In order to test our study model, uninfected or infected hTERT host cells were incubated 24 hours with different doses of RSV. Near 80% of uninfected hTERT cells remain viable at concentrations of 100 µM (Fig. 1A). In order to test the impact of RSV on T. gondii lytic cycle, transgenic RH RFP (Red Fluorescent Protein) tachyzoites were intracellularly grown during 24 hours in presence of RSV. As it can be observed, RSV showed a dose-dependent effect in intracellular T. gondii replication (Fig. 1B) with an IC50 value of 53 +/- 4 µM. This result agrees with those observed by Chen et al., , in which a 50 µM of RSV blocked T. gondii intracellular growth. Adeyemi et al.,  identified RSV as one of the 62 compounds with anti-T. gondii effect with an IC50 value of 1.03 µg/ml (4.4 µM) after 72 hours of incubation and in comparison with pyrimethamine. The authors also observed that HFF cells were 100% viable at 2 µg/ml (8.5 µM). The differences in IC50 values can be due to the exposition time with the drug. Taken bibliographic and our results together, RSV is a natural product with potential for an alternative Toxoplasma infection therapy.
Since HDAC enzymes are one of the multiple targets of RSV, we analyzed the acetylation levels of H3 and H4K16. Tachyzoites treated with RSV presented a significant weaker H3ac and H4K16ac labelling in comparison with DMSO (Fig. 1C, 2 A and additional file 1). We decided to corroborate these data by Western blot. RSV treatment reduced the H3ac and H4K16ac mark intensity when compared to DMSO control (Fig. 1D and 2B). This result is in agreement with another study in which RSV treatment decreased H3 and H4 acetylation levels in Trypanosoma cruzi . More specifically, it was recently observed that RSV reduced the H4K16ac mark . In addition, in yeast, Sir2, a target of RSV, negatively controlled the activation of DNA replication origins within heterochromatin and euchromatin by de-acetylating H4K16 . Our results suggest that RSV could be affecting T. gondii transcription and/or replication trough H3 and H4K16 deacetylation. However, further studies are required to confirm this issue.
In order to determine if the effect of RSV could be associated to DSB damage, among others, γH2A.X levels (DSB level mark) were tested by Western blot. A specific rabbit anti-T. gondii phosphorylated peptide in the SQE motif of T. gondii H2A.X C-terminal end was prepared. To confirm its specificity, a Western blot against the recombinant non-phosphorylated T. gondii H2A.X was performed. As it can be observed, the rabbit anti-TgγH2A.X antibody does not recognize T. gondii H2A.X (Fig. 3A), but reacts with an expected band of 14.5-kDa in T. gondii lysate (Fig. 3B). After treatment with RSV, the γH2A.X signal is increased compared to DMSO control (Fig. 3B), about 5.90 times (Fig. 3C).
Here we observed that the γH2A.X level is increased in presence of RSV, a mark compatible with DNA damage. In another model, RSV at 50 µM induced DNA damage, S-phase arrest and enhanced γH2AX levels in a panel of head and neck squamous cell carcinoma lines . Double strand breaks (DSB) are repaired by homologous recombination (HRR) which is triggered in the late S phase by DSB that appear during DNA replication (e.g. due to fork collapse), or non-homologous end joining (NHEJ), throughout the cell cycle . Histone H4 acetylated on its lysine in position 16 (H4K16ac) is a mark that facilitates the HRR pathway [6, 23]. Putting all the data together, we propose a possible model of RSV action, at the concentrations used, which would allow us to partially explain how it could affect T. gondii replication (Fig. 3D). Briefly, RSV could induce a deficiency in H3 and H4K16 acetylation, leading to altered transcription levels, DNA replication initiation and maybe the HRR pathway. This could affect the correct DNA replication and the progress of the cell cycle, keeping γH2A.X levels high. In turn, RSV could be inducing DNA damage, either by oxidation, generating fork collapse and DSB [24, 25], increasing the difficulty of repair via HRR in the absence of H4K16ac.
Since RSV is a multitarget drug, it is difficult to establish which targets are being affected in T. gondii. One of the targets could be a histone acetyltransferase. However, RSV already showed a sirtuin activating activity . It is possible that this is one of the targets with consequent histone deacetylation. T. gondii sirtuins sir2a and sir2b have not yet been characterized. Therefore, its specific functions (e.g. role in the acetylation of H4K16), genomic localization when is present in the nucleus, etc., have not yet been reported. This impairs the verification whether the observed effect of RSV could be linked to these sirtuins.