Authors

Discussion

Across the Apicomplexan phylum, DNA damage response pathway(s) are not well characterised. Many anti-parasitic drugs rely on causing DNA damage or interrupting DNA replication as their mode of action, so it would be very useful to learn more about how these mechanisms work. In model eukaryotes, cell cycles are closely regulated and DNA damage is flagged for repair at a series of cell-cycle checkpoints.  These checkpoints are signalled through phosphatidylinositol 3-kinase-related kinases (PIKKs). Interestingly, some apicomplexans have retained homologs of human PIKKs, such as ATM and ATR, whereas others have not. In Toxoplasma gondii (T. gondii), there are putative homologs of ATM and ATR, whereas in close relatives such as Plasmodium falciparum (P. falciparum) these are missing and the closest remaining homolog to this family is the lipid kinase phosphoinositide 3-kinase (PI3K). 

            To investigate this, we created an inducible knockdown of the putative ATM gene in T.gondii. The knockdown was found to be non-lethal so the protein is probably not essential, which was unexpected because a recent genome-wide knockdown screen reported a severe fitness defect for this gene. By contrast, our knockdown grew normally.  DNA damaging agents (e.g. the alkylating agent MMS) were added to see if the knockdown made cells more vulnerable to damage and whether this would affect growth, but knockdown parasites were not apparently sensitised to DNA damage.  Nevertheless, knockdown parasites did fail to produce a marker of DNA damage, the phosphorylation of histone 2A, in response to the topoisomerase inhibitor camptothecin – a double-strand-break-inducing agent.   Furthermore, when low levels of DNA damage were applied, T. gondii replication was slowed and the ATM knockdown reduced this effect, possibly by preventing initiation of a checkpoint.  Overall, results thus far suggest that the putative T. gondii ATM does play a role in the DNA damage response and checkpoint signalling, but is non-essential.

            Previously published work using an inhibitor of the human ATM, KU-55933 (usually used in cancer studies) showed that this inhibited histone phosphorylation after DNA damage in T. gondii. The authors therefore concluded that this inhibitor acts on the T. gondii ATM homolog.  However, our results call into question the specificity of this inhibitor: although it did prevent histone phosphorylation in response to damage, it did not phenocopy the genetic knockdown, and caused severe growth defects in parasites in the absence of DNA damage.  Off-target cytotoxic effects are possible, as is an effect on the other PIKK homolog (putative ATR) in this parasite.

            Future work will focus on the role(s) of T. gondii ATR versus ATM, and on cross-complementation studies to establish how Plasmodium responds to DNA damage in the apparent absence of either of these kinases.