Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX, a Lys49 Phospholipase A₂ Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766)

Abstract

Snake venom phospholipases A (PLA s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.

Antibacterial properties of snake venom components

“…It was proven that presence of charged and aromatic amino acids plays an important role in interaction of peptides with bacterial cell membrane (Almeida et al 2018). The antibacterial evaluation of LmutTX, Lys49 PLA 2 from Lachesis muta muta snake venom and synthetic peptides designed on its base show promising activity against Gram-negative and Gram-positive bacteria (Diniz-Sousa et al 2018). Also other C-terminal, cationic peptides derived from Lys49 PLA 2 s have been evaluated for their microbicidal and anti-tumor potential (Páramo et al 1998;Murillo et al 2007;Costa et al 2008), presenting promising results.…”

Harnessing snake venom phospholipases A2 to novel approaches for overcoming antibiotic resistance

“…PepC‐W showed an overall better performance than PepC against S. aureus , S. aureus resistant to methicillin (MRSA) and P. aeruginosa . Moreover, PepC‐W cytotoxicity assays against the C2C12 line of muscle myoblasts of mouse C3H resulted in low activity, validating its selectivity against prokaryotic cells (Diniz‐Sousa et al, ).…”

“…In addition, several variants of antibacterial Lys49 svPLA 2 s C‐terminal peptides have been synthesized, with the intention of improving their therapeutic profile, by (a) increasing activity and/or stability against degradation by proteases, (b) reducing toxicity, and (c) establish relevant SAR towards a better understanding of their mechanism of action in order to guide rational design of more potent and safer mimetics (Lomonte et al, ; Santamaria, Larios, Angulo, et al, ; Santamaria, Larios, Quiros, et al, ) (Table ). In several cases, sequence modifications have consisted in inserting hydrophobic residues known as relevant in many AMPs, especially Trp (Diniz‐Sousa et al, ; Santamaria, Larios, Angulo, et al, ). Trp and other hydrophobic residues are larger than other amino acids usually found in Lys49 svPLA 2 s C‐terminal peptides, hence their modified versions have not only variable hydrophobicity (ranging from +8.93 kcal mol −1 to +20.97 kcal mol −1 ) but also higher mass, typically between 1,600 and 2,000 Da (Shagaghi et al, ).…”

“…Still, one cannot disregard that conversion of naturally occurring/inspired bioactive peptides into therapeutically useful drugs is a difficult and laborious challenge, starting right from the preclinical stage. For instance, it has already been mentioned that an increase in Trp content can improve antibacterial activity of Lys49 PLA 2 s‐derived AMPs; however, that was also found to potentiate toxicity against eukaryotic cells, meaning that a fine sequence tuning is required to ensure a favorable balance between activity and safety (Diniz‐Sousa et al, ; Santamaria, Larios, Angulo, et al, ). Insertion of positively charged residues as Lys to promote peptide accumulation onto the negatively charged membranes should be equally used with caution: while Lys residues promote formation of a hydrophilic patch eventually allowing water entrance and diffusion through the membrane, causing its destabilization (Almeida et al, ; Yu et al, ), they also make peptides more prone to proteolytic degradation (Arias et al, ).…”

“…These invariably possess a Cys residue at position 11, and assays with p-BthTX-I and its analogue where Cys was replaced by Ser showed a decrease in antimicrobial activity, probably due to the fact that the substituted peptide was no longer able to form a dimer resembling (p-BthTX-I) 2 (Santos-Filho et al, 2015). In turn, substituted analogues in Table 3 have increased activity as compared to native sequences on which they were inspired (Diniz-Sousa et al, 2018;Santamaria, Larios, Angulo, et al, 2005). This stems from the fact that replacements were deliberately done in order to increase antimicrobial activity; in some variants, one or more specific residues, mainly Tyr, were replaced by Trp, as this amino acid is known to promote strong interactions with bacterial membranes (Arias et al, 2018;Lomonte et al, 2010).…”