Antimicrobial resistance is a severe global health problem and has become one of the main causes of morbidity and mortality in the world. The high spontaneous mutation rate and genetic recombination of the microorganisms, associated with the irrational use of antibiotics, have promoted the prompt development of multi-resistant pathogens, leading to research on new antimicrobial molecules from different natural sources [1
Scorpion venom constitutes a complex mixture of molecules with high therapeutic potential [2
]. Venom peptides with insecticide, antiviral, antimicrobial, hemolytic, antiproliferative, bradykinin potentialization, and immunomodulatory activities, which have been described in previous studies, are targeted in the prospection of novel molecules of biotechnological interest [3
]. Antimicrobial peptides (AMPs) without disulfide bridges have been identified in different scorpion species, presenting a positive net charge and a hydrophobic character, which permit interaction with microorganism cellular membranes [4
]. AMPs usually present random coil conformation in polar solvent, but show an α-helical conformation when they interact with membranes or non-polar solvents [5
]. The mechanism of action of the AMPs can be described as follows: First, the electrostatic interaction occurs between its positive amino acids with the negatively charged phospholipids in the microorganism membrane; then a displacement of lipids is induced, leading to pore formation [6
]. Accordingly, the bacterial sensitivity is directly related to the different composition and physicochemical properties of the lipids in cellular membranes [7
In eukaryotic membranes, lipids in the outer membrane are commonly neutral phospholipids, such as phosphatidylserine and sphingomyelin. However, the bacterial cellular membrane is mainly composed of negatively charged lipids, such as phosphatidylglycerol (PG), cardiolipin, and phosphatidylethanolamine (PE) [8
]. The negative net charge in bacterial membranes plays an important role in the preferential interaction of cationic AMPs and microorganism membranes [1
Several structural determinants are responsible for the antimicrobial and cytolytic activity of antimicrobial peptides. Studies have shown that increased α-helical conformation, cationic character, hydrophobicity and hydrophobic moment of native antimicrobial peptides have an effect on the spectrum of antibiotic action. [9
]. Another important factor for the use of these molecules is their amidation in the C-terminal region, as it has demonstrated a greater protection to the proteolysis and a greater structural stability of the amphipathic helix [12
]. However, a clearer understanding of the influence of these characteristics on the peptide structure may help to develop new strategies that will facilitate the creation of new pharmacological agents that improve or optimize mechanisms to suppress the capacity of pathogens to generate resistance, as well as to improve the potential therapeutic use of these peptides.
Stigmurin is an AMP without disulfide bridges with 17 amino acid residues, obtained from the transcriptome analysis of the scorpion Tityus stigmurus
venom gland by our research group. This antimicrobial peptide presented in vitro and in vivo antimicrobial activity and low hemolytic activity [13
]. Amino acid substitutions in a short peptide chain have been reported to improve the native peptide characteristics, increasing its biological activity and reducing its toxicity. This is a promising approach to develop new molecules with therapeutic potential [15
]. To design the analogs of this study, we replaced the polar and uncharged Ser and Gly residues of the native peptide with positively charged Lys residues in order to increase the positive charge of Stigmurin and observe the effect of increasing the charge on antimicrobial activity and hemolytic activity of this non-hemolytic peptide with a low positive charge (+2). Moreover, the structural conformation in silico of the two analog peptides, denominated as StigA25 and StigA31, was assessed by circular dichroism, as well as their antimicrobial activity in vitro and in silico by molecular dynamics.
Antimicrobial peptides (AMPs) are small molecules that act as part of the innate immune system against pathogenic microorganisms [18
]. These peptides usually present a general toxic effect on normal cell lines, as well as induce hemolysis in pharmacological concentrations [19
]. In this study, the amino acid substitutions on the native peptide Stigmurin for lysine residues (K) resulted in the increase of the α-helix structure, positive net charge, and hydrophobic moment percentages in the analog peptides, as analyzed by in silico methods. The structural models obtained by ab initio modeling for Stigmurin, StigA25, and StigA31 also showed a structure mainly composed of α-helix and hydrophobic residues (approximately 64.7%), with the potential to interact with non-polar compounds [20
]. The amino acid substitutions in the native sequence for lysine residues (K) confer a higher cationicity and helical conformation to the analog peptides, and these characteristics have been related to an increase in antimicrobial activity [21
]. The molecule amphipathicity is an important factor for the AMP activity in bacteria, which enables the peptide interaction with the lipid bilayer compounds, and this can compromise the bacterial membrane integrity or inhibit essential cellular compounds, reducing the development of resistance. The sum of these characteristics gives these AMPs great importance in microorganism membrane recognition and interaction [20
In our study, StigA31 (+7) showed a higher negative IPE in the in silico analysis of lipid bilayers when compared with Stigmurin (+2) and StigA25 (+5), indicating a stronger intermolecular interaction. The compilation of these results suggests that StigA31 showed the highest in vitro antimicrobial activity due to the highest IPE value (more negative value) of StigA31 compared to A25 and Stigmurin (−313.31 kcal mol−1, −258.29 kcal mol−1 and −89.25 kcal mol−1, respectively). These results indicate that the mechanism of action of these AMPs is directly associated with electrostatic (Coulomb) interactions that are more dominant than the Van der Waals (Lennard-Jones) interactions. The mutated positions play a crucial role in the antimicrobial efficiency of the peptides due to the insertion of positive charges in the side chains that make the interaction stronger with the anionic membrane. The data reaffirm the predictive capacity of MD simulations and brings the future perspective of studying new analogues through theoretical methods.
The capacity of cationic peptides without disulfide bridges to modify their structural conformation according to the environment provides a flexibility that is important to the interaction with the microbial targets, which can contribute to their antimicrobial activity [19
]. In this study, the CD analysis demonstrated the structural variability of StigA25 and StigA31 in different solvents, showing a high percentage of α-helix in hydrophobic environments similar to anionic membranes and a predominant random coil conformation when submitted to polar solvents. This structural variability was already observed for the native peptide [14
]. Furthermore, Stigmurin, StigA25, and StigA31 demonstrated structural stability in a broad temperature and pH range when analyzed by CD in the presence of SDS 20 mM, returning to its initial conformation after cooling.
StigA25 and StigA31 demonstrated broad-spectrum antimicrobial activity, which was higher than that presented by Stigmurin. The native peptide demonstrated activity against Gram-positive bacteria, but was not effective against Gram-negative bacteria at the higher tested dose (150 µM). Furthermore, the analog peptide activity was higher than that of standard antibiotics Vancomycin, Gentamicin, and Amphotericin B for the most tested strains after 24 h incubation, showing that the analogs generated in this study are promising candidates for the development of anti-infective therapeutic agents. The higher antimicrobial activity in vitro of StigA31 (+7) when compared to Stigmurin (+2) and StigA25 (+5) was also demonstrated in the theoretical analysis obtained by molecular dynamics. In the secondary structure analysis by CD we did not observe the increment in the helical conformation of Stig25 and Stig31 in comparison with Stigmurin, with the positive net charge increment being the main factor related to the action spectrum and antimicrobial activity increase.
Previous studies suggest that AMPs can interact with high affinity with lipopolysaccharide (LPS), a glycolipid present in the Gram-negative bacteria outer membrane [22
]. The potent activity of the analog peptides StigA25 and StigA31 against bacteria strains can be associated with their cationicity, compared to Stigmurin. The peptide–bacteria interaction in a complex and possibly specific process, being the product of the more efficient interaction of the peptide with some microorganism compounds [23
]. LPSs provide a protective barrier against molecules higher than 1000 Da, then acting as a defensive mechanism against antibiotics and AMPs [24
], which suggests that the peptides reported here do not act through this mechanism, since these AMPs are large for crossing the microbial pores and reach the cell membrane. The antimicrobial peptides, due to their cationic and amphiphilic nature, may accumulate on the bacteria surface, leading to cell wall rupture through the interaction with their compounds [25
]. A similar action mechanism was described for Kn2-7, an analog peptide from the native peptide BmKn2 isolated from the scorpion Mesobuthus martensii
Karsch, which promoted cell wall rupture of S. aureus
and E. coli
through the LTA and LPS binding, respectively [26
]. SEM data indicate that the mechanism of death of peptides is distinct from that of most cationic peptides, which generally influence membrane permeability or even form pores on the bacterial surface, seriously disrupting cell morphology [1
]. Our study therefore provides evidence that Stigmurin and its analogues may induce bacterial suppression by disrupting the cell wall without promoting cell lysis. In an earlier study with Bac2A peptide analogs, drastic changes were also observed in the cell surface of S. aureus
, with protuberances and cracks in the cell wall, not demonstrating interference in the inner membrane of the cells. In addition, the Bac2A peptide kills S. aureus
without promoting cell lysis [27
Regarding the antiparasitic activity, the three tested peptides inhibited the epimastigote forms of the T. cruzi
Y strain. No significant difference was observed between the inhibition caused by the analog peptides; though when compared with the native peptide Stigmurin in an earlier study, both analog peptides showed higher antiparasitic activity [15
]. Various antimicrobial peptides from aquatic animals have been isolated and tested against epimatigote forms of T. cruzi
; however, they did not show antiparasitic activity for these strains [28
], different from the analog peptides StigA25 and StigA31, which demonstrated a high inhibition rate. Similar activity was also shown for other analog peptides from the scorpion peptide Stigmurin [15
] and various analog peptides from scorpion venom with significant activity against trypanosomatides [29
]. When tested in trypomastigote forms of the T. cruzi
and Y strains we observed the same pattern for epimastigote, with the analogs peptides showing higher activity than the native peptide Stigmurin. When comparing the analog peptides with benznidazol (where the inhibition rate for the same Y strain was calculated in an earlier study), at the same concentration and exposure time, both analog peptides presented higher antiparasitic activity in the lower concentrations tested [15
]. Benznidazol is the main drug used in the treatment of Chaga’s disease, showing efficacy after 72 h incubation in tripomastigote forms of the T. cruzi
Y strain [30
]. Therefore, the analog peptides were more effective in lower incubation time with lower concentrations. Our results demonstrate that the analog peptides generated are potential candidates for additional investigation and application as therapeutic agents for Chagas disease, as an alternative or in addition to the conventional treatment, considering their antiparasitic properties.
AMPs can interact with the erythrocyte surface through the interaction with sialic acid presented in the glycoproteins or glycosphingolipids, which constitute the glycocalyx [3
]. La Salude Bea and collaborators [31
] studied analogs from the native peptide BmKn1 found in the scorpion Buthus martensii
Karsch venom. They suggested that modification of the native AMP sequence, which results in the increase of positive net charge through the addition of lysine residues, leads to the increase in the antimicrobial and hemolytic activity. Under the conditions that we evaluated here, the analog peptides presented low hemolysis rate at the concentrations at which they presented antimicrobial activity, demonstrating potential therapeutic application of these bioactive peptides.