Resveratrol induces H2A.X phosphorylation and H4K16 de-acetylation in Toxoplasma gondii

Resveratrol (RSV) is a multi-target drug that demonstrated activity against Toxoplasma gondii in macrophage and HFF cell line infection models. In addition to modulate redox homeostasis, RSV is also an activator of Sir2, a type III HDAC. RSV inhibited intracellular T. gondii tachyzoite growth at concentrations below the toxic effect on host cells. The IC50 value in a 24-hours treatment was 53 μM. After 96 hours of treatment the maximum non-toxic concentration for host cell, 20 μM, only inhibited T. gondii growth a 50%. RSV induced a reduction in H4K16 acetylation (H4K16ac), a mark associated to transcription, DNA replication and homologous recombination repair, without any effect on H3 acetylation (H3ac). RSV also enhanced the SQE motif phosphorylation on T. gondii H2A.X (termed γH2A.X), a DNA damage associated PTM. Sirtinol, a specific Sir2 inhibitor also inhibited T. gondii but did not altered the acetylation status of H3 and H4K16 as well as H2A.X phosphorylation. Our findings suggest a possible link between RSV and DNA damage or DNA repair process maybe due to DNA replication stress and/or another undetermined mechanism.


Introduction
Toxoplasma gondii is an important pathogen for animals and human health, particularly during pregnancy 19 and in immunocompromised patients [1]. The success of human infection is based on the ability of the 20 tachyzoite to rapidly infect any nucleated cell. This event starts the asexual cycle which also occurs in 21 other mammals and birds. The asexual phase is characterized by two stages, the rapidly replicating and 22 highly disseminating tachyzoite, and the bradyzoite, which replicates slowly and is located in tissue cysts 23 for the rest of the life of the animal or individual [2]. The replicative process can trigger a collapse in the 24 DNA replication fork and its consequent double strand break (DSB). This effect could also be occurring 25 at least in the T. gondii tachyzoite. On one hand, histone H2A.X is phosphorylated at the C-terminal SQEF 26 motif to generate H2AX foci in DSB sites. In T. gondii, it is possible to detect the presence of H2AX 27 under normal growth conditions [3,4]. On the other hand, the ATM kinase inhibitor, key to triggers the 28 DNA damage response (DDR), blocks H2A.X phosphorylation and stops the replication of Toxoplasma 29 [4]. Toxoplasma possesses the conserved DSB repair machinery although it lacks some key effector 30 proteins to decide the repair route: homologous recombination (HRR) or non-homologous end joining 31 (NHEJ) [5]. Also, it has been observed that most of the components of the HRR pathway are essential and 32 that a large number of drugs that are genotoxic or that inhibit DDR affect the growth of Toxoplasma in 33 vitro and in vivo [6]. Resveratrol (RSV; 3,5,4'-trihydroxystilbene) is a natural polyphenolic phytoalexin produced in 40 plants. RSV [14] observed that incubation of extracellular tachyzoites with RSV 50 during 24 hours affected their viability, probably by disturbing the redox homeostasis of the parasites. Of 51 note, in addition to fork collapse, DNA can be damaged by oxidation. In addition, they observed that 52 RSV reduced tachyzoite intracellular growth and promoted autophagy in infected macrophages. In 53 parallel, Adeyemi et al., [15] identified RSV as a putative drug repurposing candidate against 54 toxoplasmosis in a 62-compound screening. However, none of them analyzed the intracellular effect of 55 RSV on T. gondii cell tachyzoites.

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In this work, we evaluated the effect of RSV on T. gondii growth and histone PTMs alteration. T. 57 gondii expresses two type-III Sir2 histone deacetylases: TGME49_227020 and TGME49_267360, 58 orthologues of P. falciparum Sir2A and Sir2B respectively and homologues to yeast Sir2 HDAC [16,17].

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The effect of RSV on H4K16 acetylation level was analyzed. In addition, changes in H2A.X levels were 60 also analyzed in control or treated intracellular tachyzoites. Collectively, our results show that RSV 61 inhibited T. gondii growth and induced H4K16 de-acetylation. Finally, H2A.X marks were highly       RSV has already demonstrated its anti-T. gondii effect in vitro in two recent works [14,15]. In 154 order to confirm these results in our study model, uninfected or infected hTERT host cell was incubated 155 with different doses of RSV and in two incubation periods (24 and 96 hours). In the 24-hours incubation 156 experiments, 80% of the cells remain viable at concentrations of 100 M, while after 96 hours of exposure, 157 90% remain viable at 12.5 M (Fig 1). In order to test the impact of RSV on T. gondii lytic cycle,  control condition) ( Fig. 2A). In fact, Western blot analysis did not show significant differences in band 10 176 intensities (Fig. 2B). Of note, the antibody used is against H3K9acK14ac marks. Maybe, only one of these 177 marks are being modulated by some of T. gondii Sir2. Unfortunately, the anti-H3K9me3 used here did not 178 work in immunofluorescence assay (Fig. 2C). Sirtinol (Sir two inhibitor naphthol) is a sirtuin inhibitor, 179 highly specific for human SIRT1 and SIRT2 with IC 50 of 131 μM and 38 μM respectively [22]. We decided 180 to include this drug to see the opposite effect of resveratrol. Based on a viability study on hTERT cell (S1 181 Fig.), the concentration used infected cells was 100 M. Sirtinol did not present any effect on H3ac status 182 (Fig 2A). However, sirtinol affected T. gondii growth in a dose dependent manner with an IC 50 : 27 +/-2.5 183 M (S1 Fig.).   194 The presence of H4K16ac in DNA damaged by DSB is able to recruit DNA repair proteins 195 associated to the HRR pathway [7], among them ATM kinase that initiate the DNA damage response [6].

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ATM-like kinase activity was detected in T. gondii [4]. In order to determine if the effect of RSV and 197 sirtinol is associated to DSB damage, the H2A.X level (DSB level mark) was tested by Western blot. A 198 specific rabbit anti-T. gondii phosphorylated peptide in the SQE motif of T. gondii H2A.X C-terminal end 11 199 was prepared (see materials and methods). To confirm its specificity, a Western blot against the 200 recombinant non-phosphorylated T. gondii H2A.X was performed. As it can be observed, the rabbit anti-

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TgH2A.X antibody does not recognize T. gondii H2A.X ( Figure 4A), but reacts with an expected band 202 of 14.5-kDa in T. gondii lysate (Fig 4B). After treatment with RSV, the H2A.X signal is increased 203 compared to DMSO control, whereas sirtinol treatment produced a mild effect, below a 2-fold enrichment 204 (Fig. 4B). 205 Here we observed that the γH2A.X level is increased in presence of RSV 50 M, a mark 206 compatible with DNA damage. In another model, RSV at 50 µM induced DNA damage, S-phase arrest 207 and enhanced γH2AX levels in a panel of head and neck squamous cell carcinoma lines [25]. In addition, 208 RSV was associated to HRR inhibition through the inactivation of tyrosyl-tRNA synthetase, an enzyme 209 associated to protein synthesis but also to HRR gene regulation when translocate to nucleus [26,27]. If 210 that were the case, RSV could be used in combination with a drug that inhibits the DSB repair pathway in  drug RSV in addition to the redox homeostasis alteration suggested recently [14]. Finally, we considered 230 that RSV could be an interesting drug to analyze chromatin modulation in T. gondii as well as novel 231 candidate for alternative T. gondii infection treatment.

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H2A.X (arrow) and H2A.X band intensities were quantified and normalized against SAG1 band 312 intensities. After that, relative intensity bands (H2A.X/H2A.X) were calculated for each treatment.