Bacterial-Specific Aggregation and Killing of Immunomodulatory Host Defense Peptides

Abstract

This study involves the design and development of disulfide bridge-linked antimicrobial peptides using the host defense protein Angiogenin 4 (chAng4) as a template. The mini peptides derived from chAng4 (mCA4s) were evaluated for their antibacterial efficacies in various pathogenic bacterial strains, and the role of the oxidation state of thiols in the peptide sequence and its implication on antibacterial properties were explored. A remarkable property of these synthetic mCA4 peptides is their capability to flocculate bacteria and mediate bacterial-specific killing, in the absence of any other external stimulus. mCA4s were further evaluated for their cellular uptake, hemolytic activities, toxicities, and immunomodulatory activities in different eukaryotic cell lines. The results indicate that disulfide bridge-containing cationic amphipathic peptides show superior antibacterial efficacies, are nontoxic and nonhemolytic, and mediate bacterial flocculation and killing, in the absence of external stimuli.

Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping

“…Dab lactams exhibited greater antibacterial activity against both Gram-(+) and -(−) bacteria (Table ), lower aggregation propensity under physiological conditions, smaller nanoparticle sizes at higher concentrations, higher membrane permeability, and superior flocculation capacity (Figure , Figure S4). The flocculation propensity of AMPs is well documented to immobilize and trap bacteria, inhibiting growth and neutralizing endotoxins, , as seen in peptides rich in hydrophobic aromatic amino acids (Phe and Trp) which interact with lipid membranes. − Despite the similar hydrophobicities of the Dab lactams compared with other analogues, high CMC values of these peptides are attributed to the self-assembly behavior of peptides into nano- and macrostructures in aqueous solution that may be distinctly different than other analogues . Parent disulfides ( 1 and R1 ) and lactams ( 2 – 5 and R2 – R5 ) were tolerated by mammalian cells and nontoxic toward RAW 264.7 macrophages after 24 h treatment at 100 μM concentration.…”

“…Peptides were synthesized by solid-phase peptide synthesis (SPPS) using a Focus XC Peptide Synthesizer (AAPPTec) via Fmoc protection/deprotection chemistry, using rink amide resins, HBTU as an initiator, and DIPEA , according to previously established methods. , Lactamization was carried out on resin using HATU, instead of HBTU, as the initiator to prevent dimerization. Cyclization was achieved by HATU/DIPEA amide coupling between glutamate (Glu) residues and amine-containing Dap and Dab .…”

“…subtilis ATCC 6051 colonies were inoculated in NB media overnight. The confluent bacteria were centrifuged down and resuspended in PBS (pH = 7.4) to yield ∼8 × 10 8 bacterial cells per mL (OD 600 ∼ 0.8–1). , Bacteria were treated with peptide (2–50 μM) and incubated for 4 h. The OD 600 of the supernatant of the bacterial suspension was then measured.…”

“…In DMSO, mCA4-5 was shown by NMR and FTIR spectroscopy to exhibit a conformation with anti-parallel β-sheet and β-turn structure . In water above a critical concentration of 4 μM, mCA4-5 assembled to form nanoparticles of around 200–300 nm in size consisting of β-sheet structures, which induced flocculation, inhibited growth, and caused mortality of bacteria . The disulfide mCA4-5 stabilizes an active β-sheet structure, which loses antibacterial activity in the presence of reducing agents such as glutathione .…”

Identification of Crucial Amino Acid Residues for Antimicrobial Activity of Angiogenin 4 and Its Modulation of Gut Microbiota in Mice

“…For direct killing, selectivity of certain AMPs are dependent on the composition of the bacterial membrane which is composed of distinct lipid structure in species of bacteria ( Sohlenkamp and Geiger, 2015 ; Lei et al, 2021 ). On the other hand, AMPs exhibit immunomodulatory effects to clearance of bacteria or controlling inflammation through the recruitment and activation of innate immune cells such as neutrophils, monocytes, macrophage and dendritic cells, or proinflammatory cytokine suppression which could regulate gut microbiota ( Hancock and Sahl, 2006 ; Mansour et al, 2014 ; Nazeer et al, 2021a ). Thus, it is expected that Ang4 protein might interact with bacteria and host cells both to maintain intestinal bacteria composition.…”