Disrupted intracellular redox balance of the diplomonad fish parasite Spironucleus vortens by 5-nitroimidazoles and garlic-derived compounds.
The 5-nitroimidazole, metronidazole, has traditionally been employed in veterinary medicine to treat a range of infections including the diplomonad fish parasite Spironucleus. This study aims to determine the mode of action of metronidazole on Spironucleus vortens, including the specific mechanism of activation of the pro-drug and subsequent cellular targets of the drug metabolites. Due to the ban on use of metronidazole in the treatment of production animals in Europe and USA, garlic-derived compounds were also investigated as natural alternatives to metronidazole chemotherapy. Scanning electron microscopy (SEM) provided an overview of gross cellular damage caused by metronidazole and garlic derivatives. Proteomic analyses by 2D gel electrophoresis identified the proteins involved in specific covalent adduct formation with nitroimidazoles. Furthermore, thioredoxin reductase (TrxR) activity and non-protein thiol concentration were assayed in extracts of S. vortens before and after treatment with nitroimidazoles and garlic-derivatives. Metronidazole and garlic-derived compounds caused severe damage of trophozoites indicated by membrane blebbing and lysed cell debris. Analysis of the S. vortens proteome identified several proteins capable of specific nitroimidazole binding, including; uridine phosphorylase, enolase, protein disulphide isomerase, aminoacyl-histidine dipeptidase and malic enzyme. Of the compounds tested, metronidazole and the garlic-derived compound ajoene were the most effective at inhibiting TrxR activity and depleting non-protein thiols. These data suggest TrxR-mediated activation of nitroimidazoles, leading to depletion of non-protein thiols. Redox imbalance due to antioxidant failure is implicated as the mode of action of nitroimidazoles and garlic-derived compounds, ultimately leading to cell death. Possible synergy between garlic derivatives and metronidazole should be further investigated in vitro in order to determine their theoretical implications.
School of Biosciences, Cardiff University, Cardiff CF10 3AX, Wales, UK. williamscf@cf.ac.uk
C F
CF
Williams
D
D
Lloyd
D
D
Kolarich
K
K
Alagesan
M
M
Duchêne
J
J
Cable
D
D
Williams
D
D
Leitsch
D000818:Animals; D000994:Antitrichomonal Agents / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D016828:Diplomonadida / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D004220:Disulfides / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D005393:Fish Diseases / Q000188:drug therapy / Q000469:parasitology*; D005399:Fishes; D005737:Garlic / Q000737:chemistry*; D008795:Metronidazole / Q000494:pharmacology*; D008855:Microscopy, Electron, Scanning; D040901:Proteomics; D011529:Protozoan Infections, Animal / Q000188:drug therapy / Q000469:parasitology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013441:Sulfinic Acids / Q000494:pharmacology; D013454:Sulfoxides; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013879:Thioredoxins / Q000032:analysis; D014011:Tinidazole / Q000494:pharmacology; D053058:Trophozoites
D000994:Antitrichomonal Agents; D004220:Disulfides; D013438:Sulfhydryl Compounds; D013441:Sulfinic Acids; D013454:Sulfoxides; D014011:Tinidazole; D008795:Metronidazole; C006452:allicin; D013879:Thioredoxins; C048980:ajoene; D013880:Thioredoxin-Disulfide Reductase