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On-li ne ISS N 1678-9199
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ISSN 1678-9199
www.jvat.org
*Correspondence: osmindo@unb.br
https://doi.org/10.1590/1678-9199-JVATITD-2021-0017
Received: 05 February 2021; Accepted: 17 May 2021; Published online: 18 March 2022
RESEARCH OPEN ACCESS
Keywords:
Spider venom
Acanthoscurria natalensis
Acylpolyamines
Antimicrobial
Mass spectrometry
Antimicrobial activity and partial chemical
structure of acylpolyamines isolated from the
venom of the spider Acanthoscurria natalensis
Tania Barth1, Aline Silva2, Simone Setubal dos Santos3, Jane Lima Santos3, Patrícia Diniz Andrade4, Jessica Tsai5,
Eloísa Dutra Caldas4, Mariana de Souza Castro5,6, Osmindo Rodrigues Pires Júnior5*
1Laboratory of Animal Histolog y, Department of Biological Sciences, State University of Santa Cruz, Ilhéus, BA, Brazil.
2Laboratory of Microbiology, Department of Biological Sciences, St ate University of Santa Cruz, Ilhéus, BA , Brazil.
3Laboratory of Immunobiology, Department of Biological Sciences, State University of Santa Cruz, Ilhéus, BA, Brazil.
4Laboratory of Toxicology, Depar tment of Pharmac y, School of Health Sciences, University of Brasilia (UnB), Brasilia, DF, Brazil.
5Laboratory of Toxinology, Depar tment of Physiological Sciences, Institute of Biological Sciences, University of Brasilia (UnB), Brasilia, DF, Brazil.
6Laboratory of Protein Chemistr y and Biochemistry, Depar tment of Cell Biology, Institute of Biological Sciences, University of Brasilia (UnB),
Brasilia, DF, Brazil.
Abstract
Background: Acylpolyamines are one of the main non-peptide compounds present in
spider venom and represent a promising alternative in the search for new molecules
with antimicrobial action.
Methods: e venom of Acanthoscurria natalensis spider was fractionated by reverse-
phase liquid chromatography (RP-HPLC) and the antimicrobia l activity of the fractions
was tested using a liquid growth inhibition assay. e main antimicrobial fraction
containing acylpolyamines (ApAn) was submitted to two additional chromatographic
steps and ana lyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet
(UV) spect roscopy and ESI-MS/MS analysis and for minimum in hibitory concentration
(MIC) and hemolytic activity determination.
Results: Five acylpolyamines were isolated from the venom with molecular masses
between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742
and ApAn756. e analysis of UV absorption prole of each ApAn and the fragmentation
pattern obtai ned by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore
and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn
presented MIC between 128 µM a nd 256 µM against Escherichia coli and Staphylococcus
aureus, without causing hemolysis against mouse erythrocytes.
Conclusion: e antimicrobial and non-hemolytic properties of the analyzed ApAn
may be relevant for their applicat ion as possible therapeutic agents and the identication
of an unconventional chromophore for spider acylpolyamines suggests an even greater
chemical diversity.
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Background
Compounds produced by dierent organisms found in nature
represent a valuable alternative for the discovery of new
agents with therapeutic potential [1–3]. e search for agents
with antimicrobial action is especially important, given the
establishment of bacterial strains resistant to conventional
drugs [4]. In this context, a signicant number of antimicrobial
peptides (AMPs) were identied from several biological sources,
including the venom of snakes, scorpions, spiders, a mong others
[2, 5, 6]. e antimicrobial potential and information about the
results of preclinical and clinical tests obtained for several of
these peptides have been widely revised [1–3, 7–9]. Despite the
therapeutic potential of AMPs, these molecules may have some
disadvantages in terms of clinical application, including protease
degradation, hemolytic action and high production cost [1, 2].
However, in addition to AMPs, spider venoms, for example,
contain dierent biologically active molecules, including non-
peptide molecules, such as organic acids, biogenic amines and
acylpolyamines [10, 11].
Acylpolyamines are non-peptide organic molecules of
low molecular weight (> 1000 Da) and represent the most
abundant component of spider venom [12]. e combination of
techniques, such as mass spectrometry and nuclear magnetic
resonance, are i mportant for the cha racterization of the chemical
struct ure of acylpolyamines [13]. Currently, the structure of 409
acylpolyamines is available in the venoMS database [14]. e
general chemical structure of these molecules comprises four
segments, being a lipophilic aromatic acyl head, a linker a mino
acid residue, the polyam ine backbone chain and the backbone tail
[15]. Due to the possible combinations between these segments,
acylpolyamines can be structurally very diverse, varying in
length, number of amide bonds and functional groups [11, 16].
Acylpolyamines are recognized for their neuromodulatory
activity on the nervous system of vertebrates and invertebrates,
mainly a ntagonizing glutamate receptors a nd selectively block ing
cationic channels [12, 13]. However, some studies have shown
that acylpolyamines may have other biological activities,
such as antimicrobial action. Antimicrobial properties have
been identied in spider acylpolyamines since 2007, when
Pereira et al. [17] identied an acylpolyamine named mygalin,
isolated from the hemocytes of the spider Acanthoscurria
gomesiana. Subsequently, acylpolyamines with antimicrobial
activity were identied in the venom of Brachypelma smithi [18],
Nephilengys cruentata [19] and Vitalius dubius [20], being only
the acylpolyamine of V. du b iu s, called VdTX-I, fully described
in the literature and, in none of these studies, the mechanism
of antimicrobial action of acylpolyamines was investigated.
Recently, a new study has shown that the mechanism of
antimicrobial action of a synthetic version of mygalin on
Escherichia coli involves disruption of the bacterial membrane,
inhibition of DNA synthesis, the generation of reactive oxygen
species (ROS), among others actions [21]. Regarding the tara ntula
spider Acanthoscurria natalensis, the present study represents the
rst report on the presence of acylpolyamines in the venom of
this species. us, we present here the partial chemical structure
of ve acylpolyamines isolated from the venom of A. natalensis
and the antimicrobial and hemolytic activity of these molecules.
Methods
Spiders and venom extraction
Female spiders of A. natalensis (n = 30) were collected (SISBIO
license number 51803-1) from Fazenda Nossa Senhora Aparecida
(GO, Brazil). e venom (~ 30 μL/animal) was extracted by
electrostimulation (75 V for 3 s) between 1 and 2 times for each
animal [22] (SISGEN license number A826A3A). e samples
were lyophilized and stored at -20 °C until use.
Acylpolyamine purication by reversed-phase liquid
chromatography (RP-HPLC)
Acylpoliamine purification was obtained using three
chromatographic steps (step 1 a 3). For the rst step (step 1),
the crude venom was solubilized (20 mg/mL) in solvent A [0.12%
triuoroacetic acid (TFA) (v/v) in water] and centrifuged (10,000
rpm). Aliquots of 200 μL of the supernatant were applied to a
C18 reversed-phase column (Vydac 218TP54, 4.5 mm x 250 mm,
5 μm), previously equilibrated with the same solvent, using a
ow rate of 1 mL/min. e elution of fractions was obtained
using a linear gradient of 0 to 60% of solvent B [0.12% TFA
(v/v) in acetonitrile (ACN)] in 60 min. e chromatographic
fractions were tested for antimicrobial activity and the main
active fraction, named ApAn, was rechromatographed (step
2) using solvents A [0.24% TFA (v/v) in water] and B [0.24%
TFA (v/v) in methanol]. Samples of the ApAn fraction were
solubilized in solvent A and centrifuged (10,000 rpm). Aliquots
of 200 μL of the supernatant were applied to a Phenyl Hexyl C
18
reversed-phase column (Phenomenex, 2.10 mm x 300 mm, 2.6
µm) previously equilibrated with the same solvent, using a ow
rate of 1 mL/min. e elution of fractions was obtained using
a gradient from 0 to 18% B in 40 min., 18% B from 40 to 50
min. and from 18 to 30% B from 50 to 60 min. ese fractions
were also tested for antimicrobial activity and those with more
activity and better chromatographic resolution (named ApAn1
to ApAn5) were selected for the next rechromatography (step
3), performed as in step 2, but with a new elution gradient. In
step 3, for fractions ApAn1 to ApAn3, the gradient was 0 to 20%
B in 10 min. and 20% B from 10 to 40 min and for fractions
ApAn4 and ApAn5, the gradient was 0 to 30% B in 10 min.
and 30% B from 10 to 40 min. e main peak of each fraction
obtained in this step was analyzed by MALDI-TOF to verify its
molecular mass and sample homogeneity. Samples were then
accumulated for analysis by UV spectroscopy and ESI-MS/MS
and for determination of minimum inhibitory concentration
(MIC) and hemolytic activity. e eluted fractions in each step
were detected simultaneously at 216 nm and 280 nm, manually
collected, lyophilized and stored at -20oC until use.
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MALDI-TOF/TOF
e ApAn1 to ApAn5 fractions were analyzed using a SCIEX
TOF/TOFTM 5800 MALDI mass spectrometer (AB SCIEX,
Framingham, MA, USA) with α-cyano-4-hydroxycinnamic
acid (HCCA) as the matrix. e ions were detected in reector
positive mode from m/z 500 to 2000 and the mass spectra were
converted to the “.mzxml” format for data analysis using the
mMass 5.5.0 soware. According to the observed molecular
masses, the fractions ApAn1, ApAn2, ApAn3, ApAn4 and
ApAn5, were named ApAn728, Ap614a, ApAn614b, ApAn742
and ApAn756, respectively.
Ultraviolet (UV) spectroscopy
UV analyzes were performed on UV-Visible spectrophotometer
(UV-1800, Shimadzu) and the UV spectrum (200–400 nm)
of ApAn728, Ap614a, ApAn614b, ApAn742 and ApAn756
solubilized in Milli-Q water was acquired at room temperature. For
comparison, the UV spectrum of L-tyrosine, 5-hydroxytryptamine,
histamine, L-tryptophan and L-phenylalanine compounds were
also acquired under the same conditions.
ESI-MS
ESI-MS and MS/MS analysis were performed using a 4000 Qtrap
triple-quadrupole mass spectrometer (SCIEX, Framingha m, MA,
USA) tted with a Turbo Ion Spray electrospray ionization (ESI)
source. System operation and data acquisition were controlled
by Analyst® (V 1.5.1) soware (SCIEX). Samples of ApAn728,
Ap614a, ApAn614b, ApAn742 and ApAn756 (solubilized in
methanol/water, containing 0.1% formic acid) were analyzed by
direct infusion into the mass spectrometer under ow of 10 μL/
min. ESI-MS/MS was performed in positive ionization mode,
with multiple reaction monitoring (MRM). declustering potential
(DP), collision energy (CE) and collision cell exit potentia l (CXP)
were optimized for the three most abundant transitions for each
analyte. e parameters of the mass spectrometer ion source
were: entrance potential at 10 V, ion source at 500 oC, ion source
gas 1 (GS1) at 12 psi, ion spray voltage at 5500 V, curtain gas at
10 psi, and collision gas at medium.
Antimicrobial activity
Antimicrobial activ ity of chromatographic samples was evaluated
by liquid growth inhibition assays [23] against Gram-negative
Escherichia coli ATCC 25922 and Gram-positive Staphylococcus
aureus ATCC 25923 bacteria grown in Mueller-Hinton (MH)
medium at 37 °C under agitation. Aer 24 h and optical density
reached 1 to 590 nm, aliquots of 50 μL of each culture diluted
1:100 (S. aureus) and 1:50 (E. coli) were incubated with 50 μL
of chromatographic samples (in duplicate) diluted in Milli-Q
water for 24 h at 37 °C. Milli-Q water or 0.4% (v/v) formaldehyde
were used as positive and negative control, respectively. Growth
inhibition was determined by measuring absorbance at 595 nm
in a Multiskan FC plate reader (ermo Scientic).
Minimum inhibitory concentration (MIC)
MIC was determined by liquid growth inhibition assays as
described above (in the “Antimicrobial activity” section).
Aliquots of 50 μL of the samples serially diluted from 256 µM
to 4 µM for ApAn728, Ap614a, ApAn614b and 128 µM to 4 µM
for ApAn742 and ApAn756, were incubated for 24 h at 37 °C
with 50 μL of the S. aureus and E. coli dilution. MIC was dened
as the lowest concentration that causes 100% inhibition of the
bacterial growth, obtained from three or two independent
experiments performed in duplicate or triplicate, according to
the amount of sample available.
Hemolytic activity
e hemolytic act ivity of ApAn728, Ap614a, ApAn614b, ApAn742
and ApAn756 was tested against erythrocytes of SWISS mice
[24] (approved by the Ethics Committee on Animal Use, of the
University of Brasilia (CEUA-UnB), under protocol UnBDoc
number 44/2017). Erythrocytes were washed with Krebs solution
(113 mM NaCl, 1.2 mM KH2PO4, 4.0 mM KCl, 1.2 mM MgSO4,
2.5 mM CaCl2, 25 mM NaHCO3, 11.1 mM C6H12O6, pH 7. 4) to
obtain a 4% erythrocytes suspension. Aliquots of 50 µL of this
suspension were incubated with 50 µL of a serial dilution (256
µM to 0.125 µM) of ApAn728, Ap614a, ApAn614b, ApAn742
and ApAn756 in a 96-well plate for 2 h at 37 oC. Aer, samples
were centrifuged (1000 x g for 3 min) and the absorbance of
the supernatant was measured at 550 nm on Multiskan FC
(ermo Scientic). Erythrocytes incubated with Krebs solution
or 1% TritonX-100 were used as a negative and positive controls,
respectively. Hemolytic activity was expressed as a percentage of
the positive control (100% hemolysis) from three independent
experiments performed in duplicate.
Results
Acylpolyamine purication
e crude venom of A. natalensis was fractionated by RP-HPLC
(step 1) (Fig. 1) and chromatographic fractions were tested for
antimicrobial activity. e main active fraction, eluting between
12 and 22 min and containing the acylpoliamines, was named
ApAn. Due to the large number of components, this fraction
was rechromatographed (step 2) and the resulting fractions
(Fig. 2) were tested for antimicrobial activity. e ve major
active fractions (ApAn1 to ApAn5) (Fig. 2) were selected for
rechromatography (step 3) (Fig. 3), resulting in the elution of
a major peak for each fraction. ese peaks were analyzed by
M AL DI-TOF (Fig. 4) and only one major protonated ion [M+H]
+
for each peak was identied, suggesting sample homogeneity.
ese ions were identied at m/z 729 (ApAn1), m/z 615 (ApAn2),
m/z 615 (ApAn3), m/z 743 (ApAn4) and m/z 757 (ApAn5) and
according to the corresponding molecular mass, they were
nally named ApAn728, ApAn614a, ApAn614b, ApAn742 and
ApAn756, respectively (Fig. 4).
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Figure 1. Chromatographic prole (step 1) of the total venom (4 mg) of A. natalensis fractionated by RP-HPLC on a column C18, under linear gradient from 0
to 60% solvent B (0.12% v/v TFA in ACN) in 60 min and ow 1.0 mL/min. ApAn: fraction of interest containing acylpolyamines.
Figure 2. Rechromatography (step 2) of the ApAn fraction by RP-HPLC on a C18 column with optimized methodology (0 to 18% solvent B in 40 min, 18% B
from 40 to 50 min and 18 to 30% B of 50 to 60 min). Solvent B: 0.24% TFA v/v in methanol. Fractions of interest were named as ApAn1, ApAn2, ApAn3, ApAn4
and ApAn5.
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Figure 3. Rechromatography (step 3) of ApAn1, ApAn2, ApAn3, ApAn4 and ApAn5 fractions by RP-HPLC on a C18 column with optimized methodology (for
ApAn1 to ApAn3: linear gradient from 0 to 20% solvent B in 10 min and 20% B of 10 to 40 min; for ApAn4 and ApAn5: linear gradient from 0 to 30% B in 10
min and 30% B from 10 to 40 min). Solvent B: 0.24% TFA v/v in methanol.
Partial characterization of ApAn chemical structure
e partial chemical structure of ApAn728, Ap614a, ApAn614b,
ApAn742 and ApAn756 was suggested from the analysis of UV
absorption spectra and interpretation of the fragmentation
pattern obtained by the ESI-MS/MS spectra, compared
with data described in the literature and database venoMS
(https://www.venoms.ch/). Overlapping of the ApAn UV
spectra with the histamine, L-tryptophan, L-phenylalanine,
5-hydroxytryptamine and L-tyrosine spectra, showed that
ApAn presented the same tyrosine absorption prole, with
maximum values at 224 nm and 274 nm. (Fig. 5), suggesting
that the aromatic acyl group of ApAn contains a tyrosyl unit.
e analysis of ApAn ESI-MS spectra showed the presence of
ions at m/z 729, 615, 743 and 757 in the form [M+H]+ (ESI-MS
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Figure 4. MALDI-TOF/MS spectrum from ApAn1, ApAn2, ApAn3, ApAn4 and ApAn5 (isolated in step 3) obtained in positive reector mode. Samples in
α-cyano-4-hydroxycinnamic matrix. In parentheses, nomenclature assumed according to the observed molecular mass.
and other ESI-MS/MS spectra can be found in Additional
files 1–5), corresponding to the ions obtained by MALDI-TOF
(Fig. 4). e MS/MS spectrum of all ApAn (Fig. 6) showed the
presence of ions at m/z 163, m/z 136 and m/z 107, which were
interpreted as products of the fragmentation of the tyrosyl unit,
corroborating the results obtained by UV spectra. In addition,
the ion at m/z 220 suggested that the tyrosyl unit is linked to
butylamine group and that all ApAn have the same chemical
structure in this region of the molecule.
For the initial portion of the ApAn polyamine chain, ions
were observed at m/z 291 and 365 (ApAn728 and ApAn742)
and at m/z 305 and 379 (ApAn614a, ApAn614b, ApAn742 and
ApAn756), suggesting that the latter ions occur due to the
presence of an additiona l methylene unit. e main ions obser ved
above m/z 365 and m/z 379 in ApAn spectra, were related to
the fragmentation products of the intermediate portion of the
polyamine chain. However, these ions were not structurally
indicated, as no compatible structures were found.
Regarding the terminal polyamine chain, the presence of
ion pairs at m/z 129/112 (ApAn728, ApAn742) and/or m/z
143/126 (ApAn614a, ApAn614b, ApAn742, ApAn756) in the
ApAn spectra, indicated the fragmentation of this portion of
the polyamine chain and the loss of an N H
3
residue, considering
that the dierence in mass between each pair is 17 Da. ese ion
pairs suggest that the st ructure of the termi nal polyamine port ion
is consistent with the PA43 (129/112) or PA53 (143/126) units.
In the MS/MS spectrum of ApAn742, the ions at m/z 291 and
365 and at m/z 305 and 379 (for the initial polyamine cha in) were
observed, as well as the ion pairs 129/112 and 143/126 (for the
terminal polyamine chain), leading us to consider the possible
co-elution of isomeric molecules in this sample.
Based on the above considerations, Figure 6 (inset) shows the
suggested partial chemical structure for the chromophore and
for the initial and terminal polyamine chain. For ApAn742,
two structures have been suggested, but other options can also
be considered.
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Figure 5. UV absorption spectrum from ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756 compared to dierent compounds. The maximum values of
absorption at (2) 224 nm and (1) 274 nm of the samples were coincident with the values shown by L-tyrosine. The spectra were acquired between 200 and 400
nm at room temperature.
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Figure 6. ESI-MS/MS spectra of the protonated ion [M+H]+ of (A) ApAn728, (B) ApAn614a, (C) ApAn614b, (D) ApAn742 and (E) ApAn756 and suggested
partial chemical structure (inset). For (D) ApAn742, two structures were suggested due to possible co-elution of isomers in the sample.
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Minimum inhibitory concentration (MIC)
e antimicrobial activity of ApAn was tested against E. coli
and S. aureus at dierent concentrations (Fig. 7). ApAn614a and
ApAn614b showed low activity, inhibiting around 20-40% of the
growth of both bacteria, even at the highest concentration tested
(256 µM). e antimicrobial activity of ApAn728, ApAn742
and ApAn756 was dose dependent and the MIC of ApAn728
was 256 µM against S. aureus, while the MIC of ApAn742 and
ApAn756 was 128 µM against S. aureus and E. coli.
Hemolytic activity
e eect of ApAn728, ApAn614a, ApAn614b, ApAn742 and
ApAn756 on mice erythrocytes was evaluated at dierent
concentrations (256 µM to 0.125 µM) and until the concentration
of 256 µM (shown in Fig. 8), the hemolytic activity remained
around 1% only.
Discussion
Acylpolyamines represent one of the most abundant components
of the spider venom [25], reaching up to 50 dierent molecules in a
single sample [26]. In the venom of A. natalensis, such abundance
was also clearly veried by the chromatographic prole, where
the elution of ApAn fraction occurred for approximately 4
minutes and the rechromatography of this resu lted in the elution
of approximately 15 fractions not fully resolved, in addition
to ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756.
Molecular masses between 350 and 1000 Da have been reported
for acylpolyamines isolated from spider venom [25]. ApAn
showed molecular masses of 614, 728, 742 and 756 Da. Some
acylpolyamines with molecular masses similar to ApAn have
already been reported for other spider species. e ac ylpolyamine
called VdTX-I, with 728 Da, was isolated from the venom of V.
dubius, but its chemical structure has not been determined [27].
Figure 7. Antimicrobial activity of (A) ApAn728, (B) ApAn614a, (C) ApAn614b, (D) ApAn742 and (E) ApAn756 against Staphylococcus aureus (SA) and
Escherichia coli (EC), as a result of the serial dilution from 256 µM to 4 µM or 128 µM to 4 µM concentration, performed to determine the MIC. Data were
expressed as mean ± SD.
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In Aphonopelma chalcodes two acylpolyamines were identied,
with 60 0 Da (Apc600) and 728 Da (Apc728), being their chemical
structure partially characterized [28].
e chemical st ructure of acylpolyamines can be characterized
initially by the identication of its chromophore, through
the interpretation of the mass spectrum and UV absorption
pattern. The latter can be correlated with characteristic
chromophores , such as for example: λ
max
at 268, 284, a nd 292 nm
(4-hydroxyindole) and λmax at 280, 288; shoulder at λ = 270 nm
(indol e) [29]. ese patterns have been identied, for example, in
acylpolyamines isolated f rom the Argiope lobata and Nephilengys
borobonica spiders [30, 31]. However, the UV spectru m of ApAn
showed a tyrosine-like pattern with maximum absorption at
224 and 274 nm, suggesting that the chromophore of ApAn is
represented by a tyrosil unit. Although this pattern is unusual,
the acylpolyamines of the spider Aphonopelma californicum
[32] and the wasp Philanthus triangulum, named Philantotoxins
PTX-433, PTX-334 and PTX-343 [33], also have a tyrosil unit
in its composition. Já a Apc600 and Apc728 from A. chalcodes
showed a ty ramine-l ike chromophore [28]. Furthermore, based
on the analysis of the acylpolya mine mass spectra, the presence
of characteristic product ions at m/z 107, 123, 130 and 146,
correlated with the phenol, di-hydroxybenzene, indole and
mono-hydroxyindole chromophores, respectively [26, 31], can
contribute to the identication of the chromophore. Among
these, only the m/z 107 ion was identied in the ApAn spectra,
possibly originating from phenolic ring present in tyrosine. e
m/z 136 ion, observed in dierent ApAn spectra, also indicates
the presence of a tyrosine-like chromophore, as observed in the
spectra of Philantotoxins PTX-433, PTX-334 and PTX-343 [33].
e fragmentation of the polyamine chain, in turn, results in
the formation of other characteristic ions.
In the ApAn, the ion pairs m/z 129/112 and/or 143/126 were
identied. ese ions were related to fragmentation of the
terminal portion of the polyamine chain with the neutral loss
of an ammonia (NH3), as observed in N. clavata acylpolyamine
JSTX-3 [34] and/or Agelenopsis aperta [35, 36]. ese ions also
suggested that the terminal portion of the polyamine chain of
ApAn is formed by PA43 or PA53 units, which have already been
described in previous studies [29, 37]. In addition, a possible co-
elution of isomers in the sample of ApAn742 was considered,
since ion pairs 129/112 and 143/126, identied in the spectra of
ApAn742, cannot be present in the chemical structure of the
same molecule, considering that N-atoms are separated by three
to ve methylene units [38]. e polyamine chain is a common
component among acylpolyamines and its composition and
extent can vary considerably [25], due to possible methylation
and hydroxylation sites and the variable number of nitrogen
atoms [38]. ApAn728, 742 and 756 have a 14 Da mass dierence
between them, which could be attributed to the addition of a
Figure 8. Hemolytic activity of ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. Krebs: buer used as a 0% hemolysis control. Triton-X 100: used as
a 100% hemolysis control. The graph represents the results obtained for the highest concentration used (256 µM). Data were expressed as mean ± SD.
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methylene unit (CH2), similarly to that observed between the
NPTX-943 and NPTX-957 acylpololyamines [31]. However, as
it was not possible to identify the complete chemical structure
of ApAn, this information could not be conrmed, as well as
the dierence of 114 Da of ApAn614a and ApAn614b compared
with ApAn728.
Although acylpolyamines are little explored about their
antimicrobial potential, they are promising, as they have other
desirable characteristics for the formulation of therapeutic
agents, such as the sma ll size and the ease of obtaining sy nthetic
analogs [12]. ApAn were active against Gram-negative (E. coli)
and Gram-positive (S. aureus) bacteria, but the antimicrobial
activity was generally more ecient against Gram-positive
S. aureus bacteria. is may be due to the composition of the
bacterial wall, where, unlike Gram-negative bacteria, Gram-
positive bacteria lack a more resistant external membrane [39].
e acylpolyamines isolated from N. cruentata, were active
against S. aureus, Staphylococcus epidermides, Candida albicans
and Candida glabrata, however their MIC was not determined
[19]. VdTX-I acylpolyamine isolated from the venom of the spider
Vitalius dubius was evaluated for its antimicrobial activity and
also showed activity aga inst a wide spectr um of microorganisms,
including the fungus C. albicans, in concentrations ranging from
12.5-100 μM [20]. Another acylpolyamine with antimicrobial
activity, called mygalin, was isolated from hemocytes of the
spider Acanthoscurria gomesiana. is molecule, with 417 Da,
was tested against E. coli, Micrococcus luteus and C. albicans, but
it was active only against E. coli, with MIC of 85 µM [17]. e MIC
values of ApAn728 (256 µM against S. aureus) and of ApAn742
and ApAn756 (128 µM against S. aureus and E. coli), were
relatively higher compared to some antimicrobial peptides, such
as the Cupienin 1 pept ide, isolated from the venom of the spider
Cupienius salei. is peptide was active against four bacterial
strains, with MIC between 0.08 and 5.0 μM [40]. e peptides
isolated from A. gomesiana hemocytes, called Gomesina and
Acanthoscurrina-1 and -2, also showed important antimicrobial
activity. Gomesina peptide was active against Gram-positive
bacteria (0.2-12.5 µM), Gram-negative bacteria (0.4-6.25 µM)
and fungi (0.4-25 µM) [41], while Acanthoscurrina-1 and -2
peptides were active against C. albicans (1.1-2.3 µM) and E. coli
(2.3-5.6 µM) [42]. On the other hand, higher MIC values are
also found for some antimicrobial peptides, similarly to that
found for ApAn. For example, the Latarci nas (6a and 7) peptides,
isolated from the venom of the spider Lachesana tarabaevi,
showed antimicrobial activity with MIC greater than 70 µM
[43] and the peptide Licotoxin I, isolated from the venom of the
spider Lycosa carolinensis, presented MIC between 80 and 150
μM against E. coli [44]. Despite the high MIC values, the ApAn
did not show hemolytic activity against mouse erythrocytes,
even in the highest tested concentration (256 µM), dierently
from what was observed for the VdTXI acylpolyamine of V.
dubius, which under the concentration of 100 µM showed
6% of hemolysis against human erythrocytes [20]. Regarding
mygalin, a lthough no reports have been found on its hemolytic
activity, it has been shown that at concentrations between 11.9
and 95.9 µM, mygalin does not interfere with the cell viability
of macrophages and splenocytes [45]. Antimicrobial peptides,
in turn, can exhibit even more pronounced hemolytic activity,
such as Gomesina, which causes 16% of hemolysis in human
erythrocytes from low concentrations (1 µM) [41]. Licotoxin I
does not exhibit hemolytic activity up to a concentration of 30
µM, but at a concentration of 200 µM, its hemolytic activity on
rabbit erythrocytes reaches 55% [44].
Conclusion
Together, the results of UV spectroscopy and ESI-MS/MS
obtained in this work suggested that the acyl aromatic group
of acylpolyamines isolated from the A. natalensis venom is
represented by tyrosine. e identication of this unconventional
chromophore for acylpolyamines from spiders demonstrates an
even greater diversity of these molecules and that much remains
to be discovered. Our results also suggest that the terminal
polyamine chain of the ApAn is composed of structural units
PA43 or PA53. However, complementary studies using techniques
such as nuclear magnetic resonance (NMR) are still necessary
for the complete elucidation of the chemical structure of ApAn.
In addition, the antimicrobial action against E. coli and S. aureus
and non-hemolytic property of ApAn, may be relevant for the
use of these molecules as possible therapeutic agents.
Abbreviations
ACN: acetonitrile; AMPs: antimicrobial peptides; ApAn:
acylpolyamines of Acanthoscurria natalensis; ATCC: American
Type Culture Collection; CE: collision energy; CXP: collision
cell exit potential; DP: decluttering potential; ESI-MS:
electrospray ionization mass spectrometry; GS1: ion source
gas 1; HCCA: α-cyano-4-hydroxycinnamic acid; MALDI-
TOF: matrix associated laser desorption-ionization – time of
ight; MH: Mueller-Hinton grown medium; MIC: minimum
inhibitory concentration; MRM: multiple reaction monitoring;
ROS: reactive oxygen species; RP-HPLC: reverse-phase liquid
chromatography; TFA: triuoroacetic acid; UV: ultraviolet
spectroscopy.
Acknowledgments
We would like to express our sincere thanks to Dr. Diego
Madureira of the Department of Biological Basis of Health
Sciences, University of Brasilia, for making feasible the
experiments in MALDI-TOF.
Availability of data and materials
All data generated or analyzed during this study are included
in this article.
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Barth et al. J Venom Anim Toxins incl Trop Dis, 2022, 28:e20210017 Page 12 of 13
Funding
is work was funded by the National Council for scientic and
technological development (CNPq), the Brazilian Coordination
for the Improvement of Higher Education Personnel (CAPES),
Federal District Research Support Foundation (FAP-DF) and
University of Brasilia Foundation (FUB).
Competing interests
e authors declare that they have no competing interests.
Authors’ contributions
TB and ORPJ conceived this research, planned and executed
experiments, interpreted data and wrote the manuscript. JT
performed chromatographic analysis to obtain samples. PDA
and EDC performed the ESI-MS experiments. MSC made it
possible to conduct experiments by MALDI-TOF and revised
the manuscript. AS, SSS and JLS revised and corrected the
manuscript. All authors read and approved the nal manuscript.
Ethics approval
e present study was approved by SISBIO (license number
51803-1) and SISGEN (license number A826A3A) and CEUA-
UnB (license number 44/2017).
Consent for publication
Not applicable.
Supplementary material
e following online material is available for this article:
Additional le 1. ESI-MS and MS/MS spectra of ApAn728.
(A) e protonated ion [M+H]+ at m/z 729 was detected in MS
mode. (B) Fragmentation spectrum MS/MS of ion at m/z 365.
Additional file 2. ESI-MS spectrum of ApAn614a. The
protonated ion [M+H]+ at m/z 615 was detected in MS mode.
Additional file 3. ESI-MS spectrum of ApAn614b. The
protonated ion [M+H]+ at m/z 615 was detected in MS mode.
Additional le 4. ESI-MS and MS/MS spectra of ApAn742.
(A) e protonated ion [M+H]+ at m/z 743 was detected in MS
mode. (B) Fragmentation spectrum MS/MS of ion at m/z 372.
Additional le 5. ESI-MS and MS/MS spectra of ApAn756.
(A) e protonated ion [M+H]+ at m/z 757 was detected in MS
mode. (B) Fragmentation spectrum MS/MS of ion at m/z 379.
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