Marcelo Rosado Fantappié

Dnmt2-dependent methylomes lack defined DNA methylation patterns

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.

Oxidative stress fuels Trypanosoma cruzi infection in mice

Oxidative damage contributes to microbe elimination during macrophage respiratory burst. Nuclear factor, erythroid-derived 2, like 2 (NRF2) orchestrates antioxidant defenses, including the expression of heme-oxygenase-1 (HO-1). Unexpectedly, the activation of NRF2 and HO-1 reduces infection by a number of pathogens, although the mechanism responsible for this effect is largely unknown. We studied Trypanosoma cruzi infection in mice in which NRF2/HO-1 was induced with cobalt protoporphyrin (CoPP). CoPP reduced parasitemia and tissue parasitism, while an inhibitor of HO-1 activity increased T. cruzi parasitemia in blood. CoPP-induced effects did not depend on the adaptive immunity, nor were parasites directly targeted. We also found that CoPP reduced macrophage parasitism, which depended on NRF2 expression but not on classical mechanisms such as apoptosis of infected cells, induction of type I IFN, or NO. We found that exogenous expression of NRF2 or HO-1 also reduced macrophage parasitism. Several antioxidants, including NRF2 activators, reduced macrophage parasite burden, while pro-oxidants promoted it. Reducing the intracellular labile iron pool decreased parasitism, and antioxidants increased the expression of ferritin and ferroportin in infected macrophages. Ferrous sulfate reversed the CoPP-induced decrease in macrophage parasite burden and, given in vivo, reversed their protective effects. Our results indicate that oxidative stress contributes to parasite persistence in host tissues and open a new avenue for the development of anti-T. cruzi drugs.

Epigenetic Changes Modulate Schistosome Egg Formation and Are a Novel Target for Reducing Transmission of Schistosomiasis

Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.

Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone.

Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening an S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.

Role of the acidic tail of high mobility group protein B1 (HMGB1) in protein stability and DNA bending.

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling.

The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1DeltaC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1DeltaC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

Identification and characterization of an R‐Smad ortholog (SmSmad1B) from Schistosoma mansoni

Smad proteins are the cellular mediators of the transforming growth factor‐β superfamily signals. Herein, we describe the isolation of a fourth Smad gene from the helminth Schistosoma mansoni, a receptor‐regulated Smad (R‐Smad) gene termed SmSmad1B. The SmSmad1B protein is composed of 380 amino acids, and contains conserved MH1 and MH2 domains separated by a short 42 amino acid linker region. The SmSmad1B gene (> 10.7 kb) is composed of five exons separated by four introns. On the basis of phylogenetic analysis, SmSmad1B demonstrates homology to Smad proteins involved in the bone morphogenetic protein pathway. SmSmad1B transcript is expressed in all stages of schistosome development, and exhibits the highest expression level in the cercariae stage. By immunolocalization experiments, the SmSmad1B protein was detected in the cells of the parenchyma of adult schistosomes as well as in female reproductive tissues. Yeast two‐hybrid experiments revealed an interaction between SmSmad1B and the common Smad, SmSmad4. As determined by yeast three‐hybrid assays and pull‐down assays, the presence of the wild‐type or mutated SmTβRI receptor resulted in a decreased interaction between SmSmad1B and SmSmad4. These results suggest the presence of a nonfunctional interaction between SmSmad1B and SmTβRI that does not give rise to the phosphorylation and the release of SmSmad1B to form a heterodimer with SmSmad4. SmSmad1B, as well as the schistosome bone morphogenetic protein‐related Smad SmSmad1 and the transforming growth factor‐β‐related SmSmad2, interacted with the schistosome coactivator proteins SmGCN5 and SmCBP1 in pull‐down assays. In all, these data suggest the involvement of SmSmad1B in critical biological processes such as schistosome reproductive development.

CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

Background The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. Principal Findings We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. Conclusions We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

Cloning of SmNCoA-62, a novel nuclear receptor co-activator from Schistosoma mansoni: Assembly of a complex with a SmRXR1/SmNR1 heterodimer, SmGCN5 and SmCBP1 ☆

The Schistosoma mansoni nuclear receptors (NR) SmRXR1 and SmNR1 have recently been shown to form a heterodimer and to bind to canonic hormone response DNA elements. Recruitment of co-regulatory proteins to NRs is required for their transcriptional and biological activities. Here, we cloned a novel S. mansoni NR co-activator, SmNCoA-62. SmNCoA-62 is highly homologous to the human Vitamin D receptor co-activator NCoA62/SKIP. SmNCoA-62 contains the SNW nuclear receptor interaction domain and a putative C-terminus transactivation domain. By using in vitro pull-down assays, we fully mapped the interaction domains of S. mansoni NR co-activators, SmNCoA-62, SmGCN5 and SmCBP1 with SmRXR1 and SmNR1, as well as the domains that mediate interactions amongst the co-activators themselves. By mutagenesis analysis, we showed that SmCBP1 LxxLL motif 2 and LxxLL motif 3, but not LxxLL motif 1, were essential to mediate the interactions of SmCBP1 with the EF domains of SmRXR1 and SmNR1. Histone acetyltransferases SmGCN5 and SmCBP1 specifically acetylated the C/D domains of SmRXR1 and SmNR1. In addition, two acetylation sites of SmNR1 were identified. SmGCN5 and SmCBP1 also acetylated SmNCoA-62 but with significant differences in their acetylation activities. Using gel shift analysis, we were able to demonstrate, in vitro, the assembly of the co-activators on the SmRXR1/SmNR1 heterodimer bound to DNA. LxxLL motifs 2 and 3 of SmCBP1 seemed to play a crucial role for the assembly of the co-activators to the DNA-bound SmRXR1/SmNR1 heterodimer.