Identification of an Antimicrobial Peptide from the Venom of the Trinidad Thick-Tailed Scorpion Tityus trinitatis with Potent Activity against ESKAPE Pathogens and Clostridioides difficile

Abstract

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC₅₀ = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.

Keywords: Clostridioides difficile; ESKAPE pathogen; antimicrobial peptide; neurotoxin; scorpion venom.

Figure 1

Reversed-phase HPLC on a semipreparative Vydac C-18 column of venom from female T. trinitatis after partial purification on Sep-Pak C-18 cartridges. The dashed line shows the concentration of acetonitrile in the eluting solvent. The peaks denoted 1–3 contained peptides in major abundance with molecular masses in the range 5000 to 8000 Da. The peaks denoted 4–6 contained peptides with antimicrobial activity. The peptides in peaks 1–6 were purified further.

Figure 2

Purification to near homogeneity of (A) TtAP-1, (B) TtAP-2 and (C) Tt-AP3 on a semipreparative Vydac C-4 column. The triangles show where peak collection began and ended. * denotes the peak containing the antimicrobial peptide. The dashed line shows the concentration of acetonitrile in the eluting solvent.

Figure 3

Survival of A. baumannii (DSM 30008) (A) and S. aureus (ATCC 43300) (B) in Mueller–Hinton broth after the addition of 6.25 µg/mL TtAP-1 (equal to 1× MIC). Control incubations were carried out in the absence of peptide.

Figure 4

A comparison of the primary structures of (A) TtAP-1 with structurally similar peptides from other scorpion species and poneratoxin-Na1a from the ant N. apicalis, (B) TtAP-2 with peptides similar to TsAP-2 from other scorpion species and (C) TtAP-3 with peptides similar to stigmurin from other scorpion species. Conservative amino acid substitutions are shown in red and non-conservative substitutions in green. Gaps denoted by * have been inserted in some sequences to maximize structural similarity. The numbers in parentheses indicate the % identity with each of the T. trinitatis peptides, respectively.

Figure 5

A Schiffer–Edmundson wheel representation of the conformation of (A) TtAP-1, (B) TtAP-2 and (C) TtAP-3. Basic amino acids are shown in blue and strongly hydrophobic amino acids are shown in yellow. The other colours show non-charged residues with varying degrees of hydrophilicity.