Research Article: Counter-ion effect on antistaphylococcal activity and cytotoxicity of selected antimicrobial peptides

Date Published: January 6, 2018

Publisher: Springer Vienna

Author(s): Karol Sikora, Maciej Jaśkiewicz, Damian Neubauer, Marta Bauer, Sylwia Bartoszewska, Wioletta Barańska-Rybak, Wojciech Kamysz.


In view of an appreciable increase in resistance of Staphylococcus aureus to the conventional antibiotics, it is desired to develop new effective drugs. Antimicrobial peptides (AMPs) seem to be attractive candidates. In general, AMPs samples used for in vitro studies consist of a peptide, counter-ion, and water. The presence of the counter-ion could be significant as it affects peptide secondary structure and biological activity. The purpose of this study was to estimate the impact of counter-ion on antistaphylococcal activity of selected AMPs (CAMEL, citropin 1.1, LL-37, pexiganan, temporin A). To do this, three kinds of salts were prepared, namely, acetates, hydrochlorides, and trifluoroacetates. In addition, the hemolytic activity against human red blood cells (hRBCs) and cytotoxicity (HaCaT) were determined. The results indicate that there is a substantial difference between different salts, but the pattern is not consistent for the peptides. In general, the antistaphylococcal activity decreased in the order: CAMEL > temporin A > pexiganan > citropin 1.1 ≫ LL-37. The highest selectivity indexes were determined for CAMEL hydrochloride, pexiganan acetate, and temporin A trifluoroacetate. This study shows how important is to take into account the kind of counter-ions when designing novel peptide-based antimicrobials.

Partial Text

Nowadays, the solid-phase synthesis is a popular method in organic chemistry, especially in peptide synthesis (SPPS). Since 1963, when solid-phase method had been introduced by Merrifield, a lot of effort has been expended on SPPS development. Nonetheless, the main features of the method remained unchanged. In general, amino acid derivatives are subsequently attached to an elongated peptide chain and the product is released from the resin with a strong acid. The most popular is Fmoc/tert-butyl chemistry where trifluoroacetic acid (TFA) is used for cleavage and final deprotection. Moreover, it is used in RP-HPLC as an additive to mobile phase, e.g., for ion-pairing of basic side chains and N-terminus amino group (Chandrudu et al. 2013; Mäde et al. 2014). In effect, synthetic peptides purified by RP-HPLC are obtained as trifluoroacetate salts. Moreover, an excess of the trifluoroacetate ion (together with those anions that are directly bound to positively charged groups) in peptides’ lyophilizate may occur. Importantly, TFA anions are able to affect both the biological and physico-chemical properties of peptides, and for this reason, it is important to consider the counter-ions in peptide studies, including in vivo experiments. To date, several reports regarding the TFA toxicity against cells, e.g., suppression of proliferation of osteoblasts, were reported (Cornish et al. 1999). In fact, counter-ions may also affect the secondary structure of peptides and proteins (Gaussier et al. 2002). The presence of different counter-ions can affect hydrogen-bonding network and alter its structure (Blondelle et al. 1995) (Cinelli et al. 2001). Characterization of synthetic peptides, including conformational analysis, is routinely performed using IR and CD spectroscopy. However, a strong IR band at around 1670 cm−1 assigned to TFA which overlaps the amide I band of peptides (1600–1700 cm−1) may complicate analysis (Gaussier et al. 2002). Several methods of peptide TFA− counter-ion exchange are described in the literature. A popular one utilizes lyophilization from aqueous solutions of a strong acid, e.g., hydrochloric. As a matter of fact, this method is limited to those anions of acids that are stronger than TFA (with lower pKa values). Other counter-ion exchange methods are based on RP-HPLC, anion-exchange resins, and dialysis through membranes (Roux et al. 2008). Despite the documented influence of counter-ions on the structure and biological activity of molecules, studies on antimicrobial peptides (AMPs) in this area are rather sparse. Nevertheless, the counter-ion effect cannot be ignored, especially because AMPs are positively charged and can interact with anions such as trifluoroacetates. Antimicrobial peptides (AMPs) are the group of compounds that seem to be an alternative to the conventional antibiotics. These evolutionally conserved molecules play a vital role in the innate immune systems of almost all organisms (Mansour et al. 2014). They are targeting a broad spectrum of organisms including bacteria, fungi, protozoa, and viruses, and are able to trigger and coordinate multiple components of innate immunity. For this reason, much attention has been paid to the design of novel, synthetic AMPs (de novo design) with improved properties. A substantial number of AMPs exhibit the antistaphylococcal activity that makes them an object of intense research (Dawgul et al. 2016; Mohamed et al. 2016). Since Staphylococcus aureus is still one of the leading pathogens associated with nosocomial and wound infections, it is desired to develop new effective therapies. The aim of this study was to investigate the effect of counter-ion type on antistaphylococcal activity and cytotoxicity of selected AMPs. Peptides used in this study were, CAMEL (Andreu et al. 1992), citropin 1.1 (Wegener et al. 1999), LL-37 (Larrick et al. 1995), pexiganan (Ge et al. 1999), and temporin A (Simmaco et al. 1996). For estimation of the influence of different counter-ions on antistaphylococcal activity, the trifluoroacetate, acetate, and chloride salts were prepared.

Almost all the AMPs tested in this study exhibited a high activity against reference and clinical strains of S. aureus. Moreover, essential differences in antimicrobial, hemolytic activity, and cytotoxicity were found between the tested salts. In general, LL-37 showed weak antistaphylococcal properties, but three sensitive isolates were identified. The greatest antistaphylococcal activity was noticed for CAMEL with a slight superiority of chloride form. On the other hand, this peptide showed the highest hemolytic activity among all the tested peptides and a relatively high cytotoxicity. Nonetheless, pexiganan acetate and temporin A trifluoroacetate exhibited the highest selectivity index among the tested salts. Surprisingly, pexiganan acetate is the one showing the highest hemolytic activity against human RBCs. The results obtained in this study suggest that there is no simple correlation between the type of counter-ion in the peptide and the biological activity. We believe that each case should be considered individually due to peptide-dependent differences between salts. Nevertheless, our findings undoubtedly support the thesis that the kind of the counter-ion is critical for biological properties of antimicrobial peptides.




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