*Article* **Antimicrobial Activity of Snake β-Defensins and Derived Peptides**

**Nancy Oguiura 1, \*, Poliana Garcia Corrêa 1 , Isabella Lemos Rosmino <sup>1</sup> , Ana Olívia de Souza <sup>2</sup> and Kerly Fernanda Mesquita Pasqualoto <sup>3</sup>**


**Abstract:** β-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several β-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from β-defensins, were designed applying the bioisosterism strategy. The linear β-defensins were more active against *Escherichia coli*, *Micrococcus luteus*, *Citrobacter freundii*, and *Staphylococcus aureus*. The derived peptides (7–14 mer) showed antibacterial activity against those bacteria and on *Klebsiella pneumoniae*. Nonetheless, they did not present activity against *Candida albicans*, *Cryptococcus neoformans*, *Trychophyton rubrum*, and *Aspergillus fumigatus* showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake β-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

**Keywords:** β-defensins; snakes; antimicrobial activity; bioisosterism; peptides

**Key Contribution:** We tested many snake β-defensins against bacteria from oral flora, *Micrococcus luteus*, and *Escherichia coli*. This work is the first testing of the antibacterial activity of snake βdefensins besides crotamine. A bioisosterism approach was used to design the derived peptides from β-defensins from *Lachesis muta*, *Bothrops jararaca*, and *Crotalus durissus* snakes. Our results have shown that (i) the defensin's C-terminal portion seems to be crucial against bacteria; (ii) the presence of tryptophan residue into the derived peptide's sequence plays an important role for the antibacterial activity; and (iii) the Cys to Ser substitution has abolished the derived peptides' antifungal activity.

**1. Introduction**

With the frightening advent of the global increase of microbial resistance to conventional antibiotics, the search for alternatives has become of utmost importance, and the industry, as well as the regulatory authorities, are realizing the potential of antimicrobial peptides. Since the last decade, antimicrobial peptides have been trialed in clinical phases [1].

In drug development, peptides' properties have been considered more convenient due to their high affinity for the target and selective biological activities [2]. Bacterial resistance is a global health problem due to the indiscriminate use of antibiotics in humans, as well as in animals and in agricultural production that needs multipronged solutions [3]. To

**Citation:** Oguiura, N.; Corrêa, P.G.; Rosmino, I.L.; de Souza, A.O.; Pasqualoto, K.F.M. Antimicrobial Activity of Snake β-Defensins and Derived Peptides. *Toxins* **2022**, *14*, 1. https://doi.org/10.3390/toxins 14010001

Received: 15 October 2021 Accepted: 29 November 2021 Published: 21 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

contribute to this campaign, many antimicrobial peptides have been discovered and some are in the clinical development phase [4]. For instance, molecules from innate immunity, such as cathelicidins present in snake venoms, have been reported as potentially active against some bacterial strains [5–10], including antibiofilm activity [11,12].

Snakes have developed many active peptides for predation and in their venom composition, many classes of proteins as phospholipases A2 (PLA2) [13], L-amino-acid oxidase (LAAO) [14], metalloproteases [15], cathelicidins [8] and crotamine [16–18] are also described as showing antimicrobial activity. Many attempts have been made to shorten these proteins and discover the active site [19–22]. Crotamine is a small basic myotoxin present in the venom of the rattlesnake *C. durissus terrificus* and has a β-defensin structure [23,24] and antimicrobial activity [16–18]. Crotamine and small basic myotoxins from this family are unique and only present in rattlesnake venom [23].

β-defensins from various vertebrates have been studied, and little is known about these molecules and the innate immunity in snakes. β-defensin-like genes with unknown functions have also been described in Brazilian snakes using a PCR approach [25–27]. In the current study, we evaluated the antimicrobial activity of snake β-defensins, both from rattlesnake venom (crotamine) and non-venom β-defensins. Its primary sequences were deduced from genomic sequences and synthesized, except for crotamine purified from rattlesnake venom. Moreover, the amino acid sequences were used to design shorter derived peptides, which can be obtained using simpler and more economical procedures. The snake β-defensins and derived peptides were assayed against microorganisms relevant to the snake's biology and human health because they can cause opportunistic infections.
