Mayr Anna-Lena, Paunkov Ana, Hummel Karin, Razzazi-Fazeli Ebrahim, Leitsch David
VetCore Facility for Research, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Kinderspitalgasse 15, 1090, Vienna, Austria.
Int J Parasitol Drugs Drug Resist. 2024 Dec;26:100566. doi: 10.1016/j.ijpddr.2024.100566. Epub 2024 Sep 26.
The microaerophilic parasite Trichomonas vaginalis occurs worldwide and causes inflammation of the urogenital tract, especially in women. With 156 million infections annually, trichomoniasis is the most prevalent non-viral sexually transmitted disease. Trichomoniasis is treated with 5-nitroimidazoles, especially metronidazole, which are prodrugs that have to be reduced at their nitro group to be activated. Resistance rates to metronidazole have remained comparably low, but they can be higher in certain areas leading to an increase of refractory cases. Metronidazole resistance in T. vaginalis can develop in vivo in clinical isolates, or it can be induced in the laboratory. Both types of resistance share certain characteristics but differ with regard to the dependence of ambient oxygen to become manifest. Although several candidate factors for metronidazole resistance have been described in the past, e.g. pyruvate:ferredoxin oxidoreductase and ferredoxin or thioredoxin reductase, open questions regarding their role in resistance have remained. In order to address these questions, we performed a proteomic study with metronidazole-sensitive and -resistant laboratory strains, as well as with clinical strains, in order to identify factors causative for resistance. The list of proteins consistently associated with resistance was surprisingly short. Resistant laboratory and clinical strains only shared the downregulation of flavin reductase 1 (FR1), an enzyme previously identified to be involved in resistance. Originally, FR1 was believed to be an oxygen scavenging enzyme, but here we identified it as a ferric iron reductase which produces ferrous iron. Based on this finding and on further experimental evidence as presented herein, we propose a novel mechanism of metronidazole activation which is based on ferrous iron binding to proteins, thereby rendering them susceptible to complex formation with metronidazole. Upon resolution of iron-protein-metronidazole complexes, metronidazole radicals are formed which quickly react with thiols or proteins in the direct vicinity, leading to breaks in the peptide backbone.
微需氧寄生虫阴道毛滴虫在全球范围内均有出现，可引发泌尿生殖道炎症，尤其是在女性中。滴虫病每年感染人数达1.56亿，是最常见的非病毒性传播疾病。滴虫病采用5-硝基咪唑类药物治疗，尤其是甲硝唑，这些前体药物必须在其硝基处被还原才能激活。甲硝唑的耐药率一直相对较低，但在某些地区可能会更高，导致难治性病例增加。阴道毛滴虫对甲硝唑的耐药性可在临床分离株体内产生，也可在实验室中诱导产生。这两种耐药类型具有某些共同特征，但在表现出耐药性对环境氧气的依赖性方面有所不同。尽管过去已经描述了几种甲硝唑耐药的候选因素，例如丙酮酸：铁氧化还原蛋白氧化还原酶、铁氧化还原蛋白或硫氧还蛋白还原酶，但关于它们在耐药性中的作用仍存在一些未解决的问题。为了解决这些问题，我们对甲硝唑敏感和耐药的实验室菌株以及临床菌株进行了蛋白质组学研究，以确定导致耐药的因素。与耐药性始终相关的蛋白质列表出人意料地短。耐药的实验室菌株和临床菌株仅共同表现出黄素还原酶1（FR1）的下调，FR1是一种先前已确定与耐药性有关的酶。最初，FR1被认为是一种清除氧气的酶，但在此我们将其鉴定为一种产生亚铁的铁还原酶。基于这一发现以及本文提供的进一步实验证据，我们提出了一种新的甲硝唑激活机制，该机制基于亚铁与蛋白质结合，从而使它们易于与甲硝唑形成复合物。当铁-蛋白质-甲硝唑复合物分解时，会形成甲硝唑自由基，这些自由基会迅速与附近的硫醇或蛋白质反应，导致肽主链断裂。
