSB at 37 C.
2.2. Bacteriocin preparation
Enterocin AS-48 was obtained from cultured broths of the producer
strain E. faecalis A-48-32 after concentration by cation exchange chromatography and repurication by semi-preparative reversed-phase
high-performance chromatography as described elsewhere (Abriouel
et al., 2003). The puried bacteriocin was dissolved (1 mg/ml) in sterile
Milli-Q water and stored at 20 C. Before use, the puried bacteriocin
was diluted in TSB broth in order to achieve the desired nal bacteriocin
concentrations of 0.1 or 10 g/ml.
2.3. Bacteriocin treatments
2.3.1. Planktonic cells
Overnight cultures of L. monocytogenes CECT 4032 in TSB were inoculated (1% v/v, ca. 106 cfu/ml) into 10 ml of one fth-strength TSB
(6.0 g/l) supplemented or not with AS-48 at the maximum concentration not inhibiting growth (0.1 g/ml). Cultures were grown for 15 h
at 37 C. Optical density was monitored at 550 nm every 30 min using
an Absorbance Reader (Varioskan Flash Reader, Thermo Scientic).
Cells were collected by centrifugation (5000 g for 15 min) and the
cell pellets were immediately frozen in liquid nitrogen and stored at
80 C until protein extraction. Each experiment was done in triplicate
(controls and samples treated with AS-48).
2.3.2. Sessile cells
Overnight cultures were inoculated (1% v/v) onto one fth-strength
TSB and distributed on sterile Petri plates. The plates were incubated at
37 C for 48 h to allow biolm formation. Then, the cultured broths
were discarded and the biolms formed on the plates were washed
with 4 ml of sterile saline solution to remove loosely associated bacterial cells. TSB broths supplemented with or without enterocin AS-48
(10 g/ml) were added to the plates, which were then further incubated
at 37 C for 48 h, while renewing the medium (with or without AS-48)
every 24 h. The controls and treated biolms were resuspended in
10 ml of phosphate buffered saline (PBS) (Merk, Darmstadt,
Germany) and the cells were harvested by centrifugation for 15 min
at 5000 g. The cell pellets were immediately frozen in liquid nitrogen

203

and stored at 80 C until protein extraction. Each experiment was

done in triplicate (control biolms and biolms treated with AS-48).
2.4. Whole cell protein extraction
The cell pellets obtained as described above both from planktonic
and sessile L. monocytogenes cells treated or not with AS-48 were
resuspended in 2 ml of PBS and dispersed in liquid nitrogen with a
200 l micropippette to obtain cryobeads. The bacterial beads were
ground in liquid nitrogen using a cryogenic grinder (6870 Freezer/
Mill, SpexCertiPrep, Stanmore, UK) with three steps of 3 min at a rate
of 24 impacts/s. After sample centrifugation (5000 g for 5 min,
4 C), the supernatants were ltered through a 0.45-m pore size lter
(Chromal PET; Macherey-Nagel, Dren, Germany). Protein extraction
from the ltered supernatants was carried out with a Trizol reagent
(Euromedex, Souffelweyersheim, France) as previously described
(Izquierdo et al., 2009). Protein concentrations were determined using
Bradford protein assay (Bio-Rad) according to the manufacturer's
instructions.
2.5. 2-D gel electrophoresis
Protein extracts (150 g) were loaded onto 17-cm strips with a pH
range of 3 to 10 (Bio-Rad), focused for 60,000 V h, and then separated
on a 12% SDS-polyacrylamide gel as reported previously (Izquierdo
et al., 2009). The gels were stained with Bio-Safe Coomassie (Bio-Rad)
and scanned on a GS-800 Calibrated Densitometer (Bio-Rad).
2.6. Image analysis
Image analysis of the 2-DE gels was performed using PD Quest 8.0.1
software (Bio-Rad). Three gels were produced from independent cultures
of each condition and only spots that were present on the three gels were
selected for inter-condition comparison. Spot intensities were normalized
to the sum of intensities of all valid spots in one gel. For analysis of
changes in protein expression during enterocin AS-48 exposure, a protein
was considered to be under- or overproduced when changes in normalized spot intensities were at least 1.5-fold at a signicance level of
p b 0.05 (Student's t test for paired samples), as previously described
(Snchez et al., 2007). Regarding proteome comparison between different
culture conditions of L. monocytogenes CECT 4032, proteins were considered differentially produced when spot intensities passed the threshold
of a twofold difference (one-way ANOVA, p-value b 0.05), as described
previously (Izquierdo et al., 2009).
2.7. Identication of proteins by LC-MS analysis
Spots of interest were subjected to tryptic in-gel digestion (Izquierdo
et al., 2009) and analyzed by chip-liquid chromatography-quadrupole
time of ight (chip-LC-QTOF) using an Agilent G6510A QTOF mass spectrometer equipped with an Agilent 1200 Nano LC system and an Agilent
HPLC Chip Cube, G4240A (Agilent Technologies, Santa Clara, CA, USA),
as described previously (Izquierdo et al., 2009). Briey, one microliter of
the sample was injected using an injection loop of 8 l, a loading ow
rate of 3 l/min for 4 min and a solvent made of ultra-pure water and
acetonitrile (HPLC-S gradient grade, Biosolve, Valkenswaard, The
Netherlands) (97/3 v/v) with 0.1% formic acid (98100%, Merck). For
the analytical elution, a 24 min gradient from 3 to 60% of acetonitrile in
ultra-pure water with 0.1% formic acid was applied at a ow rate of
300 nl/min. ESI in positive mode with 1850 capillary voltage was used.
The data were collected in centroid mode using extended dynamic
range at a mass range of m/z 2002000 both in MS1 and MS/MS and
using two methods with different scanning speeds: one slow with a
scan rate of 1 spectra/s for both MS1 and MS/MS, and one fast scan rate
of 0.25 spectra/s for both MS1 and MS/MS. For data acquisition and data

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N. Caballero Gmez et al. / International Journal of Food Microbiology 167 (2013) 202207

export, MassHunter version B.02.0.197.0 (Agilent Technologies) was

used.
2.8. Protein identication
After data acquisition, the les were uploaded to the in house installed
version of Phenyx (Geneva Bioinformatics, Geneva, Switzerland) for
searching the NCBInr (r. 20090608) database with the following criteria:
taxonomy:bacteria; scoring model: ESI-QTOF; parent charge: +2, +3
(trust = medium); single round; methionine oxidation, cysteine
carboxyamidomethylation (cysteine treated with iodoacetamide), and
phosphorylation as partial modications; trypsin as digestion enzyme;
allowance of two missed cleavages; cleavage mode: normal; parent ion
tolerance: 0.6 Da; peptide thresholds: length 6, score threshold 5.0,
identication signicance p-value 1.0E4, accession number score
threshold 6.0, coverage threshold 0.2, identied ion series: b; b++;
y; y++; allowance of conict resolution. A publicly available MS/MS
search algorithm (open mass spectrometry search algorithm, OMSSA)
(Geer et al., 2004) was used with the same search criteria as described
above to conrm protein identities and limit the risk of false positives.
On the basis of consensus scoring, only proteins recognized by both database search algorithms at a false positive rate of 5% were considered to be
correctly identied (Kapp et al., 2005).
3. Results
Sessile and planktonic L. monocytogenes CECT 4032 were cultured in
exponential to early stationary phases under non-stressing conditions
and a stress condition with AS-48. Whole cell proteins were extracted
and analyzed from three independent biological replicates.
3.1. Response of planktonic L. monocytogenes to enterocin AS-48
To understand the physiological changes that occur in planktonic
cells of L. monocytogenes after growth in the presence of enterocin AS48, we compared the proteomes of untreated and treated planktonic
cells of L. monocytogenes CECT 4032. 2-DE analysis of AS-48-treated
cells from the early stationary phase led to the identication of ten proteins that were found to have different patterns of expression compared
to the untreated controls (Fig. 1). The expression levels of these proteins
were signicantly (P b 0.05) affected by the presence of enterocin AS48. These proteins were excised in duplicate and individually identied

by tryptic digestion and matrix-assisted laser desorption ionizationtime of ight mass spectrometry, and the results of these analyses are
summarized in Table 1.
In the presence of AS-48, four proteins were over-expressed: molecular chaperone DnaK (spot 702), enolase (spot 1043), phosphotransferase
system enzyme I (spot 1702) and bifunctional acetaldehyde-CoA/alcohol
dehydrogenase (spot 8802) (Fig. 1, Table 1). Enolase and enzyme I are implicated in carbohydrate metabolism and transport and bifunctional
acetaldehyde-CoA/alcohol dehydrogenase is implicated in energy production. The chaperone DnaK is related to stress response.
The following three proteins were found to be under-expressed:
glyceraldehyde-3-phosphate dehydrogenase (spot 4506), lin2463 (spot
6602) and glutamate dehydrogenase (spot 7502) (Fig. 1, Table 1).
Glyceraldehyde-3-phosphate dehydrogenase is related to carbohydrate
metabolism, while glutamate dehydrogenase and hypothetical protein
lin2463 (glutamate decarboxylase) are implicated in amino acid metabolism. Furthermore, three proteins were only expressed in the absence of
AS-48: molecular chaperone GroEL (spot 1705) as stress protein, elongation factor Tu (spot 2606) and pyruvate oxidase (spot 2703) (Fig. 1,
Table 1) which have been linked to protein synthesis and cell metabolism.

3.2. Response of sessile L. monocytogenes to enterocin AS-48

The proteomes of L. monocytogenes biolms formed in the presence
and absence of AS-48 were compared. Twelve proteins were differentially expressed signicantly (P b 0.05) in AS-48-treated biolms of
L. monocytogenes CECT 4032 compared to the controls without enterocin
(Fig. 2, Table 2). Among them, eleven were over-expressed: conserved
hypothetical protein (spot 101), chaperone protein DnaK (spot 701),
chaperonin GroEL (spot 702), translation elongation factor Tu (spot
1501), branched-chain amino acid aminotransferase (spot 2301),
isocitrate dehydrogenase NADP-dependent (spot 2402), glycyl-tRNA synthetase (spot 2601), translation elongation factor G (spot 2803),
transketolase (spot 3801), inosine-5-monophosphate dehydrogenase
(spot 5501) and malate dehydrogenase (quinone) (spot 5601) (Fig. 2,
Table 2). Proteins that were overexpressed in response to AS-48 treatment of L. monocytogenes CECT 4032 biolms include proteins implicated
in several cellular functions like stress proteins, carbohydrate metabolism
and transport and also amino acid synthesis. The only protein that was
not expressed in the presence of AS-48 was glyceraldehyde-3phosphate dehydrogenase (spot 4401) (Fig. 2, Table 2), which is related
with carbohydrate metabolism.

Fig. 1. Proteomes of planktonic Listeria monocytogenes CECT 4032 in the absence or presence of enterocin AS-48. Representative 2-DE gels of whole cell proteomes from early stationaryphase cells of L. monocytogenes 4032 CECT cultured without (A) or with 0.1 g/ml AS-48 (B). Spots exhibiting constitutive differential expression between growth of L. monocytogenes CECT
4032 in standard conditions and in the presence of AS-48 were identied by peptide mass ngerprinting and are labeled, and the identications of the 10 spots affected by enterocin AS-48
are listed in Table 1.

N. Caballero Gmez et al. / International Journal of Food Microbiology 167 (2013) 202207

205

Table 1
Proteins differentially expressed between planktonic Listeria monocytogenes CECT 4032 treated or not with enterocin AS-48.
Protein identity
Proteins over-expressed in the presence of AS-48
Molecular chaperone DnaK
Enolase
Phosphotransferase system enzyme I, partial
Bifunctional acetaldehyde-CoA/alcohol dehydrogenase
Proteins not expressed in the presence of AS-48
Glyceraldehyde-3-phosphate dehydrogenase
Glutamate decarboxylase (lin2463)
Glutamate dehydrogenase
Proteins not expressed in the presence of AS-48
Molecular chaperone GroEL
Elongation factor Tu
Pyruvate oxidase
a

Spot N

Accession numbera

Mascot score

Sequence coverage (%)

702
1043
1702
8802

gi|217964381
gi|46908628
gi|2623820
gi|16800743

281.42
215.73
121.14
197.94

66
72
32
29

4506
6602
7502

gi|16801615
gi|16801525
gi|116871946

107.47
88.19
48.12

45
28
19

1705
2606
2703

gi|116873505
gi|16804690
gi|46906973

107.8
64.28
158.42

31
22
41

Accession number in the NCBI database.

4. Discussion
The phenotypic and genotypic robustness of L. monocytogenes is of
particular concern to the food industry as it has a variety of encoded
proteins implicated in survival mechanisms to withstand environmental stressors such as heat, cold, salt, acidic pH, or antimicrobials. In addition, some of these stress responses can result in enhanced survival,
enhanced virulence, and even cross protection against multiple
stressors. To gain further insight into the protein expression of
L. monocytogenes CECT 4032 under different conditions, a proteomic
analysis was performed. The results obtained in the current study revealed that L. monocytogenes CECT 4032 exhibited differential protein
expression depending on its cellular state (planktonic and sessile), as
well as in the presence or absence of enterocin AS-48. The proteins regulated as a result of the effect of AS-48 are implicated in carbohydrate
and amino acid metabolism and transport, and also in stress response.
Treatment of planktonic L. monocytogenes CECT 4032 with a subinhibitory concentration of AS-48 induced the over-expression of the
major stress protein, chaperone DnaK of L. monocytogenes (spot 702,
Fig. 1, Table 1) which has been reported to be required for tolerance to
environmental stresses (Hanawa et al., 1999). Similarly, representatives
of the major molecular chaperones (belonging to the class of folding
chaperones) DnaK and GroEL were over-expressed when sessile
L. monocytogenes CECT 4032 was treated with AS-48 (spots 701 and
702, Fig. 2, Table 2). The role of chaperones rely on ATP-driven conformational changes to mediate the net refolding/unfolding of the substrates and the over-expression of DnaK in both cellular states of
L. monocytogenes CECT 4032 (planktonic and sessile) could be crucial
to protect cells from damage by protein denaturation as a result of proteolysis or aggregation as it has been reported by Liu et al. (2002). In
previous studies, AS-48 has been shown to form membrane pores

through an interaction with cytoplasmic membrane, leading to membrane permeabilization and a rapid collapse of the cytoplasmic membrane potential (Glvez et al., 1991). At sub-inhibitory concentrations,
the interaction of AS-48 with membrane lipids and proteins may induce
the over-expression of stress proteins such as DnaK and GroEL as a rst
response of the cell to carry on the degradation of abnormal or damaged
polypeptides such as those resulting from arrested protein synthesis or
misfolded membrane-associated proteins. Furthermore, it has been reported that DnaK and GroEL were successfully induced in cells preincubated with a sub-inhibitory concentration of streptomycin
(Cardoso et al., 2010) and that DnaK was important for L. monocytogenes
biolm formation and disinfectant resistance (Van der Veen and Abee,
2010) which corroborate the results obtained with AS-48 in the current
study.
Planktonic L. monocytogenes CECT 4032 treated with a sub-inhibitory concentration of AS-48 also exhibited an over-expression of proteins
involved in carbohydrate transport, phosphorylation and metabolism
like enolase (phosphopyruvate hydratase) a protein present in both
the cytoplasm and on the cell surface and enzyme I-phosphoenolpyruvate protein kinase, a component of the bacterial phosphotransferase
system. Overexpression of proteins involved in energy metabolism
could be an attempt to compensate for partially-impaired energy generation caused by sub-inhibitory concentrations of AS-48 interacting with
the bacterial cytoplasmic membrane. In another study, stress proteins
and glycolytic proteins were found to be induced in the autolytic proteome of L. monocytogenes (Pinto et al., 2012), and Yu et al. (2011) suggested that enolase is also involved in autolysis in Staphylococcus
aureus besides being a key enzyme for glycolysis. Although enterocin
AS-48 also induces a bacteriolytic effect that is associated with autolysin
activation (Glvez et al., 1990), no overexpression of autolysin was observed in the proteomes of the treated L. monocytogenes cells. In

Table 2
Proteins differentially expressed between sessile Listeria monocytogenes CECT 4032 formed in the presence or absence of enterocin AS-48.
Protein identity
Proteins over-expressed in the presence of AS-48
Conserved hypothetical protein
Chaperone protein DnaK
Chaperonin GroEL
Translation elongation factor Tu
Branched-chain amino acid aminotransferase
Isocitrate dehydrogenase, NADP-dependent
Glycyl-tRNA synthetase
Translation elongation factor G
Transketolase
Inosine-5-monophosphate dehydrogenase
Malate dehydrogenase (quinone)
Proteins not expressed in the presence of AS-48
Glyceraldehyde-3-phosphate dehydrogenase
a

Accession number in the NCBI database.

Spot N

Accession numbera

Mascot score

Sequence coverage (%)

101
701
702
1501
2301
2402
2601
2803
3801
5501
5601

gi|223043647
gi|223044356
gi|223044069
gi|223042706
gi|223042796
gi|223043575
gi|223044387
gi|223042789
gi|223043117
gi|223043434
gi|223042549

76.12
240.66
147.08
144.05
98.24
94.9
153.27
272.09
151.45
195.77
122.99

62
53
46
55
39
32
48
58
32
56
34

4401

gi|16801615

76.01

32

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N. Caballero Gmez et al. / International Journal of Food Microbiology 167 (2013) 202207

Fig. 2. Proteomes of Listeria monocytogenes CECT 4032 biolms formed in the absence or presence of enterocin AS-48. Representative 2-DE gels of whole cell proteomes from sessile L.
monocytogenes CECT 4032 without (A) or with 10 g/ml enterocin AS-48 (B). Spots exhibiting constitutive differential expression between biolms formed in standard conditions and
in the presence of AS-48 were identied by peptide mass ngerprinting and are labeled, and the identications of the 12 spots affected by enterocin AS-48 are listed in Table 2.

contrast, two proteins related to glutamate metabolism (glutamate

dehydrogenase and glutamate decarboxylase) were underexpressed
in the planktonic cells treated with AS-48. In L. monocytogenes, glutamate decarboxylase plays a role in maintaining cytoplasm pH homeostasis, enhancing the survival of the bacterium under acidic conditions
(Cotter et al., 2001a,2001b, 2005). Glutamate decarboxylase has also
been shown to play a role in L. monocytogenes nisin tolerance in the
presence of glutamate, presumably by contributing to intracellular
ATP pools (Begley et al., 2010). Glutamate dehydrogenase may provide
intracellular glutamate in the absence of available extracellular glutamate. These results would suggest that enterocin AS-48 at subinhibitory
concentrations may impair GAD-mediated survival mechanisms in L.
monocytogenes. Interestingly, enterocin AS-48 shows strong antilisterial
activity in glutamate-rich food systems (e.g. meat products) as well as in
non-meat products, suggesting that, unlike nisin, its antimicrobial activity is not inuenced by the presence of glutamate.
Sessile L. monocytogenes CECT 4032 over-expressed other proteins
implicated in carbohydrate metabolism and transport in response to
AS-48, such as isocitrate dehydrogenase NADP-dependent (spot
2402), transketolase (spot 3801) and malate dehydrogenase (spot
5601) (Fig. 2, Table 2). In this case, the proteINS over-expressed may
have a similar role as suggested above for planktonic cells to enhance
energy production, although the proteins involved in sessile cells
concerned other routes of carbohydrate metabolism (pentose and glycolysis pathways). Furthermore, over-expression of proteins implicated
in RNA translation such as translation elongation factor Tu (spot 1501),
glycyl-tRNA synthetase (spot 2601) and translation elongation factor G
(spot 2803) in sessile cells (Fig. 2, Table 2) could be related to an enhanced protein synthesis required for biolm formation. Elongation factor Tu was also overexpressed in L. monocytogenes under autolytic stress
conditions (Pinto et al., 2012) and in Escherichia coli it seems to have a
role additional to its translation elongation function in that it may act
as a chaperone-like protein protecting cells from stress (Caldas et al.,
1998). Furthermore, elongation factor Tu as well as GroEL chaperonin
has been described in association with the cell walls of lactobacilli,
where they are suspected to play a role in cell adhesion (Izquierdo
et al., 2009; Granato et al., 2004; Bergonzelli et al., 2006; Siciliano
et al., 2008) and therefore could be related to biolm formation.
Biolms of L. monocytogenes CECT 4032 did not exhibit any decrease
in protein expression in the presence of AS-48 which indicate their high

resistance to enterocin in comparison with planktonic cells. As a matter

of fact, the biolms tolerated a 100-fold higher concentration of bacteriocin compared to sessile cells. Remarkably, most proteins not expressed
by planktonic L. monocytogenes CECT 4032 in the presence of AS-48 are
proteINS over-expressed in sessile cells like molecular chaperone GroEL
and elongation factor Tu. On the contrary, glyceraldehyde-3-phosphate
dehydrogenase under-expressed by planktonic L. monocytogenes CECT
4032 was not expressed by sessile cells. The different physiological conditions in the sessile state together with the lower sensitivity to
enterocin AS-48 and the higher bacteriocin concentration used in biolm assays could account for the observed differences.
The results obtained from proteomic analysis showed clearly that
the proteome of L. monocytogenes CECT 4032 may depend on different
parameters such as the cellular state and the presence or absence of
AS-48. However, further studies carried out in food matrices could be
very useful in order to evaluate the effects of food composition, additives, preservatives, and processing technologies on the modulation of
L. monocytogenes cellular components in response to AS-48.
5. Conclusions
The present work described a comparative proteomic analysis of
planktonic and sessile L. monocytogenes CECT 4032 in the presence
and absence of AS-48. As evidenced by the proteome analysis (Fig. 3),
L. monocytogenes showed a similar pattern of stress proteins (chaperones) in both cellular states highlighting the over-expression of DnaK,
however different patterns of proteins were over-expressed in planktonic and sessile L. monocytogenes exposed to the enterocin AS-48 and
were related with carbohydrate metabolism/transport and also with
amino acid metabolism. However, planktonic cells shifted toward a
more energy-efcient catabolism in the presence of AS-48, possibly triggered by a lowered energy state in the cells induced by permeabilization
of the bacterial cytoplasmic membrane by bacteriocin.
Acknowledgments
This work was supported by research project AGL2008-01553/ALI
(MICINN, FEDER). Natacha Caballero was beneciary of a research grant
from the same institution, and a mobility grant (MECD, MHE201100091, Programas Doctorado con Mencin hacia la Excelencia). We also

N. Caballero Gmez et al. / International Journal of Food Microbiology 167 (2013) 202207

AS-48

Planktonic
L. monocytogenes

Chaperone
DnaK
Energy
production

Sessile
L. monocytogenes

Chaperone
Dna K, GroEL
Energy
production

Carbohydrate
metabolism

Carbohydrate
metabolism

Protein
synthesis

Protein
synthesis

Fig. 3. Schematic representation of the effect of AS-48 on protein expression in planktonic

and sessile Listeria monocytogenes CECT 4032.

acknowledge the University of Jaen research program and Campus de

Excelencia Agroalimentario CeiA3. The mass analysis was carried out by
Peter Horvatovich from the Department of Analytical Biochemistry,
University of Groningen, The Netherlands.

References
Abriouel, H., Valdivia, E., Martnez-Bueno, M., Maqueda, M., Glvez, A., 2003. A simple
method for semi-preparative-scale production and recovery of enterocin AS-48
derived from Enterococcus faecalis subsp. liquefaciens A-48-32. J. Microbiol. Methods
55, 599605.
Abriouel, H., Lucas, R., Ben Omar, N., Valdivia, E., Glvez, A., 2010. Potential applications of
the cyclic peptide enterocin AS-48 in the preservation of vegetable foods and beverages. Probiotics Antimicrob. Proteins 2, 7789.
Begley, M., Cotter, P.D., Hill, C., Ross, R.P., 2010. Glutamate decarboxylase-mediated nisin
resistance in Listeria monocytogenes. Appl. Environ. Microbiol. 76, 65416546.
Bergonzelli, G.E., Granato, D., Pridmore, R.D., Marvin-Guy, L.F., Donnicola, D., CorthsyTheulaz, I.E., 2006. GroEL of Lactobacillus johnsonii La1 (NCC 533) is cell surface associated: potential role in interactions with the host and the gastric pathogen
Helicobacter pylori. Infect. Immun. 74, 425434.
Caballero Gmez, N., Abriouel, H., Grande, M.J., Prez Pulido, R., Glvez, A., 2012. Effect of
enterocin AS-48 in combination with biocides on planktonic and sessile Listeria
monocytogenes. Food Microbiol. 30, 5158.
Caldas, T.D., Yaagoubi, A.El., Richarme, G., 1998. Chaperone properties of bacterial elongation factor EF-Tu. J. Biol. Chem. 273, 1147811482.
Cardoso, K., Ferreira Gandra, R., Wisniewski, E.S., Aoki Osaku, C., Kimiko Kadowaki, M.,
Felipach-Neto, V., Aby-Azar Haus, L.F., Garcia Simo, R., 2010. DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii. J. Med.
Microbiol. 59, 10611068.
Cotter, P.D., Gahan, C.G., Hill, C., 2001a. A glutamate decarboxylase system protects Listeria
monocytogenes in gastric uid. Mol. Microbiol. 40, 465475.
Cotter, P.D., O'Reilly, K., Hill, C., 2001b. Role of the glutamate decarboxylase acid resistance
system in the survival of Listeria monocytogenes LO28 in low pH foods. J. Food Prot.
64, 13621368.
Cotter, P.D., Ryan, S., Gahan, C.G., Hill, C., 2005. Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at
low pH. Appl. Environ. Microbiol. 71, 28322839.
Desai, M.A., Soni, K.A., Nannapaneni, R., Schilling, M.W., Silva, J.L., 2012. Reduction of
Listeria monocytogenes biolms on stainless steel and polystyrene surfaces by essential oils. J. Food Prot. 75, 13321337.
EFSA, 2010. Scientic Report of EFSA: the community summary report on trends and
sources of zoonoses, zoonotic agents and food-borne outbreaks in the European
Union in 2008. (Accessed on March 23, 2013, http://www.efsa.europa.eu/en/
efsajournal/pub/1496.htm).

207

El-Ziney, M.G., Jakobsen, M., 2009. Effectiveness of reuterin alone and in combination
with nisin or other food contact surfaces sanitizers and cleaners for disinfection of
stainless steel surfaces contaminated with Escherichia coli and Listeria innocua.
J. Food Agric. Environ. 7, 145149.
Glvez, A., Valdivia, E., Martnez-Bueno, M., Maqueda, M., 1990. Induction of autolysis in
Enterococcus faecalis by peptide AS-48. J. Appl. Bacteriol. 69, 406413.
Glvez, A., Maqueda, M., Martnez-Bueno, M., Valdivia, E., 1991. Permeation of bacterial cells, permeation of cytoplasmic and articial membrane vesicles, and
channel formation on bilayers by peptide antibiotic AS-48. J. Bacteriol. 173,
886892.
Gandhi, M., Chikindas, M.L., 2007. Listeria: a foodborne pathogen that knows how to survive. Int. J. Food Microbiol. 113, 115.
Geer, L.Y., Sanford, P.M., Kowalak, J.A., Wagner, L., Xu, M., Maynard, D.M., Yang, X., Shi, W.,
Bryant, S.H., 2004. Open mass spectrometry search algorithm. J. Proteome Res. 3,
958964.
Granato, D., Bergonzelli, G.E., Pridmore, R.D., Marvin, L., Rouvet, M., Corthesy-Theulaz, I.E.,
2004. Cell surface-associated elongation factor Tu mediates the attachment of
Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins. Infect.
Immun. 72, 21602169.
Guerra, N.P., Araujo, A.B., Barrera, A.M., Torrado Agrasar, A., Lpez Macas, C., Carballo, J.,
Pastrana, L., 2005. Antimicrobial activity of nisin adsorbed to surfaces commonly
used in the food industry. J. Food Prot. 68, 10121019.
Hanawa, T., Fukuda, M., Kawakami, H., Hirano, H., Kamiya, S., Yamamoto, T., 1999. The
Listeria monocytogenes DnaK chaperone is required for stress tolerance and efcient
phagocytosis with macrophages. Cell Stress Chaperones 4, 118128.
Izquierdo, E., Horvatovich, P., Marchioni, E., Aoude-Werner, D., Sanz, Y., Ennahar, S., 2009.
2-DE and MS analysis of key proteins in the adhesion of Lactobacillus plantarum, a
rst step toward early selection of probiotics based on bacterial biomarkers. Electrophoresis 30, 949956.
Kapp, E.A., Schtz, F., Connolly, L.M., Chakel, J.A., Meza, J.E., Miller, C.A., Fenyo, D., Eng, J.K.,
Adkins, J.N., Omenn, G.S., Simpson, R.J., 2005. An evaluation, comparison, and accurate benchmarking of several publicly available MS/MS search algorithms: sensitivity
and specicity analysis. Proteomics 5, 34753490.
Latorre, A., Van Kessel, A.J.S., Karns, J.S., Zurakowski, M.J., Pradhan, A.K., Boor, K.J., Jayarao,
B.M., Houser, B.A., Daugherty, C.S., Schukken, Y.H., 2010. Biolm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with
Listeria monocytogenes. J. Dairy Sci. 93, 27922802.
Lianou, A., Sofos, J.N., 2007. A review of the incidence and transmission of Listeria
monocytogenes in ready-to-eat products in retail and food service environments.
J. Food Prot. 70, 21722198.
Liu, S., Graham, J.E., Bigelow, L., Morse, P.D., Wilkinson, B.J., 2002. Identication of Listeria
monocytogenes genes expressed in response to growth at low temperature. Appl.
Environ. Microbiol. 68, 16971705.
Lundn, J.M., Miettinen, M.K., Autio, T.J., Korkeala, H.J., 2000. Persistent Listeria
monocytogenes strains show enhanced adherence to food contact surface after short
contact times. J. Food Prot. 63, 12041207.
Maqueda, M., Glvez, A., Martnez Bueno, M., Sanchez-Barrena, M.J., Gonzlez, C., Albert,
A., Rico, M., Valdivia, E., 2004. Peptide AS-48: prototype of a new class of cyclic bacteriocins. Curr. Protein Pept. Sci. 5, 399416.
McLauchlin, J., Mitchell, R.T., Smerdon, W.J., Jewell, K., 2004. Listeria monocytogenes and
listeriosis: a review of hazard characterisation for use in microbiological risk assessment of foods. Int. J. Food Microbiol. 92, 1533.
Mendoza, F., Maqueda, M., Glvez, A., Martnez-Bueno, M., Valdivia, E., 1999. Antilisterial
activity of peptide AS-48 and study of changes induced in the cell envelope properties of an AS-48-adapted strain of Listeria monocytogenes. Appl. Environ. Microbiol.
65, 618625.
Palmer, M.E., Wiedmann, M., Boor, K.J., 2009. Sigma(B) and sigma(L) contribute to Listeria
monocytogenes 10403S response to the antimicrobial peptides SdpC and nisin.
Foodborne Pathog. Dis. 6, 10571065.
Pinto, E., Marques, N., Andrew, P.W., Faleiro, L., 2012. Over-production of P60 family proteins, glycolytic and stress response proteins characterizes the autolytic prole
Listeria monocytogenes. Adv. Microbiol. 2, 181200.
Snchez, B., Champomier-Vergs, M.C., Stuer-Lauridsen, B., Ruas-Madiedo, P., Anglade, P.,
Baraige, F., de los Reyes-Gaviln, C.G., Johansen, E., Zagorec, M., Margolles, A., 2007.
Adaptation and response of Bidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach. Appl. Environ. Microbiol. 73, 67576767.
Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy, S.L., Jones, J.L.,
Grifn, P.M., 2011. Foodborne illness acquired in the United States major pathogens. Emerg. Infect. Dis. 17, 715.
Siciliano, R.A., Cacace, G., Mazzeo, M.F., Morelli, L., Elli, M., Rossi, M., Malorni, A., 2008. Proteomic investigation of the aggregation phenomenon in Lactobacillus crispatus.
Biochim. Biophys. Acta Protein Proteomics 1784, 335342.
Tadesse, T., Mretr, T., Kohler, A., Axelsson, L., Naterstad, K., 2009. Complex phenotypic
and genotypic responses of Listeria monocytogenes strains exposed to the class IIa
bacteriocin sakacin P. Appl. Environ. Microbiol. 75, 69736980.
Tadesse, T., Mretr, T., Kohler, A., Axelsson, L., Naterstad, K., 2011. Global transcriptional
analysis of spontaneous sakacin P-resistant mutant strains of Listeria monocytogenes
during growth on different sugars. PLoS One 6, e16192.
Van der Veen, S., Abee, T., 2010. HrcA and DnaK are important for static and continuousow biolm formation and disinfectant resistance in Listeria monocytogenes.
Microbiology 156, 37823790.
Yu, X., Zheng, L., Yang, J., Lei, T., Ji, Y., 2011. Characterization of essential enolase in Staphylococcus aureus. World J. Microbiol. Biotechnol. 27, 897905.