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Accepted Article
Article Type: Original Article
Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX, a
Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom
(Linnaeus, 1766)
Rafaela Diniz-Sousa1,2, Cleópatra A. S. Caldeira1,3, Anderson M. Kayano1, Mauro V.
Paloschi1,2,4, Daniel. C. Pimenta5, Rodrigo Simões-Silva1, Amália S. Ferreira1, Fernando B.
Zanchi1,2,3, Najla B. Matos1,2,6, Fernando P. Grabner7, Leonardo A. Calderon1,2,3, Juliana P.
Zuliani1,2,3,4 and Andreimar M. Soares1,2,3,7
1
Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo
Cruz, FIOCRUZ, Fiocruz Rondônia, and Departamento de Medicina, Universidade
Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
2
Programa de Pós-Graduação em Biologia Experimental, PGBIOEXP, Universidade
Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
3
Programa de Pós-Graduação em Biodiversidade e Biotecnologia, Rede BIONORTE,
Brazil.
4
Laboratório de Imunologia Celular Aplicada à Saúde, Fundação Oswaldo Cruz,
FIOCRUZ, Fiocruz Rondônia, Porto Velho-RO, Brazil.
5
Laboratório de Bioquímica, Instituto Butantan, São Paulo-SP, Brazil.
6
Laboratório de Microbiologia, Centro de Pesquisa em Medicina Tropical de
Rondônia, CEPEM, and Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia,
Porto Velho-RO, Brazil.
7
Centro Universitário São Lucas, UNISL, Porto Velho-RO, Brazil.
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/bcpt.12921
This article is protected by copyright. All rights reserved.
Accepted Article
Author for correspondence: Andreimar Martins Soares. Centro de Estudos de Biomoléculas
Aplicadas à Saúde – CEBio, Fiocruz Rondônia/FIOCRUZ. Rua da Beira, 7176, Bairro
Lagoa, 76812-245 Porto Velho, Rondônia, Brazil (e-mail: [email protected]).
Abstract: Snake venom phospholipases A2 (PLA2s) are responsible for numerous
pathophysiological effects in snakebites; however, their biochemical properties favour
antimicrobial actions against different pathogens, thus constituting a true source of potential
microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from
Lachesis muta muta venom using two chromatographic steps: Size Exclusion and Reverse
Phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase
activity on an artificial substrate. The primary structure made it possible to identify an
unpublished protein from L. m. muta venom, named LmutTX, that presented high identity
with other Lys49 PLA2s from bothropic venoms. Synthetic peptides designed from LmutTX
were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against
C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low
cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gramnegative bacteria; however, S. aureus ATCC 29213 and MRSA strains were more sensitive to
the toxin’s action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosa
ATCC 27853 strains, showing promising results. This study describes for the first time the
isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific
regions of the sequence may constitute new sources of molecules with biotechnological
potential.
Keywords: Snake venom, Lachesis muta muta, Phospholipase A2 homologue, Synthetic
peptides, Antibacterial activity.
Accepted Article
In recent years, the search for new antimicrobial agents from natural sources has intensified,
such as those from microorganisms [1], algae [2], plants [3], along with anuran [4], scorpion
[5], spider [6], wasp [7], bee [8] and snake venoms [9,10] that would constitute an important
treatment alternative against innumerable emerging or non-emerging infectious diseases that
afflict society.
Among these, snake venoms have shown promising antimicrobial activities on
different pathogens, such as bacteria, fungi and protozoa [11–13]. Of the proteins found in
these venoms, phospholipases A2 (PLA2s) represent one of the main classes of molecules
responsible for such activities [12,14–16].
A subgroup of PLA2s within this family can induce tissue damage by mechanisms
independent of catalysis. These proteins, called PLA2 homologues or Lys49 PLA2s, present a
substitution of the aspartate residue at position 49 for a lysine residue, which prevents the
coordination of Ca2+ ions in the binding loop, leading to loss of enzymatic activity [17].
Although they do not hydrolyze membrane phospholipids, PLA2 homologues can cause local
myonecrosis and oedema [18,19] and exert antimicrobial action on a variety of pathogenic
microorganisms [11,20].
Recent studies suggest that residues located in the C-terminal region of the molecule
(consisting of a combination of hydrophobic and cationic residues) may be involved in the
destabilization and perturbation of biological membranes, with a preference for anionic
phospholipids [21,22]. C-terminal peptides derived from PLA2 homologues have been
evaluated for their microbicidal and anti-tumour potential [20,23,24], presenting promising
results.
This article is protected by copyright. All rights reserved.
Accepted Article
The snake Lachesis muta muta, also known as “surucucu pico-de-jaca” or
“bushmaster”, is the largest venomous snake in the Americas. Because it inhabits remote
forest areas, this snake is difficult to find and/or keep in captivity, with few published studies
due to the difficulty in obtaining its venom [25–27].
This study describes, for the first time, the isolation and biochemical
characterization of a PLA2 homologue from Lachesis muta muta snake venom, called
LmutTX, and demonstrates that synthetic peptides derived from the primary structure of the
protein may represent possible antibacterial agents against multiresistant bacteria.
2. Material and methods
2.1 Venom
The lyophilized venom of the snake Lachesis muta muta (LmmV) was obtained from
the serpentarium BioAgents (Batatais-SP, Brazil). The study was authorized by the Brazilian
Institute of Environment and Renewable Natural Resources (license number 27131-1).
2.2. Synthetic peptides
The peptides synthesized from residues 1-12 (SLVELGKMILQE – PepN and PepNW), 41-52 (DRCCYVHKCCYK – PepS and PepS-W) and 105-116 (KKYNYLKPFCKK –
PepC and PepC-W), were obtained from Aminotech Research and Development, Diadema –
SP, Brazil, with a degree of purity greater than 97%. The modified peptides (represented by
letter W) correspond to the original sequence with the substitution of valine residue (PepNW) and tyrosine residue (PepS-W and PepC-W) by tryptophan.
Accepted Article
2.3. Isolation of the LmmV toxin
LmmV was solubilized in a 0.5 M ammonium bicarbonate buffer pH 8.0 and applied
to a Sephacryl S200 chromatography column (GE Lifescience Health Care, 10 x 30 cm),
coupled with an Akta Purifier HPLC system (GE Lifescience Health Care). The fraction of
interest was submitted to fractionation in a C18 reverse phase column (25 cm x 4.6 mm, 5 μm
- Discovery Supelco) using 0.1% trifluoroacetic acid (TFA - Sigma Aldrich, USA) (Eluent A)
and 99.9% acetonitrile (ACN - Sigma Aldrich, USA) + 0.1% TFA (Eluent B) as solutions, at
a flow rate of 1 mL/min and linear gradient of 0-70%. The absorbance was measured at 280
nm.
2.4. Measurement of protein concentration
The quantification of LmmV proteins, fractions and toxin followed the modified
Lowry method (DC Protein, Bio-Rad, USA), using bovine serum albumin (BSA) as a
standard.
2.5. One-dimensional eletrophoresis
The procedure for one-dimensional electrophoresis was performed according to
Laemmli [28]. Determination of the relative mass of the proteins was evaluated by SDSPAGE using gels in discontinuous formats with a concentrator gel (4% acrylamide in 0.5 M
Tris-HCl buffer, pH 6.8) (Sigma Aldrich, USA) and a separating gel (12.5% acrylamide in
1.5 M Tris-HCl buffer, pH 8.8). The experimental buffer solution used to fill the well
reservoirs was formed by 0.06 M Tris-Base, 0.5 M Glycine and 10% SDS (Sigma Aldrich,
USA). The samples were preheated at 95°C for 5 min. and applied to the concentrator gel
wells along with the Molecular Weight standard (7 to 175 kDa - BioLabs P7709S, USA or 9
This article is protected by copyright. All rights reserved.
Accepted Article
to 200 kDa, Amresco’s BlueStep K973, USA). In the electrophoretic experiment, a current of
15 mA per gel and free voltage was fixed for 1 hr and 40 min.
After the experiment, the gel was washed for 15 min. with a fixing solution (50%
ethyl alcohol and 12% acetic acid) and then stained with Coomassie G-250 blue solution
(Sigma Aldrich, USA) for 10-30 min. After this period, the gel was destained in bleach
solution (20% ethyl alcohol and 3% acetic acid). Images of the gels were scanned in an
ImageScanner III (GE Lifescience Health Care).
2.6. Phospholipase activity on 4N3OBA
The procedure was performed as described by Petrovic et al. [29] with
modifications. The
substrate 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) (Enzo
Lifescience, USA) (5 mg) was diluted in 5.4 mL of acetonitrile. 0.2 mL aliquots were dried
and maintained at -20°C. Each tube containing 4N3OBA was diluted in 2 mL of sample
buffer (0.01 M Tris-HCl at pH 8.0, 0.01 M CaCl2 and 0.1 M NaCl) (Sigma Aldrich, USA)
and kept on ice. In order to determine the phospholipase activity, 190 μL of 4N3OBA reagent
was combined with 10 μL of sample [crude venom and/or fractions, BthTX-2 (positive
control) or water (negative control)] and immediately incubated at 37°C; the absorbance was
measured at 425 nm for 30 min. Phospholipase activity was considered to be directly
proportional to the increase in absorbance values, expressed as mean ± standard deviation and
submitted to analysis of variance (ANOVA) followed by the Tukey post-test for p <0.05.
2.7. Sequencing and molecular modelling of LmutTX
The isolated toxin was sequenced using two techniques: i) N-terminal sequencing by
Edman degradation, in which the sequence was determined by a PPSQ-33A automatic
sequencer (Shimadzu, Kyoto, Japan), and subsequently subjected to a similarity search using
Accepted Article
BLAST software, with successive multiple alignment through CLUSTALW2; and ii) Mass
spectrometry in which the protein was digested with trypsin (Sequencing grade modified
trypsin, Promega), according to the manufacturer’s instructions (protease:protein ratio of 1:20
w/w), and the fragments obtained from the digestion were analysed in an ESI-IT-TOF
(Electrospray-Ion Trap-Time of Flight) spectrometer (Shimadzu Co., Japan) equipped with
UFLC (Ultra-Fast Liquid Chromatography) (20A Prominence, Shimadzu). The generated
MS/MS spectra were analysed using the software Peaks Mass Spectrometry (Bioinformatics
Solutions Inc., Canada) and the accuracy was calculated according to Coutinho-Neto et al.
[30].
In order to investigate structural characteristics of the amino acids, we performed
homology modelling of LmutTX’s sequence using the crystal structure of MTX-II from
Bothrops brazili (PDB code: 4DCF) as template, with a resolution of 2.7Å [31] and the
structure of an acidic PLA2 from Deinagkistrodon acutus (PDB code: 1IJL) [32]. In order to
search and retrieve the template structure, Protein Blast (http://blast.ncbi.nlm.nih.gov) [33]
and Protein Data Bank (PDB) (http://www.pdb.org) were used. The sequence alignments
were carried out in MODELLER v9.16 [34] and ClustalW [35] software. Model building was
carried out in MODELLER v9.16. A total of 1000 models were generated, and the final
model was selected based on the lowest DOPE scores calculated by the MODELLER
software. The overall stereochemical quality of the final model for LmutTX was assessed
using the program PROCHECK [36]. Interactive visualization and comparative analysis of
molecular structures were carried out in UCSF Chimera [37].
This article is protected by copyright. All rights reserved.
Accepted Article
2.8. Antimicrobial activity
The antibacterial activity was established according to the Clinical and Laboratory
Standards Institute (CLSI, 2012). The percent of inhibition of bacterial growth was
determined using a microdilution susceptibility test; the bacteria used were Staphylococcus
aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas
aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 13883) and Escherichia coli
(ATCC 25922). All bacterial strains were cultured in Luria Bertani broth (LB - BD Difco ™)
for 24 hr (exponential phase) and adjusted to a turbidity absorbance corresponding to 0.5 on
the McFarland scale, signifying 1.5 x 106 colony forming units/mL. The samples were
incubated with the bacteria at concentrations of 12.5 μg/mL for LmutTX and 125, 62.5, 31.2,
15.6 and 7.8 μg/mL for the synthetic peptides with a final volume of 200 μL and analysed in
96-well microplates for 24 and 48 hr at 37°C. The bacterial suspension in LB broth and the
antibiotics Imipenem (LmutTX) and Chloramphenicol (Peptides) (Sigma Aldrich, USA) (20
μg/mL) were used as controls. LB broth alone was used as a negative control. Inhibition of
bacterial growth was determined by spectrophotometry at a wavelength of 630 nm (TC 96
Elisa Microplate Reader). The results were expressed as mean ± standard deviation and
submitted to analysis of variance (ANOVA) followed by the Tukey post-test with a
significance level of p <0.05. The experiments were performed in triplicate (n = 3).
2.9. Cytotoxicity on differentiated C2C12 muscle cells in myotubes
The procedure was performed according to Lomonte et al. [39] with modifications.
C2C12 cells (murine myoblast cell line ATCC® CRL-1772™) were grown in DMEM
(Dulbecco's modified Eagle's medium, Sigma Aldrich, USA) supplemented with 10% foetal
bovine serum, (FBS, Sigma Aldrich, USA), 2 g/L HEPES, 3.7 g/L bicarbonate and 50 µg/mL
of gentamicine (Sigma Aldrich, USA) in a humidified atmosphere with 5% CO2 at 37ºC. An
Accepted Article
initial suspension of 2 x 104 cells was plated in 96-well plates and incubated for 4 days. After
this period, the growth medium was replaced by DMEM with 1% FBS, for 4-6 days. For the
cytotoxicity assay, the protein and peptides were diluted in PBS and added to the cell culture,
in a total volume of 200 μL/well. Controls of 0% and 100% toxicity consisted of cell medium
and Triton X-100 (Sigma Aldrich, USA), respectively. After 3 hr of incubation, 150 μL
aliquots of the supernatant were collected to determine the activity of lactic dehydrogenase
(LDH), released due to cell damage, using a commercial kit (LDH Ref. K014, Bioclin,
Brazil). The results were expressed as mean ± standard deviation and submitted to analysis of
variance (ANOVA) followed by the Tukey post-test with a significance level of p <0.05. The
experiments were performed in triplicate (n = 3).
3. Results
3.1. Purification and physical-chemical characterization of a PLA2 homologue from Lachesis
muta muta venom (LmmV)
LmmV was fractionated in two chromatographic steps: Size Exclusion and Reverse
Phase. In the first step, four fractions, named LmS-1 through LmS-4, respectively (Fig. 1),
were eluted. The fractions LmS-3 and LmS-4 presented proteins with molecular weights of
approximately 13 kDa (Fig. 1A) (compatible with snake venom PLA2s) and when submitted
to an evaluation of the phospholipase activity on an artificial substrate, neither presented
significant activity (Fig. 1B). Due to the low amount of protein in the LmS-4 fraction, the
study was directed to the LmS-3 fraction (which corresponds to approximately 2% of the
protein content of the venom). In the second step, the LmS-3 fraction was chromatographed
on a reverse phase column, where six fractions (P1 to P6) were eluted. 12.5% SDS-PAGE of
the fractions in reducing conditions showed that the fraction P6 consisted of a single protein
band (< 19 kDa), whereas the other fractions showed no visible bands (Fig. 2A), possibly
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Accepted Article
because they contain peptide fractions. Evaluation of the phospholipase activity of the P6
fraction showed that it was devoid of enzymatic activity, since there was no statistically
significant difference between the negative control and P6 (Fig. 2B), possibly being a PLA2
homologue. The sample was subjected to a second step of chromatographic fragmentation in
a reverse phase column in order to ascertain its purity, which was confirmed by the elution of
a single fraction (Fig. 2C) in 36% Eluent B and the presence of a single protein band on 15%
SDS-PAGE. The precise determination of the molecular mass of P6 was performed by mass
spectrometry, where the protein presented an m/z value of 13,890 and only one peak in the
mass spectrum (Fig 2D). The purified protein coreesponded to 0.8% of the venom protein
content and was named LmutTX.
3.1.1 Sequencing and molecular modelling of LmutTX
The primary structure of LmutTX was determined using two techniques: i) Edman
degradation sequencing, obtaining the first 57 amino acid residues from the N-terminal
region of the molecule, and ii) Mass spectrometry by analysing the peptides’ fragmentation
spectra generated from enzymatic digestion, with the elucidation of 63 amino acid residues.
A total of 120 residues were determined, completing 98.36% of the sequence, with only 2
residues located in the non-sequenced C-terminal region. The fragments obtained from
enzymatic digestion, with their respective regions in the primary structure and molecular
masses are listed in Table 1.
The similarity search and multiple alignment of the complete sequence in the
database proved that the protein possesses high identity with snake venom Lys49 PLA2
homologues of the genus Bothrops, such as MTX-II – B. brazili (95.1%), MjTX-II – B.
moojeni (92.6%), BnSP-7 – B. pauloensis (92.4%), PrTX-II – B. pirajai, Blk-PLA2 – B.
leucurus (90.9%) and BaTX – B. alternatus (86.8%) (Fig. 3). This is the first description of a
Accepted Article
snake venom Lys49 PLA2 from the genus Lachesis. The amino acid sequence of LmutTX
showed conserved residues within Lys49 PLA2s, such as His48, Asp99, Tyr52 and Tyr73
(active site) and the Tyr28 → Asn, Gly32 → Leu and Asp49 → Lys substitutions (Ca2+
binding loop), according to the numbering system proposed by Renetseder et al. [40].
The template 4DCF showed 95% similarity to the LmutTX sequence. Despite the high
similarity, the modelling is important for the verification of the amino acid positions of the
active site. The analysis of the Ramachandran plot [41] showed that more than 90.4% of the
amino acids of the LmutTX were in favourable regions and RMSD between structures
resulted in 0.52Å. Thus, the models are reasonably good and quite similar to the template
(Fig. 3B).
LmutTX’s structural model presented all conserved secondary structures for group
IIA PLA2s, such as the N-terminal helix (α1), the two long antiparallel helices (α2 and α3),
one short helix, the two antiparallel β-sheets (β-wing) and the C-terminal region.
Since many studies with Lys49 PLA2s [20,23,24,42] have suggested the involvement
of specific protein regions in the perturbation of biological membranes, six peptides were
designed and synthesized, three identical to the sequence (PepC - C-terminal region; PepN N-terminal region; PepS – region containing the catalytic site) along with three peptides that
were modified from the original peptide sequence (PepC-W, PepN-W and PepS-W), by
replacing Tyrosine residues by Tryptophan residues (Tyr
→ Trp), in order to increase the
degree of hydrophobicity of the designed peptides (Fig. 3B).
3.2 Functional characterization of LmutTX
3.2.1 Cytotoxicity of LmmV, LmutTX and synthetic peptides against differentiated C2C12
cells in myotubes
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Accepted Article
To evaluate the cytotoxic effect of LmmV, LmutTX and synthetic peptides, a line of
murine myoblasts (C2C12) was used in different sample concentrations. LmmV was
cytotoxic at all concentrations tested (25-300 μg/mL), showing maximal action at
concentrations above 50 μg/mL, whereas LmutTX was only cytotoxic above 200 μg/mL (Fig.
4A). The peptides showed low cytotoxicity on the cells, reaching approximately 30% of
maximum cell damage at 600 μg/mL with the peptide PepC-W (Fig. 4B). The other peptides
presented cell damage below 20% at all concentrations tested.
3.2.2 Antimicrobial activity of LmutTX and synthetic peptides against different Grampositive and Gram-negative bacteria
In order to observe LmutTX’s antibacterial action, it was tested against Grampositive (S. aureus and MRSA) and Gram-negative strains (E. coli, P. aeruginosa and K.
pneumoniae) at a concentration of 12.5 μg/mL over 24 and 48 hr. The protein was able to
inhibit 60% of growth of S. aureus and MRSA strains for 24- and 48-hr periods. An
inhibition of ~35% was observed for P. aeruginosa in 24 hr, increasing this percentage to
50% in 48 hr; ~30% of K. pneumoniae growth was inhibited by the toxin in both incubation
periods (Fig. 5). E. coli bacteria were not susceptible to the toxin’s action at the concentration
evaluated. Since S. aureus, MRSA and P. aeruginosa were most susceptible to LmutTX, they
were selected for antimicrobial activity tests with synthetic peptides.
Of the six synthesized peptides, only those corresponding to the C-terminal region
(PepC and PepC-W) showed significant activity against the selected bacteria. PepC inhibited
60% of MRSA growth at concentrations of 125 and 62.5 μg/mL in 24 hr (Fig. 6A), while
PepC-W inhibited 100-95% of MRSA growth at concentrations of 125 to 7.8 μg/mL over a
period of 24 hr, maintaining this activity for 48 hr (Fig. 6B). The same action was observed
for PepC-W against P. aeruginosa, where the peptide was able to inhibit 100% of bacterial
Accepted Article
growth at a concentration of 125 μg/mL and ~ 90% at 62.5 μg/mL after 24 hr, retaining
approximately 80% inhibition for 48 hr (Fig. 6D). PepC had no significant activity on P.
aeruginosa (Fig. 6C).
4. Discussion
Among the sources of biodiversity, snake venoms are proving to be conducive to the
discovery of compounds or biological activities with therapeutic potential due to their variety
of proteins, enzymes and peptides [43,44]. Few proteins have been isolated from Lachesis
muta muta venom [45–49], which allows for the identification of many unknown molecules
that are common to other genera. This study describes the isolation and characterization of
the first Lys49 PLA2 for L. m. muta venom, called LmutTX. Purification of the protein
followed methodologies commonly used to obtain Asp49 PLA2s from laquetic venoms, such
as LmTX-I, LmTX-II, LM-PLA2-I and LM-PLA2-II [50,51]. Variability in the expression of
PLA2s in snake venoms of the genus Lachesis, as reported by Sanz et al. [25], may have been
one of the factors that hindered the prior purification of Lys49 PLA2s from these venoms, as
well as their identification using transcriptomic studies, as mentioned by Junqueira-deAzevedo et al. [52]. LmutTX presented a molecular mass of 13,889 Da and was devoid of
phospholipase activity on an artificial substrate. Its primary structure had high identity with
other PLA2 homologues from bothropic venoms [11,53], only differing at 5 residues (Val19,
Thr20, Ser34, Tyr119 and Tyr121), as well as at 2 non-sequenced residues (positions 117 and
118).
The residues that form the catalytic site of Asp49 PLA2s (His48, Asp99, Tyr52 and
Tyr73) are conserved in Lys49 PLA2s, along with the natural mutations at the residues Tyr28
→ Asn, Gly32 → Leu and Asp49 → Lys, which make it impossible to coordinate Ca2+ in the
ion-binding site [53,54].
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Accepted Article
Lys49 PLA2s induce myonecrosis by mechanisms independent of phospholipid
hydrolysis [55]. Several mechanisms have been proposed to explain its toxic action on cell
membranes [53,56,57]; however, understanding the structure-function relationship of these
peculiar toxins is not a simple task. Fernandes et al. [53] suggested a mechanism of action
based on the C-terminal region of these myotoxins, where the presence of hydrophobic and
cationic residues in this segment would aid in the interaction with and perturbation of
biological membranes, which would induce the release of ions and molecules, intensifying
tissue damage. Disruption of the bilayer can trigger, as a consequence, the influx of Ca2+ and
the release of K+ and ATP to the extracellular medium [58,59], involving the participation of
purinergic receptors [60]. Kini et al. [61] reported that the variation in the toxic effects of
PLA2s is based on the differences in protein membrane penetrability. This variation may be
closely linked to key changes in the primary structure located in regions that are crucial for
the action of these proteins.
LmutTX showed a low cytotoxic effect on differentiated C2C12 cells in myotubes
compared to other myotoxins from bothropic venoms [39], since 100 μg/mL was not able to
induce LDH release significantly, being only observed at concentrations above 200 μg/mL.
Differences were also found in MT-II (Lys49) from B. asper and LmTX-I (Asp49) from L. m.
muta, where MT-II was cytotoxic at concentrations ≥ 30 μg/mL, while LmTX-I did not affect
cell viability at any of the tested concentrations (70 to 270 μg/mL) [62,63]. The cytolytic
action of Lys49 PLA2s is not restricted to muscle cells but also to other cell types, such as
endothelial cells [64], macrophages [60], lymphocytes [65] and tumour cell lines [66,67].
Amphipathic substances are characterized by their cytotoxic activity on eukaryotic
and prokaryotic cells [68]. Based on this principle, the peptide PepC-W, modified with the
insertion of hydrophobic residues (substitution of Tyr → Trp and Tyr → Phe) and containing
a positive net charge of +5, presented a discrete increase in the cytotoxic effect on C2C12
Accepted Article
myotubes, as observed by Lomonte et al. [68]. The peptide PepC-W exhibited a different
spatial distribution of hydrophobic residues from that of the peptide p115-W3 as described by
these authors, as well as fewer residual positive charges, which may have led to different
cytotoxic conformations and potential. Regarding the spatial positioning of amino acid
residues, Lomonte, Angulo and Santamaría [69] showed that some of the peptides in
positions 115-129 may have their toxic activities abolished when their amino acid sequences
are randomly positioned, suggesting the importance of preserving certain domains in the
molecule in order to maintain its activity.
C-terminal peptides derived from Lys49 PLA2s also present cytotoxicity on tumour
cell lines originating from lymphoid leukaemia (Jurkat), adenocarcinoma (SKBR3),
melanoma (B16F10), sarcoma (S180) [67], myeloma (P3X) and breast cancer (EMT6) [70],
in addition to extensive activity on bacterial cells [20,71,72].
The antibacterial evaluation of LmutTX and synthetic peptides showed promising
activity. LmutTX presented action against Gram-negative and Gram-positive bacteria. The
antibacterial action of PLA2 homologues has been well described, such as BnuTX-I (B. n.
urutu) with activity on P. aeruginosa [73]; MTX-II (B. brazili) on E. coli [23]; CoaTX-II (C.
o. abyssus) on P. aeruginosa, E. coli and S. aureus [14]; and MjTX-II on E. coli [11]. Among
the synthetic peptides from LmutTX that were tested, PepC-W completely inhibited bacterial
growth of P. aeruginosa and MRSA strains. The peptide PepC also had action on MRSA,
though less than that of the modified peptide. This difference in activity may be associated
with the level of hydrophobicity between the two C-terminal peptides (PepC → 25% and
PepC-W → 41.67%) or even with favourable conformational changes originating from the
modification of Tyr/Phe → Trp residues in PepC-W, since the number of positive charges is
evenly arranged between them.
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Accepted Article
Peptides derived from the C-terminal region of PLA2 homologues have been the
focus of many studies in order to elucidate their contribution in the mechanism of myotoxic
action of these proteins and/or in their use as possible antimicrobial [20,23] and anti-tumour
agents [66,67,70,74].
Páramo et al. [20] showed that a C-terminal peptide, named p115-129 from B.
asper’s myotoxin II, had potent activity on strains of E. coli and S. aureus, via interaction
with LPS and lipoteichoic acid molecules, respectively, with irreversible morphological
changes in the membrane and cell death. Santamaría et al. [72] also demonstrated the
interaction of C-terminal peptides with LPS molecules, suggesting possible mechanisms of
action for cationic peptides.
Among the peptides evaluated in the present study, PepC-W showed activity against
Gram-positive (MRSA) and Gram-negative bacteria (P. aeruginosa), though it was more
selective for Gram-positive bacteria, since it not only completely inhibited their growth at
low concentrations but also maintained its action for 48 hr.
The mechanism of action of this peptide is unknown, but its cationic and
hydrophobic characteristics may favour electrostatic interactions between components of the
outer membrane and the cell wall of Gram-positive bacteria, similar to teicoic acids, and
assist in the insertion of hydrophobic residues in the bilayer. On the other hand, a more
specific action cannot be ruled out since S. aureus and MRSA must share extracellular
components, such as lipids, carbohydrates, peripheral or transmembrane proteins,
glycoconjugates, among others, [75] with physicochemical or structural properties propitious
to PepC-W activity; however, S. aureus strains were less sensitive to the peptide (data not
shown), suggesting that other factors (or molecular targets) may be involved in the activity
against MRSA.
Accepted Article
The therapeutic application of peptides is still a challenge to be overcome due to the
numerous difficulties in oral administration of peptide drugs, which include the acidic and
enzymatic degradation of these molecules in the gastrointestinal tract, as well as their passage
through intestinal mucosal cells via active transport or passive diffusion [76]. However,
several chemical strategies have been used to stabilize the secondary structures of drugcandidate peptides, such as cyclization, N-methylation, addition of staples and construction of
hydrophobic surfaces [76,77].
Even though it is in preliminary in vitro tests, the antibacterial potential of PepC-W
is notorious. Its efficiency in inhibiting the growth of resistant bacteria to conventional drugs
makes it a promising candidate in the fight against pathogenic microorganisms.
The characterization of LmutTX, the first Lys49 PLA2 to be described from venoms
of the genus Lachesis, and its synthetic peptides, with antibacterial activity on resistant
strains and low cytotoxicity in murine C2C12 cells, shows the relevance of the present study
and reveals new possibilities for the application of peptides derived from proteins as potential
antimicrobial agents.
Acknowledgements
The authors express their gratitude to Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação Rondônia de
Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas and to the Pesquisa do
Estado de Rondônia (FAPERO), Brasil for the financial support. The authors thank the
Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for the use
of its facilities.
This article is protected by copyright. All rights reserved.
Accepted Article
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This article is protected by copyright. All rights reserved.
Accepted Article
LEGENDS FOR FIGURES
Figure 1: Fractionation of LmmV and phospholipase activity of the fractions obtained in size
exclusion chromatography. (A) Size Exclusion Chromatography (SEC) of Lachesis muta
muta venom (LmmV) in a Sephacryl S-200 column. Four fractions were eluted from this
chromatography, named LmS-1 to LmS-4, respectively. On the upper right is the
electrophoretic profile of the fractions under reducing conditions (15% SDS-PAGE), where a
predominance of high molecular weight (LmS-1 and 2) and low molecular weight (LmS-3
and 4) proteins are observed. 1: LmS-1; 2: LmS-2; 3: LmS-3; 4: LmS-4. (B) Phospholipase
activity of the fractions obtained in the SEC with the substrate 4N3OBA. 10 μg of the sample
or controls were incubated with 190 μL (298 μM) of substrate for 30 min. at 37°C, and the
absorbance measured at 425 nm. The gels were stained with Coomassie Blue G250 and the
results expressed as mean ± standard deviation (n=3) and submitted for analysis of variance
(ANOVA) followed by the Tukey post-test. (*) Significant values for p<0.05; (#) statistically
different from the negative control. WM: BioLabs P7709S, USA.
Figure 2: Isolation of a protein devoid of catalytic activity from LmmV. (A) Reverse Phase
Chromatography (RP) of the fraction LmS-3 on a C18 column (P1 to P6) and analysis of the
fractions using 12.5% SDS-PAGE. (B) A second chromatographic step of the P6 fraction in
RP obtaining a single fraction. Next, 15% SDS-PAGE of the isolated protein. The gels were
stained with Coomassie Blue G250. WM: AMRESCO’S BlueStep K973, USA. (C)
Phospholipase activity of the isolated protein with the substrate 4N3OBA, using BthTX-II
and water as positive and negative controls, respectively. The results were expressed as mean
± standard deviation, followed by analysis of variance with ANOVA and Tukey post-test.
(***) Statistically significant in relation to the positive control (p <0.05); (#) Statistically
different from the negative control. (D) Determination of the molecular mass of the fraction
Accepted Article
P6 with MALDI-TOF, using synapinic acid as the ionization matrix. A single peak is
observed in the mass spectrum at 13,889 Da, as result of the subtraction of m/z value by H+
mass, corresponding to the isolated protein.
Figure 3: Multiple alignment of LmutTX’s sequence with toxins from bothropic venoms and
design of the synthetic peptides. (A) The sequence was analysed in the UNIPROT database,
where it showed identity level above 86.8% compared to snake venom PLA2s of the genus
Bothrops, like B. brazili (MTX-II: I6L8L6), B. moojeni (MjTX-II: Q9I834), B. pauloensis
(BnSP-7: Q9IAT9), B. pirajai (PrTX-II: P82287), B. leucurus (BlK-PLA2: P86975) and B.
alternatus (BaTX: P86453), respectively. (*) same amino acid residue; (.) one altered
residue; (:) two altered residues. In yellow: residues that form the Ca2+-binding site; in blue:
residues that form the catalytic site. The gaps represent the alignment according to the
numbering system proposed by Renetseder et al. (1985), using bovine pancreatic PLA2 as the
structural model. (B) Selected regions used to design the peptides.
Figure 4: Cytotoxicity in differentiated C2C12 cells in myotubes. (A) LmmV, LmutTX
and (B) the synthetic peptides were incubated with differentiated C2C12 cells in myotubes
for 3 hr at different sample concentrations at a final volume of 200 μL/well. Controls of 0%
and 100% toxicity consisted of cell medium and Triton X-100, respectively. The activity was
performed in triplicate wells, for three individual experiments. (*) Statistically significant
relative to the positive control (0.1% Triton X-100) for p <0.05. Negative control (C-): cell
culture medium.
This article is protected by copyright. All rights reserved.
Accepted Article
Figure 5: Screening of LmutTX against Gram-positive and Gram-negative bacteria.
12.5 μg/mL of LmutTX was incubated with Staphylococcus aureus ATCC 29213 (SA),
MRSA, Pseudomonas aeruginosa ATCC 27853 (PA), Klebsiella pneumoniae ATCC 13883
(KP) and Escherichia coli ATCC (EC) for 24 and 48 hr. Imipenem was used as a positive
control. The assay was performed in triplicate from two individual experiments. The results
were expressed as mean ± standard deviation, followed by analysis of variance using
ANOVA and Tukey post-test considering the level of significance as p <0.05. (*) in relation
to growth control.
Figure 6: Antimicrobial activity of the peptides derived from LmutTX against Grampositive and Gram-negative bacteria over 24- and 48-hr periods. The peptides were
diluted at concentrations of 125; 62.5; 31.2; 15.6 and 7.8 μg/mL and incubated with S. aureus
ATCC 29213, MRSA and P. aeruginosa ATCC 27853 over 24- and 48-hr periods.
Chloramphenicol was used as a positive control. The assay was performed in triplicate from
three individual experiments, and the results were expressed as mean ± standard deviation,
followed by analysis of variance using ANOVA and Tukey post-test for p<0.05 (*) in
relation to the growth control.
Accepted Article
Figure 1
B
LmS-1
mAU
kDa
1
2
3
72
52
38
33
24
1500
LmS-2
0.3
#
12
LmS-3
LmS-4
41.71
0
0.0
10.0
20.0
30.0
40.0
Time
50.0
#
**
17
1000
500
4
Abs 425 nm
Abs 280 nm
A
60.0
70.0
min
0.2
#
***
0.1
***
0.0
C+
C-
***
***
LmS-1 LmS-2 LmS-3 LmS-4
PLA2 activity (4N3OBA)
kDa
1500
Abs 280 nm
P6 P5 P4 P3 P2 P1
120
91
62
46
38
26
P2
mAU
B
%B
0.6
#
80
19
60
1000
P1
40
P6
Abs 425 nm
A
(----) % ACN
Accepted Article
Figure 2
0.4
0.2
500
P4
P5
20
0.0
P3
C+
0
0
10.0
20.0
30.0
40.0
50.0
***
C-
P6
PLA2 activity (4N3OBA)
min
Time
***
%Int.
13890
C
D
13890.38
mAU
kDa
100
%B
1400
1200
800
72
52
38
33
24
17
80
80
70
60
12
600
40
(----) % ACN
Abs 280 nm
1000
90
60
50
40
30
400
20
20
200
10
0
0
10.0
20.0
30.0
40.0
min
0
10000
Time
12000
14000
m/z
16000
18000
Accepted Article
Figure 3
A
15
LmutTX
MTX-II
MjTX-II
BnSP-7
PrTX-II
Blk-PLA2
BaTX
28
32
48-49 52
57-58
62-66
73
SLVELGKMILQETG-KNPVTSYGAYGCNCGVLGSGKPKDATDRCCYVHKCCYKKLT--D-C-----DPKKDRYSYSWKDK
SLVELGKMILQETG-KNPAKSYGAYGCNCGVLGRGKPKDATDRCCYVHKCCYKKLT--D-C-----DPKKDRYSYSWKDK
SLFELGKMILQETG-KNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYKKLT--G-C-----DPKKDRYSYSWKDK
-SFELGKMILQETG-KNPAKSYGAYGCNCGVLGRGQPKDATDRCCYVHKCCYKKLT--G-C-----DPKKDRYSYSWKDK
SLFELGKMILQETG-KNPAKSYGAYGCNCGVLGRGKPKDATDRCCYVHKCCYKKLT--G-C-----NPKKDRYSYSWKDK
SLFELGKMILQETG-KNSVKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYKKLT--G-C-----DPKKDRYSYSWKDK
SLFELGKMILQETG-KNPAKSYGAYYCYCGWGGQGQPKDATDRCCYVHKCCYKKLT--G-C-----NPKKDRYSYSWKDK
.*********** ** ..***.* * ** * *:******************** . *
:*************
85
123
99
% Identity
LmutTX
MTX-II
MjTX-II
BnSP-7
PrTX-II
Blk-PLA2
BaTX
TIVC-GENNSCLKELCECDKAVAICLRENLDTYNKK--YNYL-KPFCKKADPC
TIVC-GENNSCLKELCECDKAVAICLRENLDTYNKKYRNNHL-KPFCKKADPC
TIVC-GENNSCLKELCECDKAVAICLRENLDTYNKKYRYNYL-KPFCKKADPC
TIVC-GENNPCLKELCECDKAVAICLRENLGTYNKKYR-YHL-KPFCKKADPC
TIVC-GENNPCLKELCECDKAVAICLRENLGTYNKKYR-YHL-KPFCKKADDC
TIVC-GENNPCLKELCECDKAVAICLRENLGTYNKKYR-YHL-KPFCKKADPC
TIVC-GENNSCLKELCECDKAVAICLRENLNTYNKKYR-YYL-KPLCKKADAC
**** **** ********************.******
:* **:***** *
PepS
LmutTX
B
95.1
92.6
92.4
90.9
90.9
86.8
PepS-W
42-53
Helix α3
Helix α2
Ca2+-binding loop
β-wing
Peptides from LmutTX
C-terminal
Helix α1
1-13
PepN-W
PepN
Short helix
115-128
PepC
PepC-W
B
A
100
LDH Release (%)
LmutTX
LmmV
100
LDH Release (%)
75
50
25
PepC
PepC-W
80
PepN
60
PepN-W
PepS
40
PepS-W
20
0
0
25
50
100
200
37.5
300
75
150
μg/mL
μg/mL
Figure 5
LmutTX
100
Imipenem
80
60
***
40
*** ***
***
***
***
***
***
20
24 hours
P
EC
K
M SA
R
SA
PA
P
EC
K
M SA
R
SA
PA
0
SA
% Bacterial Inhibition
Accepted Article
Figure 4
48 hours
300
600
Accepted Article
Figure 6
Table 1. Mass-to-charge ratio (m/z) of thhe fragments obtained from LmutTX sequencing.
Number
Sequenced fragment
Region
1
2
3
4
5
6
7
8
KLTDCDPKKDR
YSYSWK
DKTIVCGENNSCLK
ELCECDK
AVAICLR
ENLDTYNKK
YNYLKPFCK
KADPC
53-63
64-69
70-83
84-90
91-97
98-106
109-117
118-122
*ppm: parts per million
Measured
Mass
1374.6925
832.3755
1636.7549
952.3630
801.4531
1123.5509
1231.6060
532.2315
Theoretical
Mass
1374.69
832.38
1636.75
952.36
801.45
1123.55
1231.60
532.23
uracy
Accu
(pp
pm)*
-1.81
5..40
-2
2.99
-3.15
-3.86
-0
0.80
-4
4.87
-2
2.81
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