Figure 1.
Smp14 promoter-dependent activation by the SmRXR1/SmNR1 heterodimer and the HATs SmGCN5 and SmCBP1.
The luciferase gene reporter assays were performed in HEK293 cells. The Smp14 promoter-target sequence was cloned upstream of the luciferase reporter gene in the vector pGL4.23 (Smp14/3X-pGL4.23). This vector was co-transfected with the SmRXR1/SmNR1 heterodimer and/or co-transfected with SmCBP1 or SmGCN5 (A–H). The HDAC inhibitors NaB (5 mM) and TSA (2 µM) were tested (B). The dose-dependent transcriptional activation of the Smp14 promoter by SmCBP1 or SmGCN5 is shown (C and D). Small synthetic HAT inhibitors (2 µM) were tested in the transfected cells (E and F): PU139 (a), PU141 (b), SF7 (c), SF18 (d) and SF19 (e). Dose-dependent inhibition (0.5 nM, 1 µM and 2 µM) of SmCBP1 and SmGCN5 by the most potent inhibitor, PU139 (G and H). Results were plotted in relation to the firefly luciferase activity obtained from cells transfected with the Smp14/3X-pGL4.23 vector alone. Graphs are pooled from three independent experiments. Student's t-test was applied, with *p<0.05, **p<0,01, ***p<0.001 and ***p<0.001. Western blot data are representative of three independent experiments.
Figure 2.
HAT inhibition impairs Smp14 transcription and translation in S. mansoni adult worms.
Ten adult worm pairs were cultivated with increasing concentrations of the HAT inhibitor PU139 or DMSO as vehicle. PU139 concentrations between 0.01 and 20 µM were solubilized in 0.1% DMSO, and 50 µM PU139 was only soluble in 0.5% DMSO. (A) qRT-PCR analysis of Smp14 mRNA levels after a two-day incubation time. (B) Western blot analysis of total protein extract from a two-day worm pair culture using polyclonal antibodies against Smp14 (upper panels) and monoclonal antibodies against α-tubulin (lower panels) as loading control. (C) qRT-PCR of Smp14 after a longer period (four days) of PU139 (20 µM) treatment. (D) Smp14 protein levels at the fourth day of treatment with 20 µM PU139. Graphs are pooled from three independent experiments. Student's t-test was applied, with *p<0.05 and ***p<0.001. Western blot data are representative of three independent experiments.
Figure 3.
PU139 can reverse histone acetylation, keeping the Smp14 promoter in a repressed state.
Ten adult worm pairs were cultivated with 20 µM PU139 or vehicle for 48 h. (A) Western blot analysis of total worm extract. The levels of S. mansoni acetylated histone H3 and H4 are shown. (B–E) ChIP analysis of S. mansoni histone modifications at the Smp14 promoter. Fifty worm pairs were treated with 20 µM PU139 or vehicle at different time points and submitted to chromatin extraction. Chromatin was immunoprecipitated with antibodies directed against acetylated H3 and H4, RNA pol II (markers of transcriptionally active chromatin) and H3K27me3 (a marker of repressed chromatin). ChIP DNA (Smp14 promoter) was quantified by real-time PCR and normalized as a percentage of input DNA. Results are pooled from four independent experiments. Student's t-test was applied, with *p<0.05, ***p<0.001 and ****p<0.0001.
Figure 4.
HAT inhibition affects the normal development of S. mansoni eggs.
Ten adult worm pairs were cultivated with 20 µM PU139 or vehicle up to four days, and the number of eggs was counted on a daily basis (A). The estimated areas (length and width) of these same eggs were measured (B). Student's t-test was applied, with ***p<0.001. The morphology of the eggs was analyzed under optical microscopy. Scale bars: 10 µm (C). Adult worm pairs under the same treatment were fixed and stained with hydrochloric carmine for confocal laser scanning microscopy (CLSM) analysis. ne: normal egg; ae: abnormal egg; ot: ootype; vc: vitelline cells; u: uterus. The arrowhead in “ae” points to a fissure. tt: tegument tubercles. Scale bars: 20 µm (D).
Figure 5.
HAT inhibition compromises eggshell integrity.
(A) Scanning electron microscopy of eggs laid by worm pairs cultivated for two days with vehicle (panels a and b) or 20 µM PU139 (panels c, d, e and f). A normal S. mansoni egg is shown (panel a) with the typical smooth coat of microspines on the eggshell (Es, panel b) seen at higher magnification (boxed area in panel a). PU139 treatment severely affected eggshell formation and integrity (panel c). Gross structural defects on the eggshell can be visualized at higher magnification (panel d; NEs: normal eggshell, AEs: abnormal eggshell). A remarkable phenotypic defect observed in eggs laid by PU139-treated parasites was the presence of holes in the eggshell (arrows in panel e). At a higher magnification, it can be clearly observed that, besides the holes (panel e and f, arrows), a large fissure in the eggshell was formed (panel f, F), leading to leakage of egg contents (panel f, arrowheads). Scale bars 10 µm. (B). Transmission electron microscopy of eggs laid by worm pairs cultivated for two days with vehicle (panels a, c and e) or 20 µM PU139 (panels b, d, and f). Normal eggs (panels a, d and e) show a thick and continuous eggshell (Es). The characteristic microspines (ms) of S. mansoni eggs are indicated by the arrows. The eggshells of the eggs laid by PU139-treated females (panels b, d and f) revealed remarkable differences in their structure, when compared with the control eggs, showing much thinner and discontinuous (arrows) eggshells. The holes in the eggshells were confirmed by TEM.
Figure 6.
HAT inhibition compromises the reproductive system of female worms.
Ten adult worm pairs were cultivated with 20 µM PU139 (panels b, d and f) or vehicle (panels a, c and e) for two days. The worms were fixed and stained with Certistain (Merck) for confocal laser scanning microscopy (CLSM) analysis. ne: normal egg; ae: abnormal egg; ot: ootype; ov: ovary; mo: mature oocytes; io: immature oocytes; vd: vitelline ducts; vc: vitelline cells; rs: receptaculum seminis. The PU139 treatment provoked several negative phenotypes: 1 - the morphology of the ovaries and oocyte maturation (panel b); 2 - the vitellaria size and the number of vitelline cells (panel d); 3 – The vitelline duct and ootype depleted of vitelline cells (panel f). Scale bars: 20 µm.
Figure 7.
SmCBP1 and SmGCN5 play a key role in Smp14 expression and female sexual reproductive development.
A dsRNAi experiment was carried out on sixteen adult worm pairs cultivated for seven days. Worms were electroporated and soaked with dsRNAi from SmCBP1 or SmGCN5 or with the non-specific dsRNAi LUC. On the seventh day of culture, the mRNA levels of SmCBP1, SmGCN5 or Smp14 (graphs A and B) were determined by qRT-PCR, normalized by the α-tubulin transcript levels. The results are depicted in relation to the non-specific dsRNAi (dsLUC) mRNA levels. The effects of the dsRNAi on targeted-protein synthesis were assessed by Western blot (panels C and D). The total number of eggs laid by the same parasites that received the dsRNAi was counted (graphs E and F). Adult worms from the dsRNAi experiments were analyzed by confocal laser scanning microscopy (G). Worms received either the control dsRNAi LUC (panels a and d) or the dsRNAi for SmCBP1 (panels b and e) or SmGCN5 (panels c and f). Panels a–c, and d–f, reveal the effect of the dsRNAi in the ovary, and vitellaria, respectively. OV: ovary; mo: mature oocytes; io: immature oocytes; v: vitellaria; rs: receptaculum seminis. Results are pooled from three independent experiments. Western blots were repeated three times. The confocal microscopy images are representative of several parasites analyzed. Scale bars 10 µm. Student's t-test was applied, with *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Figure 8.
Proposed model of the acetylation-dependent activation of Smp14 transcription and eggshell formation.
The nuclear receptor heterodimer SmRXR1/SmNR1 binds to a specific DNA response element present in the Smp14 promoter [14] and recruits the two histone acetyltransferases SmGCN5 and SmCBP1. Chromatin is then remodeled, RNA Pol II is attracted to the promoter and transcription occurs. Synthesis of Smp14 proteins will lead to eggshell formation, which is a prerequisite for egg dissemination and granuloma formation. Interfering with the histone acetyltransferase activities of either SmCBP1 or SmGCN5 reverses chromatin decondensation, with no access for RNA Poll II. Transcription of the Smp14 gene is halted, compromising eggshell integrity, which can ultimately lead to a reduction in the transmission and immunopathology of schistosomiasis.