Antimicrobial activity, membrane interaction and structural features of short arginine-rich antimicrobial peptides

Abstract

Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

A Reliable Multifaceted Solution against Foodborne Viral Infections: The Case of RiLK1 Decapeptide

“…Therefore, based on these preliminary results, further insights into the virucidal activity of both RiLK1/RilK3 and the other peptides were provided in this study (Tables 2 and 3). With respect to RiLK1 and RiLK3, the results obtained on HAV at 4 °C were comparable to those previously obtained at room temperature [46] (Table 2). As regards MNV-1 (Table 3), a virucidal effect was observed only for RiLK1 after treatment at room temperature (LRV of 1.2 and 1.0 at 80 µM and 40 µM, respectively), while RiLK3 was not effective regardless of the concentration or temperature condition applied.…”

“…In our previous studies, the structural characterization as well as the antibacterial and antifungal activities of the peptides 1018-K6, MTP1, RiLK1, and RiLK3 were demonstrated [42][43][44][45][46], thus leading to investigations of their possible antiviral effects that have not been studied so far, considering that the ultimate goal of research is to find agents that exhibit a broad-spectrum antimicrobial activity and are therefore attractive for manufacturing applications. Moreover, two further peptides were considered in our study to expand the panel of potential antiviral compounds (Table 1), the newly designed RiLK30 and the already-known HIV inhibitor AVP2 [47], which was used as a reference.…”

“…These analyses were performed in presence of the negatively charged SDS that is commonly used as biomimetic membrane model to study the structural features of membranotropic molecules. In previous studies, the structure and the conformational stability of 1018-K6, MTP1, RiLK1 and RiLK3 have been already elucidated in detail [42][43][44][45][46]. Briefly, all these four peptides exhibited a high structural stability across a wide range of pH (2-11) and temperature (4-90 °C) but they assumed different conformations in mimicked membrane solutions such as mixed α-helix/β-sheet for MTP1, β-sheet for 1018-K6 and higher-ordered self-aggregates, assembling into bigger oligomeric species in equilibrium with partially structured monomers for RiLK1 and RiLK3.…”

“…In a previous work, the antiviral effects of RiLK1 and RiLK3 were already investigated against the hepatitis A virus (HAV) following the treatment at room temperature [46] and the obtained results have been reported in Table 2. Briefly, RiLK1 was able to reduce the HAV infectivity more than 1 log in comparison with the untreated virus, displaying a Log Reduction Value (LRV) of 1.4 at 80 µM and of 1.1 at 40 µM concentration [46]. Conversely, RiLK3 was unable to inhibit HAV infection at the same concentrations tested.…”

“…We have recently demonstrated the antibacterial activity of a panel of designed AMPs against food pathogen contaminants, including Gram-positive and Gram-negative bacteria and fungi but not viruses [42][43][44][45][46]. The structural properties and mechanism of action of these peptides have been elucidated previously and these compounds were also found to be non-cytotoxic [42][43][44][45][46].…”