Comparative proteomic analysis of metronidazole-sensitive and resistant Trichomonas vaginalis suggests a novel mode of metronidazole action and resistance

Abstract

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.

Graphical abstract

Fig. 1

Strategy of the proteomic analysis. In two independent analyses the proteome profiles of control T. vaginalis C1, 9 × 50 μM bipyridyl-treated C1, and highly in vitro resistant C1 were compared. Only proteins that were found to be differentially up- or downregulated twice in resistant C1 but not bipyridyl-treated C1 were matched to the proteins differentially expressed in highly in vitro resistant T1 vs. control T1. The resulting subset of proteins (highlighted in yellow) was considered as specific for in vitro metronidazole resistance in T. vaginalis (Table 4). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Ferric iron reductase activity of FR1. FR1 activity was measured by determining the formation of ferrous iron (Fe²⁺) from ferric iron (Fe³⁺) through the quantification of complex formation of Fe²⁺ with bipyridyl. Iron-bipyridyl complexes absorb strongly at λ₅₂₂. The absorption values of known concentrations (10–60 μM) of ferrous iron sulphate with bipyridyl were used to make a calibration curve (A). Subsequently, the absorption values obtained with FR1 in the presence of 50 μM or 100 μM ferric iron chloride (FeCl₃) were matched against the calibration curve (B).

Fig. 3

Damage to proteins in T. vaginalis C1 cell extracts. Cell extracts were incubated at RT for 30 min with NADPH (4 mM), FMN (20 μM), and 500 μM FeSO₄ either in absence (A) or presence (B) of 1 mM metronidazole. Afterwards, proteins were precipitated with TCA and resolubilized in 2DE buffer to perform 2D gel electrophoresis. The gels were Coomassie-stained and the damage to proteins assessed by comparing the protein profiles (pH range 5–8). When cells had been preincubated with 10 μM diphenyleneiodonium (DPI) prior to preparation of the cell extract, the damage inflicted by metronidazole was greatly reduced (C). When iron was omitted and 300 μM deferoxamine (DFO) was added to cell extracts the damage inflicted by metronidazole was minimal (D). The central sections of the 2D gels are shown (approximately 30–60 kDa range; pH range approx. 5.5 to 6.5). For orientation, three proteins identified in an earlier study (Leitsch et al., 2009, 2012) are indicated: 1, thioredoxin reductase (Uniprot ID: A0A8U0WQ27); 2, cytosolic malate dehydrogenase (Uniprot ID: Q27819); 3, enolase (Uniprot ID: A2E269). The respective sizes are given in brackets. The respective entire gel images are shown in Supplementary Fig. 1.

Fig. 4

No damage to proteins can be observed when using cell extracts of metronidazole-resistant T. vaginalis B7268. Cell extracts of B7268 were prepared as described for C1 (Fig. 3) and incubated either in absence (A) or presence (B) of 1 mM metronidazole. No damage to proteins could be observed on 2D gels. The central sections of the 2D gels are shown (approximately 30–60 kDa range; pH rage approx. 5.5 to 6.5). For orientation, three proteins identified in an earlier study (Leitsch et al., 2009, 2012) are indicated: 1, thioredoxin reductase (Uniprot ID: A0A8U0WQ27); 2, cytosolic malate dehydrogenase (Uniprot ID: Q27819); 3, enolase (Uniprot ID: A2E269). The respective sizes are given in brackets. The respective entire gel images are shown in Supplementary Fig. 1.

Fig. 5

Novel model of metronidazole activation and resistance. In normal T. vaginalis cells FR1 reduces ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) [1] which then binds to cysteines of cytoplasmic proteins to form iron-cysteine complexes [2], constituting the labile iron pool. When metronidazole enters the cells [3] it forms a triple complex with these complexes leading to the formation of metronidazole radicals upon their resolution (Willson and Searle, 1975). The metronidazole radicals react with the nearby protein leading to the formation of adducts and to breaks in the peptide backbone [4], thereby destroying the proteins. In metronidazole-resistant cells, FR1 is not, or only weakly active and levels of ferrous iron are low. Consequently, the cysteines in cytoplasmic proteins are not bound by ferrous iron and not primed for attack by metronidazole. Created with BioRender.com.