Figure 1.
2DE of E. histolytica Cell Extracts Reveals Adduct Formation upon Metronidazole Treatment
(A) The adducts formed can be identified as shifts to more basic pI values on 2D gels. Cells were treated with 50 μM metronidazole for 2 h or left untreated. Isoelectric focusing was performed between pH 5 and pH 8; SDS PAGE was run on 12.5% polyacrylamide gels. The proteins are marked on a section of the 2D gel of the untreated sample (upper gel image) and of the treated sample (lower gel image).
Protein 1: metronidazole target protein 1 (two shifts); protein 2: superoxide dismutase (one shift); protein 3: purine nucleoside phosphorylase (one shift); protein 4: thioredoxin reductase (two shifts); and protein 5: thioredoxin (one shift). Unmodified proteins are marked by blue dotted circles, and shifted proteins are indicated by red circles.
(B) The same proteins in a close-up: unmodified proteins are encircled, and the respective modified isoforms are indicated by arrows.
Table 1.
List of the Proteins Found to Be Shifted to Higher pI on 2D Gels upon Metronidazole Treatment of E. histolytica
Figure 2.
Different Nitroimidazoles Shift the Same Target Proteins to a Different Extent
(A) The nitroimidazoles used in this study are shown: the 5-nitroimidazoles metronidazole (1), tinidazole (2), and ornidazole (3), and the 2-nitroimidazole azomycin (4). Different side chains result in different contributions of the compounds to the pI shifts on 2D gels.
(B) Adduct formation of the same proteins with different nitroimidazoles. The same proteins were shifted with the 5-nitroimidazoles metronidazole, ornidazole, and tinidiazole, and with the 2-nitroimidazole azomycin. The width of the shifts in pI depended on the total charge of the nitroimidazole used, which is determined by its side chains. Shifts are exemplified by two of the proteins found: superoxide dismutase and metronidazole target protein 1. The unmodified proteins are encircled, and shifted proteins are indicated by arrows.
Figure 3.
Interdependence of Non-Protein Thiols and Metronidazole Toxicity
(A) Metronidazole diminishes non-protein thiol levels in the cell. Determination of total non-protein sulfhydryl groups (fmol/cell), i.e., free thiols, in the cell after treatment of E. histolytica cells for 2 h with either additional 50 mM cysteine (67 mM in total) or 50 μM metronidazole alone, or both in combination. The experiment was independently performed three times. Vertical bars indicate standard deviations.
(B) The decrease of non-protein thiols is most pronounced under anaerobic conditions when treating cells with metronidazole and azomycin. Cells were either treated with 50 μM of metronidazole (left) or azomycin (right) for 2 h under anaerobic (0% O2 and 18% CO2), microaerophilic (5% O2 and 8% CO2), and aerobic (21% O2 and 0.4% CO2) conditions. Values are given as percentages of non-protein thiol concentrations of the respective untreated samples.
(C) Cysteine counteracts metronidazole toxicity. E. histolytica cells were treated with either 30 μM or 50 μM of metronidazole in the presence or absence of additional 50 mM cysteine for 20 h. Viability of cells was determined by trypan blue exclusion. The experiment was independently repeated three times. Numbers of viable cells are given as percentages of total cell counts. Vertical bars indicate standard deviations.
(D) Cysteine reduces adduct formation of proteins with metronidazole. Cells were treated with 50 μM of metronidazole either in the presence or absence of additional 50 mM cysteine for 2 h. Afterwards, cell lysates were prepared, and the extent of adduct formation of superoxide dismutase (1), thioredoxin reductase (2), thioredoxin (3), metronidazole target protein 1 (4), and purine nucleoside phosphorylase (5) with metronidazole was visualized by 2DE. Unmodified proteins are encircled, and shifted proteins are indicated by arrows.
Figure 4.
RecEh Trx Increases in Mass upon Modification with Metronidazole or Tinidazole
(A) Detection of mass increment by LC-ESI-QTOF-MS. Total protein mass was determined using LC-ESI-QTOF-MS after His-Tag purification of the protein. When BL21 (DE3) cells were not exposed to metronidazole or tinidazole during expression of recEh Trx, a single peak in the deconvoluted mass spectrum was observed. This peak was in good agreement with the expected mass of 12,322 Da when taking into account that methionine at position 1 is removed, six histidines are attached at the C-terminus, and one cysteine-disulfide bridge is formed. When BL21 (DE3) cells were exposed to metronidazole or tinidazole during recombinant protein expression, recEh Trx modified with metronidazole or tinidazole eluted slightly earlier on the LC than unmodified recEh Trx. The determined mass increments, i.e., 141 Da with metronidazole treatment and 217 Da with tinidazole, support the reaction scheme for 5-nitroimidazoles as proposed by Wislocki and colleagues [33]. Additional peaks in the spectra of modified recEh Trx correspond to mass increments of approximately 16 Da or 32 Da, and can be attributed to bound oxygen (a single oxygen or two).
(B) Model of adduct formation of 5-nitroimidazoles with sulfhydryl groups. After the uptake of 5-nitroimidazoles into the cell (1), the nitro group is reduced to form a nitro radical anion (2). According to the model of Wislocki and colleagues [33], the nitro radical anion is further reduced (−16 Da) to a highly reactive 5-nitrosoimidazole (3) which forms adducts (−2 Da) with sulfhydryl groups via the C4 of the imidazole ring (4). Adduct formation is accompanied by reduction (2 e−) of the nitroso group to a hydroxylamine group (+2 Da). Subsequently (5), the hydroxylamine group is further reduced (2 e−) to an amino group (−16 Da), resulting in the stable and non-reactive 5-aminoimidazole adduct. At physiological pH or under the conditions applied during MS, the amino group is protonated (+1 Da) (6). A total of six e− is necessary at the nitrogen of the former nitro group to obtain the aminoimidazole adduct which, compared to the corresponding nitroimidazole, decreases in mass by 30 Da. The bound protein or thiol loses one proton during adduct formation. Thus, in total, nitroimidazole adducts gain the molecular mass of the respective nitroimidazole less 31 Da.
Figure 5.
Metronidazole Binding Diminishes Thioredoxin Reductase Activity of recEh TrxR
After expression of recEH TrxR and recEH Trx in the presence or absence of metronidazole for 3 h, proteins were isolated and thioredoxin reductase activity of recEh TrxR was determined by reduction of DTNB via recEh Trx as measured at λ = 412 nm (OD412). Diamonds (♦) indicate the use of unmodified forms of both recEh TrxR and recEh Trx, crosses (x) the use of unmodified recEH TrxR and metronidazole-modified recEh Trx. Squares () indicate the use of metronidazole-modified recEh TrxR and unmodified recEh Trx, and triangles (▴) the use of modified forms of both recEh TrxR and recEh Trx.
Table 2.
Nitroreductase Activity of RecEh TrxR
Figure 6.
Model of Metronidazole Activation and Metronidazole Action in E. histolytica
After uptake by the cell, metronidazole is reduced by thioredoxin reductase (TrxR) (as shown in this study; black arrow), possibly by purine nucleoside phosphorylase (hypothesized in this study; dotted arrow) and, probably, by ferredoxin (shown to reduce metronidazole in T. vaginalis and G. intestinalis; interrupted arrow). After activation, metronidazole can develop its toxicity in a 2-fold way, either as a nitroradical anion or, if further reduced, as a reactive nitrosoimidazole (red arrows). The nitroradical anion can reduce O2 and thereby generate reactive oxygen species, which are highly detrimental to the microaerophilic E. histolytica cell. Alternatively, the nitrosoimidazole is generated, which forms adducts with non-protein thiols and/or proteins, resulting in the depletion of non-protein thiols and the modification of thioredoxin reductase (TrxR), thioredoxin (Trx), superoxide dismutase (SOD), metronidazole target protein 1 (Mtp1), and purine nucleoside phosphorylase (PNP). Presumably, these five proteins become targets of modification due to their close spatial proximity to the site of metronidazole activation, i.e., thioredoxin reductase. The formation of covalent metronidazole adducts with proteins involved in antioxidant defense is likely to render the cells more vulnerable to oxidative stress, thereby exacerbating metronidazole toxicity to E. histolytica in the presence of oxygen [43].