Mode of Action of Antimicrobial Peptide P45 on Listeria Monocytogenes - Sirtori - 2008 - Journal of Basic Microbiology - Wiley Online Library

Journal of Basic Microbiology 2008, 48, 393–400 393

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Mode of action of antimicrobial peptide P45 on Listeria monocytogenes

Lisana Reginini Sirtori, Amanda de Souza da Motta and Adriano Brandelli

Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

The mode of action of an antimicrobial peptide produced by Bacillus sp. P45 isolated from the

intestine of the Amazonian basin fish Piaractus mesopotamicus was investigated. The anti-

microbial peptide was purified from culture supernatants by precipitation with ammonium

sulfate and gel filtration chromatography. The peptide has an EC50 of 300 AU (activity units)

ml–1 and kills all viable cells of Listeria monocytogenes with a concentration of 800 AU ml–1. A

decrease in OD was observed when L. monocytogenes cultures were treated with the peptide,

suggesting that cells were lysed. Transmission electron microscopy showed damage of the cell

envelope and loss of protoplasmic material. The peptide P45 was bactericidal and bacteriolytic

to L. monocytogenes. There is evidence that the mode of action is interefering at cell membranes

and the cell wall. The knowledge of the mode of action of antimicrobial peptides is an essential

step to consider their utilization in food or clinic.

Keywords: Bacteriocin / Bacillus / Listeriosis / Mode of action / Electron microscopy

Received: December 28, 2007; accepted: March 06, 2008

DOI 10.1002/jobm.200700406

Introduction*

Production of antimicrobial peptides is found as a

widespread strategy used by plants, animals and micro-

organisms to combat pathogenic microorganism. Be-

sides the variable structural characteristics, these pep-

tides are mostly cationic, showing an amphipathic

nature, containing about 30 to 100 aminoacid residues

in a linear or cyclic arrangement [1]. These peptides are

divided in several groups according their molecular

mass, secondary and tertiary structures, presence or

absence of disulfide bridges. Diverse biological activities

have been associated with these peptides like bacteri-

cidal, antifungal, antiviral and antitumoral [2–4].

The emergence of multidrug-resistant pathogens that

caused serious problems in hospitals worldwide has

intensified the search for novel drugs, in order to re-

place or to be used in complement with the existing

antibiotics. In this concern much interest has been

focused on a group of antimicrobial peptides, so-called

Correspondence: Adriano Brandelli, ICTA-UFRGS, Av. Bento Gonçal-ves 9500, 91501-970, Porto Alegre, Brazil E-mail: [email protected] Fax: +5551 3316 7048

bacteriocins. These peptides share some common phys-

ico-chemical properties, such as small molecular mass,

cationic and amphiphilic character, and being often

membrane active [5]. Some studies have demonstrated

that their mechanism of activity is associated with

membrane permeabilization, although different pep-

tides may act in different ways and the exact mecha-

nisms are only beginning to be elucidated [6]. Most

antimicrobial peptides kill target cells by membrane

permeabilization through peptide-lipid interactions.

Various mechanisms have been proposed, including the

formation of the discrete channels that dissipate ion

gradients across the membrane [7], disturbance of the

lipid bilayer as a result of carpet-like peptide binding

[8], phase separation due to specific peptide-lipid inter-

action [9], and detergent-like solubilization of the

membrane [10].

Some species of Bacillus have an extensive history of

safe use in the industry, and products from Bacillus spp.

are already used as food additives [11]. Several antimi-

crobial peptides representing diverse chemical struc-

tures are produced by Bacillus, being cyclic peptides like

gramicidin S and bacitracin, and lipopeptides like

iturins, bacilomycins and fengicins typical secondary

394 L. R. Sirtori et al. Journal of Basic Microbiology 2008, 48, 393–400


metabolites produced by this genus [12]. Production of

bacteriocins or bacteriocin-like substances (BLS) has

been described for several species of Bacillus such as

B. amyloliquefaciens, B. cereus, B. coagulans, B. licheniformis

among other [13–15]. B. subtilis produce diverse antimi-

crobial substances, some of them synthesized during

the exponential phase like subtilosin, subtilin, sub-

lancin, TasA, and other during the stationary phase,

like surfactin, bacilysin and iturins [12, 16].

The Amazon region contains an enormous biological

diversity, including about 40% of fresh water fishes

[17]. Despite the huge biological diversity of Amazonian

microbial life, relatively few studies have been devel-

oped to investigate other aspects than microbial ecol-

ogy [18]. More recently, some investigations have

focused the searching for microorganisms with bio-

technological interest, such as novel enzymes [19, 20],

and antimicrobials [21]. We recently described a new

Bacillus sp. isolated from the intestines of the teleost

fish Piaractus mesopotamicus of the Brazilian Amazon

basin. This strain produces an antimicrobial peptide

that was purified and characterized as a surfactin-like

peptide [22]. The aim of this work was to investigate the

effect and the mode of action of the antimicrobial pep-

tide P45, and to evaluate its potential use as antimicro-

bial to prevent the proliferation of L. monocytogenes.

Materials and methods

Bacterial strains The microorganism Bacillus sp. P45, isolated from the

fish Piaractus mesopotamicus, was used for peptide P45

production [22]. The indicator strain was Listeria monocy-

togenes ATCC 7644 (American Type Culture Collection,

Rockville, USA). Other strains were from the culture

collection of the Food Science and Technology Institute

(Porto Alegre, Brazil). Strains were stored at –20 °C in

BHI broth (Merck, Darmstadt, Germany) containing

20% (v/v) glycerol. Bacteria were propagated twice in

fresh BHI medium before they were used. The cultiva-

tion of strains was performed aerobically.

Production of antimicrobial substance The strain P45 was cultivated in 250 ml Erlenmeyer

flasks containing 50 ml of BHI broth at 30 °C for 48 h in

an orbital shaker (Cientec, Piracicaba, Brazil) operating

at 125 rpm. The culture was centrifuged for 15 min at

10,000 g and the resulting culture supernatant fluid

was sterilized by filtration through 0.22 µm cellulose

membranes (Millipore, Bedford, MA, USA). This culture

supernatant was used to evaluate the inhibitory effect

on growth of L. monocytogenes. Antimicrobial activity

was further purified by precipitation with ammonium

sulfate and gel filtration chromatography as described

elsewhere [22]. Alternativelly, the column was eluted

with 10 mmol l–1 phosphate buffer pH 7.0 containing

1.5 mol l–1 NaCl. Soybean trypsin inhibitor (21 kDa) and

bovine serum albumin (66 kDa) were also eluted to

obtain reference values for elution volumes.

Antimicrobial activity The antimicrobial activity was determined by the serial

two-fold dilution method [23]. Aliquots of 20 µl were

applied into cellulose discs on agar plates previouly

innoculated with a swab submerged in 8.5 g l–1 NaCl

solution containing 108 CFU ml–1 of L. monocytogenes

ATCC 7644. Plates were incubated at 37 °C for 24 h. The

antimicrobial activity titre was determined by a serial

dilution method previously described [24] and activity

was defined as the reciprocal of the dilution after the

last serial dilution giving a zone of inhibition and was

expressed as activity unit (AU) per milliliter.

Hemolytic activity The hemolytic activity was determined on sheep blood

agar plates [25]. An isolate of Staphylococcus aureus with

known hemolytic activity was used as a positive control.

Effect on the growth of L. monocytogenes An overnight culture of L. monocytogenes was obtained in

BHI at 37 °C for °C 18 h. A 500 µl sample of this culture

containing 105 CFU ml–1 was inoculated in tubes con-

taining 16 ml of BHI and incubated at 37 °C. The

growth was monitored at 2 h intervals by optical

density (OD) at 600 nm and by viable cell counts (CFU

ml–1). Crude BLS P45 preparation (final concentration

200 AU ml–1) was added to cultures of L. monocytogenes

after 4.5 h of cultivation, and the effect of crude BLS

P45 on turbidity and on the number of viable cells was

determined at 2 h intervals.

Determination of MIC and MBC The minimal inhibitory concentration (MIC) and the

minimal bactericidal concentration (MBC) of BLS P45

were determined as described previously [26]. Sterile

96-well microplates (Corning, New York, NY, USA) were

filled with 100 µl of serial dilutions of BLS P45 (concen-

trations ranging from 1600 AU ml–1 to 25 AU ml–1) and

then a standardized number of bacteria (100 µl of a

106 CFU ml–1 suspension) were added into each well. A

positive control was done using sterile NaCl solution

instead BLS P45. Negative controls were developed with

sterile BHI plus different concentrations of BLS P45.

Journal of Basic Microbiology 2008, 48, 393–400 Mode of action of antimicrobial peptide P45 on Listeria monocytogenes 395


Microplates were incubated at 37 °C for 24 h, and then

the MIC was determined as the last dilution at which

no increase in turbidity was observed. MBC was deter-

mined inoculating an aliquot of 20 µl from the wells

presenting negative growth to another microplate con-

taining 100 µl of sterile BHI. Controls were also

reinoculated. Microplates were incubated at 37 °C for

24 h. The last dilution where no growth was observed

was considered as MBC.

Dose-response curve A dose response curve was determined using different

concentration of BLS P45 (between 50 AU ml–1 and 6400

AU ml–1) and an initial inoculum of 106

CFU ml–1. Viable

counts were determined after incubation at 37 °C for

120 min. Kinetics of the BLS P45 effect on L. monocyto-

genes were determined at 37 °C with a BLS P45 concen-

tration of 1600 AU ml–1 and an initial inoculum of

106 CFU ml–1. The viable cell counts (CFU ml–1) and OD

at 600 nm were determined after 2, 4, 10, 20, 30, 40, 50,

60, 75, 90 e 105 min of incubation.

Transmission electron microscopy Listeria monocytogenes was incubated for 60 min with

1600 AU ml–1 of BLS P45. Cells were harvested by

centrifugation and washed twice with 0.1 mol l–1

phosphate buffer pH 7.3. The cells were fixed with

2.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde

in 0.12 mol l–1 phosphate buffer for 10 days and were

then postfixed in 2% (w/v) osmium tetroxide in the

same buffer for 45 min. Dehydration was done in a

graded acetone series (30% to 100%, v/v) and embedding

in Araldite-Durcopan® for 72 h at 60 °C. Thin sections

(Ultramicrotomo UPC 2.0, Leica) were mounted on grids,

covered with collodium film, and poststained with

uranyl acetate and Reynold’s lead citrate. Preparations

were observed with a Phillips EM 208-5 electron micro-

scope (Phillips) operating at 80 kV, or a JEOL JEM

1200ExII electron microscope (JEOL, Tokyo, Japan) oper-

ating at 120 kV.

Results

Antimicrobial activity was detected in culture super-

natant of Bacillus sp. P45. The effect of the crude pep-

tide P45 on the survival of L. monocytogenes ATCC 7644 is

shown in Fig. 1. Complete inhibition of growth was

observed after 90 min of BLS P45 addition. The bacterio-

lytic effect was observed by the inhibition of L. monocy-

togenes growth with the decreased OD600 during incuba-

tion time.

Figure 1. Effect of crude extract of BLS P45 on survival of Listeria monocytogenes ATCC 7466 at 37 °C. Optical density at 600 nm (�, �) and viability (CFU ml-1) (�, �) were monitored in control (�, �) and treated (�, �) cells with a concentration of 200 AU ml–1. The arrow indicates the time of BLS P45 addition. Each poit represents the mean of three independent experiments.

BLS P45 was partially purified and tested in vitro

against L. monocytogenes. The substance was purified

about 20-fold, with a yield of 16% and specific activity

of 3,048 AU mg–1. This substance was active against

L. monocytogenes. MIC was determined as 400 AU ml–1,

whereas a dose of 800 AU ml–1 was needed to achieve a

bactericidal effect (MBC). MIC values were similar for

other strains of Listeria, but higher for other bacteria

like Bacillus spp and Staphylococcus spp. (Table 1).

The hemolytic activity was assayed on sheep blood

agar plates and negative reactions were observed with

crude and purified fractions of BLS P45 (data not

shown).

The effect of BLS P45 concentration on the survival of

L. monocytogenes is shown in Fig. 2. The number of viable

cells was reduced upon increased the concentration of

Table 1. Minimal inhibitory concentration (MIC) of antimicrobial peptide P45 for selected bacteria.

Indicator bacteria MIC (AU ml–1)

Listeria monocytogenes ATCC 7644 400 Listeria monocytogenes 4C 400 Listeria monocytogenes 78/03 200 Listeria innocua 33090 400 Listeria innocua 1572 400 Listeria sp. (clinical isolate) 400 Bacillus cereus (food isolate) 800 Bacillus subtilis (food isolate) 800 Corynebacterium fimi NCTC 7547 400 Leuconostoc mesenteroides 400 Rhodococcus sp. (clinical isolate) >1600 Enterococcus faecalis (food isolate) >1600 Staphylococcus aureus 4059 >1600 Staphylococcus intermedius (clinical isolate) >1600



Figure 2. Effect of BLS P45 concentration on cell viability of L. monocytogenes ATCC 7644. Viable cell counts were determined after treatment with different BLS P45 concentrations during 120 min at 37 °C. The initial inoculum was 106 CFU ml–1. Each poit represents the mean ± SEM of three independent experiments.

the antimicrobial substance. Complete inhibition of

growth was observed with 800 AU ml–1. The EC50, con-

sidered the concentration that caused half-reduction of

viable counts, was around 300 AU ml–1.

The kinetics of the BLS P45 effect on growth of

L. monocytogenes was investigated. The addition of BLS

P45 (3200 AU ml–1) to a cell suspension of L. monocyto-

genes resulted in a decrease in viable counts of 4 log

cycles related to the control within 10 min of incuba-

tion (Fig. 3). After 20 min, complete cell death was ob-

served. The inhibition of L. monocytogenes growth also

resulted in decreased OD600 during the incubation time.

L. monocytogenes harvested from culture were incu-

bated with 1600 AU ml–1 of BLS P45 for 60 min. After

Figure 3. Kinetics of the BLS P45 effect on L. monocytogenes ATCC 7644. Viability was monitored in control (�) and treated (�) cells with a BLS P45 concentration of 1600 AU ml–1. Each poit represents the mean ± SEM of three independent experiments.

Figure 4. Transmission electron microscopy of L. monocytogenes. Control cells (upper panel) and after treatment for 60 min with 1600 AU ml–1 of BLS (lower panel).

incubation, the microorganisms were prepared for

transmission electron microscopy (TEM). L. monocyto-

genes cells treated with BLS P45 showed vesiculization

of the protoplasm, pore formation, desintegration of

the cells and extensive damage of the cell envelope

(membranes and cell wall) (Fig. 4).

The BLS P45 was submitted to gel filtration chroma-

tography under different conditions. When the column

was eluted with phosphate buffer, the antimicrobial

activity was eluted near the void volume of the column,

corresponding to a Mr of about 150 kDa (Fig. 5). The

addition of 1.5 mol l–1 NaCl to this same buffer resulted

in the detection of antimicrobial activity within the

resolution volume of the column (Fig. 5). In this last



Figure 5. Elution profile of BLS P45 during gel filtration chroma-tography on Sephadex G-100. The column was eluted with 10 mmol l–1 phosphate buffer (�, �) or this same buffer containing 1.5 mol l–1 NaCl (�, �). Fractions were monitored for absorbance at 280 nm (�, �) and antimicrobial acitivty (�, �).

case, the elution volume of BLS P45 was closer to that

observed for soybean trypsin inhibitor (21 kDa). The

antimicrobial activity was maintained after treatment

with Tween 20 and Tween 80 and sodium deoxycholate

(residual activity was 70%).

Discussion

Only a few bacteria producing bacteriocins have been

isolated from fish intestines, like Carnobacterium diver-

gens V41 and C. piscicola V1 from rainbown trout [27],

Vibrio sp. NM10 and Bacillus sp. NM12 [28] from coastal

japonese fishes. More recently bacteriocin-like peptides

produced by bacteria isolated from Amazonian fishes

have been described [15, 21]. Among those bacteria, the

strain P45 produces a surfactin-like peptide that was

previously purified and characterized [22]. In this work,

the effect and the mode of action of BLS P45 on indica-

tor bacteria were addressed, particularly using L. mono-

cytogenes as the target organism.

Antimicrobial activity eluted at the void volume of a

Sephadex G-100 suggesting a molecular mass of about

150 kDa. However in the presence of high ionic

strenght the activity eluted in the resolution volume of

the column, corresponding to a molecular mass near to

20 kDa, suggesting that the secreted peptide form large

extracellular aggregates. This fact agrees with the mo-

lecular mass of the purified peptide, determined as

1,450 Da by mass spectroscopy [22]. This behavior has

been described for some bacteriocins and other antimi-

crobial peptides, such as the linocin M18, produced by

Brevibacterium linens [29]. The organization of bacterio-

cins as large aggregates has been associated to the

highly hydrophobic nature of the peptides [14].

Different experiments were carried out with the aim

of elucidate the mode of action of the BLS produced by

Bacillus sp. P45. The results suggest that this substance

has a bactericidal effect on L. monocytogenes, based on

the decrease in the number of viable cells after the

addition of the BLS P45, with complete cell death

within 20 min. The rapid bacterial death suggests that

the BLS targets to the cell wall or the cytoplasmic

membrane. The bacteriolytic effect was also indicated

by the rapid reduction of OD600, indicating that the cells

of L. monocytogenes were lysed. These observations were

corroborated by transmission electron microscopy

which clearly showed that the cell lysis had occured

after treatment with the antimicrobial substance. The

effect of the antimicrobial substance depends on the

conditions of the experiment, like the dose and degree

of purity of the bacteriocin, the indicator strain and its

cellular concentration [30]. In this regard bacteriostatic

may become a bacteriolytic effect by simply increasing

the bacteriocin dose [24]. In this context, some authors

suggest that bacteriocin-induced lysis could be due to

the liberation of autolytic enzymes, that are usually

electrostatically bound to anionic polymers (teichoic

and lipoteichoic acids) of the cell wall, that are dis-

placed by cationic bacteriocins from their binding sites

[31].

Transmission electron microscopy showed that the

effect on L. monocytogenes must be regarded as indicative

of cytoplasmic membrane alteration induced by BLS

P45, since untreated cells were not injured. Further-

more, since the cytoplasmic membrane is cooperative

in the cell wall synthesis and turnover, perturbation of

this membrane may also affect cell wall integrity and

autolysis regulation [32].

The damage of cell membranes agrees with the

mechanism proposed for some bacteriocins of Bacillus.

Subtilosin A binding to lipid bilayers is modulated by

the lipid composition of the membranes [33]. Mem-

brane bound subtilosin A may adopt an orientation in

which the hydrophobic region of the molecule is im-

mersed into the bilayers and the negative charged re-

gion is exposed. It may be that these characteristics

supplement or enhance the interaction of subtilosin A

with a membrane or receptor, which may lead to the

activity of the peptide [34]. The mechanism of mem-

brane interaction of two amphipathic peptides, MSI-78

and MSI-594, derived from magainin 2 and melittin has

been studied. Both the peptides show excellent antimi-

crobial activity and adopt α-helical conformation upon



binding to membrane and that peptide-induced disor-

der depend on the lipid composition of bilayers [35].

The results of Ramammorthy et al. [36] on antimicrobial

peptide G15 suggest that the peptide prefers to bind

with anionic lipid bilayers altering the conformation

of the lipid head group as reported in the literature

[37].

Some bacteriocins are well established as antimicro-

bials for potential application in food preservation sys-

tems. Nisin A already showed effective inhibitory activ-

ity on the Listeria monocytogenes growth in cheese [38].

Enterocin, when inoculated in ham, pig meat, meat

chicken and sausage, showed inhibitory capacity on the

L. monocytogenes growth. Lactocin also had inhibitory

capacity on the same microorganism when applied in

ground meat [38, 39]. Nisin presents desirable proper-

ties for food preservation, like the absence of toxi-

city and undesired taste or flavor, heat and storage

stability, is degraded by digestive enzymes, is naturally

produced by Lactococcus lactis, and has prominent anti-

microbial spectrum against Gram-positive microorgan-

isms [40].

Bacteriocins produced by different Bacillus species

showed variable antimicrobial spectrum. The bacterio-

cin of B. thuringiensis HD9 showed inhibitory activity on

other B. thuringiensis strains and antifungical activity

against Aspergillus nidulans and Fusarium graminis [41].

Although BLS P45 inhibited only Kluyveromyces marx-

ianus among fungi [22], it showed a broad inhibitory

spectrum against bacteria, resembling subtilosin A

[42].

Listeriosis is one of the most important food-borne

diseases, and several regulatory agencies have made

efforts to control this disease worldwide [43]. L. monocy-

togenes is widely distributed in the environment, and is

therefore present in several foods from both animal

and vegetal origin [43, 44]. L. monocytogenes is sensitive

to several bacteriocins, like nisin, pediocin and lactoco-

cin [45]. Indeed, the use of bacteriocins or bacteriocino-

genic strains to inhibit this pathogen in dairy and meat

products have been described [38]. However, this bacte-

rium can become highly resistant to some bacteriocins,

such as nisin and pediocin [46, 47], indicating the rele-

vance of searching for new antimicrobials to control

this pathogen.

The growing increase in the bacterial resistance to

conventional antimicrobials and the searching for new

preservation methods for the food chain raise up the

interest for these antimicrobial substances. This BLS

P45 has potential application as a natural biopreserva-

tive to control pathogenic and spoilage microorgan-

isms.

Acknowledgements

Authors thank the Center of Electron Microscopy (CME-

UFRGS) for technical support in TEM experiments. This

work received financial support of Conselho Nacional

de Desenvolvimento Científico e Tecnológico (CNPq,

Brazil).

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