Starynets Kateryna, Paunkov Ana, Wagner Anja, Kratochwill Klaus, Klotz Christian, Leitsch David
Institute for Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Kinderspitalgasse 15, A-1090, Vienna, Austria.
Core Facility Proteomics, Medical University of Vienna, A-1090, Vienna, Austria.
Int J Parasitol Drugs Drug Resist. 2025 Apr;27:100585. doi: 10.1016/j.ijpddr.2025.100585. Epub 2025 Feb 1.
The microaerophilic/anaerobic protist Giardia lamblia is a world-wide occurring parasite of the human small intestine. It causes giardiasis which manifests as diarrhoea accompanied by other sequelae. Giardiasis is most commonly treated with either the 5-nitroimidazole metronidazole or the benzimidazole albendazole. Unfortunately, the number of refractory cases is increasing, which is probably caused, at least in part, by drug resistance. However, most attempts to isolate metronidazole-resistant G. lamblia strains from patients have failed so far because the parasites were not resistant when tested in vitro. We hypothesized that this failure might be caused by drug assay conditions which are standardly anaerobic, and performed metronidazole susceptibility testing with two well studied strains, i.e. WB C6 and BRIS/87/HEPU/713 (strain 713) under microaerophilic conditions. Indeed, 713 proved to be less susceptible to metronidazole under microaerophilic conditions as compared to anaerobic conditions, and residual growth was even noted at concentrations of metronidazole similar to those in the serum of treated patients (i.e. about 100 μM). Further experiments showed that 713 also grows much faster under microaerobic conditions than WB C6. Reduced susceptibility to metronidazole under microaerobic conditions was also observed in a clinical isolate from a refractory giardiasis case. Two-dimensional gel electrophoresis showed that microaerobic growth was accompanied by the upregulation of superoxide reductase, a pyridoxamine 5'-phosphate oxidase putative domain-containing protein, and a TlpA-like protein in 713 but not in WB C6. All three proteins are known, or can be predicted to have antioxidant functions. Indeed, overexpression of pyridoxamine 5'-phosphate oxidase in WB C6 from a plasmid carrying the respective gene behind the arginine deiminase promoter significantly improved growth of the transfected cell line under microaerobic conditions. Moreover, similarly overexpressed superoxide reductase conferred significant protection against metronidazole. Our results suggest that oxygen concentrations can affect the outcomes of metronidazole treatment against G. lamblia.
微需氧/厌氧原生生物蓝氏贾第鞭毛虫是一种在全球范围内寄生于人类小肠的寄生虫。它会引发贾第虫病，表现为腹泻并伴有其他后遗症。贾第虫病最常用5-硝基咪唑类的甲硝唑或苯并咪唑类的阿苯达唑进行治疗。不幸的是，难治性病例的数量正在增加，这可能至少部分是由耐药性引起的。然而，到目前为止，大多数从患者中分离耐甲硝唑蓝氏贾第鞭毛虫菌株的尝试都失败了，因为这些寄生虫在体外测试时并不耐药。我们推测这种失败可能是由通常为厌氧的药物检测条件导致的，并在微需氧条件下对两个经过充分研究的菌株，即WB C6和BRIS/87/HEPU/713（713菌株）进行了甲硝唑敏感性测试。事实上，与厌氧条件相比，713在微需氧条件下对甲硝唑的敏感性较低，并且在与接受治疗患者血清中相似浓度的甲硝唑（即约100μM）下甚至观察到有残余生长。进一步的实验表明，713在微需氧条件下的生长速度也比WB C6快得多。在一例难治性贾第虫病病例的临床分离株中也观察到在微需氧条件下对甲硝唑的敏感性降低。二维凝胶电泳显示，微需氧生长伴随着713中超氧化物还原酶、一种含有磷酸吡哆醛5'-磷酸氧化酶假定结构域的蛋白质和一种类TlpA蛋白的上调，而WB C6中则没有。这三种蛋白质都已知或可预测具有抗氧化功能。实际上，在精氨酸脱亚氨酶启动子后面携带相应基因的质粒使WB C6中的磷酸吡哆醛5'-磷酸氧化酶过表达，显著改善了转染细胞系在微需氧条件下的生长。此外，类似地过表达的超氧化物还原酶赋予了对甲硝唑的显著保护作用。我们的结果表明，氧浓度会影响甲硝唑治疗蓝氏贾第鞭毛虫的效果。
