Antimicrobial and Antibiofilm Activity of Mauriporin,a Multifunctional Scorpion Venom Peptide

Ammar Almaaytah • Shadi Tarazi • Fawzi Alsheyab •

Qosay Al-Balas • Tareq Mukattash

Accepted: 24 March 2014

� Springer Science+Business Media New York 2014

Abstract Many pathogenic free living and biofilm

forming bacterial organisms can cause serious infections

to humans that could consequently have devastating

effects on human health. A significant number of these

microbial organisms are resistant to almost all known

conventional antibiotics and the ability of some these

strains to form sessile communities of biofilms increases

the resistance ability of bacteria to antibiotic treatment.

Global research is currently focused on finding novel

therapies to counteract the threat of bacterial and biofilm

infections rather than using conventional antibiotics.

Mauriporin, a novel cationic a-helical peptide identified

from the venom derived cDNA library of the scorpion

Androctonus mauritanicus was reported to display selec-

tive cytotoxic and anti-proliferative activity against pros-

tate cancer cell lines. In the present study, we investigated

the antimicrobial and antibiofilm activities of Mauriporin.

Our results show that Mauriporin displays potent antimi-

crobial activities against a range of Gram-positive and

Gram-negative planktonic bacteria with MIC values in the

range 5 lM to 10 lM. Mauriporin was also able to pre-

vent Pseudomonas aeruginosa biofilm formation while

showing weak hemolytic activity towards human eryth-

rocytes. Studies on the mechanism of action of Mau-

riporin revealed that the peptide is probably inducing

bacterial cell death through membrane permeabilization

determined by the release of b-galactosidase enzyme from

peptide treated Escherichia coli cells. Moreover, DNA

binding studies found that Mauriporin can cause potent

binding to intracellular DNA. All these results indicate

that Mauriporin has a considerable potential for thera-

peutic application as a novel drug candidate for eradi-

cating bacterial infections.

Keywords Antimicrobial peptide � Antibiofilm activity �Mauriporin � Membrane-permeation � Scorpion non-

disulfide bridged peptide � Molecular modelling

Introduction

Bacterial infections represent a major cause of morbidity

and mortality worldwide and are considered a heavy

health burden facing the global human population (de

Kraker et al. 2011; Kwong et al. 2012). The discovery of

antibiotics in the last century revolutionized medicine and

allowed mankind for the first time in history to success-

fully manage infectious diseases caused by microbial

pathogens (Davies and Davies 2010). Unfortunately and

due to the excessive overuse and misuse of antibiotics, a

significant proportion of pathogenic microorganisms

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10989-014-9405-0) contains supplementarymaterial, which is available to authorized users.

A. Almaaytah (&)

Department of Pharmaceutical Technology, Faculty of

Pharmacy, Jordan University of Science and Technology,

Irbid, Jordan

e-mail: amalmaaytah@just.edu.jo

S. Tarazi � F. Alsheyab

Department of Applied Biological Sciences, Faculty of Science

and Arts, Jordan University of Science and Technology,

Irbid, Jordan

Q. Al-Balas

Department of Medicinal Chemistry, Faculty of Pharmacy,

Jordan University of Science and Technology, Irbid, Jordan

T. Mukattash

Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan

University of Science and Technology, Irbid, Jordan

123

Int J Pept Res Ther

DOI 10.1007/s10989-014-9405-0

acquired the ability to resist antibiotics that are conven-

tionally used for the treatment of human infections (Ku-

tateladze and Adamia 2010; Leung et al. 2011). A

significant number of bacterial strains that cause noso-

comial infections are found to be resistant to one or more

conventional antibiotics (Rolain et al. 2012). Additionally,

some strains have been reported to exhibit multi-drug

resistance and others were also reported to resist almost

all approved antibiotics currently found in the clinic

(Rolain et al. 2012). Some bacterial strains have also

acquired the ability to form sessile communities of bio-

films on medical devices leading to serious untreatable

noscomial infections in immune-compromised and criti-

cally ill patients (Donlan 2002; Gotz 2002). This situation

imposes a huge pressure on the medical community to

identify novel agents capable of eradicating multidrug

resistant strains of bacteria and biofilm related infections.

Scorpions are 400 million years old and are consid-

ered to be one of the oldest animal species living on the

planet (Jeyaprakash and Hoy 2009). As a result of the

intensive environmental pressure applied on these

organisms and their long evolutionary existence, scorpi-

ons managed to develop venom cocktails rich in bioac-

tive peptides. These peptides are classified into disulfide

bridged peptides (DBPs) and the non-disulfide bridged

peptides (NDBPs). The NDBPs are functionally diverse

and display numerous biological activities (Almaaytah

and Albalas 2014; Almaaytah et al. 2012). Recently we

have identified a novel scorpion NDBP through molec-

ular cloning from the venom derived cDNA library of

the scorpion Androctonus mauritanicus. Named Mau-

riporin, this peptide was found to be composed of 48

residues and displayed a cationic alpha helical structure

(Almaaytah et al. 2013). Mauriporin was found to exhibit

selective cytotoxic and anti-proliferative activity against

prostate cancer cell lines in a dose dependent manner.

Multiple sequence alignment and sequence similarity

analysis revealed that Mauriporin belongs to the family

of multifunctional scorpion NDBPs. Members of this

group including Bmkbpp, Parabutoporin and Im-1 were

reported to display antimicrobial, insecticidal and

immune-modulatory activities. On the basis of such

findings, we decided to investigate the antimicrobial and

antibiofilm activity of Mauriporin against a range of

Gram-positive and Gram-negative bacterial strains.

Additionally, we have assessed the antibiofilm activity of

Mauriporin against biofilm growing cells of Pseudomo-

nas aeruginosa and also analyzed the underlying

molecular mechanisms responsible for the antimicrobial

activity of the peptide in addition to molecular modelling

of the peptide itself. To the best of our knowledge, this

is the first study to try to asses to antibiofilm activities of

a scorpion antimicrobial peptide.

Materials and Methods

Peptide Synthesis and Purification

The peptides employed in this study were synthesized by

solid-phase methods using N-(9-fluorenyl) methoxycar-

bonyl (Fmoc) chemistry (G.L. Biochem, China) The solvent

used for synthesis was dimethylformamide (DMF). 2-(1H-

Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-

phosphate (HBTU) was used as an activator. Fmoc was

deprotected by 20 % piperidine, and Wang resin was cleaved

using trifluoroacetic acid (TFA). The peptides were precip-

itated from dry ether. The identity and purity of the synthetic

peptide were confirmed by high performance liquid chro-

matography and ESI–MS mass spectrometry (Supplemen-

tary material).

Microorganisms

To examine the spectrum of activity of Mauriporin against

Gram-positive and Gram-negative bacteria, the antimicro-

bial activity of Mauriporin was tested and determined

against a number of standard model bacterial strains that

were acquired from the American type tissue culture col-

lection (ATCC) including Listeria ivanovii strain (ATCC

19119), Staphylococcus epidermidis (ATCC 12228),

Escherichia coli (ATCC 25922), Salmonella enterica

(ATCC 10708), Salmonella typhimurium (ATCC 29629),

Pseudomonas aeruginosa (ATCC 27853), Pseudomonas

aeruginosa (ATCC 9027), Acinetobacter baumannii

(ATCC 19606) and Klebsiella pneumoniae (ATCC

700603).

Bacterial Susceptibility Assay

The antimicrobial activity of Mauriporin was tested against

two Gram-positive bacteria and seven Gram-negative

bacteria and the Minimum inhibitory concentrations (MIC)

of Mauriporin were determined by adapting the microbroth

dilution method outlined by the Clinical and Laboratory

Standards Institute (CLSI) guidelines (Komatsuzawa et al.

2000; Ouhara et al. 2008). Briefly, bacterial cells were

grown overnight in Muller Hinton Broth and diluted to

106 CFU/ml in culture medium prior to use. Twofold serial

dilutions of Mauriporin were prepared in culture medium at

a volume of 50 ll/well in 96-well plates. Mauriporin was

tested against planktonic bacteria within the concentration

range of 0.5–30 lM. Mauriporin concentrations in the

range stated, were inoculated with microorganism cultures

(105 colony forming units (CFU)/ml), and placed into

96-well microtiter cell culture plates. Plates were incubated

for 18 h, at 37 �C, in a humidified atmosphere. Following

this, the growth of bacteria was determined by means of

Int J Pept Res Ther

123

measuring optical density (OD) at k = 570 nm by an

ELISA plate reader. For the minimum bactericidal con-

centration (MBC), 10 ll of the well contents was spread on

agar and grown at 37 �C for 24 h or 48 h. The MBC was

determined as the lowest concentration that resulted in

\0.1 % survival of the subculture. All MIC and MBC

determinations were made in triplicate.

Antibiofilm Activity

Biofilm formation was performed as previously described

in (Ceri et al. 2001; Falciani et al. 2012; Feng et al. 2013;

Luca et al. 2013) employing the Calgary biofilm device

(Innovotech, Canada). Lyophilised synthetic Mauriporin

was prepared as described in the previous section. Briefly,

P. aeruginosa (ATCC 27853) was incubated in Mueller–

Hinton broth (MHB) for 20 h at 37 �C, and cultures were

diluted in the same medium to achieve a concentration of

107 CFU/ml. 150 ll of this bacterial culture were added to

the 96 pegs-lids on which biofilm cells can build up and the

pegs were incubated for 20 h under a rotation of 125 rpm

at 35 �C to allow biofilm formation on the purpose-

designed pegs. Once the biofilms were allowed to form, the

pegs were rinsed twice with phosphate buffered saline

(PBS) to remove planktonic cells. Each peg-lid was then

transferred into a ‘‘challenge 96-well microtiter plate’’

containing two hundred microlitres of each Mauriporin

concentration and the peg lids containing the biofilms were

incubated for 2 h at 37 �C. The ability of Mauriporin to

inhibit biofilm formation was determined by three different

methods and these include the visual observation of post

biofilm bacterial growth, colony count and biofilm biomass

evaluation.

For the visual observation method, the pegs were

removed from the challenge plates and rinsed twice with

PBS. This was followed by transferring the pegs into a

96-well recovery plate containing 200 ll MHB. The

recovery plate was incubated at 37 �C for 4 h and the

plates were observed visually for any visible growth of

bacteria detached from the peptide treated peg carrying the

biofilms. Growth of bacteria in the individual wells is

indicative for biofilm survival after peptide treatment. In

addition to the visual growth, the peptide treated biofilms

were assessed for their minimum biofilm eradication con-

centration (MBEC) as this parameter is defined as the

minimum concentration needed to inhibit the regrowth of

biofilms after 4 h of peptide treatment. For colony count

determination, the pegs were removed from the plates and

transferred into tubes containing 500 ll of PBS. Sonication

was employed to remove the bacterial cells from the pegs

and 10 ll aliquots of bacterial suspension were tenfold

serially diluted, plated into sterile nutrient agar plates and

incubated overnight at 37 �C for counting. The minimum

bactericidal concentration (MBECb) was defined as the

lowest concentration to eradicate 3 log10 of the viable

microorganisms in a biofilm (99.9 % killing) after 2 h of

incubation and the CFU were expressed as the percentage

of colony growth in respect to the control which was

identified as the (peptide—untreated biofilms). The

reduction of MTT to its insoluble product formazan was

also used for the determination of viable biofilm cells.

Briefly, the pegs were washed with PBS after peptide

treatment and the pegs were transferred into a new 96 well

plate each well containing 200 ll of 0.5 mg/ml MTT in

PBS. The new 96 well plate was incubated at 37 �C for 4 h.

Afterwards, 50 ll of 25 % sodium dodecyl sulphate (SDS)

were added to each well to dissolve formazan crystals.

Bacterial viability was determined by absorbance mea-

surements at 595 nm using an ELISA plate reader and

calculated with respect to control cells (bacteria untreated

with the peptide). Finally, the biofilm biomass was deter-

mined by transferring the peptide treated pegs into a new

96 well plate containing 200 ll of 99 % methanol each,

and the plate was incubated for 15 min at room tempera-

ture in order to fix the biofilm into the pegs. Staining of the

peg lids was achieved by transferring the pegs into a new

96 well plate with each well containing 200 ll of a crystal

violet (CV) solution (0.05 % in water) for 20 min. After-

wards the excess CV was removed by washing the pegs

with water. The bound CV was released from the pegs by

transferring the peg lids into a new 96 well plate with each

well containing 200 ll of 33 % acetic acid and then the

absorbance was measured at 600 nm using an ELISA

plate reader.

Erythrocyte Hemolysis Assay

In order to determine the potential toxicity of Mauriporin

against human erythrocytes, its hemolytic activity was

examined and compared against Melittin a well studied

antimicrobial peptide in literature. The hemolytic activity

of both peptides was assessed by incubating a 4 % sus-

pension of human erythrocytes with the peptide for 60 min.

The human erythrocytes were washed several times with

0.9 % NaCl by centrifugation, then incubated for 60 min,

at 378 C in 0.9 % NaCl as the negative control, 0.1 %

Triton X-100 as the positive control and with the different

Mauriporin concentrations dissolved in 0.9 % NaCl solu-

tion. The cells were then centrifuged at 9009g for 5 min.

200 ll of the supernatnant were transferred from each

sample as 4 replicates into a 96-well plate and their

absorbance measured at 570 nm. The percent hemolysis

was calculated using the following equation: hemoly-

sis = (A – A0)/(AX - A0) 9 100, where A is OD570 with

the peptide solution, A0 is OD 570 in NaCl, and AX is OD

570 nm with 0.1 % Triton X-100.

Int J Pept Res Ther

123

Time Kill Studies of Exponential Phase Growing

Bacteria

The killing kinetics of Mauriporin against bacterial strains

was analyzed by time–kill assays as previously described

with a slight modification (Eckert et al. 2006). Time–kill

experiments were performed at 37 �C with shaking at

220 rpm. Log-phase bacteria (3 9 107 CFU/ml) were

incubated with different concentrations at 1 9 MIC,

2 9 MIC, 3 9 MIC and 4 9 MIC of Mauriporin in Muller

Hinton media. 10 ll culture aliquots were withdrawn at

different time points (0, 15, 30, 60, 120, 300 and 480 min),

serially diluted and plated into pre-sterilized nutrient agar

plates, afterwards the plates were incubated overnight at

37 �C.

Time Kill Studies of Stationary Phase Growing

Bacteria

To maintain the bacteria in slow growing stationary phase,

a nutrient depleted MHB was prepared according to the

method described previously with minor modifications

(Mascio et al. 2007). Briefly, the bacterial strains were

cultivated in MHB at 37 �C for 46 h. The bacterial strains

were centrifuged at 4,0009g for 10 min and then the

supernatant was discarded and the bacterial pellet was

washed three times with 1 9 PBS buffer. Stationary-phase

cells (1 9 106 CFU/mL) were separately incubated in

MHB in the presence of different concentrations of Mau-

riporin. The time–kill assay was performed as detailed in

the previous section.

b-Galactosidase Assay

Mauriporin’s ability to inflict significant damage to the

cytoplasmic membrane of bacterial cells and its ability to

induce membrane permeabilization was assessed by mea-

suring the activity of the peptide on the release of cyto-

plasmic beta-galactosidase from E. coli (ML-35) cells into

the culture medium using ONPG as substrate. E. coli

(ML-35) were harvested, washed and centrifuged for

10 min (1.49g) and resuspended in PBS solution with an

absorbance of 0.8 at 420 nm. About 1 9 106 cells (in

100 ll PBS) were incubated with different concentrations

of Mauriporin (1 9 to 4 9 MIC). The hydrolysis of ONPG

to O-nitro phenol by beta-galactosidase enzyme was

monitored at 405 nm using an ELISA plate reader over

different time points (10, 20, 30, 40, 50, 60 and 120 min).

Extraction of Genomic DNA

The isolation of genomic DNA from E. coli bacteria was

performed according to the Wizard� Genomic DNA

Purification Kit (Promega, USA) protocol. Extracted DNA

was considered to be pure if an optical ratio of DNA was in

the range of OD260/OD280 C1.8. The concentration of

purified E. coli DNA, the absorbance at 260 nm and the

absorbance at 280 nm were calculated using Thermo sci-

entific Nano drop 1000 and DNA rehydration solution

(Tris–EDTA) was used to calibrate the Nano drop device.

DNA Gel Retardation

The assay was performed according to the method descri-

bed previously with minor modifications (Imura et al.

2007). Briefly, 500 ng of the genomic DNA was mixed

with increasing amounts of Mauriporin in 30 ll binding

buffer (10 mM Tris–HCl, 1 mM EDTA buffer, pH 8.0).

The migration of DNA was assessed by gel electrophoresis

using a 0.8 % agarose gel prepared by adding 0.4 g of

agarose into 50 ml of 19 tris borate EDTA (TBE) buffer

with the bands detected by fluorescence of eithidium bro-

mide (EB). Finally, Gel retardation was visualized under

UV illumination using a Gel Doc_IT 310 imaging system.

In Silico Analysis and Molecular Modelling

The physicochemical parameters of Mauriporin were

evaluated using the ProtParam software from the ExPASy

server (Gasteiger et al. 2003). The HHpred (HHsearch 2.0)

software was used to find the best template for homology

modelling and structure prediction by HMM–HMM com-

parison (Soding et al. 2005). The reliable template with the

nearest homology was found with the Set2 Rpb1-interact-

ing (SRI) domain of the Huntingtin interacting protein B

and showed a score of 20.72. PROSA II was used for

analyzing packing and solvent exposure characteristics

(Wiederstein and Sippl 2007). I-TASSER was also utilized

for confirmation of the model reliability (Zhang 2008). The

RAMPAGE: Assessment of the Ramachandran Plot soft-

ware was also used for three dimensional structure vali-

dation (Sali and Blundell 1994).

The final model was visualized using Accelrys� Dis-

covery studio software.

Results

In Vitro Antimicrobial Activity of Mauriporin

Mauriporin exhibited potent antimicrobial activity against

all bacterial strains studied. MICs against the bacterial

strains are summarized in Table 1. Among these tested

strains, L. ivanovii, S. enterica, P. aeruginosa (ATCC

27853), A. baumannii and K. pneumonia were the most

sensitive to the peptide with a MIC value of 5 lM.

Int J Pept Res Ther

123

Additionally, Mauriporin displayed moderate activity with

a MIC value of 7.5 lM against both Gram-negative strains

of E .coli and S. typhimurium while the weakest activity

was reported against S. epidermidis and P. aeruginosa

(ATCC 9027) with MICs of 10 lM. The MBC values were

equal to the corresponding MIC values (Data not shown).

This behaviour indicates the Mauriporin is exerting a

bactericidal rather than bacteriostatic activity against

planktonic bacterial strains.

Antimicrobial Activity of Mauriporin Against Biofilm

Cells of P. aeruginosa

The antibiofilm activity of Mauriporin was assessed by

three independent methods (Experimental section) against

a pre-formed biofilm of P. aeruginosa (ATCC 27853)

which is a well studied biofilm forming bacterial strain

(Mah et al. 2003; Matsuzaki 2009; Falciani et al. 2012).

The methods utilized for evaluating Mauriporin’s antibio-

film activity included the visual observation of post biofilm

bacterial growth, colony count and biofilm biomass eval-

uation. As shown in Fig. 1, The MBEC value defined as the

minimum peptide concentration needed to inhibit regrowth

of bacterial biofilms after peptide treatment was found to

be equal to 200 lM which is 20 fold higher than the cor-

responding MIC value of Mauriporin against the planktonic

form of P. aeruginosa. Additionally, the minimum Mau-

riporin concentration needed to reduce the number of via-

ble biofilm cells to almost zero (99.9 % killing) (MBECb)

was found to be 500 lM which was 2.5 fold higher than the

MBEC value (Fig. 1). The percentage of viable biofilm

cells after peptide treatment was also determined using the

MTT assay and colony count method. As shown in

Table 2, The viable count method was more sensitive in

detecting the number of viable bacterial cells when com-

pared to the MTT assay and complete eradication of bio-

film viability was achieved at 500 lM which is consistent

with the MBECb value, while at a concentration of 200 lM

which corresponds to the MBEC value, the percentage of

viability using the previous methods was reported to be of

38 %. Finally, the biofilm biomass percentage after peptide

treatment was detected by CV staining and percentages

were calculated with respect to control samples (bacterial

biofilm not treated with Mauriporin), from a concentration

of 350 lM up to 500 lM of Mauriporin, the biofilm bio-

mass percentages ranged from 29 to 7 %. These concen-

trations were responsible for almost total microbial death

as detected by MTT reduction assay (Fig. 1; Table 2).

Table 1 Minimum inhibitory concentrations (MICs) of Mauriporin

against the test microorganisms employed in this study

Gram-positive strains MIC value (lM)

Listeria ivanovii 5

Staphylococcus epidermidis 10

Gram-negative strains MIC value (lM)

Salmonella enterica 5

Pseudomonas aeruginosa (ATCC 27853) 5

Acinetobacter baumannii 5

Klebsiella pneumoniae 5

Escherichia coli 7.5

Salmonella typhimurium 7.5

Pseudomonas aeruginosa (ATCC 9027) 10

0 50 100 150 200 250 300 350 400 450 5000

10

20

30

40

50

60

70

80

90

100Antibiofilm activity

Concentration (µM)

%

Colony count

MTT

Biofilm biomass

Fig. 1 Mauriporin antibiofilm activity against P. aeureginosa using

three interdependent methods. The activity was analyzed by calcu-

lating the percentage of viable bacterial cell counts after peptide

treatment. (MTT, colony counts) and also by calculating the biofilm

biomass. Y-axis indicates the total percentage with respect to control

samples (bacterial biofilm not treated with the peptide). Data are

representative of three independent experiments

Table 2 Antibiofilm activity of Mauriporin at different concentrations

Peptide

concentration (lM)

Viable Biofilm cellsa (%) Biofilm

biomassb (%)Colony

counts

MTT

reduction

500 0.1 2 7

450 0.7 3 18

400 4.3 2 27

350 8 2 29

300 11 4 30

250 14 6 37

200 38 38 51

150 68 63 63

100 79 69 98

50 98 98 98

The antibiofilm activities of Mauriporin was evaluated by assessing

the peptide’s activity on the number of viable cells and biofim bio-

mass. The experiments were performed in triplicates for all values

mentioned abovea Percentage of viable biofilm cells was determined using the viable

colony count method or by the reduction of MTT to formazanb The biofilm biomass percentage was determined using crystal violet

Int J Pept Res Ther

123

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 4800

1

2

3

4

5

6

7

8

9

10

11

E.coli

Time (min)

Log

CFU

/ml

7.5 (µM)

15 (µM)

22.5 (µM)

30 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11S.typhi

Time (min)

Log

CFU

/ml

7.5 (µM)

15 (µM)

22.5 (µM)

30 (µM)

Positive

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 4800

1

2

3

4

5

6

7

8

9

10

11 A.baumannii

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15 (µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11 K.pneumoniae

Time (hr)

Log

CFU

/ml

5 (µM)

10 (µM)

15 (µM)

20 (µM)

Positive

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0

1

2

3

4

5

6

7

8

9

10

11S.enterica

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15 (µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11P.aeruginosa

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15 (µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11P.aeruginosa (ATCC 9027)

Time (min)

Log

CFU

/ml

10 (µM)

20 (µM)

30 (µM)

40 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11

S.epidermis

Time (min)

Log

CFU

/ml 10 (µM)

20 (µM)

30 (µM)

40 (µM)

Positive

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 4800

1

2

3

4

5

6

7

8

9

10

11L.ivanovii

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15 (µM)

20 (µM)

Positive

Fig. 2 Time-kill assays for Mauriporin against exponentially grow-

ing bacterial strains of E. coli, S. typhi, A. baumannii, K. pneumoniae,

S. enterica, P. aeruginosa-ATCC 27853, P. aeruginosa-ATCC 9027,

the S. epidermidis and L. ivanovii. Viability was counted at the

indicated time points by serial dilution plating. Values are the mean of

independent tests performed in duplicates

Int J Pept Res Ther

123

0

1

2

3

4

5

6

7

8

9

10

11 E.coli

Time (min)

Log

CFU

/ml

7.5 (µM)

15 (µM)

22.5 (µM)

30 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11

S.typhi

Time (min)

Log

CFU

/ml

7.5 (µM)

15 (µM)

22.5 (µM)

30 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11

A.baumannii

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15(µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11 K.pneumoniae

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15(µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11S.enterica

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15(µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11P.aeruginosa

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15(µM)

20 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11P.aeruginosa ( ATCC 9027)

Time (min)

Log

CFU

/ml

10 (µM)

20 (µM)

30 (µM)

40 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11 S.epidermis

Time (min)

Log

CFU

/ml

10 (µM)

20 (µM)

30 (µM)

40 (µM)

Positive

0

1

2

3

4

5

6

7

8

9

10

11L.ivanovii

Time (min)

Log

CFU

/ml

5 (µM)

10 (µM)

15(µM)

20 (µM)

Positive

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 4800 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 0 30 60 12090 150 180 210 240 270 300 330 360 390 420 450 480

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

Fig. 3 Time-kill assays for Mauriporin against stationary growing

bacterial strains of E. coli, S. typhi, A. baumannii, K. pneumoniae,

S. enterica, P. aeruginosa-ATCC 27853, P. aeruginosa-ATCC 9027,

the S. epidermidis and L. ivanovii. Viability was counted at the

indicated time points by serial dilution plating. Values are the mean of

independent tests performed in duplicates

Int J Pept Res Ther

123

Killing Kinetics

The results of the killing kinetics analysis of Mauriporin

against exponentially growing bacterial strains of L. iva-

novii, S. epidermidis, E. coli, S. enterica, S. typhimurium,

P. aeruginosa (ATCC 27853), P. aeruginosa (ATCC

9027), A. baumannii and K. pneumoniae are shown in

Fig. 2. Mauriporin managed to totally eradicate (99.9 %

killing) almost all strains of bacteria tested within 30 min

of incubation when exposed to the corresponding MIC

concentrations of the peptide. At 2-fold MIC, E. coli were

killed within 30 min of exposure while at threefold MIC,

S. epidermidis were killed within 60 min. Finally, at

fourfold MIC, 300 min of exposure were needed to totally

kill K. pneumonia. For the slow growing stationary bac-

teria, Mauriporin displayed bactericidal effects (99.9 %

killing) at MIC values of the peptide within 30 min of

incubation against all bacterial strains with the exception of

K. pneumonia which needed a threefold MIC concentration

of the peptide to kill cells within 180 min (Fig. 3). This

behaviour is consistent with several other cationic antimi-

crobial peptides reported earlier and highlights the bacte-

ricidal nature of the Mauriporin against bacteria (Feng

et al. 2013; Luca et al. 2013). Additionally, the fast rate of

killing which is observed with Mauriporin excludes the

possibility of having an intracellular target as the major

mechanism of action for Mauriporin and indicates that the

peptide is possibly killing bacteria through membrane

permeabilization.

Hemolytic Assay

In order to examine the potential toxicity of Mauriporin

against human mammalian cells and compare its toxicity

against reported antimicrobial peptides in literature, the

hemolytic activity of Mauriporin was compared against the

antimicrobial peptide Melittin, an extensively studied

peptide with potent antimicrobial activities. For both pep-

tides the hemolytic activity was studied as a measure of

toxicity against human erythrocytes at a concentration

ranging from 1 to 100 lM. Results of the hemolytic

activity are presented in Table 3. At the MIC & MBC

concentrations of Mauriporin which were reported to be in

the range of 5–10 lM against all bacterial strains evaluated

previously, Mauriporin exhibited minimal hemolysis of

(3–5.8 %) when incubated with human RBCs for 1 h while

the hemolytic activity of Melittin at the same concentra-

tions were significantly higher as the percentage hemolysis

was in the range of (17–32.8 %). When the concentration

of Mauriporin was increased to a concentration of 100 lM

which is tenfold higher than the MIC & MBC values

obtained for all tested strains, Mauriporin caused 15.4 % of

hemolysis and the percentage hemolysis reported for

Melittin was 51.4 % (Table 3). The results obtained from

the hemolytic studies clearly display that Mauriporin is not

causing significant hemolysis at antimicrobial concentra-

tions and the peptide is significantly less toxic than the

antimicrobial peptide Melittin.

Cytoplasmic Membrane Permeability

An increase in cytoplasmic permeability and leakage of

intracellular components as a result of membrane damage

causes b-galactosidase enzyme to convert ONPG to gal-

actose and ortho-nitrophenol which can be monitored

spectrophotometrically at 405 nm. As shown in Fig. 4,

Mauriporin at different concentrations induced significant

change in the permeability of E. coli cells as indicated by

the rapid increase of OD values over time which corre-

sponds with o-nitrophenol formation. The absorbance

increased in a dose dependent manner and it reaches it

Table 3 Hemolytic effect of Mauriporin and Melittin on human

erythrocytes after 60 min of incubation

Peptide

concentration (lM)

Hemolysis (%)

(Mauriporin)

Hemolysis (%)

(Melittin)

1 0.2 0.16

5 3 17.1

10 5.8 32.8

20 9.1 45.9

40 11.8 46.6

60 12 49.9

80 12.5 50.5

100 15.4 51.4

Time (min)

OD

(42

0)

0 10 20 30 40 50 60 70 80 90 100 110 1200.5

0.6

0.7

0.8

0.9

1.07.5 (µM)

15 (µM)

30 (µM)

PBS

Fig. 4 Release of cytoplasmic b-galactosidase from E. coli bacterial

cells after treatment with four concentrations of Mauriporin (7.5, 15,

22.5 and 30 lM) or PBS (negative control). The Y-axis represents the

optical density (OD) at 405 nm. Data are representative of two

independent experiments

Int J Pept Res Ther

123

maximum after 60 min of incubation for all four concen-

trations employed.

DNA-Binding Activity

The DNA-binding capability of Mauriporin was evaluated

by analyzing the electrophoretic mobility of DNA bands at

various ratios of peptide to DNA. As shown in Fig. 5.

Mauriporin managed to cause significant retardation in the

movement of the DNA in a peptide/DNA ratio dependent

manner. Mauriporin managed to completely suppress DNA

mobility of the different peptide/DNA samples starting at a

ratio of 1.27 and up to a ratio of 3.82.

Molecular Modelling of Mauriporin

The theoretical analyses of Mauriporin revealed that the

peptide is hydrophilic in nature scoring a grand average of

hydropathicity of -0.586, calculated as the sum of

hydropathy values of all amino acids divided by the number

of residues in the sequence. Mauriporin’s theoretical model

assessment revealed the peptide to exhibit an alpha helix

conformation as generated by two different methodologies

described previously (Fig. 6). The three dimensional model

generated for Mauriporin was validated using Ramachan-

dran plot and confirmed that 98 % of the modelled amino

acid residues were in acceptable regions in relation to the

phi and psi torsion angles of the secondary structure for-

mation model. Additionally, the PROSA II software gen-

erated a z-score value of -2 for Mauriporin which indicated

the generated Mauriporin model is of good quality.

Discussion

Antimicrobial peptides (AMPs) represent an attractive

group of molecules for development as novel therapeutics

for the purpose of combating microbial infections and

specifically the ones that are caused by resistant forms of

bacteria. Over the last few decades, there has been a pro-

gressive shrinkage and decline in the number of effective

antibiotics available in the clinic due to the excessive

overuse and misuse of this class of drugs, issues that

heavily contributed the global problem of microbial resis-

tance (Ouhara et al. 2008; Davies and Davies 2010; Leung

et al. 2011; Rolain et al. 2012). Mauriporin is a novel

cationic-alpha helical peptide that was identified through

molecular cloning from the venom derived cDNA library

of the scorpion A. mauritanicus and was found to exhibit

potent selective antiproliferative activities against prostate

cancer cell lines. Multiple sequence alignment and

sequence similarity analysis revealed that Mauriporin

belongs to the multifunctional group of scorpion non-

disulfide bridged peptides (NDBPs) which are known to

display multifunctional activities including antimicrobial,

immune-modulatory and bradykinin potentiating activities.

In this study we have functionally characterized the

antimicrobial and antibiofilm activities of Mauriporin. Our

studies revealed that Mauriporin exhibits potent broad

spectrum antimicrobial activities with MIC values in the

range of 5–10 lM against different strains of Gram-posi-

tive and Gram-negative bacteria. The MBC values of

Mauriporin were equal to the MIC values suggesting that

the peptide exerts a bactericidal rather than a bacteriostatic

activity against the different microbes that were examined

in this study. These results are consistent with the antimi-

crobial activities of other scorpion NDBPs sharing high

sequence homology with Mauriporin such as Im-1,

Parabutoporin and BmKbpp and thus confirming the mul-

tifunctional nature of this group of molecules (Miyashita

et al. 2010; Remijsen et al. 2010; Zeng et al., Zeng et al.

2012). Additionally, the killing kinetics of Mauriporin

against bacterial strains in both exponential and stationary

Fig. 5 Gel retardation assays. Binding was assayed by the inhibitory

effect of Mauriporin on the migration of DNA bands. Various

amounts of peptides were incubated with 500 ng of E. coli genomic

DNA at room temperature for 10 min, and the reaction mixtures were

applied to a 0.8 % agarose gel. The gel was visualized after ethidium

bromide staining and UV irradiation. The numbers below the lanes

represent the weight ratio (peptide/DNA)

Fig. 6 Three-dimensional structural modelling of Mauriporin. Red

regions correspond to helical structures within the peptide while the

green and white regions represent hinged regions and unordered

conformations, respectively. The structure was visualized using

Accelrys Discovery studio software (Color figure online)

Int J Pept Res Ther

123

growing phase revealed that the peptide is exhibiting fast

killing kinetics, a behaviour that is correlated with a

membrane permeabilization mode of action reported with

most AMPs in contrast to the slow killing kinetics dis-

played by antibiotics and antimicrobial agents targeting

intracellular bacterial components (Schneider et al. 2010).

However, the fast killing antimicrobial behaviour reported

with Mauriporin could lead to serious patient adverse

effects if the peptide is to be considered for parentral

antimicrobial therapy. The fast killing of bacterial cells can

lead to the release of a significant amount of bacterial cell

wall products such as lipopolysaccharide (LPS) and li-

poteichoic acid, resulting in potentially lethal sepsis.

The membrane permeabilization mode of action pro-

posed for Mauriporin was also confirmed and verified by the

dose dependent increase in plasma membrane permeability

and OPNG influx as determined by the beta-galactosidase

assay described previously. This increase in cytoplasmic

membrane permeability is indicative of a significant loss of

the membrane’s structural integrity and is consistent with

previous studies performed against tumorigenic cell lines

treated with Mauriporin which showed significant increase

in intracellular lactate dehydrogenase LDH permeability

(Almaaytah et al. 2013). The antibiofilm activity of Mau-

riporin was also investigated against P. aeureguinosa

bilofilms grown on pegs-lids supplied by the Calgary bio-

film device. A 20 fold increase in MIC concentration of

Mauriporin (MBEC) was required to inhibit the re-growth

of biofilms within 4 h of peptide treatment. The tolerance of

P. aeruginosa biofilm cells towards Mauriporin may be

explained by the inherent structural resistance of sessile

biofilm bacteria towards AMPs. P. aeureguinosa biofilms

are reported to display a significant amount of b-D-man-

nuronate and a-L-guluronate polymer alginates on their

extracellular polymeric matrix; these alignates can decrease

the dielectric constant of aqueous mediums and conse-

quently decrease the polarity and solubility of cationic

AMPs and eventually lead to peptide–peptide aggregation

within the biofilm microenvironment (Chan et al. 2004).

Moreover, the anionic alignates have been reported to

neutralize the positive charge carried by cationic AMPs

(Sutherland 2001). These factors contribute collectively in

deactivating the antimicrobial activity of cationic AMPs

and could form the molecular basis of the inherent resis-

tance displayed by biofilms towards this class of molecules.

However, the MBEC/MIC ratio reported for Mauriporin

was 20, whereas some of the antibiotics used in the clinic

for treating human infections such as aminoglycosides,

fluoroquinolones, and tetracyclines require a concentration

that is 100-fold higher the MIC in order to kill sessile

bacteria (Olson et al. 2002; Høiby et al. 2011), this indicated

that Mauriporin is still more effective than antibiotics in

retarding bacterial biofilm growth.

Three different methods for biofilm quantification were

evaluated in this study, as reported in the previous section

the minimum Mauriporin concentration needed to reduce

the number of viable biofilm cells to almost zero (99.9 %

killing) (MBECb) was found to be 500 lM which is con-

sistent with the values obtained from the colony count

method for biofilm quantification and clearly displays that

this method is most suitable for biofilm quantification when

compared to CV staining and MTT reduction to insoluble

formazan (Table 2) as several inconsistencies in biofilm

biomass percentage were reported with these techniques.

The hemolytic activity of Mauriporin was also assessed

and compared with Melittin against human erythrocytes

and the results confirm the high selectivity and low toxicity

of the peptide against mammalian cells as reported in

previous studies and in agreement with several selective

membrane active peptides reported in literature (Zasloff

1987; Matsuzaki 2009; Wang et al. 2008). Several AMPs

have been reported to cross the plasma membrane and bind

intracellular targets after disrupting the cytoplasmic

membrane’s structural integrity (Joshi et al. 2010; Zhang

et al. 2010). Accordingly, we assessed the ability of

Mauriporin to bind bacterial genomic DNA and the results

indicate that the peptide exhibits significant potency in

causing DNA gel retardation at different peptide/DNA

ratios, although several additional studies are needed to

explore the ability of Mauriporin to cross the plasma

membrane, our initial results display the peptide could

display additional intracellular activities. However, the

ability of Mauriporin to significantly inhibit DNA retar-

dation could also be indicative of Mauriporin’s ability to

cause significant genotoxicity to mammalian cells if the

peptide is to be considered for clinical development. Future

studies addressing the cytotoxicity and genotoxicity of the

peptide should be performed. Previously we have reported

that Mauriporin exhibits an alpha-helical structure using

CD spectroscopy, to confirm the helical nature of the

peptide we have reconstructed a 3D model of Mauriporin

using in silico homology modelling and the model clearly

displays a stable alpha helical 3D structure in confirmation

with the CD results. The three dimensional structural

analysis of Mauriporin can lead to a better understanding of

the structural determinants responsible for the antimicro-

bial activity and target selectivity. Additionally, the natu-

rally identified peptide sequence of Mauriporin can provide

a potential template for peptide optimization in future

studies for the purpose of generating novel synthetic ana-

logs of the parent peptide with enhanced antimicrobial

activity and decreased toxicity.

Finally we report the functional characterization of the

antimicrobial and antibiofilm activities of Mauriporin. To

the best of our knowledge, this is the first scorpion venom

peptide reported to display antibiofilm activities. Although

Int J Pept Res Ther

123

Mauriporin did not display potent antibiofilm activities when

compared with its activity against planktonic bacteria,

Mauriporin could prove to be a useful platform for effective

antimicrobial drug design. Additionally, the membrane

perturbation activities of Mauriporin could be exploited for

use in combination regimens with intracellular targeting

antibiotics to facilitate their entry across bacterial plasma

membranes.

Conflict of interest The authors of this manuscript certify that they

have no affiliations with or involvement in any organization or entity

with any financial interest (such as honoraria; educational grants;

participation in speakers’ bureaus; membership, employment, con-

sultancies, stock ownership, or other equity interest; and expert tes-

timony or patent-licensing arrangements) in the subject matter or

materials discussed in this manuscript.

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