Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance

Abstract

The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

Figure 1

Flavin reductase activity in T. vaginalis gel extracts as visualized by NBT staining in native polyacrylamide gels (12.5% PAA). The lowest band, marked by an arrow, was missing or at least weaker in all metronidazole-resistant clinical isolates and the highly metronidazole-resistant C1 cell line, C1res. Fourty μg of protein were loaded per isolate. Flavin reductase activites as measured and published before (Leitsch et al., 2012) are given below the gels for comparison.

Figure 2

A, Activities of recombinantly expressed FR1-7. Activities in relation to FR1 are given below the bars in %. All measurements were performed with 1 μg ml^-1 enzyme and 50 μM FMN in 100 mM potassium phosphate buffer, pH 5.5. All measurements with FR1, FR5 and FR6 were performed at least three times. Error bars indicate SEM. B, Activities with of FR1, FR5, FR6 with 10 μM FMN. Activities in relation to FR1 are given below the bars in %. All measurements were performed with 1 μg ml^-1 enzyme in 100 mM potassium phosphate buffer, pH 5.5. All measurements were performed at least three times. Error bars indicate SEM.

Figure 2

A, Activities of recombinantly expressed FR1-7. Activities in relation to FR1 are given below the bars in %. All measurements were performed with 1 μg ml^-1 enzyme and 50 μM FMN in 100 mM potassium phosphate buffer, pH 5.5. All measurements with FR1, FR5 and FR6 were performed at least three times. Error bars indicate SEM. B, Activities with of FR1, FR5, FR6 with 10 μM FMN. Activities in relation to FR1 are given below the bars in %. All measurements were performed with 1 μg ml^-1 enzyme in 100 mM potassium phosphate buffer, pH 5.5. All measurements were performed at least three times. Error bars indicate SEM.

Figure 3

Twenty, 30, and 50 μM NADPH were oxidized by recombinant flavin reductase. Afterwards generated hydrogen peroxide was measured and compared to standards. Hydrogen peroxide is the only quantitative product of FR1, and NADPH is oxidized in amounts equimolar to the hydrogen peroxide generated. All measurements were performed three times. Error bars indicate SEM.

Figure 4

Flavin reductase activity in the fully metronidazole-suscpetible isolates G3 and C1 (black bars), the intermediately metronidazole-resistant isolate Fall River (dark grey bars), and the highly metronidazole-resistant isolates CDC085, LA1, B7268, and C1res (light grey bars) was measured at an FMN concentration of 100 μM or 10 μM, respectively. Percentages below the bars indicate FR activity in relation to FR activity in G3 extract with 100 μM FMN. Note that FR activity is approximately halved in G3, C1, and Fall River if 10 μM FMN are used instead of 100 μM. In CDC085 and LA1, FR activities are reduced to a third if 10 μM FMN instead of 100 μM are used, indicating a weaker affinity of the respective FR to FMN than observed in G3 and C1. B7268 and C1res do not display any FR activity. All measurements were performed at least three times with 20 μg ml^-1 (mg protein)^-1 extract and in 100 mM potassium phosphate buffer, pH 5.5. Error bars indicate SEM.

Figure 5

A, Anti-FR1 polyclonal antiserum (1:1000) was tested on 1 μg of purified recombinant FR1-7. All FRs cross-reacted with anti-FR1 antiserum. All FRs migrate faster in the gel than predicted by their nominal mass (approx. 27 kDa), FR1-3 were migrating faster in the gel than FR4-7. B, Anti-FR1 polyclonal antiserum was used to detect FRs in gel extracts of T. vaginalis (100 μg protein/lane). As shown in the left panel, C1res does not express any FR. In the right panel, the other strains were tested: 1, G3; 2, C1; 3, JH31A; 4, Tv2; 5, IR78; 6, Fall River; 7, CDC085; 8, LA1; 9, B7268.

Figure 5

A, Anti-FR1 polyclonal antiserum (1:1000) was tested on 1 μg of purified recombinant FR1-7. All FRs cross-reacted with anti-FR1 antiserum. All FRs migrate faster in the gel than predicted by their nominal mass (approx. 27 kDa), FR1-3 were migrating faster in the gel than FR4-7. B, Anti-FR1 polyclonal antiserum was used to detect FRs in gel extracts of T. vaginalis (100 μg protein/lane). As shown in the left panel, C1res does not express any FR. In the right panel, the other strains were tested: 1, G3; 2, C1; 3, JH31A; 4, Tv2; 5, IR78; 6, Fall River; 7, CDC085; 8, LA1; 9, B7268.

Figure 6

NADH-oxidase activity was measured in T. vaginalis G3 (metronidazole-sensitive), either untreated (-) or treated (+) with 200 μM metronidazole for two hours. Cells had been incubated in cysteine-free medium in order to guarantee elevated oxygen levels. For comparison, also flavin reductase activity was measured, either in absence (-) or presence (+) of metronidazole (200 μM, 2h). All measurements were repeated at least twice. Bars indicate SEM.

Figure 7

A, Western blot against T. vaginalis B7268 cell preparations with an anti-HA antibody (1:5000) which recognizes the HA-tag fused C-terminally to FR1 and FR4. Preparations of 1 × 10⁴ parasites each were loaded on 17.5% SDS PAGE gels. EV, empty vector. B, Summary of the metronidazole-susceptibility assays conducted with B7268 transfectants bearing the empty vector (EV, black circles), FR4 (blue squares), and FR1 (red triangles). Each shape represents one assay performed over 48 h. Seven independent experiments were performed for each of the transfectants. Each shape in the summary represents the minimum lethal concentration (MLC) determined for the transfectant in one of the experiments. The MLC was determined for each transfectant after 48 hours of incubation with a series of concentrations (0-256 μg/ml) of metronidzaole for each experiment. The range of MLCs is given alongside the column.

Figure 7

A, Western blot against T. vaginalis B7268 cell preparations with an anti-HA antibody (1:5000) which recognizes the HA-tag fused C-terminally to FR1 and FR4. Preparations of 1 × 10⁴ parasites each were loaded on 17.5% SDS PAGE gels. EV, empty vector. B, Summary of the metronidazole-susceptibility assays conducted with B7268 transfectants bearing the empty vector (EV, black circles), FR4 (blue squares), and FR1 (red triangles). Each shape represents one assay performed over 48 h. Seven independent experiments were performed for each of the transfectants. Each shape in the summary represents the minimum lethal concentration (MLC) determined for the transfectant in one of the experiments. The MLC was determined for each transfectant after 48 hours of incubation with a series of concentrations (0-256 μg/ml) of metronidzaole for each experiment. The range of MLCs is given alongside the column.