Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Abstract

In most aerobic organisms hemoperoxidases play a major role in H(2)O(2)-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H(2)O(2). To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

Figure 1

Sequence analysis of T. cruzi APX. The sequence of TcAPX was aligned with other ascorbate-dependent peroxidases; each of the different subcellular isoforms in Arabidopsis thaliana and the pea cytosolic form. The residues that are common with the TcAPX sequence are represented by dots; dashes represent gaps in the sequence made to optimize the alignments. Differences between the sequences when compared with TcAPX are indicated. Amino acids implicated in the redox activity of APXs are noted in capital letters above each aligned block and d represents residues involved in electrostatic interactions between dimers in the pea cytosolic APX. The boxed region in TcAPX represents a hydrophobic domain and the three conserved segments that form the ascorbate-interacting surface are highlighted. The arrow corresponds to the primer used for expression of the recombinant protein (Materials and Methods). The sequences aligned are: 1, T. cruzi TcAPX (AJ457987). 2, A. thaliana stromal APX (CAA67425). 3, A. thaliana thylakoid APX (CAA67426). 4, A. thaliana cytosolic, soluble APX (Q05431). 5, A. thaliana membrane associated APX (CAA66640). 6, P. sativum cytosolic, soluble APX (P48534).

Figure 2

Investigating the ascorbate-dependent activity of TcAPX. (A) Fractions obtained during the purification of TcAPX were resolved on a 10% SDS/PAGE gel and visualized by Coomassie staining. A clarified fraction of an E. coli (pTrcHis-APX) cell lysate after 3 h isopropyl β-d-thiogalactoside induction (lane 1) was loaded onto a Ni-NTA column and the flowthrough collected (lane 2). The column was washed extensively with 10 mM imidazole (lanes 3 and 4). TcAPX eluted at 200 mM imidazole (lane 5). Markers show molecular mass in kilodaltons. The band of 30 kDa (indicated) corresponds to recombinant TcAPX. (B) TcAPX activity was assayed by following the oxidation of ascorbate (25–300 μM) in the presence TcAPX (0.1 μM) and H₂O₂ (0–10 μM). At each ascorbate concentration, the app V_max was calculated and then examined further by secondary plot analysis. (C) TcAPX activity was assayed by following the oxidation of ascorbate [25 (●), 50 (○), 100 (▾), and 300 (▿) μM] in the presence TcAPX (0.1 μM) and H₂O₂ (0–10 μM). All assays were initiated by the addition of the H₂O₂. TcAPX activities are expressed as nanomoles of ascorbate oxidised per minute per milligram, while [ascorbate] and [H₂O₂] are expressed in micromolar. The postulated scheme for the metabolism of H₂O₂ in T. cruzi by an ascorbate peroxidase pathway is shown. Trypanothione disulfide (TS₂) is reduced to dihydrotrypanothione (T[SH]₂) by the NADPH-dependent TR. TcAPX can reduce H₂O₂ to H₂O by using T[SH]₂ as electron donor in the presence of ascorbate.

Figure 3

TcAPX is a hemoperoxidase. (A) The absorption spectra from 400 to 620 nm are shown for 10 μΜ TcAPX in 50 mM sodium phosphate buffer (pH 6.5), 30 μM ascorbate before (a) and after (b) the addition of 100 μM H₂O₂. The shift in the Soret peak from 413 to 421 nm is indicated. The inhibition of TcAPX (2 μM) activity was examined in the presence of 0 (●), 10 (○), and 100 (▾) μM H₂O₂ (B) or 0 (●), 100 (○), and 1000 (▾) μM sodium azide (C) in 50 mM sodium phosphate buffer (pH 6.5). At time intervals 50-μl samples were assayed for APX activity in the presence of 100 μM ascorbate and 20 μM H₂O₂. The data are expressed as a percentage of activity compared with the initial activity.

Figure 4

Immunolocalization of TcAPX to the ER in T. cruzi epimastigotes. (A) Specificity of the c-myc (9E10) antiserum was examined by probing a blot containing 10 μg of cell lysates from T. cruzi wild-type (lane 1) and T. cruzi expressing epitope-tagged TcAPX (lane 2). Protein loading was judged by amido black staining. An arrow indicates the c-myc (9E10)-tagged TcAPX (33 kDa). (B) T. cruzi epimastigote cells expressing epitope-tagged TcAPX were stained with TOTO-3 (blue; 1), anti-c-myc (9E10; green; 2), and anti-BiP (red; 3) and the pattern of TcAPX and BiP colocalization observed (yellow; 4). A phase image of the cell (5) was obtained, on which all images were superimposed (6).

Figure 5

Susceptibility of pTEX-APX-transformed and wild-type T. cruzi epimastigotes to H₂O₂. Wild-type (shaded symbols) and TcAPX-overexpressing (open symbols) parasites were grown in the presence of different concentrations of H₂O₂ and the cell numbers determined (Materials and Methods). The concentration of H₂O₂ that inhibited parasite growth by 50% (IC₅₀) was established. The differences observed in sensitivity toward H₂O₂ in the wild-type and transformed cell lines were statistically significant (P < 0.01), as assessed by Student's t test.