Aquiluscidin, a Cathelicidin from Crotalus aquilus, and the Vcn-23 Derivative Peptide, Have Anti-Microbial Activity against Gram-Negative and Gram-Positive Bacteria

Abstract

Anti-microbial peptides play a vital role in the defense mechanisms of various organisms performing functions that range from the elimination of microorganisms, through diverse mechanisms, to the modulation of the immune response, providing protection to the host. Among these peptides, cathelicidins, a well-studied family of anti-microbial peptides, are found in various animal species, including reptiles. Due to the rise in anti-microbial resistance, these compounds have been suggested as potential candidates for developing new drugs. In this study, we identified and characterized a cathelicidin-like peptide called Aquiluscidin (Aq-CATH) from transcripts obtained from the skin and oral mucosa of the Querétaro's dark rattlesnake, Crotalus aquilus. The cDNA was cloned, sequenced, and yielded a 566-base-pair sequence. Using bioinformatics, we predicted that the peptide precursor contains a signal peptide, a 101-amino-acid conserved cathelin domain, an anionic region, and a 34-amino-acid mature peptide in the C-terminal region. Aq-CATH and a derived 23-amino-acid peptide (Vcn-23) were synthesized, and their anti-microbial activity was evaluated against various species of bacteria in in vitro assays. The minimal inhibitory concentrations against bacteria ranged from 2 to 8 μg/mL for both peptides. Furthermore, at concentrations of up to 50 μM, they exhibited no significant hemolytic activity (<2.3% and <1.2% for Aquiluscidin and Vcn-23, respectively) against rat erythrocytes and displayed no significant cytotoxic activity at low concentrations (>65% cell viability at 25 µM). Finally, this study represents the first identification of an antimicrobial peptide in Crotalus aquilus, which belongs to the cathelicidin family and exhibits the characteristic features of these peptides. Both Aq-CATH and its derived molecule, Vcn-23, displayed remarkable inhibitory activity against all tested bacteria, highlighting their potential as promising candidates for further antimicrobial research.

Keywords: Crotalus aquilus; Gram-negative bacteria; Gram-positive bacteria; anti-microbial peptide; cathelicidin.

Figure 1

(A) Identification of the open reading frame in the Translate server. The nucleotide sequence is shown above the amino acid sequence, indicating the codons corresponding to each amino acid; the dash represents the stop codon. (B) The result of the domain prediction by the SMART server, showing the one belonging to the cathelicidin family (Pfam).

Figure 2

Alignment of the cathelin domain of cathelicidins identified in different snake species obtained in Clustal Omega and visualized in MView. Coverage (cov) and identity (pid) percentages are indicated in comparison to Aquiluscidin. Amino acids are shown in colors.

Figure 3

α-helix conformation predicted for C. aquilus cathelicidin on the HeliQuest server. Basic amino acids are shown in blue, nonpolar amino acids in yellow, and polar amino acids in purple; the arrow indicates the hydrophobic side of the helix. * Determined in Peptide Property Calculator.

Figure 4

Result of the cytotoxicity assay of cathelicidin peptides in HEK293 cells, showing the different concentrations used in µM range and the percentage of viable cells corresponding to each treatment. The mean and bars corresponding to the standard error of triplicates are shown.

Figure 5

Hemolytic activity of peptides with rat erythrocytes. Different concentrations (µM) were evaluated. The black line corresponds to Vcn-23; the red line to Aquiluscidin. The mean and bars corresponding to the standard error of triplicates are shown.