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. 2018 Jun 28;14(6):e1007107.
doi: 10.1371/journal.ppat.1007107. eCollection 2018 Jun.

Methyl-CpG-binding (SmMBD2/3) and Chromobox (SmCBX) Proteins Are Required for Neoblast Proliferation and Oviposition in the Parasitic Blood Fluke Schistosoma Mansoni

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Methyl-CpG-binding (SmMBD2/3) and Chromobox (SmCBX) Proteins Are Required for Neoblast Proliferation and Oviposition in the Parasitic Blood Fluke Schistosoma Mansoni

Kathrin K Geyer et al. PLoS Pathog. .
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While schistosomiasis remains a significant health problem in low to middle income countries, it also represents a recently recognised threat to more economically-developed regions. Until a vaccine is developed, this neglected infectious disease is primarily controlled by praziquantel, a drug with a currently unknown mechanism of action. By further elucidating how Schistosoma molecular components cooperate to regulate parasite developmental processes, next generation targets will be identified. Here, we continue our studies on schistosome epigenetic participants and characterise the function of a DNA methylation reader, the Schistosoma mansoni methyl-CpG-binding domain protein (SmMBD2/3). Firstly, we demonstrate that SmMBD2/3 contains amino acid features essential for 5-methyl cytosine (5mC) binding and illustrate that adult schistosome nuclear extracts (females > males) contain this activity. We subsequently show that SmMBD2/3 translocates into nuclear compartments of transfected murine NIH-3T3 fibroblasts and recombinant SmMBD2/3 exhibits 5mC binding activity. Secondly, using a yeast-two hybrid (Y2H) screen, we show that SmMBD2/3 interacts with the chromo shadow domain (CSD) of an epigenetic adaptor, S. mansoni chromobox protein (SmCBX). Moreover, fluorescent in situ hybridisation (FISH) mediated co-localisation of Smmbd2/3 and Smcbx to mesenchymal cells as well as somatic- and reproductive- stem cells confirms the Y2H results and demonstrates that these interacting partners are ubiquitously expressed and found within both differentiated as well as proliferating cells. Finally, using RNA interference, we reveal that depletion of Smmbd2/3 or Smcbx in adult females leads to significant reductions (46-58%) in the number of proliferating somatic stem cells (PSCs or neoblasts) as well as in the quantity of in vitro laid eggs. Collectively, these results further expand upon the schistosome components involved in epigenetic processes and suggest that pharmacological inhibition of SmMBD2/3 and/or SmCBX biology could prove useful in the development of future schistosomiasis control strategies.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. SmMBD2/3 contains amino acid residues critical for binding to 5mC templates.
(A) Diagrammatic representation of the 314 amino acid SmMBD2/3 (encoded by Smp_138180) illustrating the methyl-CpG (mCpG) binding domain (PF01429), two putative predicted bipartite nuclear localisation signals (NLS), a coiled-coil domain (CC) and a C-terminal domain of methyl CpG binding protein 2 and 3 (PF140489). Amino acid positions are indicated in bold numbering. (B) A multiple sequence alignment of the methyl-CpG (mCpG) binding domains (PF01429) collected from SmMBD2/3 (italics and contained in a blue box) and MBD homologs was generated. MBDs unable to bind 5mC are indicated in red. Highly conserved residues are highlighted in turquoise and moderately conserved residues are shaded grey. A ‘*’ indicates amino acid residues that contribute to 5mC binding as assessed by mutational studies (summarised in Table 1). A ‘#’ signifies additional amino acid residues that directly interact with 5mC and a ‘:’ indicates amino acid residues that interact with the DNA phosphate backbone [21]. Amino acid insertions in AmMBD1 and DmMBD2/3 are indicated in black boxes above and below the alignment, respectively. AmMBD1: XP_003250634.1, HsMBD1: NP_002375.1, MmMBD1: NP_038622.2, HsMeCP2: NP_001104262.1, MmMeCP2: NP_001075448.1, XlMeCP2: NP_001081854.1, HsMBD4: NP_001263201.1, MmMBD4: NP_034904.2, HsMBD2: NP_003918.1, MmMBD2 NP_034903.2, GgMBD2: NP_001012403.1, HsMBD3: NP_001268382.1, MmMBD3: NP_038623.1, XlMBD3: AAD55389.1, BmMBD2/3: XP_004929675.1, ApMBD2/3: ACF05483.1, HpMBD2/3: ACF05485.1, AdMBD2/3: [25], SmMBD2/3: CCD59176.1, DmMBD2/3: NP_731370.1. (C) Ribbon representation of SmMBD2/3 homology model (green) depicts 5mC-interacting residues (stick representation: amino acids in red, 5mC in orange) found within PF01429. The SmMD2/3 model was generated using the solved crystal structure of G. gallus MBD2 co-complexed with methylated DNA as detailed in the Materials and Methods. The tetra-amino acid archetypal binding pocket is indicated (red).
Fig 2
Fig 2. SmMBD2/3 is preferentially localised to nuclei in transfected NIH-3T3 fibroblasts.
(A) Representative NIH-3T3 fibroblast cells transfected with full-length SmMBD2/3-FLAG and RFP-FLAG constructs are illustrated. Scale bar = 10 μm. DAPI represents transfected cells visualised with a 405 nm blue diode laser; SmMBD2/3 represents SmMBD2/3-FLAG transfected cells visualised with a 488 nm argon laser; RFP represents RFP-FLAG transfected cells visualised with a 561 nm diode-pumped, solid state laser; Merge represents cellular superimposition of DAPI/SmMBD2/3-FLAG or DAPI/RFP signals. (B) Relative SmMBD2/3-FLAG and RFP-FLAG nuclear (Nuc) vs cytoplasmic (Cyto) fluorescence was quantified from LSCM images collected from a total of 40 transfected cells/construct. SmMBD2/3-FLAG was found to be significantly enriched within nuclear compared to cytoplasmic compartments (p < 0.05). SmMBD2/3-FLAG nuclear localisation was also found to be significantly greater than RFP-FLAG nuclear localisation in the transfected cells (p < 0.01).
Fig 3
Fig 3. Schistosome nuclear protein extracts and recombinant SmMBD2/3 both contain 5mC binding activities.
(A) The 5mC binding capacity of nuclear protein extracts derived from adult male and female worms were quantified using the Epigentek MBD2 binding activity/inhibition assay. NIH-3T3 nuclear protein extracts and BSA were included as positive and negative controls, respectively. A significant difference in 5mC binding (in the CpG context) amongst protein samples was found. (B) IPTG-induced rSmMBD2/3-His6 protein (arrowhead; 36.5 kDa) was produced in E. coli, purified by Ni2+-NTA column chromatography and subjected to MALDI-TOF MS (Materials and Methods; 22 peptides covering 67% of full length SmMBD2/3 identified). An un-induced sample was also produced and similarly processed. (C) The 5mC binding activity (within a CpG context) of purified rSmMBD2/3-His6 was measured using the Epigentek MBD2 binding activity/inhibition assay and compared to un-induced bacterial and BSA protein samples. Significant differences in 5mC binding were observed between rSmMBD2/3-His6 and both the BSA and un-induced samples.
Fig 4
Fig 4. SmMBD2/3 interacts with the nuclear chromobox protein SmCBX (Smp_179650).
(A) A truncated version of Smp_179650 (delta 1–160) was repeatedly (5/14 times or 36% of all hits; S2 Table) identified as an interacting partner of SmMBD2/3 in Y2H assays. This truncated version of Smp_179650 contained the chromo shadow domain (CSD; blue oval, PF01393), a region associated with protein-protein interactions [50]. Full-length Smp_179650 also contains the chromodomain (CD; yellow rectangle, PF00385) and a monopartite nuclear localisation signal (NLS, 109VPEPAKKKRTS119). Amino acid positions are indicated (bold numbers). (B) The SmMBD2/3 –SmCBX (Δ1–160) interaction strength was quantified using the X-β-gal based (PXG) assay [35]. Experimental controls included: p53 + SV40 large T antigen (positive) and SmMBD2/3 + pGADT7 (empty prey vector), pGBKT7 (empty bait vector) + SmCBX/ Δ1–160, pGBKT7 + pGADT7 (all negative). (C) DNA microarray analysis of Smcbx expression throughout 15 lifecycle stages. Bar chart represents normalised mean fluorescent intensities + standard deviation (n = 3 replicates/lifecycle stage except adult female, where n = 2) of Smcbx transcript abundance derived from oligonucleotide CONTIG6649 as described previously [36]. Inset drawing represents SmCBX (Smp_179650) gene organisation (4 exons–yellow boxes; 3 introns–black lines) and localisation of oligonucleotide CONTIG6649 to exon 3 (SchistoGeneDB v5.2).
Fig 5
Fig 5. Smmbd2/3 and Smcbx are broadly expressed in male and female schistosomes.
(A) Expression of Smmbd2/3 and Smcbx in male somatic tissues, (B) testes and (C) ovaries relative to Smhistone H2B. Both genes are broadly expressed in somatic tissues, including the histone H2B+ neoblasts and in most cell types (histone H2B-) within the male and female germ line. Scale bars = 20 μm. Blue = DAPI. Magenta and green = pseudocoloured antisense RNA probes for Smmbd2/3, Smcbx and Smhistone H2B. White = co-localisation of two RNA probes. Where illustrated, delineated areas (dashed white boxes) are magnified in the insets (solid white boxes).
Fig 6
Fig 6. RNAi-mediated knockdown of Smmbd2/3 and Smcbx affects the proliferation of schistosome stem cells.
(A) Seven-week old adult male and female schistosomes were electroporated with 5 μg siRNA duplexes targeting luciferase (siLuc), Smcbx (siSmcbx) or Smmbd2/3 (siSmmbd2/3). Following 48 hr, total RNA was harvested and subjected to qRT-PCR. Percent knockdown (KD) and statistical significance (Student’s t test, two tailed, unequal variance) is indicated. All siRNA and qRT-PCR DNA sequences are included in S1 Table. (B) Representative anterior ends and ovaries of female schistosomes treated with siRNA duplexes at day seven post treatment. Blue = DAPI; Green = EdU+ cells. Bar = 50 μM. Column scatter plot (horizontal bars = mean and +/- StDev of mean) represents the percentage of proliferating cells remaining in female worms treated with siRNA duplexes for seven days (siLuc, n = 11; siSmcbx, n = 11; siSmmbd2/3 = 12). The percentage of proliferating cells affected by knockdown (in comparison to siLuc control worms) is indicated where significant (one-way ANOVA followed by Tukey HSD test).
Fig 7
Fig 7. SmMBD2/3 and SmCBX are both required for schistosome oviposition.
(A) Seven-week old adult male and female schistosome pairs were electroporated with 5 μg siRNA duplexes targeting luciferase (siLuc), Smcbx (siSmcbx) or Smmbd2/3 (siSmmbd2/3). At day 7 post treatment, eggs were collected from wells (5 worm pairs/well; n = 3) and counted. A one-way ANOVA followed by Tukey HSD test was performed to identify statistically significant treatments. (B) Percentage of eggs (n = 14 for siLuc, n = 20 for siSmcbx, n = 15 for siSmmbd2/3) demonstrating abnormal (grey) versus normal (black) phenotypes. Normal = oval eggs with lateral spine, containing regular surface autofluorescence. (C) Representative fluorescent images of eggs collected from wells of siLuc, siSmcbx and siSmmbd2/3 treated worm pairs. Green = eggshell autofluorescence; blue = DAPI+ cells. Bar = 20 μm.

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