Alkyl Hydroperoxide Reductase as a Determinant of Parasite Antiperoxide Response in Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that is widely parasitic in the nucleated cells of warm-blooded animals. Bioinformatic analysis of alkyl hydroperoxide reductase 1 (AHP1) of T. gondii is a member of the Prxs family and exhibits peroxidase activity. Cys166 was certified to be a key enzyme active site of TgAHP1, indicating that the enzyme follows a cysteine-dependent redox process. TgAHP1 was present in a punctate staining pattern anterior to the T. gondii nucleus. Oxidative stress experiments showed that the ∆Ahp1 strain was more sensitive to tert-butyl hydroperoxide (tBOOH) than hydrogen peroxide (H2O2), indicating that tBOOH may be a sensitive substrate for TgAHP1. Under tBOOH culture conditions, the ∆Ahp1 strain was significantly less invasive, proliferative, and pathogenic in mice. This was mainly due to the induction of tBOOH, which increased the level of reactive oxygen species in the parasites and eventually led to apoptosis. This study shows that TgAHP1 is a peroxisomes protein with cysteine-dependent peroxidase activity and sensitive to tBOOH.


Introduction
T. gondii is an obligate intracellular parasitic protozoan that infects a wide variety of warm-blooded animals [1], which are widely distributed across the world, both in terms of their geographical location and the diversity of their host range. Infection by this parasite causes congenital diseases and abortions in farm animals, which can lead to significant reproductive losses and consequent economic losses [2].
In most eukaryotes, oxygen is usually required for the participation of normal cellular physiological functions. This part of oxygen ions is partially reduced to superoxide anions and then reduced to other reactive oxygen species (ROS) [3], such as hydrogen peroxide and hydroxyl radicals [4]. In the early stages of parasite invasion, macrophages and natural killer cells mainly transmit reactive oxygen species (ROS) to eliminate T. gondii [5]. For the elimination of ROS, the path-ogen had a specific ROS detoxification mechanism. It has been detected that protozoa contain some major antioxidant enzymes, which are the main components of the enzymatic detoxification pathway and act downstream of superoxide dismutase (SOD) to prevent the formation of hydroxyl radicals by detoxifying H 2 O 2 . The low concentration of ROS participates in the cellular signal transduction process. However, superabundant ROS often induces oxidative stress and an unbalanced redox system. Oxidative damage causes cell damage and apoptosis and ultimately leads to tissue and organ damage and cell death [6].
Peroxiredoxins (Prxs), such as hydrogen peroxide and alkyl peroxides, are a ubiquitous family of antioxidant enzymes that protect cells from the erosion of reactive oxygen species and participate in a series of cell signaling pathways. Peroxiredoxin is a highly conserved thiol-dependent protein that detoxifies various reactive oxygen species (ROS) and is essential for the maintenance of cellular redox homeostasis [7]. Among the antioxidant proteins, Prx systems were the most recently discovered, involving nonheme peroxidases that catalyze the reduction of H 2 O 2 , peroxynitrite, and alkyl hydroperoxides via Trx and other thiol-containing reducing agents as electron donors. Unlike other peroxidases, the redox process of Prxs did not require the participation of cofactors, and it relied mainly on cysteine with redox activity to exert its effect [8]. At present, Prxs have been identified in almost all organisms, and their expression levels and catalytic rates are relatively high.
On the basis of structural similarity and sequence homology, Prxs have been divided into six subfamilies: AhpC/Prx1, Prx6, Prx5, Tpx, BCP/PrxQ, and AhpE [9]. Based on the catalytic process, Prxs have also been classified into three types: typical 2-Cys, atypical 2-Cys, and 1-Cys Prxs [10]. All types of Prxs relied on cysteine to exert their catalytic effect and referred to as cysteine peroxides (CP), which are usually located in the N-terminal region of Prxs. The catalytic process can be divided into three steps. In the first step, CP is directly responsible for attacking the residues of the oxide, releasing alcohol or water molecules; the second step involves a second cysteine with redox activity, called dissociated cysteine (CR), which is usually located in the C-terminal region, and CR is responsible for attacking the oxidized CP to form a CP-CR disulfide bond. Of course, 1-Cys Prxs lack this step due to the deletion of CR residues. The third step is the reduction and regeneration of oxidized Prxs by thiol-containing electron donors, such as thioredoxins (Trxs), glutathione, or glutathione transferase [11].
In this study, the enzyme active site of the TgAHP1 protein was identified, leading to the determination of alkyl hydroperoxide reductase (Ahp1), which belongs to the Prx5 subfamily and is a cysteine-dependent peroxidase. Our data indicated that TgAHP1 was more sensitive to the tBOOH, and the presence of AHP1 reduced the oxidative stimulation of tBOOH in parasites.

Ethical Statement.
The experiments using mice were approved by the Ethics Committee for Animal Experiments of the Laboratory Animal Center of Shandong Agricultural University, China.

Strain Types and Culture
Conditions. RHΔKu80 tachyzoites were cultured in DF-1 cells (a spontaneously immortalized chicken embryo fibroblast cell line) and Vero cells (African green monkey kidney cells) at 37°C and 5% CO 2 . Both DF-1 cells and Vero cells were stored in our laboratory.

Parasite Survival
Assays. For all assays, tachyzoites were pretreated with tBOOH (OUHE Technology) diluted to 10 -4 M with DMEM for 2 h. Parasite plaque, synchronous invasion, and replication assays were performed as described in previous report [12]. The tachyzoites were stained and observed by the immunofluorescence assay (IFA). The number of parasitophorous vacuoles (PV) and the number of host cells were counted in triplicates from three independent biological replicates, and the results were statistically analyzed to assess the invasion efficiency of different strains stimulated by different oxidants. The invasion efficiency was calculated by the number of infected cells/total host cells. One hundred vacuoles were selected, and the number of tachyzoites in each vacuole was counted to evaluate the proliferation efficiency. Real-time images were obtained through a microscope in three independent experiments, and two observers were responsible for statistics and calculation.

Mice Pathogenicity Test.
Seven-week-old female BALB/c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and bred under the conditions stipulated by the Shandong Provincial Laboratory Animal Affairs Administration. Freshly purified tachyzoites were injected intraperitoneally, and the survival and symptoms of mice were monitored daily.

Intracellular
Oxidative Activity Measurement. Intracellular reactive oxygen species (ROS) levels in T. gondii were determined using the probe 2′,7′-dichlorofluorescein diacetate (DCF-DA, Solarbio). RHΔKu80 and ΔAhp1 tachyzoites were collected, pretreated with tBOOH for different times, and then incubated with 10 μM DCF-DA at 37°C for 20 min. Tachyzoites were washed 3 times with PBS and detected with a fluorescence microplate reader. The excitation and emission wavelengths were 488 and 525 nm, respectively.

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Oxidative Medicine and Cellular Longevity 2.9. Determination of Apoptosis Level. The annexin V/propidium iodide (PI) method was used to detect the apoptosis rate of the parasites, and PI is responsible for staining the nucleus of apoptotic parasites. RHΔKu80 and ΔAhp1 tachyzoites were pretreated with tBOOH for different times and washed twice with PBS, and PI was added and then incubated for 10 min. Tachyzoites were observed using a laser scanning confocal microscope, with apoptosis rates calculated for different treatment groups.
2.10. Statistical Analysis. Prism 5 (GraphPad Software, Inc., CA, USA) was used for statistical comparison, while Student's t-test was used to carry out a two-way analysis of variance. Statistical data are expressed as the mean value of the standard error of the mean (SEM). P < 0:05 and P < 0:01 were considered statistically significant and extremely significant, respectively.

Results
3.1. Bioinformatic Analysis of T. gondii Ahp1. BLAST was performed in the T. gondii database based on the yeast AHP1 amino acid sequence to obtain an amino acid sequence with a lower E value, and the gene with the accession number TGGT1_286630 (ToxoDB database) was found. The Ahp1 gene of T. gondii contains a redoxin domain (Figure 1(a)) and is homologous to Hammondia hammondi and Neospora caninum through the analysis of a phylogenetic tree (Figure 1(b)). The sequence of TgAHP1 was compared with other species, and two conserved amino acid residues were found at the Cys 166 and Cys 191 sites (Figure 1(c)). Based on the sequence alignment results, the AHP1 of T. gondii is attributed to an alkyl hydroperoxide with peroxidase activity.

Ahp1 Localized in the Cytoplasm of T. gondii Tachyzoites.
To observe the expression of Ahp1 in T. gondii, 10 HA tags were fusion expressed in the C-terminal of TgAhp1 (Figures 2(a) and 2(b)). The localization of TgAHP1 in T. gondii was determined using an immunofluorescence assay, and the AHP1 protein was found to be presented primarily in a punctate staining pattern anterior to the parasite nucleus ( Figure 2(c)).   (Figure 3(d)). The CAT enzyme activities of these proteins were calculated (Figure 3(e)). The AHP1-Gly 191 -Arg protein still retained peroxidase activity, while the AHP1-Gly 166 -Arg and double mutation Gly 166 -Arg and Gly 191 -Arg proteins lost their enzymatic activities, indicating that Cys 166 is the key enzyme active site of the AHP1 protein in T. gondii.

An TgAHP1
Protein-Sensitive Peroxide of tBOOH. For screen-sensitive peroxides of T. gondii, the RHΔKu80 strain was treated with different concentrations of H 2 O 2 and tBOOH, respectively, and the invasion and proliferation assays were performed for RHΔKu80 strain. The statistical results showed that there was a significant difference in the invasion and proliferation of the strain after treatment with H 2 O 2 or tBOOH (Figures 4(a)-4(c)), indicating that the RHΔKu80 strain is more sensitive to peroxides. RHΔKu80 tachyzoites were treated with tert-butyl hydrogen peroxide (tBOOH) and hydrogen peroxide (H 2 O 2 ), and the expressions of the TgAhp1 gene were detected by RT-qPCR at dif-ferent time points. The transcription levels of TgAhp1 increased when treated with tBOOH and H 2 O 2 , but were higher than that of H 2 O 2 within 20 h of tBOOH stimulation, indicating that TgAHP1 is more sensitive to tBOOH. After 20 h, the gene expression trend to converge (Figure 4(d)), which may be due to the fact that the host cells and tachyzoites had degraded the peroxides.
3.5. Survival Efficiency of Ahp1 Knockout Strain. The Ahp1 gene of T. gondii was deleted using the CRISPR/Cas9 system ( Figure 5(a)), and ΔAhp1 was verified by Western blot (Figure 5(b)). The knockout of Ahp1 not obviously affected the invasion efficiency of T. gondii ( Figure 5(c)). Both the ΔAhp1 strain and the RHΔKu80 strain contained 4 or 8 parasites at 24 h for each parasitophorous vacuole, and there was no significant difference in statistical analysis ( Figure 5(d)), indicating that TgAhp1 is not essential for Toxoplasma's survival efficiency.
3.6. Survival Efficiency of Ahp1 Knockout Strain Treated with tBOOH. The RHΔKu80 and ΔAhp1 strains were treated with 10 -4 M tBOOH, respectively, and the invasion and proliferation assays were performed. The statistical results showed that the knocking-out Ahp1 gene weakened the invasion ability of T. gondii with 10 -4 M tBOOH treatment (Figure 6(a)). Under the stimulation of 10 -4 M tBOOH, the RHΔKu80 strain had significantly more octoploids than the ΔAhp1 strain ( Figure 6(b)), inferring that the RHΔKu80 strains has obvious proliferation. Plaque numbers and sizes were determined for 7 days. By statistical analysis, no significant difference could be observed in the size of the plaque in the untreated control group (Figure 6(c)). More plaques and a larger plaque area (Figures 6(d) and 6(e)) were formed by  Oxidative Medicine and Cellular Longevity the RHΔKu80 strain than ΔAhp1 strain after treatment with 10 -4 M tBOOH. No significant changes were found in the mice pathogenicity test between the ΔAhp1 and RHΔKu80 strains ( Figure 6(f)). However, tBOOH reduced the pathogenicity of ΔAhp1 strains in mice ( Figure 6(f)). These indicate that the presence of the Ahp1 gene weakens the oxidative stimulation effect of tBOOH on tachyzoites. Therefore, tBOOH may be related to the redox reaction of TgAHP1.

Induction of Excessive ROS and Apoptosis in Ahp1
Knockout Strains by tBOOH. Reactive oxygen levels were detected in parasites stimulated with tBOOH, and a more rapid increase in DCF was observed in the ΔAhp1 strain compared to the RHΔKu80 strain. Until 15 min of stimulation, the trend towards equilibrium (Figures 7(a) and 7(b)) revealed that reactive oxygen species accumulated more in the ΔAhp1 strain. The possibility of knocking-out the Ahp1 gene promotes the

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Oxidative Medicine and Cellular Longevity production of ROS in the parasites, which are related to the survival of the parasites in the host cells. Excessive ROS often induce oxidative stress in parasites, resulting in oxidative damage and possibly apoptosis. The tBOOH-induced tachyzoites of RHΔKu80 and ΔAhp1 strains were observed with a fluorescence microscope, and the apoptotic tachyzoites were stained red by PI (Figure 7(c)). The number of apoptotic tachyzoites was counted, and it was found that the apoptosis rate of ΔAhp1 tachyzoites stimulated with tBOOH was significantly higher than that of RHΔKu80 tachyzoites (Figures 7(d) and 7(e)), indicating that tBOOH tends to cause apoptosis in Ahp1 knockout strain.

Discussion
Several reports have shown that parasites such as T. gondii require an effective antioxidant system to maintain the critical balance between antioxidants and prooxidants for their survival in the host cell. The use of dichlorofluorescein diacetate (an endogenous oxidative stress detector) to verify that walnut wood ketone and phenazine methyl sulfate are potentially toxic to T. gondii without affecting host cells suggests that T. gondii is susceptible to oxidative damage caused by the breakdown of redox balance [13]. Through the phylogenetic analysis of peroxidases from different species, TgAhp1 was similar  Oxidative Medicine and Cellular Longevity to HHA_286630 of Harmon coccidia and NCLIV_014020 from Neospora, indicating that the peroxidase AHP1 not only exists in T. gondii but also in other protozoa. T. gondii possesses three types of peroxidases, including one type of 1-Cys peroxidase and two types of 2-Cys peroxidases [14]. Proteins of the Prx family reduced peroxides at the expense of their free cysteine residues, thereby forming disulfide bonds or peroxydisulfuric acid [15]. Studies have shown that disulfide bonds can be formed between the two cysteines of Ahp1, which play a corresponding role in the enzymatic activity. The amino acid sequence of TgAHP1 includes six cysteines, and multiple sequence alignments showed that the Cys 166 and Cys 191 sites are conserved with Prx5 subfamily proteins in several species of fungi and bacteria [16]. The results of our enzyme activity assay showed that no peroxidase activity was detected in the TgAHP1 protein when Cys 166 was mutated. However, the TgAhp1 protein still retained peroxidase activity even when Cys 191 was mutated, indicating that Cys 166 is one of the active sites of the enzyme, whereas Cys 191 is not. Therefore, the oxidation-reduction reaction occurring in TgAHP1 depends on the Cys 166 site; nevertheless, based on the present results, the redox process of TgAHP1 (relying on 1-Cys or 2-Cys) is still uncertain.
The reactive oxygen decomposition system is a powerful cytoplasmic antioxidant system in T. gondii, which helps the parasite to cope with the oxidative stress encountered in the intracellular parasitic life [17]. As a peroxidase, Ahp1 is a receptor for alkyl hydroperoxides (including lipid peroxides) and is responsible for resisting unsaturated lipids [16]. Saccharomycetes are sensitive to tert-butyl hydroperoxide with Ahp1 knocking out [18], and yeast Ahp1 is considered an important cytoplasmic thioredoxin-dependent alkyl hydroperoxide reductase, which is involved in oxidative stress [19]. Thess reports are consistent with TgAHP1 for tBOOH sensitivity. The presence of the TgAhp1 gene may be associated with a reduction in hydroperoxides, which in turn reduces the oxidative stimulation of T. gondii. The forma tion of disulfide bridges was progressively prevented when the quantity of alkyl hydroperoxides was high and when the enzyme was incubated with all the components required to sustain its catalytic cycle [20]. Knocking out the TgAhp1 gene led to the accumulation of reactive oxygen species in the parasite and induced apoptosis, thus confirming this speculation. TgAhp1 may play a certain protective role against oxidative damage to DNA. Parasites survive in a redox equilibrium state under normal circumstances; however, an imbalance occurs when the concentration of free radicals increases due to internal or external factors [21], which can lead to peroxidation of biological macromolecules, such as oxidative DNA damage, and then affect the growth and survival of cells [22]. The  9 Oxidative Medicine and Cellular Longevity comet assay, also known as single cell gel electrophoresis (SCGE), is a technique for detecting DNA damage [23], which shows DNA damage by calculating the migration rate of parasite DNA in the gel [18]. However, in our results, it was difficult to accurately determine the oxidative protection of the TgAHP1 protein on parasite DNA (date unpublished) by the comet assay. The relevance of TgAHP1 to DNA protective function needs further confirmation.
Some reports have shown that knocking out the Ahp1 gene enhances oxidant damage to somatic cells. In Saccharomyces cerevisiae and some bacteria, the Ahp1 protein participated in oxidative stress in the cytoplasm [24], which is same to TgAHP1 and located in the cytoplasm of T. gondii. Catalase is the characteristic marker enzyme of peroxisomes and is highly conserved across species. Regardless of the CAT's importance in detoxifying H 2 O 2 , this enzyme is lacking in most pathogenic protozoans [20]. The deduced amino acid sequence of T. gondii catalase has typical features of eukaryotic catalases [4,25]. A cytosolic catalase can act on the detoxification of the majority of host born peroxides due to its high substrate turnover [4,26].
The Urm1 ubiquitin-like system of Drosophila is one of the oldest protein functional regulatory systems in eukaryotes, which is related to cell growth and development, sulfur transport, nitrogen synthesis, and antioxidative stress [27]. Several reports have shown that this system specifically regulates the activity of Ahp1 (Prx5 family) and affects the antioxidant response in Drosophila [28]. Members of the Urm1 family had conserved functions in the oxidative stress response, and Urm1 target proteins had been identified from yeast, fungi, Drosophila, and human cells [29]. In addition, Ahp1 forms a dimer with a complete redox center that is closely related to Urm1 receptor activity in the uric acidification of S. cerevisiae [30]. When a mutation occurs in Ahp1, two residues near the redox active center will greatly reduce the level of urea ification without compromising the protective effect of tBOOH [31]. There is a possibility that the residue Lys of TgAhp1 can covalently bind to the ubiquitin-related modifier Urm1 to regulate the oxidative stress response. Studies from our laboratory have shown that the Urm1 ubiquitin system exists in T. gondii (date unpublished), and the TgUrm1 ubiquitin regulatory system may also regulate TgAhp1, thereby affecting the oxidative stress of T. gondii. Currently, TgURM1 and TgAHP1 have been confirmed as a pair of interacting proteins (date unpublished), and other works are in progress.
Substrates of Ahp1 also have a deleterious effect on the enzyme activity. The decrease in enzyme activity is linked to the irreversible modification of one of the two cysteines involved in the disulfide-linked dimerization process. The antioxidative stress response of TgAHP1 depends on the formation and breaking of disulfide bonds. The inactivation of Ahp1 during oxidative stress may have important biological implications.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there was no conflict of interest during the development of this research.