A Novel Proline-Rich Cathelicidin from the Alpaca Vicugna pacos with Potency to Combat Antibiotic-Resistant Bacteria: Mechanism of Action and the Functional Role of the C-Terminal Region

Abstract

Over recent years, a growing number of bacterial species have become resistant to clinically relevant antibiotics. Proline-rich antimicrobial peptides (PrAMPs) having a potent antimicrobial activity and a negligible toxicity toward mammalian cells attract attention as new templates for the development of antibiotic drugs. Here, we mined genomes of all living Camelidae species and found a novel family of Bac7-like proline-rich cathelicidins which inhibited bacterial protein synthesis. The N-terminal region of a novel peptide from the alpaca Vicugna pacos named VicBac is responsible for inhibition of bacterial protein synthesis with an IC50 value of 0.5 µM in the E. coli cell-free system whereas the C-terminal region allows the peptide to penetrate bacterial membranes effectively. We also found that the full-length VicBac did not induce bacterial resistance after a two-week selection experiment, unlike the N-terminal truncated analog, which depended on the SbmA transport system. Both pro- and anti-inflammatory action of VicBac and its N-terminal truncated variant on various human cell types was found by multiplex immunoassay. The presence of the C-terminal tail in the natural VicBac does not provide for specific immune-modulatory effects in vitro but enhances the observed impact compared with the truncated analog. The pronounced antibacterial activity of VicBac, along with its moderate adverse effects on mammalian cells, make this molecule a promising scaffold for the development of peptide antibiotics.

Genomic Insights into Bacterial Resistance to Proline-Rich Antimicrobial Peptide Bac7

“…Similar to our previous experiments with other short PrAMPs such as VicBac 1-22 and a panel of mini-ChBac7.5Nα analogs [3,15,26], ≥32-fold increases in MIC values were registered just after initial 3-4 passages subjected to selection by Bac7 1-22 . The obtained Bac7 1-22 -resistant strains were named as B1 and B2.…”

“…Thioredoxin (Trx) was used as the fusion partner to ensure high yield of the peptide in the native conformation. The gene encoding pexiganan was obtained by annealing of two primers Pex-f and Pex-r (Table S1) followed by one-round DNA-polymerase extension and then cloned into pET-based vector as described previously [15]. The oligonucleotides used in this work were designed on the basis of E. coli K-12 codon usage bias.…”

“…The selection of resistant strains makes it possible to elucidate both the key targets of AMPs and mechanisms of the resistance development. Here, we used the MDR uropathogenic E. coli 1057 strain isolated from a patient with UTI and sequenced in our previous study [15]. This strain bears the plasmid-mediated acquired resistance to betalactams (extended spectrum beta-lactamase (ESBL) producer, blaCTX-M-15), macrolides, and sulfamethoxazole (Figure S2).…”

γ-Core Guided Antibiotic Design Based on Human Enteric Defensin 5

“…It is known that AMPs may inactivate microbes through an intracellular mechanism in addition to membrane disruption. For instance, proline-rich AMPs can kill bacteria by binding to the ribosome and disrupting protein synthesis [ 45 , 46 ], and polyphemusin-I, a β-hairpin AMP from horseshoe crabs, may target bacterial nucleic acid-associated proteins [ 47 ]. We previously also found that HD5d5, a flexible derivative of HD5, could translocate into the cytoplasm of MDRAB and attenuate the activities of superoxide dismutase and catalase, causing a lethal accumulation of reactive oxygen species [ 15 ].…”

Designing double-site lipidated peptide amphiphiles as potent antimicrobial biomaterials to combat multidrug-resistant bacteria

“…AMPs are small molecular active peptides and have been widely isolated and characterized from animals, plants, bacteria, fungi, protists, and archaea (Wang et al, 2016). Different AMPs show different biological functions, such as antibacterial (Shao et al, 2021;Panteleev et al, 2022;Wang et al, 2022), antibiofilm (Zai et al, 2021), antivirals (Ji et al, 2018), antifungal (Chou et al, 2021), anticancer and immunomodulatory (Lugo et al, 2019;Mookherjee et al, 2020). More than 22,400 AMPs have been isolated from nature or created synthetically in the lab as of this writing (Shi et al, 2022).…”