A highly thermostable antimicrobial peptide from Aspergillus clavatus ES1: biochemical and molecular characterization.

Abstract

Antimicrobial peptides (AMPs) are extremely attractive candidates as therapeutic agents due to their wide spectrum of antimicrobial activity and mechanism of action, which differs from that of small-molecule antibiotics. In this study, a 6.0-kDa antimicrobial peptide from Aspergillus clavatus ES1, designated as AcAMP, was isolated by a one-step heat treatment. AcAMP was sensitive to proteolytic enzymes, stable between pH 5.0 and 10.0, and heat resistant (15 min at 100 degrees C). The acamp gene encoding AcAMP peptide was isolated by reverse-transcriptase polymerase chain reaction (RT-PCR) and cloned in pCRII-TOPO vector. Sequence analysis of the complementary DNA (cDNA) acamp gene revealed an open reading frame of 282 bp encoding a peptide of 94 amino acid residues consisting of a 21-aa signal peptide, a 22-aa pro-peptide, and a 51-aa mature peptide. The deduced amino acid sequence showed high identity with other ascomycete antifungal peptides. AcAMP belongs to the group of small, cysteine-rich, basic proteins with antimicrobial activity. In addition to its antifungal activity, AcAMP is the first fungal peptide exhibiting antibacterial activity against several Gram-positive and Gram-negative bacteria. Based on all these features, AcAMP can be considered as a promising new member of the restraint family of ascomycete antimicrobial peptides that might be used in biological control of plant diseases and also for potential applications in food preservation.

Full text

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J Ind Microbiol Biotechnol (2010) 37:805–813
DOI 10.1007/s10295-010-0725-6
123
ORIGINAL PAPER
A highly thermostable antimicrobial peptide from Aspergillus
clavatus ES1: biochemical and molecular characterization
Mohamed Hajji · Kemel Jellouli · Noomen Hmidet ·
RaWk Balti · Alya Sellami-Kamoun · Moncef Nasri
Received: 12 January 2010 / Accepted: 11 April 2010 / Published online: 4 May 2010
© Society for Industrial Microbiology 2010
Abstract
attractive candidates as therapeutic agents due to their wide
spectrum of antimicrobial activity and mechanism of
action, which diVers from that of small-molecule antibiotics.
In this study, a 6.0-kDa antimicrobial peptide from
Aspergillus clavatus ES1, designated as AcAMP, was iso-
lated by a one-step heat treatment. AcAMP was sensitive to
proteolytic enzymes, stable between pH 5.0 and 10.0, and
heat resistant (15 min at 100°C). The acamp gene encoding
AcAMP peptide was isolated by reverse-transcriptase poly-
merase chain reaction (RT-PCR) and cloned in pCR®II-
TOPO vector. Sequence analysis of the complementary
DNA (cDNA) acamp gene revealed an open reading frame
of 282 bp encoding a peptide of 94 amino acid residues
consisting of a 21-aa signal peptide, a 22-aa pro-peptide,
and a 51-aa mature peptide. The deduced amino acid
sequence showed high identity with other ascomycete anti-
fungal peptides. AcAMP belongs to the group of small,
cysteine-rich, basic proteins with antimicrobial activity. In
addition to its antifungal activity, AcAMP is the Wrst fungal
peptide exhibiting antibacterial activity against several
Gram-positive and Gram-negative bacteria. Based on all
these features, AcAMP can be considered as a promising
new member of the restraint family of ascomycete antimi-
crobial peptides that might be used in biological control of
plant diseases and also for potential applications in food
preservation.
Antimicrobial peptides (AMPs) are extremely
Keywords
Antibacterial · Antifungal · Thermostable
AcAMP · Aspergillus clavatus ES1 ·
Introduction
During recent decades, bacteria resistance to most clinically
available antimicrobial agents has emerged at an alarming
rate. Consequently, discovery of new antimicrobials has
become increasingly crucial [29]. Most research has
focused on investigation of natural products as sources of
novel bioactive molecules. Antimicrobial peptides (AMPs),
widely expressed by a diverse range of organisms, repre-
sent a new family of antibiotics that have stimulated
research and clinical interest as new therapeutic options for
infections caused by multidrug-resistant bacteria and fungi
[29]. It has been shown that AMPs possess antibacterial,
antiviral, antifungal, antitumor, and immunomodulatory
activities [11]. These peptides are generally short, with a
broad spectrum of antimicrobial activity, strong thermal
stability, and low immunogenicity [31]. AMPs are gener-
ally deWned as peptides of fewer than 100 amino acid resi-
dues with an overall positive charge imparted by the
presence of multiple lysine and arginine residues and a sub-
stantial portion (¸30% or more) of hydrophobic residues.
To date, more than 800 AMPs with several diVerent
sequence motifs have been isolated from a wide range of
organisms including bacteria, fungi, plants, and vertebrates
[10, 13]. A common feature shared among the cationic
AMPs is their ability to fold into amphipathic conforma-
tions, often induced by interaction with membranes or
membrane mimics [11]. Alongside their direct antimicro-
bial activities against Gram-positive and Gram-negative
bacteria, these peptides play additional roles as antibiotics
against fungi [23] and protozoa [1].
M. Hajji (&) · K. Jellouli · N. Hmidet · R. Balti ·
A. Sellami-Kamoun · M. Nasri
Laboratoire de Génie Enzymatique et de
Microbiologie, Ecole Nationale d’Ingénieurs de Sfax,
1173-3038 Sfax, Tunisia
e-mail: hajjimed1989@yahoo.fr

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806J Ind Microbiol Biotechnol (2010) 37:805–813
123
Filamentous fungi are valuable sources of small anti-
fungal peptides. A limited number of peptides have so far
been isolated from this restraint family. The Wrst Wlamen-
tous fungus described as a potent producer of an antifungal
peptide, called AFP, was Aspergillus giganteus [16, 19].
An antifungal peptide, named PAF, was then isolated from
Penicillium chrysogenum [20], and later another antifungal
peptide, termed AnAFP, was identiWed from Aspergillus
niger [18]. In addition, NAF peptide was isolated from
Penicillium nalviogense and found to be identical to PAF
[10]. In fact, AFP showed 35.6% and 47.1% homology to
AnAFP and PAF/NAF, respectively. Recently, AcAFP
antifungal peptide was isolated from A. clavatus VR1 strain
[27]. All these reports dealt with the antifungal activity of
isolated peptides, whereas no antibacterial activity was
described for these strains, especially for A. clavatus
species.
In this paper, we report for the Wrst time the isolation of a
potent antibacterial peptide from A. clavatus ES1. We also
investigate its biochemical properties. The gene encoding
the antibacterial peptide was cloned and sequenced.
Materials and methods
Strains, plasmids, and growth conditions
A. clavatus ES1 was isolated from wastewater (Sfax, Tunisia).
It was identiWed on the basis of 860 bp of the 18S ribosomal
RNA (rRNA) analysis (EF151929) [12]. The strain was prop-
agated on potato dextrose agar (PDA) plates at 30°C, and
conidia were prepared from 7-day-old colonies by Xooding
with 10 ml sterile distilled water and scraping the agar plates.
Escherichia coli TOP10 (F¡ mcrA ?(mrr-hsdRMS-
mcrBC) ?80lacZ?M15 ?lac?74 recA1 araD139 ?(ara-
leu) 7697 galU galK rpsL (StrR) endA1 nupG) was used as
a host for plasmid propagation. The strain was purchased
from Invitrogen Life Technologies. Culture of diVerent
E. coli clones was done in Luria–Bertani (LB) medium
composed of (g l¡1): peptone, 10; yeast extract, 5; and
NaCl, 5.
The plasmid pCR®II-TOPO® (Invitrogen Corporation,
USA) was used for gene cloning. Wizard® Plus SV Mini-
preps DNA PuriWcation System kit (Promega, USA) was
used for plasmid preparation.
Production of AcAMP peptide
Production of AcAMP was carried out in medium contain-
ing (g l¡1): yeast extract, 10; peptone, 5; NaCl, 5; glucose,
20; pH 7.0. Culture media were inoculated with 107 spores
ml¡1 and incubated on a rotatory shaker (150 rev min¡1) for
96 h at 30°C in 300-ml Erlenmeyer Xasks with working
volume of 50 ml. The culture supernatant was obtained by
Wltration and centrifugation at 8,000 rpm during 15 min.
The resultant supernatant was used as crude peptide prepa-
ration.
One-step puriWcation of AcAMP and N-terminal amino
acid sequence
The thermostable character of AcAMP was exploited in the
puriWcation procedure. The supernatant was incubated at
100°C for 10 min, and then thermolabile and insoluble
materials were removed by centrifugation at 13,000 rpm for
15 min. The purity of the AcAMP was examined by 15%
(w/v) sodium dodecyl sulfate–polyacrylamide gel electro-
phoresis (SDS–PAGE) under denaturing conditions as
described by Laemmli [17]. The molecular weight of the
puriWed AcAMP was estimated using a low-molecular-
mass calibration kit as markers consisting of bovine serum
albumin (66 kDa), egg white ovalbumin (45 kDa), glyceral-
dehyde-3-P dehydrogenase (36 kDa), bovine carbonic
anhydrase (29 kDa), bovine trypsinogen (24 kDa), soybean
trypsin inhibitor (20.1 kDa), and bovine a-lactalbumin
(14.2 kDa). The peptide concentration was measured by the
method of Bradford [5] using the Bio-Rad protein assay kit
with bovine serum albumin as a standard.
The region containing the AcAMP band was excised and
electrophoretically transferred from an SDS–PAGE to a
polyvinylidene diXuoride (PVDF) membrane. Then, the
N-terminal amino acid sequence was determined by the
Edman degradation method using an ABI Procise 494 pro-
tein sequencer (Applied Biosystems).
Antimicrobial assay
Antibacterial activities of puriWed AcAMP were tested
against seven bacterial strains: Staphylococcus aureus
(ATCC 25923), Micrococcus luteus (ATCC 4698), Esche-
richia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853), Klebsiella pneumonia (ATCC 13883),
Bacillus cereus (ATCC 11778), and Enterococcus faecalis
(ATCC 29212). Antifungal activities were tested using
Aspergillus niger, Fusarium solani, and F. oxysporum. The
strains were kindly provided from the Center of Biotech-
nology, Sfax, Tunisia.
The antibacterial and antifungal activities were assessed
according to the method described by Berghe and Vlietinck
[4]. BrieXy, 200 ?l of tested microorganisms [106 colony-
forming units (cfu) ml¡1 of bacteria cells and 108 spores
ml¡1 of fungal strains] was spread on Luria–Bertani (LB)
agar and PDA medium, respectively. Then, wells (3 mm
depth, 4 mm diameter) were made using a sterile borer and
were loaded with 50 ?l peptide solution at desired concen-
trations as estimated by Bradford assay. Supernatant from

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123
uninoculated medium was used as negative control. The
Petri dishes were kept, Wrstly for 1 h at 4°C, and then were
incubated for 24 h at 37°C for bacteria and 72 h at 30°C for
fungal strains. Antimicrobial activity was evaluated by
measuring the growth inhibition zone diameter in millime-
ters (including well diameter of 4 mm). Measurements of
inhibition zones were carried out for three sample replica-
tions, and values are the average of three replicates.
EVects of enzymes, heat, and pH on AcAMP activity
In order to evaluate the sensitivity of AcAMP to proteolytic
enzymes, a solution of the isolated peptide (200 ?g ml¡1)
was incubated at 37°C for 1 h with 2 mg ml¡1 Wnal concen-
tration of the following enzymes: trypsin, proteinase K,
Alcalase, and chymotrypsin in 100 mM Tris–HCl buVer
(pH 8.0) and pepsin in 100 mM sodium acetate buVer (pH
4.0). The sensitivity of AcAMP to EndoH, Thermamyl®,
and lipase was also investigated. Samples were then boiled
for 2 min to inactivate the enzyme. The antimicrobial activ-
ities were then measured by the agar well diVusion method
against Bacillus cereus (ATCC 11778) as bacterial indica-
tor strain. Trypsin, proteinase K, chymotrypsin, pepsin,
EndoH, and lipase were purchased from Sigma Chemical
Co. (St. Louis, MO, USA). Thermamyl® and Alcalase were
purchased from Novozymes A/S, Bagsvaerd-Denmark.
To investigate thermal stability, puriWed AcAMP pep-
tide was incubated at 50°C, 70°C, 90°C, and 100°C for 5,
10, and 15 min. After cooling the treated samples on ice for
10 min, antimicrobial activity against B. cereus was mea-
sured as mentioned above.
pH stability was determined by adjusting samples of puri-
Wed AcAMP peptide with 5 M NaOH or 5 M HCl to diVerent
pH values ranging from 3.0 to 11.0, followed by incubation
for 1 h at room temperature. Antimicrobial activity against
B. cereus was then assayed as mentioned above.
Determination of minimum inhibitory concentration (MIC)
MIC values were determined by microwell dilution method
[8]. The puriWed AcAMP peptide was subjected to dilution
series in a 96-well microtiter plate. The antibiotic gentamy-
cin was used as positive standard and AcAMP free solution
was used as negative control. Each well of the microplates
included 40 ?l growth medium, 10 ?l inoculum (106
cfu ml¡1), and 50 ?l diluted AcAMP peptide. Then, the
microplates were incubated overnight at 37°C. As an indi-
cator of microorganisms growth, 40 ?l p-iodonitrotetrazo-
lium violet (INT) dissolved in water was added to the wells
and incubated at 37°C for 30 min. The colorless tetrazolium
salt acts as an electron acceptor and is reduced to a red-col-
ored formazan product by biologically active organisms
[8]. Where microbial growth was inhibited, the solution in
the well remained clear after incubation with INT. Evalua-
tion of MIC values was carried out in triplicate.
RNA isolation and reverse transcription
A. clavatus ES1 was grown in the medium described above.
After 96 h, mycelia from a 50-ml culture were harvested by
Wltration through no. 1 Whatman Wlter, washed with sterile
distilled water, quickly frozen in liquid nitrogen, and dis-
rupted by grinding; total RNA was extracted with Trizol
reagent (Invitrogen). The RNA pellet was collected by cen-
trifugation (14,000g, 15 min) and washed twice with 75%
ethanol (v/v). The RNA preparation was resuspended in
Diethylenepyrocarbonate (DEPC)-treated water, and the
purity of the preparation was estimated by calculating
A260 nm/A280 nm ratios. Reverse-transcriptase polymerase
chain reaction (RT-PCR) was performed using the
SuperScript™III Reverse Transcription System (Invitro-
gen). Total RNA (1.0 ?g), random primers (250 ng) in
nuclease-free water, 10 mM dNTP mix (1.0 ?l), and sterile
distilled water to 13 ?l were preheated for 5 min at 70°C
and then chilled in ice-water for 5 min. The following
reagents were then added: £5 First-Strand buVer (250 mM
Tris HCl pH 8.3, 375 mM KCl, 15 mM MgCl2) (4.0 ?l),
0.1 M 1,4-Dithiothreitol (DTT) (1.0 ?l), RNaseOUT™
Recombinant RNase Inhibitor (Invitrogen) (1.0 ?l), and
SuperScript™III Reverse Transcriptase (200 units ?l¡1)
(1.0 ?l). The reaction was incubated at 50°C for 50 min,
and the enzyme was then inactivated at 70°C for 15 min.
The resultant cDNA was used as template.
Cloning and cDNA sequencing of acamp gene
The primers used for isolation, sequencing, and cloning of
the open reading frame (ORF) encoding AcAMP peptide
were designed according to A. clavatus NRRL1 gene
(accession number: XM_001272037). PCR was performed
with forward primer AcAMPF: 5?-GCACAACCCCCGCC
GCAGTTT-3? and reverse primer AcAMPR: 5?-ACTC
CGCCCTTTCGCCAGCTCC-3?. PCR was carried out in a
Thermo Hybaid PCR Express thermocycler. The ampliWca-
tion reaction mixtures (50 ?l) contained 20 ng cDNA as
template with 1.0 unit Go Taq DNA polymerase, 10 mM
dNTP, £5 Go Taq buVer, and 20 ?M of each primer.
AmpliWcation of DNA was carried out under the following
conditions: denaturation (1 min at 94°C), annealing (45 s at
53°C), and extension (1 min at 72°C) for 35 cycles, fol-
lowed by 10 min at 72°C. AmpliWed PCR products were
analyzed on a standard 1% (w/v) agarose gel containing
ethidium bromide and puriWed by PureLink™ PCR puriWca-
tion kit (Invitrogen).
The puriWed PCR product was cloned in pCR®II-TOPO
plasmid vector according to the manufacturer’s protocol

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808J Ind Microbiol Biotechnol (2010) 37:805–813
123
(Invitrogen Corporation). Competent cells of E. coli TOP10
were transformed with the ligation mixture. The recombi-
nant plasmids were then puriWed with Wizard® Plus SV
Minipreps DNA PuriWcation System kit (Promega, USA).
The presence of the appropriate insert was checked by
PCR.
The nucleotide sequence of acamp gene was determined
by dideoxynucleotide chain termination method using an
automated DNA sequencer ABI Prism_3100-Avant Genetic
Analyzer (Applied Biosystems) using M13 primers from
both strands. In order to conWrm the Wdelity of the sequence,
the gene encoding acamp of A. clavatus ES1 was sequenced
three times using recombinant plasmids from three diVerent
clones as templates, in each case, to exclude potential
sequencing errors that can occur during PCR reaction.
Database searches were performed online with the pro-
grams blastx, blastp, and blastn provided by the BLAST
E-mail server [2]. CLUSTALW was used to align the amino
acid sequences. All parameters were set at their default values.
Nucleotide sequence accession number
The nucleotide sequence of acamp gene has been submitted
to the GenBank database and assigned the accession num-
ber GU390689.
Statistical analysis
Values are expressed as mean § standard deviation. Analy-
sis of variance (ANOVA) was conducted, and diVerences
between variables were tested for signiWcance by one-way
ANOVA using the Statistical Package for the Social Sci-
ences (SPSS, version 11) program. DiVerences at P · 0.05
were considered statistically signiWcant. Correlation and
regression analysis was carried out using Excel software
(Microsoft Corporation, USA).
Results
PuriWcation and N-terminal sequence of AcAMP peptide
An antimicrobial peptide from A. clavatus ES1, named
AcAMP, was puriWed in one step. The thermostable charac-
ter of the AcAMP peptide was exploited after having con-
Wrmed this feature in culture supernatant. Heat treatment
for 10 min at 100°C eVectively removed a large amount of
inactive and thermosensitive proteins from the crude super-
natant. After removing the precipitated proteins, the result-
ing supernatant contained only one small protein with
molecular mass of about 6.0 kDa as estimated by
SDS–PAGE (Fig. 1). A 50-?l sample of the obtained frac-
tion, at 200 ?g ml¡1, was used to test antimicrobial activity
against B. cereus as indicator strain. A large inhibition zone
(diameter about 22 mm) was detected, showing that the 6.0-
kDa peptide had antimicrobial activity. It is interesting to
note that AcAMP peptide was produced only when cultures
were incubated at 30°C. No antimicrobial activity was
detected in the supernatant from culture incubated at 37°C.
This observation suggests that the acamp gene could be
regulated by a thermosensitive activator.
The N-terminal sequence of the Wrst 19 amino acid resi-
dues of the puriWed AcAMP peptide was found to be
ATYDGKCYKKDNICKYKAQ (Table 1). Findings showed
that the N-terminal sequence of the mature AcAMP peptide
is identical to that of antifungal peptide (AcAFP) from
A. clavatus VR1 [27]. A single change at the fourth residue
was detected compared with AFP from A. giganteus: Asp in
AcAMP peptide is replaced by Asn in AFP [24]. The N-ter-
minal sequence of AcAMP peptide exhibits a lower degree
of homology with PAF peptide from P. chrysogenum at 58%
identity. Furthermore, AnAFP peptide from A. niger has no
similarity with AcAMP peptide from A. clavatus ES1. No
antibacterial activity was reported for these peptides from
described strains.
Antimicrobial spectrum of AcAMP peptide
AcAMP peptide from A. clavatus ES1 was tested against
various pathogenic strains by agar well diVusion method.
The puriWed AcAMP peptide exhibited inhibitory activities
Fig. 1 Sodium dodecyl sulfate–polyacrylamide gel 15% (w/v) elec-
trophoresis of the puriWed AcAMP peptide: lane 1 molecular mass
marker (Amersham Biosciences); lane 2 puriWed AcAMP peptide
obtained after heat treatment for 15 min at 100°C
1 2
66
45
36
29
24
20
14

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123
towards Gram-positive bacteria including S. aureus,
B. cereus, M. luteus, and E. faecalis. PuriWed AcAMP pep-
tide was also active against some Gram-negative bacteria
such as P. aeruginosa and E. coli, but was not active
towards K. pneumonia (Table 2). MIC was also assessed by
liquid growth inhibition assay. Antibacterial activity was
greater against Gram-positive bacteria, especially B. cereus
and M. luteus strains with MIC value of 10 ?g ml¡1, which
was similar to gentamycin as positive control. E. coli and
P. aeruginosa were also sensitive toward puriWed AcAMP
peptide, with MIC values of 30 and 50 ?g ml¡1, respec-
tively. The highest MIC value occurred with K. pneumonia
(>200 ?g ml¡1) (Table 2).
The antifungal activity of puriWed AcAMP peptide
was also evaluated against A. niger, F. solani, and
F. oxysporum. The results showed inhibitory eVects on the
growth of all studied fungi (Table 2). The inhibition diame-
ter zones were 11, 15, and 12 mm, using 50 ?l at
200 ?g ml¡1, respectively. F. solani was found to be the
most sensitive fungal strain to AcAMP peptide. As can be
seen in Table 2, the inhibitory eVects of AcAMP peptide
increased with increasing concentration.
EVects of enzymes, heat treatment, and pH on antibacterial
activity of AcAMP peptide
After puriWed AcAMP peptide was treated with several
enzymes, heat, and pH, its antimicrobial activity was
assayed against B. cereus. As reported in Table 3, the
antimicrobial activity of AcAMP was totally lost only
when treated with the proteolytic enzymes Alcalase, tryp-
sin, chymotrypsin, pepsin, and proteinase K, indicating
that the antimicrobial substance was proteinaceous. How-
ever, antimicrobial activity was unaVected by Thermamyl
and EndoH, suggesting that the peptide was not glycosyl-
ated or its activity was independent of glycosylation
(Table 3).
Table 1 N-terminal sequence
comparison of AcAMP with
AcAFP, AFP, PAF, and AnAFP
antifungal peptides
Peptide MicroorganismN-terminal sequence Reference
AcAMP
AcAFP
AFP
PAF
AnAFP
A. clavatus ES1
A. clavatus VR1
A. giganteus
P. chrysogenum
A. niger
ATYDGKCYKKDNICKYKAQ
ATYDGKCYKKDNICKYKAQ
ATYNGKCYKKDNICKYKAQ
AKYTGKCTKSKNECKYKND
LSKYGGECSLEHNTCTYRK
Present report
[27]
[25]
[20]
[18]
Table 2 Spectrum of antimi-
crobial activity of AcAMP pep-
tide from A. clavatus ES1
Microorganism Inhibition zone (mm)MIC (?g ml¡1)
50 ?g ml¡1
100 ?g ml¡1
200 ?g ml¡1
AcAMP peptideGentamycin
S. aureus
B. cereus
M. luteus
E. faecalis
E. coli
P. aeruginosa
K. pneumonia
A. niger
F. solani
F. oxysporum
11.0 § 1.0
15.0 § 2.0
12.0 § 1.0
10.0 § 1.5
12.0 § 1.0
8.0 § 1.0
0
7.0 § 1.0
10.5 § 2.0
10.0 § 1.0
16.0 § 0.5
18.0 § 1.0
14.0 § 0.5
12.0 § 1.0
13.0 § 2.0
11.0 § 1.0
0
8.0 § 1.0
12.0 § 1.0
10.0 § 1.0
19.0 § 1.0
22.0 § 1.0
18.0 § 1.0
13.0 § 1.0
16.0 § 1.0
12.5 § 1.0
0
11.0 § 0.5
15.0 § 1.0
12.0 § 1.0
20.0
10.0
10.0
50.0
30.0
50.0
>200.0
NT
NT
NT
08.0
08.0
12.0
10.0
20.0
10.0
16.0
NT
NT
NT
NT not tested
Table 3 EVects of enzymes and pH on the activity of puriWed AcAMP
peptide from A. clavatus ES1 toward the indicator strain B. cereus
(ATCC 11778) at concentration of 200 ?g ml¡1
Inhibition diameter zone (mm)
Control
Enzymes
Alcalase
Trypsin
Chymotrypsin
Pepsin
Proteinase K
Thermamyl®
Lipase
EndoH
pH
3.0–5.0
6.0–10.0
11.0
22.0 § 1.0
0
0
0
0
0
22.0 § 1.0
22.0 § 1.0
22.0 § 1.0
15.0 § 1.0
20.0 § 1.0
13.0 § 1.0

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123
AcAMP peptide exhibited high thermal stability (Fig. 2).
Antimicrobial activity against B. cereus remained stable
after 15-min incubation at 100°C. Heat stability is poten-
tially a useful characteristic during food preservation
because many food-processing procedures involve a heat-
ing step.
AcAMP remained stable after incubation for 1 h at pH
values from 6.0 to 10.0, yielding a similar inhibition zone
of about 20 mm using 50 ?l at 200 ?g ml¡1. However, its
activity was slightly reduced at pH 3.0, 4.0, 5.0, and 11.0,
with an inhibition zone of 15 mm at acidic conditions and
13 mm at alkalophilic conditions (Table 3).
Cloning of acamp gene and sequence analysis
Using the acafp gene sequence [28] and the codon usage
database of A. clavatus strains at www.kazusa.org.jp/
codon, two oligonucleotide primers called AcAMPF and
AcAMPR were designed and used to amplify the gene
encoding AcAMP peptide. AmpliWcation from cDNA gave
a fragment of about 300 bp (Fig. 3). Subsequently, the PCR
product was cloned in pCR®II-TOPO plasmid vector and
transferred into E. coli TOP10. The nucleotide sequence of
the cDNA acamp and the deduced primary structure of the
protein encoded by this gene are shown in Fig. 4. Analysis
of the nucleotide sequence of the cDNA acamp gene
revealed the presence of an ORF of 285 bp which encodes a
pre-pro-AcAMP with an ATG start codon and a TAG stop
codon. The ORF encodes a pre-pro-peptide of 94 amino
acid residues with calculated molecular mass of 9874.4 Da
and was predicted to belong to the antifungal peptide fam-
ily (Fig. 4). This ORF was conWrmed as the gene encoding
AcAMP peptide, since the 19 residues of the N-terminal
amino acid sequence of the puriWed AcAMP reported in
Table 1 perfectly matched the deduced amino acid
sequence.
SignalP version 3.0 predicted a signal peptide of 21 aa
(M1-A21) and a pro-peptide of 22 aa (S22-Q43). The
cleavage sites of the pre- and the pro-peptide were con-
served among other antimicrobial peptides. Based on amino
acid analysis by Protparam software in Expasy (http://
ca.expasy.org/cgi-bin/protparam), the mature peptide, con-
sisting of 51 aa (A44-C94), has predicted molecular weight
of 5777.7 Da and an isoelectric point estimated at 9.06.
Under physiological conditions, therefore, the AcAMP
peptide was positively charged. The most signiWcant fea-
ture of the amino acid composition of the mature AcAMP
peptide is its high Lys and Cys content. The mature
AcAMP contains 11 Lys residues and 8 Cys residues, cor-
responding to 21% and 15%, respectively.
Sequence alignment revealed 34–97% identity between
the amino acid sequence of the deduced AcAMP pre-pro-
peptide and other ascomycete antifungal peptides (Fig. 5).
A more advanced search revealed a high degree of identity
at the amino acid level with AcAFP from A. clavatus VR1.
Both contained a high amount of hydrophobic and cationic
residues, and eight Cys residues. Nevertheless, there were
two and nine diVerences between the AcAMP peptide from
A. clavatus ES1 and AcAFP and AFP peptides from
A. clavatus and A. giganteus, respectively. In the case of
AcAFP, the two diVerences are in the pre-sequence (F3V
and V20A). However, in the case of AFP, three modiWca-
tions were located in the pre-sequence (F3V, A19V, and
V20A), four in the pro-sequence (T22S, E25D, T30A, and
L43Q), and two in the mature peptide (N47D and K75V).
Furthermore, the PAF peptide from P. chrysogenum and
Fig. 2 EVect of heat treatment on puriWed AcAMP peptide activity.
The same amount of puriWed AcAMP peptide was heated for 5, 10, and
15 min at 50°C, 70°C, 90°C, and 100°C. A sample without heat treat-
ment was used as positive control
100 °C
90 °C
70 °C
50 °C
0 min
5 min 10 min
15 min
Fig. 3 AmpliWcation of a DNA fragment encoding the pre-pro-
AcAMP peptide of A. clavatus ES1: lane 1 DNA marker (low DNA
molecular marker kit, Invitrogen); lane 2 PCR product from cDNA
1 2
300 pb
250 pb

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J Ind Microbiol Biotechnol (2010) 37:805–813811
123
AnAFP peptide from A. niger contained only six Cys resi-
dues, with Cys69 and Cys92 being missing (Fig. 5).
Discussion
A highly thermostable antibacterial and antifungal pep-
tide from A. clavatus ES1, named AcAMP, was puriWed
by one-step heat treatment and was characterized bio-
chemically. The puriWcation process diVered from those
previously described for other ascomycete antifungal
peptides that employed ultraWltration and cation
exchange chromatography [19, 27]. A single band of
about 6.0 kDa was observed on SDS–PAGE analysis.
This molecular mass was close to the AcAFP peptide
from A. clavatus VR1 [27] and the AFP peptide from
A. giganteus [16], determined to be 5.77 and 5.8 kDa,
respectively. AnAFP peptide from A. niger and PAF
peptide from P. chrysogenum have molecular mass of
6.6 and 6.3 kDa, respectively [21]. All peptides reported
from ascomycetes had only antifungal activity. To our
knowledge, this is the Wrst description of an antibacterial
activity from an A. clavatus strain.
The puriWed AcAMP peptide from A. clavatus ES1 had
pronounced antibacterial activity against Gram-positive
bacteria including S. aureus, B. cereus, and E. faecalis. The
AcAMP peptide was also active against some Gram-nega-
tive bacteria such as P. aeruginosa and E. coli but was not
active toward K. pneumonia. These Wndings diVered from
those related to the inhibition data for biWdocin B, a bacte-
riocin produced by BiWdobacterium biWdum [30]. Indeed,
biWdocin B was unable to inhibit S. aureus, Clostridium
butyricum, Streptococcus thermophilus, and Gram-negative
bacteria. On the other hand, the AcAMP peptide had some
similar inhibition activities to those of biWdin [3] and biW-
long [15], two bacteriocin types able to inhibit some Gram-
positive and Gram-negative bacteria.
The eVects of various enzymes on puriWed AcAMP
peptide were evaluated. Complete inactivation of AcAMP
inhibition activity was observed after treatment with
proteolytic enzymes. These results diVered from those of
Cheikhyoussef et al. [6, 7], who reported partial inactivation
of a 3.0-kDa bacteriocin of B. infantis BCRC 14602 after
treatment with proteolytic enzymes. Enzyme treatment with
Thermamyl, lipase, and EndoH did not aVect inhibition
activity of AcAMP peptide, suggesting that carbohydrate
Fig. 4 Nucleotide sequence of cDNA acamp gene of A. clavatus ES1
and its deduced amino acid sequence. Numbers on the left side of the
amino acid and nucleotide sequences denote amino acid and nucleotide
positions, respectively. Nucleotide sequence numbered from the Wrst
base initiation codon. The Wrst amino acid Met of the enzyme is count-
ed as +1. The putative starting residues of the pre-peptide (pre),
pro-peptide (pro), and mature peptide (mature) are indicated. Asterisk
indicates a stop codon
1 atg aag gtc gtt tct ctc gct tct ctg ggt ttc gcc ctc gtc gct gcc ctt ggc gtg gca
1 M K V V S L A S L G F A L V A A L G V A
Pro
61 gcc agc ccc gtg gat gcc gat tct ctc gcc gca ggt ggt ctg gac gca aga gac gag agc
21 A S P V D A D S L A A G G L D A R D E S
Mature
121 gcc gtt caa gcc aca tac gac ggt aaa tgc tac aag aag gac aat atc tgc aag tat aag
41 A V Q A T Y D G K C Y K K D N I C K Y K
181 gca cag agc ggc aag acg gcc att tgc aag tgc tat gtc aaa gta tgc ccc cga gac ggc
61 A Q S G K T A I C K C Y V K V C P R D G
241 gcg aag tgc gag ttt gac agc tac aag ggc aag tgc tac tgc tag
81 A K C E F D S Y K G K C Y C *
Pre
Fig. 5 Amino acid sequence alignment of pre-pro-peptide AcAMP
with antifungal peptides from A. clavatus [27], A. giganteus [19],
P. chrysogenum [10], and A. niger [18]. Asterisk indicates that the
residues in that column are identical in all sequences in the alignment.
Colon indicates that conserved substitutions have been observed. Dot
indicates that semiconserved substitutions are observed. X indicates
amino acid changes between AcAMP and other fungal peptides
AcAMP_A.clavatusES1 MKVVSLASLGFALVAALGVAASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK 60
AcAFP_A.clavatusVR1 MKFVSLASLGFALVAALGVVASPVDADSLAAGGLDARDESAVQATYDGKCYKKDNICKYK 60
AFP_A.giganteus MKFVSLASLGFALVAALGAVATPVEADSLTAGGLDARDESAVLATYNGKCYKKDNICKYK 60
PAF_P.chrysogenum MQITTVALF---LFAAMGGVATPIES---VSNDLDARAEAGVLAKYTGKCTKSKNECKYK 54
AnAFP_A.niger MQLTSIAII---LFAAMGAIANPIAA---EADNLVAR--EAELSKYGGECSVEHNTCTYL 52
*:..::* : *.**:* *.*: : :..* ** . :.* *:* ..* *.*
AcAMP_A.clavatusES1 AQSGKTAICKC---YVKVCPRDGAKCEFDSYKGKCYC---- 94
AcAFP_A.clavatusVR1 AQSGKTAICKC---YVKVCPRDGAKCEFDSYKGKCYC---- 94
AFP_A.giganteus AQSGKTAICKC---YVKKCPRDGAKCEFDSYKGKCYC---- 94
PAF_P.chrysogenum NDAGKDTFIKCPKFDNKKCTKDNNKCTVDTYNNAVDCD--- 92
AnAFP_A.niger K-GGKDHIVSCPSAANLRCKTERHHCEYDEHHKTVDCQTPV 92
.** : .* * : :* * :: *
Pre
Pro
Mature

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812J Ind Microbiol Biotechnol (2010) 37:805–813
123
and lipid moieties are not required for its activity. Similar
results of enzyme treatments with proteases, lipases, and
amylases were reported for other antimicrobial peptides,
including bacteriocin produced by Pediococcus pentosace
[14], Pseudomonas sp. strain 4B [9], and Bacillus sp. [24].
PuriWed AcAMP peptide was highly heat-stable in the
range from 50°C to 100°C (Fig. 2). AcAMP peptide exhib-
ited similar heat stability to AcAFP from A. clavatus VR1
[27]. The pH stability proWle of AcAMP peptide is in
agreement with the Wndings by Meghrous et al. [22]. Bacte-
riocins from 13 strains of BiWdobacteria were active at pH
values ranging from 2.0 to 10.0. AcAMP peptide exhibited
a wider pH stability range compared with the partially puri-
Wed biWdin from B. biWdum 1452, which had optimum pH
of 4.8 and only a maximum inhibition activity in the pH
range of 4.8 to 5.5 [3]. AcAMP was relatively stable at pH
values below 6.0 and above 10.0, while biWdocin B showed
maximum pH stability in the range from 2.0 to 12.0 [30].
The heat and pH stability and the wide spectrum of activity
are very useful characteristics for the potential application
of AcAMP peptide as a food preservative [18].
The cDNA acamp gene that encoded AcAMP peptide
was isolated by RT-PCR from total RNA. The PCR prod-
uct was cloned in pCR®II-TOPO vector, and the entire
nucleotide sequence of the open reading frame was deter-
mined. Alignment and comparison of the protein sequence
deduced from cDNA gene with sequences from the protein
databases using BLAST program showed high similarity
to other ascomycete antifungal peptides. The acamp ORF
encodes a 94-amino-acid precursor protein (pre-pro-
AcAMP). The Wrst 21 amino acids correspond to a pre-
dicted signal sequence. The pre-sequence of AcAMP from
ES1 diVered from AcAFP produced by A. clavatus VR1 by
two amino acids: Val and Ala at positions 3 and 20 in
AcAMP peptide were modiWed to Phe and Val in AcAFP,
respectively [28]. The 22 amino acids from residues 22 to
43 constitute a pro-sequence that would be removed before
or during release of mature AcAMP [20]. Pro-sequence
plays an important role by preventing protein activity
before secretion. The mature protein would adopt its
active conformation after the pro-sequence is cleaved.
Antimicrobial peptides produced as pre-pro-proteins from
ascomycetes include AFP, AcAFP, and PgAFP from
A. giganteus, A. clavatus VR1, and P. chrysogenum,
respectively [25, 26, 28].
AcAMP peptide had an overall positive charge (+7)
imparted by the presence of 12 lysine and arginine residues
and a substantial portion of hydrophobic residues (about 40).
Furthermore, the predicted tertiary structure of the mature
AcAMP peptide using AcAFP as template had Wve antiparal-
lel ?-strands, deWning a small and compact ?-barrel which
was stabilized by four internal disulWde bridges (data not
shown). This property could explain the high thermostability
of the AcAMP peptide. Similar three-dimensional (3D)
structures were obtained with antifungal peptides from asco-
mycetes such as AcAFP [28], PcAFP [26], and AFP [25].
The common feature shared among the cationic antimicro-
bial peptides is their ability to fold into amphipathic confor-
mations in the presence of hydrophobic amino acid residues.
AcAMP peptide amphipathic character could be due to the
presence of Y29, V30, Y45, Y50, and a cationic K9 and K10
residues [28]. These conformations could induce interaction
with membranes or membrane mimics [11]. In addition to
their direct antimicrobial activities against Gram-positive and
Gram-negative bacteria, these peptides play additional roles
as antibiotics against fungi [23].
In conclusion, this work attempted to show evidence for
the high thermostability and strong antibacterial activity of
a 6.0-kDa peptide from A. clavatus ES1. AcAMP peptide
showed characteristics common to the group of small,
basic, cysteine-rich antifungal proteins from molds. An
ORF of 282 bp encoding a 94-amino-acid protein was char-
acterized. The protein sequence deduced from acamp gene
exhibited high identity with peptides from other fungi
strains. Considering the heat and pH stability and the wide
spectrum of action against fungi and bacteria, AcAMP pep-
tide can be an excellent candidate for application as a food
preservative and for biological control of plant diseases.
Acknowledgments
Montpellier University II for fruitful discussion and help with cloning.
The authors would like to thank Mr. Ayadi Hajji from the Faculty of
Letters and Humanities of Kairouan for his help with English. This
work was funded by the Ministry of Higher Education and ScientiWc
Research, Tunisia.
We are grateful to Mr. Philippe Roch from
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