TOXOPLASMA HISTONE VARIANTS AND THEIR ROLE IN DIFFERENT PARASITE PROCESSES

SERGIO O. ANGEL; LAURA VANAGAS; SILVINA S. BOGADO

Mar del Plata

Congreso; Reunión Conjunta 2016 entre la Sociedad Argentina de Investigación Clínica (SAIC), la Sociedad Argentina de Inmunología (SAI) y la Sociedad Argentina de Farmacología Experimental (SAFE); 2016

Toxoplasmagondii is a coccidian protozoan parasite that belongs to the phylumApicomplexa. It is estimated that toxoplasmosis exists as a chronicasymptomatic form in 5 hundred million to 1 billion of the world human population.Although infection with T. gondii is usually asymptomatic in most individuals,it is of great medical significance for pregnant women and immunocompromisedpatients. In humans, T. gondii infection is characterized by two stages, therapidly growing tachyzoites, and the latent bradyzoite tissue cysts.Tachyzoites are responsible for acute illness and congenital birth defects. T.gondii tachyzoites contain basal levels of γH2A.X, a marker of double strand break (DSB)damage, even in normal conditions lacking a DNA damaging stress. This parallelswhat is seen due to fork collapse under replication-associated DNA stress incancer cells. Bradyzoites form cysts that remain latent for many years but arestill capable of converting into the destructive tachyzoite form if hostimmunity decreases. These two developmental stages are essential for cause andpropagation of disease. Tachyzoite to bradyzoite conversion, and vice-versa,includes a high number of gene expression modifications. It is believed that theepigenetic control of gene regulation is crucial for parasite development, aprocess that relies on the posttranslational modification (PTM) of histones andhistone variant exchange. T. gondii possess the four canonical histones H2A,H2B, H3 and H4 and variant histones of H3 and H2A families. Concerning the H2Afamily, the parasite has H2AZ and H2A.X variants, a feature that is not shared byother apicomplexan parasites in which H2AX is not present. In highereukaryotes, the role of H2A.Z is associated to transcriptional regulation,genome stability, and blocking the spread of heterochromatin, whereas H2A.X isinvolved in DNA repair being recruited at double strand break (DSB) site, aprocess that requires the phosphorylation of the serine present at C-terminalmotif SQEY/F. Interestingly, T. gondii has a variant of H2B, that has been namedH2B.Z since it forms dimers mainly with H2A.Z. Double variant H2A.Z/H2B.Znucleosome and H2A.X/H2Ba are not present in the same nucleosome as it was observedby ChIP-qPCR and ChIP-seq. These findings reveal that nucleosomal arrangementsare not random in protozoa, highlighting their relevance in chromatincomposition and regulation. H2A.Z and H2B.Z have shown to be highly acetylatedat their N-terminal tails, a marker of active chromatin. H2A.Z and H2B.Z geneshave shown to be essential. The over-expression of different H2B.Z mutants,that are unable to acetylate the N-tail, has shown little effect in tachyzoitereplication rate but an important alteration in the differentiation process. Onthe other hand, proteomic analysis confirms the presence of gH2A.X innormal conditions suggesting that tachyzoites may be subjected to fork collapseand DSB, situations that activate the homologous recombination repairmachinery. H2A.X is phosphorylated at its SQE motif by ATM kinase at the initialstep of HRR pathway. Based on that, the impact on tachyzoite replication ofCPT, which generates DSB during DNA replication, and KU55933, a highly specificATM inhibitor was analyzed. Both, CPT and KU55933 produced a significant effecton parasite replication suggesting their inhibition effect may be blocking T.gondii DNA replication and/or activating cell cycle checkpoints eitheraffecting T. gondii ATM directly or through HFFATM inhibition. As expected, the combination of both  drugs generated a serious blocking ofreplication rates with a large number of tachyzoites that were not able to carryout the first event of replication. Taken together the results show thathistone variants and their PTM are important epigenetic regulators in differentprocesses of the parasite life cycle.