Die Untersuchung von Allicin aus Knoblauch als Alternative zu konventionellen Antibiotika

• Investigation of allicin from garlic as an alternative to conventional antibiotics

Reiter, Jana; Slusarenko, Alan (Thesis advisor); Rink, Lothar (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020

Abstract

Allicin is an antimicrobial substance, produced by garlic tissue upon wounding as a defence against pathogens and pests. Allicin is a reactive sulfur species (RSS) that oxidizes accessible cysteines in proteins and the redox buffer glutathione (GSH), thus inhibiting essential enzymes, and if the concentration is high enough, causing cell death. The aim of this work was to investigate allicin’s potential for medical use as an antibiotic for the treatment of bacterial lung infections. A differential isotopic labelling method (OxICAT) was used to identify allicin targets in the bacterial proteome. The proteomes of allicin-susceptible Pseudomonas fluorescens Pf0 1 and allicin-resistant PfAR 1 were compared after sublethal allicin exposure. Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation. Protein oxidation increased after exposure to allicin, and only 54% of proteins from allicin-susceptible Pf0 1, but 66% from allicin-tolerant PfAR 1, remained less than 20% oxidised. DNA gyrase was one of the proteins oxidized by allicin. Because it is only found in prokaryotes, DNA gyrase is a popular candidate target for antibiotics. Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria, however, after allicin treatment the degree of Cys433 oxidation increased to 56% in sensitive Pf0 1 but only to 11% in resistant PfAR 1. Allicin inhibited E. coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid, the first described DNA gyrase inhibitor. Purified PfAR 1 DNA gyrase was inhibited to greater extent by allicin in vitro than the Pf0 1 enzyme. Substituting PfAR 1 GyrA into Pf0 1 rendered the exchange mutants more susceptible to allicin than the Pf0 1 wild type. Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-resistant PfAR 1 background, rather than the PfAR 1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0 1 GyrA subunit per se. The growth inhibitory effect of allicin to clinical isolates of lung pathogenic bacteria from the genera Acinetobacter, Klebsiella, Pseudomonas, Streptococcus, and Staphylococcus, including MDR (multiple drug resistant) strains, was demonstrated. Minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were determined and compared to clinical antibiotics using standard EUCAST (European Committee on Antimicrobial Susceptibility Testing) procedures. The cytotoxicity of allicin to human lung and colon epithelial and murine fibroblast cells was tested in vitro and shown to be ameliorated by glutathione (GSH). Similarly, the sensitivity of rat precision-cut lung slices (PCLS) to allicin was decreased by raising the [GSH] to the approximate blood plasma level of 1 mM. Because allicin inhibited bacterial growth (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pneumoniae and Haemophilus influenzae) as a vapour, it could be used to combat bacterial lung infections via direct inhalation. Since there are no volatile antibiotics available to treat pulmonary infections, allicin, particularly at sublethal doses in combination with oral antibiotics, could make a valuable addition to currently available treatments. For the simulation of the allicin treatment of infected lungs, a lung model was developed in cooperation with Institute of Aerodynamics (RWTH Aachen University). The lung model represents the human lung from the 2nd to the 5th bronchial generation. To simulate bacterial infection the inner model surface was covered with 1 mm thick bacteria-seeded agar layer. The deposition of antimicrobial aerosols on the modelled bronchial surfaces was followed in preliminary tests without the need for animal experiments. The differential sensitivity of the test bacteria to different antibiotics and the dose-dependency of inhibition was shown using the model. Furthermore, a synergistic effect of allicin vapour and ethanol in inhibiting bacterial growth was demonstrated. The modelling of the axial velocity air-flow distribution correlated with the regions showing inhibition of bacterial growth, demonstrating that the model has predictive value and can reduce the requirement for animal sacrifice in pre-clinical trials of novel antibiotics.