Transcriptome profile of a cell replication arrest of Trypanosoma cruzi epimastigotes induced by overexpression of a small zinc finger protein

WESTERGAARD, G; LAVERRIERE, M; REVALE, S; REINERT, M; DE GAUDENZI, JG; JÄGER, AV; VAZQUEZ, MP

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

The TcZFPs are a family of small zinc finger proteins harboring WW domains or Proline rich motifs. In Trypanosoma brucei, ZFPs are involved during stage specific differentiation. TcZFPs interact with each other using the WW domain (ZFP2 and ZFP3) and the proline rich motif (ZFP1). The tcZFP1b member is exclusive to Trypanosoma cruzi and it is only expressed in trypomastigote stage. We used a tetracycline inducible vector to overexpress ectopically tcZFP1b in the epimastigote stage. Upon induction of tcZFP1b, the parasites stopped dividing completely after five days. Visual inspection showed abnormal distorted-morphology (monster) cells with multiple flagella and increased DNA contents. We were interested in investigate global transcription changes occurred during the generation of this abnormal phenotype. Thus, we performed RNA-seq transcriptome profiling with a 454 pyrosequencer to analyze the global changes after ectopic expression of tcZFP1b. The total mRNAs sequenced from induced and non-induced control epimastigotes showed, after filtering the data, a set of 70 genes having equal or more than 3X fold change upregulation, while 35 genes showed equal or more than 3X fold downregulation. Interestingly, several trans-sialidase-like genes and pseudogenes were upregulated along with several genes in the categories of amino acid catabolism and carbohydrate metabolism. On the other hand, hypothetical proteins, fatty acid biosynthesis and mitochondrial functions dominated the group of downregulated genes. Our data showed that several mRNAs sharing related functions and pathways changed their levels in a concerted pattern resembling post-transcriptional regulons. We also found two different motifs in the 3UTRs of the majority of mRNAs, one for upregulated and other for downregulated genes. An interesting fact observed in the genome-wide analysis was the 2.4X fold downregulation of -tubulin. Since new microtubules are constantly needed in order to progress to cytokinesis, this downregulation could be one of the hints pointing to the observed cytokinesis arrest. Non-dividing forms, such as trypomastigotes, would have more stabilized microtubules in the sub-pellicular corset than the fast dividing forms (epimastigotes and amastigotes). Cause or consequence of epimastigotes stopping cell division several hours upon induction was the fact that PGF2 synthase mRNAs were downregulated by 2.6 and 2.5X fold. It is known that production of PGF2 decreased significantly in non-dividing forms [1]. One of the components of the flagellum, paraflagellar rod component par4, was also downregulated 3X fold. It is not clear the function of this component in the flagellum biology but it is tempting to speculate that its downregulation could be linked to the observed phenotype. Notably, several genes involved in amino acid catabolism that use pyridoxal phosphate as cofactor were upregulated between 13X and 3X fold while the pyridoxal kinase mRNA was concomitantly up by 11X fold. Several genes related to the glyoxylate and dicarboxylate metabolism were also upregulated. Interestingly, amino acid catabolism is linked to the former metabolism through the production of hydroxypyruvate and glyoxylate. One important conclusion obtained from this work is that post-transcriptional regulons are evident in T. cruzi, as it also seems to emerge from other works in T. brucei [2, 3]. The finding of common sequence motifs in the 3UTRs reinforces the idea of the regulons model. In this work, we found two different high-confidence motifs, up-h12 in the upregulated and down-h12 in the downregulated genes. Both motifs presented conserved sequence cores exposed in loops. Interestingly, down-h12 resembles a classical AU-rich (ARE) element previously involved in the instability of mRNAs in T. cruzi. The list of down-h12 containing genes included fatty acid elongase, fatty acid desaturase, ATPase beta subunit, cytochrome C oxidase and amastin among others. Important to note is that most of the upregulated genes belonged to the family of trans-sialidase like members, a family expressed almost exclusively in trypomastigotes, the parasite form that interact with the mammalian host. The expression of trans-sialidase pseudogenes was also evident. Expression of pseudogenes was shown to have a role in gene expression via the RNA interference (RNAi) system in T. brucei. However, since RNAi is lacking in T. cruzi , the upregulation of these pseudogenes remains puzzling. A general view of the genome-wide analysis seemed to point to the activation of at least part of a program to differentiate to trypomastigotes, although the sole overexpression of tcZFP1b might not be sufficient to accomplish the task. Upregulation of proline racemase (7.1X fold) is remarkable in this context since it was shown that its ectopic expression in epimastigotes resulted in enhanced differentiation to trypomastigotes and enhanced infectivity [4, 5]. In addition, the upregulation of several trans-sialidase like mRNAs, the possible stabilization of the microtubule sub-pellicular corset to enter a nondividing form, the upregulation of a mechanism of energy production through the glycosome and amino acid catabolism, and the possible downregulation of mitochondrial functions are compelling evidence pointing towards that direction. With this genome-wide analysis in hand, one of the challenging tasks for the near future would be to dissect the chain of events unleashed after overexpression of tcZFP1b in epimastigotes, beginning by looking for direct mRNA targets of its zinc-finger motif. Since the tcZFP2 proteins interact with tcZFP1b, it will be interesting also to look for direct mRNA targets of their zinc-fingers as well.