Analysis of the interactome of <i>Schistosoma mansoni</i> histone deacetylase 8

<div><p>Background</p><p>Histone deacetylase 8 from <i>Schistosoma mansoni</i> (<i>Sm</i>HDAC8) is essential to parasite growth and development within the mammalian host and is under investigation as a target for the development of selective inhibitors as novel schistosomicidal drugs. Although some protein substrates and protein partners of human HDAC8 have been characterized, notably indicating a role in the function of the cohesin complex, nothing is known of the partners and biological function of <i>Sm</i>HDAC8.</p><p>Methodology/Principal findings</p><p>We therefore employed two strategies to characterize the <i>Sm</i>HDAC8 interactome. We first used <i>Sm</i>HDAC8 as a bait protein in yeast two-hybrid (Y2H) screening of an <i>S</i>. <i>mansoni</i> cDNA library. This allowed the identification of 49 different sequences encoding proteins. We next performed co-immunoprecipitation (Co-IP) experiments on parasite extracts with an anti-<i>Sm</i>HDAC8 antibody. Mass spectrometry (MS) analysis allowed the identification of 160 different proteins.</p><p>Conclusions/Significance</p><p><i>Sm</i>HDAC8 partners are involved in about 40 different processes, included expected functions such as the cohesin complex, cytoskeleton organization, transcriptional and translational regulation, metabolism, DNA repair, the cell cycle, protein dephosphorylation, proteolysis, protein transport, but also some proteasome and ribosome components were detected. Our results show that <i>Sm</i>HDAC8 is a versatile deacetylase, potentially involved in both cytosolic and nuclear processes.</p></div

Confirmation of interactions by candidate-specific Y2H experiments.

<p>In order to confirm the screening results, the isolated clones CtBP, MBD2, tensin and actin-1 (cloned into pGADT7 vector) transformed in the AH109 strain were mated with the bait construct <i>Sm</i>HDAC8 pGBKT7 transformed in the Y187 strain. After incubation, diploid yeasts were plated on selective medium lacking leucine and tryptophan (left panel) and then on another high stringency selective medium lacking adenine, histidine, leucine and tryptophan (right panel) showing, as expected, that <i>Sm</i>HDAC8 interacts with the clones tested.</p

Biological processes involving the proteins identified by Y2H and Co-IP/MS as <i>Sm</i>HDAC8 interactors.

<p>Histogram (left for Y2H and right for Co-IP/MS) showing the biological processes in which the identified protein partners of <i>Sm</i>HDAC8 are involved. The processes were defined using the Blast2GO software. Bars represent the protein percentage belonging to one biological process. Red and black bars highlight common and specific processes between Y2H and Co-IP/MS, respectively.</p

Venn diagram presenting the proteins identified by Y2H and Co-IP/MS as <i>Sm</i>HDAC8 interactors.

<p>Forty-nine and 160 different proteins were identified by Y2H and Co-IP/MS respectively. Four proteins are common between Y2H and Co-IP/MS: the Proliferation-associated protein 2G4, 38kDa (PA2G4, n°G4LXR6), Cathepsin-B1 (SmCB1, n°Q8MNY2), putative NADH-ubiquinone oxidoreductase (n°G4VK53) and microsomal glutathione <i>S</i>-transferase 3 (GST-3, n°G4VH65).</p

<i>Schistosoma mansoni</i> Sirtuins: Characterization and Potential as Chemotherapeutic Targets

<div><p>Background</p><p>The chemotherapy of schistosomiasis currently depends on the use of a single drug, praziquantel. In order to develop novel chemotherapeutic agents we are investigating enzymes involved in the epigenetic modification of chromatin. Sirtuins are NAD+ dependent lysine deacetylases that are involved in a wide variety of cellular processes including histone deacetylation, and have been demonstrated to be therapeutic targets in various pathologies, including cancer.</p><p>Methodology, Principal Findings</p><p>In order to determine whether <i>Schistosoma mansoni</i> sirtuins are potential therapeutic targets we first identified and characterized their protein sequences. Five sirtuins (SmSirt) are encoded in the <i>S. mansoni</i> genome and phylogenetic analysis showed that they are orthologues of mammalian Sirt1, Sirt2, Sirt5, Sirt6 and Sirt7. Both SmSirt1 and SmSirt7 have large insertion in the catalytic domain compared to their mammalian orthologues. SmSirt5 is the only mitochondrial sirtuin encoded in the parasite genome (orthologues of Sirt3 and Sirt4 are absent) and transcripts corresponding to at least five splicing isoforms were identified. All five sirtuins are expressed throughout the parasite life-cycle, but with distinct patterns of expression. Sirtuin inhibitors were used to treat both schistosomula and adult worms maintained in culture. Three inhibitors in particular, Sirtinol, Salermide and MS3 induced apoptosis and death of schistosomula, the separation of adult worm pairs, and a reduction in egg laying. Moreover, Salermide treatment led to a marked disruption of the morphology of ovaries and testes. Transcriptional knockdown of <i>SmSirt1</i> by RNA interference in adult worms led to morphological changes in the ovaries characterized by a marked increase in mature oocytes, reiterating the effects of sirtuin inhibitors and suggesting that SmSirt1 is their principal target.</p><p>Conclusion, Significance</p><p>Our data demonstrate the potential of schistosome sirtuins as therapeutic targets and validate screening for selective sirtuin inhibitors as a strategy for developing new drugs against schistosomiasis.</p></div

Freshly perfused adult couples were maintained in culture for 6 days and incubated with 10 µM or 20 µM of Salermide (A), Sirtinol (B), MS3 (C), HR103 (D), MS13 (E) and CS13 (F).

<p>DMSO solvent was use as the control. Percentages of paired worm were determined daily. The results are expressed as mean % paired worms (± SEM, three independent experiments).</p

Effects of <i>SmSirt1</i> transcript knockdown in adult worms.

<p>RNA interference was carried out by the electroporation of adult worms with dsRNA for <i>SmSirt1</i> or <i>luciferase</i> (negative control) as described in the Methods section. (A) RNAi led to a reduction of about 80% in the level of transcripts for <i>SmSirt1</i> compared to controls knocked down for the irrelevant <i>luciferase</i> gene. (B) Microscopic examination was carried out 5 days after RNAi treatment as previously. This treatment caused no obvious morphological changes to male worm testes (B) compared to the control (A). Testes are indicated by red arrows. In contrast, RNAi treatment led to a marked increase in mature oocytes in the ovaries (D) compared to the control (C) particularly in the posterior part (MO). A mature oocyte in the anterior part of the ovary (IO), normally containing only immature oocytes (D) is arrowed. Results shown are representative of four independent experiments.</p

Total number of eggs laid during 5 days treatment with sirtuin inhibitors at 10 µM and 20 µM.

<p>Results obtained with (A) Salermide, (B) Sirtinol, (C) MS3, (D) HR103, (E) MS13 and (F) CS13 were expressed as mean % of eggs laid by treated worms compared to controls (± SD, three independent experiments). Statistical analyses were performed using student's t-test, and significance is displayed as follows: ** p<0.01, ***p<0.001.</p

Alignment of the protein sequences of <i>S.</i><i>mansoni</i> sirtuins catalytic domains with orthologues from other species.

<p>Sequences used in the alignment of sirtuin class I (Figure A) in addition to SmSirt1 and SmSirt2 are <i>Clonorchis sinensis</i> Sirt1 (CsSirt1 Accession No: GAA56043.1) Sirt2 (CsSirt2 GAA29763.1), <i>Homo sapiens</i> Sirt1 (HsSirt1: AAH12499.1) Sirt2 (HsSirt2: NP_036369.2), <i>Drosophila melanogaster</i> Sirt1 (DmSirt1: NP_477351.1) Sirt2 (DmSirt2: NP_650880.2), <i>Caenorhabditis elegans</i> Sirt1 (CeSirt1: NP_001255484.1) Sirt2 (CeSirt2: NP_001024673.1). Sequences used in the alignment of sirtuin class III (Figure B) in addition to SmSirt5 are <i>C. sinensis</i> Sirt5 (CsSirt5: GAA47482.1), <i>H. sapiens</i> Sirt5 (HsSirt5: NP_036373.1), <i>Aplysia californica</i> Sirt5 (AcSirt5: NP_001191422.1), <i>Camponotus floridanus</i> Sirt5 (CfSirt5: EFN62888.1). Sequences used in the alignment of sirtuin class IV (Figure C) in addition to SmSirt6 and SmSirt7 are <i>C. sinensis</i> Sirt7 (CsSirt7: GAA293.65.2), <i>H. sapiens</i> Sirt6 (HsSirt6: CAG33481.1) Sirt7 (HsSirt7: NP_057622.1), <i>Xenopus laevis</i> Sirt6 (XlSirt6: NP_001085592.1), <i>C. floridanus</i> Sirt6 (CfSirt6: EFN65179.1) Sirt7 (CfSirt7: EFN70308.1), <i>D. melanogaster</i> Sirt6 (DmSirt6: NP_649990.2) Sirt7 (NP_651664.2). Solid lines above the sequence alignment indicate which regions of the proteins compose the Rossmann-fold domain (red), cofactor binding loop (black), small domain (blue) and loop regions (green). Highlighted residues in the sequence alignment indicate which amino acids are involved in NAD+ binding (green), zinc binding (blue) and acetyl-lysine peptide binding (red).</p

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

<div><p>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 <i>S. mansoni</i> 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 <i>Smp14</i> 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 <i>Smp14</i> promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the <i>Smp14</i> 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 <i>Smp14</i> promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the <i>Smp14</i> promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of <i>Smp14</i> 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 <i>Smp14</i> 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 <i>Smp14</i> expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control <i>S. mansoni</i> egg development.</p></div