International Journal of Peptide Research and Therapeutics
https://doi.org/10.1007/s10989-022-10383-4
(2022) 28:73
Deciphering the Limitations and Antibacterial Mechanism
of Cruzioseptins
Fernando Valdivieso‑Rivera1 · Sebastián Bermúdez‑Puga1 · Carolina Proaño‑Bolaños1,2 · José R. Almeida2
Accepted: 15 February 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022
Abstract
Antimicrobial peptides consist mainly of membrane-active sequences, which are potentially relevant to antibiotic resistance
era. In this context, a novel family of peptides isolated from the skin secretion of Cruziohyla calcarifer have been recently
identified with wide and efficient antimicrobial effects. However, the mechanism underlying their antibacterial action remains
unknown, as well as their activity under salt concentrations. For the primary purpose, spectrofluorometric and microscopic
assays were performed using fluorescent intercalating agents. In silico study also were performed aiming to confirm the
nature and energetics of the membranolytic interactions. The influence of proteolytic enzymes and salt concentrations was
accessed by broth microdilution approach, mimicking physiological conditions. Cruzioseptins showed detergent-like properties, acting by a similar lytic mechanism characterized for others cationic peptides. An increase of up to three times the
minimum inhibitory concentration was observed in presence of salts or serum. This represents an important challenge for
the clinical use of peptide-based drugs. Overall, we ratify the antimicrobial potential of cruzioseptins and suggest paradigms
that should be considered for translational medicine.
Graphical Abstract
Keywords Antimicrobial · Membranolytic · Physiological condition · Peptides
* José R. Almeida
rafael.dealmeida@ikiam.edu.ec
Extended author information available on the last page of the article
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International Journal of Peptide Research and Therapeutics
Introduction
The lack of new antibiotic agents and the emergence of
resistant bacteria remain critical and unsolved problems,
becoming a clear threat to public health (Roca et al. 2015).
Particularly, methicillin-resistant Staphylococcus aureus
infections are common, difficult to treat and potentially
serious causing high morbidity and mortality (Yang et al.
2018). A change in transmission dynamics has been
reported, highlighting the pressing need to develop new
antimicrobial therapies (Di Ruscio et al. 2019). Despite
the disinterest of large biomedical companies, several
investigations have proposed solutions focused on natural
products, mainly peptides (Lei et al. 2019; Plackett 2020).
Peptides constitute valuable chemical structures with
unique properties to face post-antibiotic era (Rončević
et al. 2019). To date, more than 3000 unique antimicrobial
peptide (AMP) sequences have been reported, forming a
rich library of opportunities for the pharmaceutical market
(Mishra et al. 2020). However, most of these molecules
remain in preclinical tests due to their high toxicity, low
bactericidal effect, proteolytic degradation in serum, and
low stability under physiological conditions (Ong et al.
2014; Mohamed et al. 2016; Boöttger et al. 2017). Only
2.5% of AMPs are clinically available and listed as FDAapproved drugs (Chen and Lu 2020). On the other hand,
the number of unique peptide sequences and applications
have grown (Lau and Dunn 2018). The development of
omic tools has accelerated the discovery of compounds
from different natural sources and motivated several therapeutic and research uses (Fosgerau and Hoffmann 2015;
Robles-Loaiza et al. 2021).
Amphibians possess a diverse collection of bioactive peptides with functions that can be explored from a
biotechnological point of view (Pierre et al. 2000; Wang
2020). For instance, cruzioseptins are a family of 21–23mer cationic peptides isolated from skin secretion of Cruziohyla calcarifer (Proaño-Bolaños et al. 2016), with high
antimicrobial activity against bacteria, fungi, Leishmania
spp. and low toxicity toward normal mammalian cells
(Mendes et al. 2020). However, the mechanism of action
and the stability in physiological conditions is unknown.
The lung of cystic fibrosis (CF) patients presents an
approximate concentration of 120–150 mM NaCl due to
the deficiency of CF transmembrane conductance regulator (CFTR; Zhang et al. 2005). In this environment, various peptides such as hBD-1, indolicins, magainins, and
gramicidins are inactivated (Goldman et al. 1997; Zhang
et al. 2005; Chu et al. 2013). Elevated levels of M
­ g2+ and
2+
­Ca ions have also been reported (Sanders et al. 2006).
In consequence, the evaluation of the potential of AMPs
against staphylococcal infections should mimic these high
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(2022) 28:73
salt loading conditions. The understanding of the precise
mode of action, limitations and hurdles are key elements
in peptide-based drug development. Based on this, the aim
of the present study was to investigate the antibacterial
activity and stability of cruzioseptins in physiological conditions. In addition, the possible membranolytic effect of
cruzioseptins in S. aureus was evaluated.
Materials and Methods
Solid Phase Peptide Synthesis (SPPS)
Three members of cruzioseptin family: CZS-1: GFLDIVKGVGKVALGAVSKLF-amide, CZS-2: GFLDVIKHVGKAALGVVTHLINQ-amide, and CZS-3: GFLDVVKHIGKAALGAVTHLINQ-amide were chemically obtained by
solid phase Fmoc chemistry using an Automated Microwave
Peptide Synthesizer, Liberty Blue (CEM Corporation). Their
purity levels were analyzed by reverse-phase high performance liquid chromatography (RP-HPLC) and MALDITOF mass spectrometry (Axima Confidence, Shimadzu).
The synthetic peptides used in the next steps showed more
than 90% purity.
In Vitro Time‑Kill Assay
The bactericidal activity of the cruzioseptins against S.
aureus ATCC 25293 was assessed by in vitro time kill assay
using the method described by Mangoni et al. (2008) with
some modifications. Briefly, bacterial suspensions were prepared with organisms in log phase growth. Posteriorly, the
suspensions were centrifuged, washed in phosphate-buffered saline (PBS) and adjusted to obtain a density of 1 × ­106
colony forming units (CFU)/ml. Bacteria was incubated at
37 °C with peptides at 1 × MIC. Aliquots were taken at 0,
20, 40, 60, 80 min and seeded into Mueller–Hinton agar. The
number of CFU was determined after 24 h of incubation at
37 °C. A control without peptide was performed under the
same experimental conditions.
Antibacterial Activity of Peptides in Presence
of Salts and Serum
The antibacterial effect of cruzioseptins in different physiological conditions including salts and serum was evaluated through broth microdilution assay. The procedure was
based on Mohamed et al. (2016). In brief, serial dilutions
of each peptide in dimethylsulphoxide (DMSO) were
prepared to obtain concentrations of 512, 256, 128, 64,
32, 16, 8, 4, 2, 1 mg/l. S. aureus in logarithmic growth
phase was diluted in MHB supplemented with serum 5%
and specific concentrations of salts: NaCl 150 mM, NaCl
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300 mM, ­M gCl 2 1 mM, and ­C aCl 2 8 µM, to obtain the
equivalent of 1 × ­106 CFU/ml, which were determined by
plate count method. Later, 2 μl of each peptide dilution
were transferred to a 96 well sterile plate and 198 μl of the
microorganism were added. As controls, 2 μl of DMSO
was included instead of peptide and 200 μl of MHB in
another well. Plates were incubated at 37 °C for 18–22 h.
Growth was monitored at 550 nm in an ELISA plate reader
G (Glomax Discover System, Promega). Later, 10 μl of
each concentration without visual growth was sub-cultured
on Mueller–Hinton agar plates. Plates were incubated at
37 °C overnight. Minimum bactericidal concentration
(MBCs) was recorded as the minimal concentration without any growth.
Membrane Permeability Assay
To evaluate whether cruzioseptins can induce bacterial
cell membrane damage, the propidium iodide (PI) uptake
assay was employed, as previously described (Kwon et al.
2019). Briefly, S. aureus ATCC 25923 (growing logarithmic-phase culture in MHB) was diluted to ­OD600 nm≈0.27.
After, bacteria were mixed with PI at a final concentration
of 10 µg/ml. Later, 50 µl of bacteria and PI was added to
the well. Posteriorly, 50 µL of peptides (CZS-1, CZS-2 and
CZS-3) at 1 × MIC was added. As negative control, bacteria were incubated in the absence of cruzioseptins. As
positive control, cells were treated with 70% isopropanol
(v/v). PI fluorescence was measured at excitation and
emission wavelengths of 580 and 620 nm, respectively.
The percentage of membrane permeabilization was calculated as the relative fluorescence intensity vs. the positive
control.
Fluorescence Microscopy
The ability to permeabilize Gram-positive membranes was
also confirmed using the microscopy technique described
by Li et al. (2014). In briefly, S. aureus was incubated in
logarithm-phase with CZS-1, CZS-2 and CZS-3 (1 × MIC)
during 30 min and centrifuged at 3000 × g × 15 min. The
pellet was stained with PI (Sigma) and DAPI (Sigma) during
15 min each one in the dark at 0 °C. PI was used as marker of
damage, since it only permeabilizes bacteria with compromised plasma membrane. DAPI was employed as membrane
permeable compound. The control was bacteria without
peptide. The cells were observed under the combination of
fluorescence and differential interference contrast (DIC) in
a Nikon Eclipse Ni microscope. The fluorescence area were
calculated based on images examined using ImageJ software
(Schneider et al. 2012).
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In Silico Molecular Docking
To evaluate the possible membranolytic effect, a molecular
docking study was performed. Thus, a mimic of Gram-positive bacterial membrane was build using the CHARMMgui server (Jo et al. 2008). The model system contains a
lipid bilayer with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero3-phosphoethanolamine (POPE) lipids in a 3:1 ratio, respectively (whose composition was taken from Kyriakou et al.
2016). This lipid mixture has also been used in other investigations (Chugunov et al. 2013; Mulholland et al. 2016).
The three-dimensional structure of the cruzioseptins was
obtained using Pymol (Schrödinger 2017). The peptides
were relaxed and optimized with UCSF Chimera (Pettersen
et al. 2004) and AutoDockTools. Finally, molecular docking
study between bacterial membrane and cruzioseptins was
carried out using AutoDock VINA (Trott and Olson 2009).
The interaction energy between the membrane and peptide
was expressed as affinity (kcal/mol).
Statistical Analysis
All data represented from triplicate individual experiments.
Data were analyzed using analysis of variance (ANOVA),
followed by post-hoc Tukey multiple comparison. Data are
presented as means ± standard error, and P < 0.05 was considered significant.
Results
Time‑Kill Assay of Cruzioseptins Against S. aureus
The killing effect of cruzioseptins against S. aureus was
evaluated at 1 × MIC of peptides. As depicted in Fig. 1, CZS1, CZS-2, and CZS-3 present a potent bactericidal effect
against S. aureus. Indeed, CZS-1 and CZS-3 showed an
irreversible reduction in the CFUs after 20 min of exposure,
while CZS-2 exerted killing activity at 40 min.
Antimicrobial Activity in Physiological
Concentrations of Salts and Serum
Under challenging physiological conditions, such as high
salt levels and serum, cruzioseptins maintain its antibacterial activity, but require higher concentration (Table 1). In
summary, in the presence of 150 mM NaCl and 300 mM
NaCl; CZS-1, CZS-2, and CZS-3 showed a twofold increase
in the minimum inhibitory concentration. On other hand,
in the presence of divalent cation salts (­ MgCl2 and ­CaCl2),
CZS-1 increased its minimal inhibitory concentration
onefold, while CZS-2 and CZS-3 increased its minimum
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Fluorescence Microscopy
Staphylococcus aureus were stained with DAPI, which bind
to genetic material regardless of cell viability. Without treatment with cruzioseptins, a small number of cells showed PI
staining. High levels of red fluorescence were only observed
in the presence of cruzioseptins. Fluorescence microscopy
images suggest that the S. aureus membrane was disrupted
by peptides (Fig. 3). The degree of membrane permeabilization (PI stained cells) was higher when S. aureus was incubated with CZS-1 than CZ-2 and CZS-3. Peptides induced
a visible aggregation of injured bacteria.
In Silico Molecular Docking
Fig. 1 Killing kinetics for cruzioseptins (CZS-1, CZS-2, CZS-3) at
1 × MIC against S. aureus. Control (PBS) represent bacteria in the
absence of peptide. Bactericidal activity was defined as a decrease
of > ­3log10 in CFUs at any of the incubation times tested according to
Mangoni et al. (2008). The dotted lines represent the 3 ­log10 border.
CZS-1 and CZS-3 showed a reduction in the CFU after 20 min. The
results are presented as means ± SD (n = 3)
inhibitory concentration twofold. In a similar way, cruzioseptins exhibited a lower antibacterial effect in the presence
of 5% serum. As shown in Table 1, some divalent cations
and serum reduced antimicrobial activity of cruzioseptins,
altering their MIC values.
Membrane Permeability Assay
The PI uptake assay evidenced that synthetic cruzioseptins
exert their antibacterial effect against S. aureus by a membrane-disruption mechanism. Negative control showed a low
PI uptake. The cell membrane of S. aureus was significantly
compromised by exposure to CZS-1, CZS-2, and CZS-3, as
demonstrated by the rapid increase in PI fluorescence within
3 min (Fig. 2). The PI uptake in CZS-1 is the most prominent than CZS-2 and CZS-3, suggesting that CZS-1 caused
larger membrane injuries. After only 3 min of exposure of
the bacteria with CZS-1 an increase in PI fluorescence is
observed (Fig. 2). No significant difference was observed
between CZS-2 and CZS-3.
Table 1 Minimum inhibitory
concentration (MIC) and
minimum bactericidal
concentration (MBC) of
cruzioseptins in the presence of
salts (NaCl, ­MgCl2 and ­CaCl2)
and human serum (5%) against
S. aureus
Peptides
CZS-1
CZS-2
CZS-3
a
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In vitro results were corroborated by computational techniques. From a phenomenological point of view, in silico
investigation showed that cruzioseptins interact with
mimetic bacterial membrane (Fig. 4). In addition, there is a
high binding affinity between cruzioseptins and the bacterial membrane. The docking score for CZS-1, CZS-2, and
CZS-3 in POPE/POPG were − 9.81, − 9.01, and − 11.61 kcal/
mol, respectively.
Discussion
AMPs have been extensively studied and proposed as alternative antibiotics in the last decade owing to their ability to induce the death of antibiotic-resistant bacteria of
clinical relevance (Mahlapuu et al. 2016; Proaño-Bolaños
et al. 2019). Their characteristics and diversity offer promising opportunities for the development of new antibacterial hits. However, like any drug candidate, some challenges must be addressed, such as: the loss of activity in
the presence of salts or enzymatic degradation caused by
serum enzymes (Mohamed et al. 2016). Indeed, most of
the AMPs in ongoing clinical trials are based on topical
application (Andersson et al. 2016). Due to this, a comprehensive understanding of the antimicrobial activity of
synthetic peptides under physiological conditions is highly
recommended to facilitate the development of successful
peptide-based drugs, as well as to guide the rational design
MIC (MBC) (mg/l)
Controla
NaCl 150 mM
NaCl 300 mM
MgCl2 1 mM
CaCl2 8 µM
Serum 5%
8 (16)
16 (64)
32 (128)
32 (32)
64 (64)
128 (128)
32 (32)
64 (64)
128 (256)
16 (16)
64 (128)
128 (128)
16 (16)
64 (64)
64 (128)
64 (64)
128 (256)
256 (512)
Previously reported in Proaño-Bolaños et al. (2016) and confirmed in our study
International Journal of Peptide Research and Therapeutics
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and the discovery of suitable antibacterial candidates
(Deslouches et al. 2020; Klubthawee et al. 2020).
Our group has previously demonstrated the antimicrobial
effect induced CZS-1, CZS-2, and CZS-3 and their potential
for drug development of antibacterial, antifungal or leishmanicidal compounds. For example, CZS-1 was highly
active against of Escherichia coli, S. aureus, and Candida
albicans and induced low hemolysis at MIC. Additionally,
CZS-1 display high activity and selectivity against of Leishmania (V.) braziliensis (Proaño-Bolaños et al. 2016; Mendes
et al. 2020).
In the present study, we evaluate the stability of cruzioseptins under various physiological conditions. The salt
sensitivity of peptides was confirmed. Cruzioseptins maintained their antibacterial activity against S. aureus under
high salt concentrations, although there was an increase of
until twofold in the MICs. Some works have demonstrated
that monovalent and divalent cations ­(Na+, ­Ca2+, ­Mg2+) can
directly and strongly affect the activity of cationic peptides.
Several authors have proposed a competition between cationic peptides and ions in the binding process to the bacterial
membrane surface (Ying et al. 2019; Ko et al. 2020). Probably, this event occurred when the cruzioseptins were analyzed in the presence of salts, leading to an increase in MIC
values. Our results demonstrate that simulation of ionic environments reduce the antibacterial action of cruzioseptins.
Similar findings were observed in the antibacterial activity
of DM-PC and their analogs in presence ­Mg2+ (Ying et al.
2019). Whereas RR and its peptide derivates maintained the
antibacterial in ­CaCl2 8 µM (Mohamed et al. 2016).
On the other hand, peptides are natural substrates of proteolytic enzymes, which constitute an important barrier for
the oral application of these prototypes. In serum, there are
a large number of proteases with different specificity able
to recognize and cleave peptide bonds (Werle and BernkopSchnürch 2006; Boöttger et al. 2017). In presence of serum
5%, the CZS-1, CZS-2, and CZS-3 activity decreased threefold in comparison with control. This suggests that peptides
Fig. 3 Representative fluorescence micrographs of S. aureus treated
with cruzioseptins at 1 × MIC. A DAPI (blue) and PI (red) fluorescence staining for the evaluation of membrane damage in S. aureus
induced by synthetic cruzioseptins. Gram-positive bacteria were incu-
bated with peptides followed by staining with fluorescent nucleic acid
binding dyes. Signals were examined in a Nikon Eclipse Ni microscope. B Percentage relative to DAPI staining. Data are presented as
the mean ± SD. *P ≤ 0.05
Fig. 2 Membrane permeabilization induced by CZS-1, CZS-2 and
CZS-3 at 1 × MIC in S. aureus by PI uptake assay. Relative fluorescence intensities represent mean ± SD of two independent experiments performed in triplicate and were calculated taking the positive
control (bacteria treated with 70% isopropanol) as maximal permeabilization (100%). No significant difference was observed between
CZS-2 and CZS-3 (ANOVA followed by Tukey, P < 0.05)
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Fig. 4 Molecular interactions of cruzioseptins (cyan color) with bacterial cell membrane. A CZS-1 showed a score of − 9.81 kcal/mol, B CZS-2
presented a score of − 9.01 kcal/mol and C CZS-3 exhibited a score of − 11.61 kcal/mol. Docking score values represent the binding energy
evaluated are probably excised by these enzymes generating
smaller fragments less active against Gram-positive bacteria
evaluated. There is probably a cleavage of C-terminal of
basic amino acids from cruzioseptins by carboxypeptidase
U (EC 3.4.17.20) or lysine carboxypeptidase (EC 3.4.17.3).
However, for further research several strategies could be
adopted to protect cruzioseptins against proteases such as
N-terminus acetylation, substitution by unnatural amino
acids, and peptide cyclization (Werle and Bernkop-Schnürch
2006; Nguyen et al. 2010; Ong et al. 2014).
Most AMPs-derived drugs have the membrane components as their primary target (Chen and Lu 2020). Due to
their cationic amphipathic structures, in earlier studies we
initially hypothesized that cruzioseptins kill microorganisms
through membrane disruption (Proaño-Bolaños et al. 2016;
Mendes et al. 2020). Here, this molecular event was confirmed by fluorescence microscopic and PI uptake assays.
DAPI (277.3 g/mol) binds to DNA without damaging the
membranes. This justifies the blue fluorescence seen in the
presence and absence of cruzioseptins. PI (668.6 g/mol)
does not enter through the intact bacterial membranes (Wu
et al. 2012; Li et al. 2014), converting it into a selective
marker of injured cells (Almeida et al. 2022). As can be
seen, PI was not able to enter the bacterial cells without
treatment, highlighting that the S. aureus membranes remain
intact. A significant increase in red fluorescence is strongly
associated with membrane damage. The PI uptake in the
presence of cruzioseptins suggests that they act via a membrane permeabilization pathway. This principle of action was
the same induced by other AMPs isolated from amphibians
such as dermaseptins (Song et al. 2020) and Japonicin-2LF
(Yuan et al. 2019). Cruzioseptins caused the aggregation
of S. aureus, probably associated with loss of the membrane potential (Niu et al. 2012). In general, studies have
demonstrated that destabilized bacteria are prone to clusters (Chen et al. 2010). Additionally, this nature of action
is supported by molecular docking analysis. Our negative
binding energy values indicate the favorable membrane–peptide interactions, similar to computational studies with frog
13
skin-derived peptides (Wu et al. 2018; Cuesta et al. 2021).
Interestingly, our in vitro results show functional and stability differences between effects caused by cruzioseptins.
Remarkably, when the bacteria were exposed to CZS-1, the
time killing was short and the PI uptake was the highest,
evidencing the cell lysis caused by CZS-1.
Taken together, these results suggest that CZS-1 present higher antibacterial and membrane-disturbing activity.
Cruzioseptins associated with the S. aureus membrane in
a salt-sensitive manner, despite a relative resistance when
compared to other biologically active peptides. Therefore,
further investigations are needed to estimate the structural
determinants and biochemical properties of cruzioseptins
that favor this tolerance.
Conclusions
In conclusion, our findings enhance our understanding of
the molecular mechanism of antibacterial cruzioseptins,
which are membrane-lytic cationic peptides. Their biological activity is influenced by salt environments and serum.
Based on translational medicine, our study reinforces the
need to evaluate AMPs according to the clinical context that
will be used. Future works should concentrate on the design
of tolerant membrane-damaging analogs based on the primary structure of cruzioseptins with higher overall success
probabilities.
Acknowledgements Part of this research was made possible by a support of the Project “Conservation of Ecuadorian amphibian diversity
and sustainable use of its genetic resources” which involves Ministerio del Ambiente y Agua del Ecuador and Universidad Regional
Amazónica Ikiam, among others supported by the Global Environmental Facility (GEF) and “Programa de las Naciones Unidas para el
Desarrollo” (PNUD). We also thank the support of the Laboratory of
Microscopy of the Universidad Regional Amazónica Ikiam. We gratefully acknowledge the help provided by Mgs. Bruno Mendes and M.Sc.
Jacqueline Noboa M.Sc. in the technical assistance with the microscopy analysis. We also thank M.Sc. Andrea Carrera for her ongoing
collaboration with constructive comments.
International Journal of Peptide Research and Therapeutics
(2022) 28:73
Data Availability All data generated or analysed during this investigation are included in this published article. Additional data will be made
available on reasonable request.
Declarations
Conflict of interest The authors declare no conflict of interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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Authors and Affiliations
Fernando Valdivieso‑Rivera1 · Sebastián Bermúdez‑Puga1 · Carolina Proaño‑Bolaños1,2 · José R. Almeida2
1
Laboratory of Molecular Biology and Biochemistry,
Universidad Regional Amazónica Ikiam, Km 7 Via Muyuna,
Tena, Napo, Ecuador
13
2
Biomolecules Discovery Group, Universidad Regional
Amazónica Ikiam, Km 7 Via Muyuna, Tena, Napo, Ecuador