Marina M. Mourão

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.

Phenotypic screen of early-developing larvae of the blood fluke, schistosoma mansoni, using RNA interference.

RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1α), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced (∼1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

Role of the Endogenous Antioxidant System in the Protection of Schistosoma mansoni Primary Sporocysts against Exogenous Oxidative Stress

Antioxidants produced by the parasite Schistosoma mansoni are believed to be involved in the maintenance of cellular redox balance, thus contributing to larval survival in their intermediate snail host, Biomphalaria glabrata. Here, we focused on specific antioxidant enzymes, including glutathione-S-transferases 26 and 28 (GST26 and 28), glutathione peroxidase (GPx), peroxiredoxin 1 and 2 (Prx1 and 2) and Cu/Zn superoxide dismutase (SOD), known to be involved in cellular redox reactions, in an attempt to evaluate their endogenous antioxidant function in the early-developing primary sporocyst stage of S. mansoni. Previously we demonstrated a specific and consistent RNA interference (RNAi)-mediated knockdown of GST26 and 28, Prx1 and 2, and GPx transcripts, and an unexpected elevation of SOD transcripts in sporocysts treated with gene-specific double-stranded (ds)RNA. In the present followup study, in vitro transforming sporocysts were exposed to dsRNAs for GST26 and 28, combined Prx1/2, GPx, SOD or green-fluorescent protein (GFP, control) for 7 days in culture, followed by assessment of the effects of specific dsRNA treatments on protein levels using semi-quantitative Western blot analysis (GST26, Prx1/2 only), and larval susceptibility to exogenous oxidative stress in in vitro killing assays. Significant decreases (80% and 50%) in immunoreactive GST26 and Prx1/2, respectively, were observed in sporocysts treated with specific dsRNA, compared to control larvae treated with GFP dsRNA. Sporocysts cultured with dsRNAs for GST26, GST28, Prx1/2 and GPx, but not SOD dsRNA, were significantly increased in their susceptibility to H2O2 oxidative stress (60–80% mortalities at 48 hr) compared to GFP dsRNA controls (∼18% mortality). H2O2-mediated killing was abrogated by bovine catalase, further supporting a protective role for endogenous sporocyst antioxidants. Finally, in vitro killing of S. mansoni sporocysts by hemocytes of susceptible NMRI B. glabrata snails was increased in larvae treated with Prx1/2, GST26 and GST28 dsRNA, compared to those treated with GFP or SOD dsRNAs. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antioxidant enzymes serve a direct role in protection against external oxidative stress, including immune-mediated cytotoxic reactions. Moreover, these findings illustrate the efficacy of a RNAi-type approach in investigating gene function in larval schistosomes.

Regulation of Schistosoma mansoni Development and Reproduction by the Mitogen-Activated Protein Kinase Signaling Pathway

Background Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. Methodology/Principal Findings We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasites MAPK signaling cascade. Conclusions We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaca (alembic´s cachaca), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaca production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaca fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

In vitro manipulation of gene expression in larval Schistosoma: a model for postgenomic approaches in Trematoda.

With rapid developments in DNA and protein sequencing technologies, combined with powerful bioinformatics tools, a continued acceleration of gene identification in parasitic helminths is predicted, potentially leading to discovery of new drug and vaccine targets, enhanced diagnostics and insights into the complex biology underlying host-parasite interactions. For the schistosome blood flukes, with the recent completion of genome sequencing and comprehensive transcriptomic datasets, there has accumulated massive amounts of gene sequence data, for which, in the vast majority of cases, little is known about actual functions within the intact organism. In this review we attempt to bring together traditional in vitro cultivation approaches and recent emergent technologies of molecular genomics, transcriptomics and genetic manipulation to illustrate the considerable progress made in our understanding of trematode gene expression and function during development of the intramolluscan larval stages. Using several prominent trematode families (Schistosomatidae, Fasciolidae, Echinostomatidae), we have focused on the current status of in vitro larval isolation/cultivation as a source of valuable raw material supporting gene discovery efforts in model digeneans that include whole genome sequencing, transcript and protein expression profiling during larval development, and progress made in the in vitro manipulation of genes and their expression in larval trematodes using transgenic and RNA interference (RNAi) approaches.

Recent advances in Schistosoma genomics.

Schistosome research has entered the genomic era with the publications reporting the Schistosoma mansoni and Schistosoma japonicum genomes. Schistosome genomics is motivated by the need for new control tools. However, much can also be learned about the biology of Schistosoma, which is a tractable experimental model. In this article, we review the recent achievements in the field of schistosome research and discuss future perspectives on genomics and how it can be integrated in a usable format, on the genetic mapping and how it has improved the genome assembly and provided new research approaches, on how epigenetics provides interesting insights into the biology of the species and on new functional genomics tools that will contribute to the understanding of the function of genes, many of which are parasite‐ or taxon specific.

Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaca and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaca samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

Background During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. Methodology/Principal Findings In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present. Conclusion Taken together, these results strongly support the hypothesis that SmZF1 acts as a transcription factor in S. mansoni.

Trypanosoma cruzi gene expression in response to gamma radiation.

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.