Peptides 42 (2013) 144–148

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Peptides
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Short communication

In vivo antimicrobial evaluation of an alanine-rich peptide derived from

Pleuronectes americanus
Leandro D. Teixeira, Osmar N. Silva, Ludovico Migliolo, Isabel C.M. Fensterseifer, Octavio L. Franco ∗
Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: In several organisms, the first barrier against microbial infections consists of antimicrobial peptides
Received 4 January 2013 (AMPs) which are molecules that act as components of the innate immune system. Recent studies have
Received in revised form 4 February 2013 demonstrated that AMPs can perform various functions in different tissues or physiological conditions.
Accepted 4 February 2013
In this view, this study was carried out in order to evaluate the multifunctional activity in vivo of an
Available online 13 February 2013
alanine-rich peptide, known as Pa-MAP, derived from the polar fish Pleuronectes americanus. Pa-MAP was
evaluated in intraperitoneally infected mice with a sub-lethal concentration of Escherichia coli at standard
Keywords:
concentrations of 1 and 5 mg kg−1 . At both concentrations, Pa-MAPs exhibited an ability to prevent E. coli
Antimicrobial peptides
Promiscuity
infection and increase mice survival, similar to the result observed in mice treated with ampicillin at
Immunomodulatory 2 mg kg−1 . In addition, mice were monitored for weight loss. The results showed that mice treated with
in vivo infections Pa-MAPs at 1 mg kg−1 gained 0.8% of body weight during the 72 h of experiment. The same was observed
with Pa-MAP at 5 mg kg−1 , which had a gain of 0.5% in body weight during the treatment. Mice treated
with ampicillin at 2 mg kg−1 show a significant weight loss of 5.6% of body weight. The untreated group
exhibited a 5.5% loss of body weight. The immunomodulatory effects were also evaluated by the quan-
tification of IL-10, IL-12, TNF-␣, IFN-␥ and nitric oxide cytokines in serum, but no immunomodulatory
activity was observed. Data presented here suggest that Pa-MAP should be used as a novel antibiotic
against infection control.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction is a facultative anaerobe able to colonize the human large intestine

and can be divided in virulent and avirulent strains. Virulence
Bacterial infection control in hospitalized patients is an enor- factors that differentiate these strains are commonly acquired on
mous challenge due to numerous contamination sources including mobile genetic elements by horizontal gene transference. Further-
invasive procedures and devices such as mechanical ventilators more, these virulence factors confer upon E. coli strains the ability to
[10], ultrasound probes [50] and catheters [58]. Aiming to con- resist to human host defenses [20,39]. E. coli strains are attributed
trol such microorganisms, permanent surveillance protocols are to cause nosocomial infections and a wide number of human
adopted in hospitals informing about preventive strategies to diseases, such as sepsis, meningitis, and diarrhea [30,35,36,47].
reduce infection [9,52]. According to the World Health Organization Otherwise, the application of novel antimicrobials seems to be
(WHO), 8.7% of hospitalized patients of 55 hospitals in 14 countries an alternative for infectious disease treatment including the devel-
in 4 WHO regions (Europe, Eastern Mediterranean, South-East Asia opment of antimicrobial peptides (AMPs) [7,23]. AMPs consist of
and Western Pacific) and 1.4 million people world-wide suffer from peptides from 12 to 100 amino acid residues length, which exert
nosocomial infections [53]. Moreover, nosocomial infections have activity against Gram-positive and -negative bacteria, fungi and
a direct impact on country costs due to increases in length of hos- viruses through multiple mechanisms [12,26,43]. These peptides
pitalization, number of physician visits and deaths [15,33]. can be isolated from various organisms such as plants [48], insects
Enterobacteriacea is one of the most prevalent bacterial families [45], amphibians [57], fishes [1] and mammals [18]. Despite their
in nosocomial infections mainly represented by Pseudomonas aeu- different origins, AMPs may show some common properties includ-
ruginosa, Klebsiella pneumoniae and Escherichia coli [10,28]. E. coli ing cationic surfaces and amphipathic structures [49]. Furthermore,
some peptides also show promiscuity as they attach to differ-
ent targets such as membranes, cell walls, cytosolic proteins and
∗ Corresponding author at: Pós-Graduação em Ciências Genômicas e Biotecnolo- nucleic acids [7,27,49]. This property could lead to multifunction-
gia, Universidade Católica de Brasília, Campus Avançado Asa Norte – SGAN 916 ality derived from a single protein molecule. This process could
Avenida W5 – CEP: 70790-160 Brasília, DF, Brazil. Tel.: +55 61 34487167/34487220; also occur due to a specific stimulus, such as pH or protein con-
fax: +55 61 33474797.
E-mail address: [email protected] (O.L. Franco).
centrations. This property is commonly found in plant and animal

0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.peptides.2013.02.001
 L.D. Teixeira et al. / Peptides 42 (2013) 144–148 145

defense peptides, in which a wide number of different functions from HPLC-8 with the following sequence: H-His-Thr-Ala-Ser-Asp-
must be generated by several structural homologs with identical Ala-Ala-Ala-Ala-Ala-Ala-Leu-Thr-Ala-Ala-Asn-Ala-Ala-Ala-Ala-
structures [16]. Moreover, cationic AMPs conformation seems to Ala-Ala-Ala-Ser-Met-Ala-NH2 , with the stepwise solid-phase
interact with anionic microorganism membranes by electrostatic method using the N-9-fluorenylmethyloxycarbonyl (Fmoc) strat-
interactions in a first step. AMPs inset into membrane bilayers and egy with a Rink amine resin (0.52 mmol g−1 ), and purified by
aggregate, forming pores and leading to an efflux of intracellular reversed-phase high-performance liquid chromatography (HPLC)
ions [40,64]. with purity degree >95% [6,34]. Pa-MAP molecular mass was
Additionally, some studies have shown the relation between determined using matrix assisted laser desorption/ionization
resistance to certain infectious diseases and AMPs secretion. time of flight mass spectrometry (MALDI-ToF MS/MS) analysis
Cipriano et al. [8] showed that AMPs secreted in fish exter- on UltraFlex III, Bruker Daltonics, Billerica, MA. Purified peptides
nal mucus may confer resistance to Aeromonas salmonicida in were dissolved in a minimum volume of water that was mixed
salmonids. Likewise, in Teleostei marine polar fish, some pep- with an ␣-cyano-4-hydroxycinnamic acid saturated matrix solu-
tides are commonly secreted into the blood and tissues depending tion (1:3, v:v), spotted onto a MALDI target plate and dried at
on sub-zero temperature [13,31]. These peptides are known as room temperature for 5 min. The ␣-cyano-4-hydroxycinnamic
antifreeze peptides (AFP), and the type I AFP family is commonly acid matrix solution was prepared at 50 mM in H2 O:ACN:TFA
found in winter flounder (Pleuronectes americanus), named HPLC- (50:50:0.3, v:v:v). Peptide monoisotopic mass was obtained in
6 and HPLC-8 [18]. Comparing AMPs and AFPs, similar structural the reflector mode with external calibration using the Peptide
and physical–chemical properties have been found, such as the Calibration Standard II for mass spectrometry (up to 4000 Da mass
hydrophobic ratio, hydrophobic moment and specific amino acid range, Bruker Daltonics, Billerica, MA).
composition [61]. Migliolo et al. [34] studied a synthetic peptide
named Pa-MAP, a derivate of the HPLC-8 peptide [25]. Addition- 2.2. Bacterial strains and growth conditions
ally, Pa-MAP primary sequence was selected from the AFP HPLC-8
produced by the polar fish P. americanus with length (decreased Escherichia coli (ATCC 8739) strains were cultivated in solid
from 37 residues to 26) and residue modifications, such as lysine Muller–Hinton medium. An isolated colony was transferred to 5 mL
7 and 18 substituted by alanine, valine 2 and 13 by treonine, and of liquid Luria–Bertani (LB) medium and grown in a rotating drum
glutamic acid 11 by alanine. The first amino acid residue in HPLC- at 37 ◦ C with aeration during 24 h. Posteriorly, 100 ␮L of this pre
8 is aspartic acid, also substituted by histidine [34]. Surprisingly, inoculum was transferred to 4.9 mL of LB medium and grown at the
Pa-MAP is devoid of arginine and lysine cationic residues, which same conditions for 2 h. The absorbance was evaluated to identify
seems to be important for antimicrobial activity [19,41]. Indeed, the late log phase, when the OD at 600 nm was 1.0, corresponding
the peptide has mostly hydrophobic amino acid residues suggest- a concentration of 1 × 108 UFC mL−1 (5 × 108 UFC at final volume).
ing that that Pa-MAP antimicrobial activity could be attributed These cells were centrifuged for 6 min at 1200 rpm and the sedi-
mostly to hydrophobic interaction. Furthermore, it shows the abil- ment was resuspended in 5 mL of phosphate buffered saline (PBS)
ity of inhibiting the HSV virus, the development of mycellar fungi and equalized to a concentration of 1 × 106 UFC at final volume for
T. mentagrophytes and T. rubrum, and deleterious activity against virulence and immunomodulatory assays [63].
E. coli, besides cytotoxic effects in tumor cells. Moreover, Pa-MAP
did not show any cytotoxic effects against human red blood cells,
2.3. Escherichia coli in vivo bioassays
enabling this peptide for in vivo assays. An additional activity was
the antitumoral effects against Caco-2 (human epithelial colorectal
In vivo experiments were performed with 6–10 weeks old
adenocarcinoma cells), HCT-116 (human colorectal carcinoma cell
female BALB/c mice from University of Campinas (Campinas/SP).
lines) and MCF-7 (human breast cancer cells) [34].
Mice were housed and used in accordance with guidelines
One of the main challenges of AMP utilization has been related
established by the Ethical Committee of Animal Use of Univer-
to peptide stability in such models. Several studies have demon-
sity of Brasília (Brasilia/DF), registered under protocol number
strated that the activity of AMPs in vitro was not the same as in vivo
UnBDOC:83931/2011, and all efforts were made to minimize ani-
models, and these controversial results may be attributed to certain
mal suffering. Mice were divided into 5 groups of 5 animals each
proteases present in serum [22]. Another cause of in vivo inactivity
(Table 1). As described above, groups were infected via intraperi-
is the high polar property of some AMPs, resulting in a reduction
toneal (IP) injection with E. coli suspension equalized and diluted
in membrane crossing or in an irregular distribution into mam-
in cold PBS to a sub lethal concentration of 1 × 105 UFC (50 ␮L in
malian cells, losing activity against intracellular microorganisms
each animal) [54]. Treatments of infected mice were performed
[59]. Moreover, as revised by Brinch et al. [3], in vivo AMP activity
with Pa-MAP at 1 and 5 mg kg−1 , both dissolved in 100 ␮L of PBS,
may also be impeded by poor drug distribution and AMP degrada-
respectively. PBS was utilized as the negative control, and ampi-
tion by increased metabolism inside the cell. AMPs also can induce
cillin at 2 mg kg−1 dissolved in 100 ␮L of PBS was utilized as the
the immune system to produce anti-AMP antibodies [2], reducing
positive control. Moreover, an uninfected control was also per-
their effectiveness
formed. All mice were housed with constant water and food in an
In this view, this study evaluated the in vivo antimicrobial activ-
air-filtered environment maintained at 20 ± 2 ◦ C during 72 h and
ity of the synthetic multifunctional peptide Pa-MAP. Mice infected
with E. coli strains were used as experimental models. Moreover,
the serum was obtained and cytokines were evaluated in order to Table 1
determine a possible immunomodulatory effect. Experimental groups distribution.

Experimental groups (n = 5) E. coli infection Treatments

1 – Infected Yes Ampicillin 2 mg kg−1

2. Materials and methods 2 – Infected and untreated Yes No
3 – Non infected No No
2.1. Peptide synthesis and mass spectrometry analyses 4 – Infected and treated group 1 Yes Pa-MAP 1 mg kg−1
5 – Infected and treated group 2 Yes Pa-MAP 5 mg kg−1
The Pa-MAP peptide was synthesized by China Peptides Groups 1, 2, 4 and 5 were infected with E. coli at 1 × 105 UFC sublethal concentration.
(Shanghai, China) based on two 11-residue repeating segments Drug treatments were injected 24 and 48 h after infection.
146 L.D. Teixeira et al. / Peptides 42 (2013) 144–148

Fig. 1. (A) MALDI-TOF mass spectrometry analysis of Pa-MAP monoisotropic mass [M+H+ ] = 2212.86. (B) After E. coli sub lethal infection, mice were treated with ampicillin
at 2mg kg−1 ( ), Pa-MAP at 1 ( ) and 5 mg kg−1 ( ) at 24 h and 48 h. At 24 h, 20% of mice of infected and untreated group ( ) died and another 20% had died at
48 h. ( ) represent the Untreated group. (a) represent Logrank test with p < 0.05, Chi square = 11.67, compared to negative control. (C and D) Weight loss was evaluated at
the beginning and at the end of experiment. (C) Mice lost weight in infected and untreated group and mice treated with ampicillin at 2 mg kg−1 . Mice treated with Pa-MAP
gained weight until the end of the experiment, as well as mice that were uninfected and untreated. (D) Difference of weight from each group at 72 h after procedures. P > 0.05.

further treated as described above (Table 1). Treatments occurred sub-lethal E. coli mice IP infection. Two concentrations of Pa-MAP
24 h and 48 h after infection. Moreover, all mice were weighed at (1 mg kg−1 and 5 mg kg−1 ) treatment were tested. Ampicillin at
the beginning and at the end of the experiment. 2 mg kg−1 was used as a positive control. During the first 24 h after
infection, the infected and untreated group decreased 20%, fur-
2.4. Cytokines evaluation ther decreasing to 60% 48 h after infection. In contrast, all mice
treated with Pa-MAP in both concentrations survived at the end of
Mice were anesthetized by xilazine and ketamine at 10 mg kg−1 experiment. The same pattern was observed in mice treated with
and 50 mg kg−1 , respectively, after 72 h. Blood collection was per- ampicillin (Fig. 1B).
formed by decapitation and serum obtained by centrifugation Mice weights were further evaluated in the beginning and at the
and stored at −20 ◦ C. The cytokines interleukin-10 (IL-10), IL-12, end of experiment. Infected and untreated mice lost 5.5% of their
interferon gamma (IFN-␥), tumor necrosis factor alpha (TNF-␣), body weight after 72 h of experiment. In contrast, mice treated with
and nitric oxide (NO) were measured in serum by enzyme-linked Pa-MAP at 1 mg kg−1 gained 0.8% of their body weight, similar to
immunosorbent assays (ELISA) using ELISA kit (Peprotech) accord- Pa-MAP at 5 mg kg−1 , which gained 0.5% of their body weight dur-
ing to the manufacturer’s instructions. ing the same period. Non-infected mice gained slightly more body
weight (2.7%) compared to the Pa-MAP treatment groups. Infected
2.5. Statistical Analysis mice treated with ampicillin at 2 mg kg−1 also had lost weight,
equivalent to 5.6% of their initial body weight (Fig. 1C and D).
The statistical significance of the experimental results was
determined by one-way Student’s t-test or one-way analysis of 3.3. Immunomodulatory activity
variance (ANOVA) followed by Dunnett’s test. Values of P < 0.05
were considered statistically significant. Graphpad Prism version Some cytokines were evaluated in attempt to identify an
6.0 was used for all statistical analyses. immunomodulatory effect of Pa-MAP in the mice immunologic sys-
tem. This evaluation of immunomodulatory activity in vivo was
3. Results investigated by quantification of IL-10, IL-12, TNF-␣ and NO in
serum. Pa-MAP used as treatment was evaluated at 1 mg kg−1 , cor-
3.1. Mass spectrometry analysis responding to a concentration of twice the minimum inhibitory
concentration (MIC) of 512 ␮g mL−1 [34], and 5 mg kg−1 , corre-
MALDI-ToF evaluation showed an ion with an m/z of 2212.86, sponding 10 times the MIC encountered in early study with
corresponding to the calculated value for the peptide sequence, Pa-MAP. Ampicillin at 2 mg kg−1 was used as a positive control.
above 95% in purity. All further bioassays were performed using These concentrations of Pa-MAP were unable to modify IL-10
purified Pa-MAP (Fig. 1A). release when compared to the non-infected and untreated mice
group. Similar data were observed for IL-12 and TNF-␣ production
3.2. Antimicrobial activities in all treatments groups (Supplementary Fig. 1).
See Supp Figure S1 as supplementary file. Supplementary
In order to confirm the in vitro protective effects of Pa-MAP material related to this article found, in the online version, at
against E. coli, in vivo antibacterial activity was evaluated by a http://dx.doi.org/10.1016/j.peptides.2013.02.001.
 L.D. Teixeira et al. / Peptides 42 (2013) 144–148 147

4. Discussion One of the most documented effects of E. coli infection is pro-

gressive weight loss, mainly due to water loss during infection
Antimicrobial resistance mechanisms developed by bacteria [44]. This symptom is also caused by heat-labile toxin (LT) and
is a serious worldwide threat to public health, particularly for heat-stable toxin (ST) presented in enterotoxigenic E. coli infec-
immunocompromised patients and those under immunosuppres- tion [55]. In the current study, 5.5% of mice body weight loss was
sion therapy, e.g. patients after organ transplant [29]. Moreover, observed in the infected and untreated group. A study developed
infections caused by antibiotic-resistant microorganisms have con- in murine model, mice infected with non-pathogenic and entero-
tributed to increases in patient mortality, especially for those hemorrhagic E. coli (NPEC and EHEC) demonstrated a clear weight
whose treatment with currently available drugs has become less loss of about 6% [5]. Furthermore, mice treated with ampicillin at
efficient [14]. Due to these facts, peptides with antimicrobial activi- 2 mg kg−1 also showed 5.6% weight loss, demonstrating that ampi-
ties have become extremely attractive for microorganisms control, cillin can eliminate bacterial infection, but did not exhibit ability to
mainly due their low toxicity effects into mammalian cells [24]. inhibit weight loss. In contrast, Pa-MAP exhibited protective effects
In our study, an alanine-rich peptide designed from a polar fish, against E. coli and body weight loss in both concentrations, preven-
P. americanus, with two repeat antifreeze motifs and clear in vitro ting this pathological effect. Similar data was observed in a study
deleterious activity against E. coli, with identical purification degree with the AMP IB-367, a protegrin peptide, evaluated to prevent oral
(see Fig. 1A) previously reported by Migliolo et al. [34] was evalu- mucositis in hamsters. In this study, animals treated with IB-367 at
ated in vivo. 0.12–2.0 mg mL−1 showed body weight gain in comparison with
Some peptides were designed to develop a multifunctional mice treated with placebo, and became significantly greater during
product, able to eliminate microbes and increase the immune the passing days [38]. Soni et al. [56] evaluated in vivo the efficacy
response, involving systematic variations in the structure of a of two combined antibiotics, ceftriaxone and vancomycin, against
base molecule, i.e. cationic charge, hydrophobicity and hydropho- E. coli intra-abdominally infected mice. Infected mice showed sig-
bic moment [21]. Moreover, some cationic peptides are known to nificant weight loss during infection and became normalized after
be able to induce some immunomodulatory effect [37,62]. Some vancomycin and ceftriaxone treatment.
studies demonstrated that only a fraction of the peptide was pre-
sented to the immune system, and these peptides should be able 5. Conclusions
to overcome some “bottlenecks” and only those with specific char-
acteristics would be able to induce the immune system [32,46]. Due to increasing number of cases of multi-resistant bacterial
Nevertheless, Pa-MAP seems to be unable to interact with immune disease against a variety of antimicrobial drugs, antimicrobial pep-
system cells to induce cytokine production. Data presented here tides have a great and considerable potential to become the new
shows that Pa-MAP neither significantly stimulates nor inhibits generation of bioactive products. Here, a peptide with an antimicro-
some cytokine production, despite of others could be modified by bial novel effect in vivo was confirmed but any immunomodulatory
the presence of peptide. This result is similar to the Fritsche et al. activity was observed indicting that action mechanism is only
studies [17]. In their studies, it was demonstrated that a short, related to a direct antimicrobial activity. This peptide demonstrated
proline-rich antimicrobial peptide has direct antibacterial action a protective effect against E. coli at lower concentrations in compar-
in vivo, but was unable to stimulate cytokine production. ison to other antimicrobial peptides and synthetic pharmacological
Despite the absence of immunomodulatory activity, data antibiotics [42,60]. Moreover, weight loss in mice was prevented
reported here shows strong evidence that the peptide Pa-MAP during treatment with Pa-MAP, in contrast with other treatments,
could be useful for pharmaceutical design once it shows the abil- i.e. ampicillin and other AMPs. In the future, Pa-MAP could be used
ity to perform E. coli inhibition in vivo. Pa-MAP demonstrated in in the development of a novel biopharmaceutical against microor-
vivo activity against E. coli at low concentrations when compared ganisms.
to other antimicrobial peptides. Schaal et al. [51] demonstrated
similar effect with rhesus ␪-defensin (RTD), a macrocyclic antimi- Acknowledgements
crobial peptide expressed in leukocytes of Old World monkeys.
This RTD peptide was administrated at a single subcutaneous dose This work was granted by CNPq, CAPES, FAPDF and UCB. The
at 5 mg kg−1 in mice previously intraperitoneally infected with authors also thank Tania Paula Garcez de Lucena Santana and
E. coli and resulted in an increase in mice survival. Vingsbo et al. the team of UCB bioassays laboratory for animal care. Moreover,
[60] demonstrated that the novel synthetic polymyxin derivatives authors also thanks Simoni C. Dias for the critical reading of this
NAB737 and NAB739 are as effective as polymyxin B, an effective manuscript.
antibiotic against Gram-negative bacterial infections, in effectively
treating E. coli peritoneal infection in mice at 1, 2 and 4 mg kg−1 . Appendix A. Supplementary data
In another study, a non-natural AMP named M33 (with 9 amino
acid residues long) showed the ability at 12.5 and 25.0 mg kg−1 to Supplementary data associated with this article can be found,
protect 100% of mice infected with lethal doses of E. coli and P. in the online version, at http://dx.doi.org/10.1016/j.peptides.
aeruginosa. Lower concentrations were unable to protect mice [42]. 2013.02.001.
Although antimicrobial activity, the mechanism of action has
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