TOXOPLASMA HISTONE VARIANTS AND THEIR ROLE IN DIFFERENT PARASITE PROCESSES

ANGEL SO*; VANAGAS L; BOGADO SS

Mar del Plata

Mesa redonda; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2016

SAIC

Toxoplasma gondii is a coccidian protozoan parasitethat belongs to the phylum Apicomplexa. It is estimatedthat toxoplasmosis exists as a chronic asymptomaticform in 5 hundred million to 1 billion of the world humanpopulation. Although infection with T. gondii is usuallyasymptomatic in most individuals, it is of great medical significancefor pregnant women and immunocompromisedpatients. In humans, T. gondii infection is characterizedby two stages, the rapidly growing tachyzoites, and thelatent bradyzoite tissue cysts. Tachyzoites are responsiblefor acute illness and congenital birth defects. T. gondiitachyzoites contain basal levels of γH2A.X, a marker ofdouble strand break (DSB) damage, even in normal conditionslacking a DNA damaging stress. This parallels whatis seen due to fork collapse under replication-associatedDNA stress in cancer cells. Bradyzoites form cysts thatremain latent for many years but are still capable of convertinginto the destructive tachyzoite form if host immunitydecreases. These two developmental stages are essentialfor cause and propagation of disease. Tachyzoite tobradyzoite conversion, and vice-versa, includes a highnumber of gene expression modifications. It is believedthat the epigenetic control of gene regulation is crucial forparasite development, a process that relies on the posttranslationalmodification (PTM) of histones and histonevariant exchange. T. gondii possess the four canonicalhistones H2A, H2B, H3 and H4 and variant histones of H3and H2A families. Concerning the H2A family, the parasitehas H2AZ and H2A.X variants, a feature that is not sharedby other apicomplexan parasites in which H2AX is notpresent. In higher eukaryotes, the role of H2A.Z is associatedto transcriptional regulation, genome stability, andblocking the spread of heterochromatin, whereas H2A.Xis involved in DNA repair being recruited at double strandbreak (DSB) site, a process that requires the phosphorylationof the serine present at C-terminal motif SQEY/F.Interestingly, T. gondii has a variant of H2B, that has beennamed H2B.Z since it forms dimers mainly with H2A.Z.Double variant H2A.Z/H2B.Z nucleosome and H2A.X/H2Ba are not present in the same nucleosome as it wasobserved by ChIP-qPCR and ChIP-seq. These findingsreveal that nucleosomal arrangements are not random inprotozoa, highlighting their relevance in chromatin compositionand regulation. H2A.Z and H2B.Z have shown tobe highly acetylated at their N-terminal tails, a marker ofactive chromatin. H2A.Z and H2B.Z genes have shownto be essential. The over-expression of different H2B.Zmutants, that are unable to acetylate the N-tail, has shownlittle effect in tachyzoite replication rate but an importantalteration in the differentiation process. On the other hand,proteomic analysis confirms the presence of gH2A.X innormal conditions suggesting that tachyzoites may besubjected to fork collapse and DSB, situations that activatethe homologous recombination repair machinery. H2A.Xis phosphorylated at its SQE motif by ATM kinase at theinitial step of HRR pathway. Based on that, the impacton tachyzoite replication of CPT, which generates DSBduring DNA replication, and KU55933, a highly specificATM inhibitor was analyzed. Both, CPT and KU55933produced a significant effect on parasite replication suggestingtheir inhibition effect may be blocking T. gondiiDNA replication and/or activating cell cycle checkpointseither affecting T. gondii ATM directly or through HFFATM inhibition. As expected, the combination of bothdrugs generated a serious blocking of replication rateswith a large number of tachyzoites that were not able tocarry out the first event of replication. Taken togetherthe results show that histone variants and their PTM areimportant epigenetic regulators in different processesof the parasite life cycle.