TiO₂‑Based Phosphoproteomic Analysis of Schistosomes: Characterization of Phosphorylated Proteins in the Different Stages and Sex of <i>Schistosoma japonicum</i>

Protein phosphorylation is an important posttranslational modification in many organisms that regulates numerous cellular processes. However, it remains poorly characterized in schistosomes, the causative agent of schistosomiasis in humans and related animals. In the present study, we characterized phosphorylated proteins in different stages and sex of <i>Schistosoma japonicum</i> (<i>S. japonicum</i>) including schistosomula (14 days), adult females (35 days), and adult males (35 days) by a titanium dioxide (TiO₂) based phosphoproteomic method. A total of 180 phosphopeptides were identified in 148 proteins. Our further studies revealed that heat shock protein 90 (Hsp90), one of the phosphoproteins codetected in the different stage and sex of schistosomes, may play an important role in the regulation of schistosome development by directly or indirectly interacting with other codetected signal molecules. Additionally, some phosphoproteins were shown to be detected in a gender-specific manner, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time reverse transcription polymerase chain reaction (RT-PCR) at transcript levels between male and female schistosomes. In summary, these findings as well as the providing of an inventory of phosphoproteins are expected to provide new insights into schistosome development and sexual maturation and then may result in the development of novel interventions against schistosomiasis

TiO₂‑Based Phosphoproteomic Analysis of Schistosomes: Characterization of Phosphorylated Proteins in the Different Stages and Sex of <i>Schistosoma japonicum</i>

Protein phosphorylation is an important posttranslational modification in many organisms that regulates numerous cellular processes. However, it remains poorly characterized in schistosomes, the causative agent of schistosomiasis in humans and related animals. In the present study, we characterized phosphorylated proteins in different stages and sex of <i>Schistosoma japonicum</i> (<i>S. japonicum</i>) including schistosomula (14 days), adult females (35 days), and adult males (35 days) by a titanium dioxide (TiO₂) based phosphoproteomic method. A total of 180 phosphopeptides were identified in 148 proteins. Our further studies revealed that heat shock protein 90 (Hsp90), one of the phosphoproteins codetected in the different stage and sex of schistosomes, may play an important role in the regulation of schistosome development by directly or indirectly interacting with other codetected signal molecules. Additionally, some phosphoproteins were shown to be detected in a gender-specific manner, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time reverse transcription polymerase chain reaction (RT-PCR) at transcript levels between male and female schistosomes. In summary, these findings as well as the providing of an inventory of phosphoproteins are expected to provide new insights into schistosome development and sexual maturation and then may result in the development of novel interventions against schistosomiasis

Expression profile of SjTeg-20 and its localization in <i>S</i>. <i>japonicum</i>.

<p>(A) qRT-PCR analysis of the expression profiles of <i>SjTeg-20</i> at different stages of <i>S</i>. <i>japonicum</i> relative to <i>SjNADH</i>. Data illustrate representative results and show the mean and standard errors derived from triplicate experiments. (B) Immunohistochemistry analysis of SjTeg-20 expression in the tegument (arrows) of adult schistosomes. Scale bars: 50 μm.</p

SjIAP and SjTeg-20 synergistically regulate the apoptotic process and maintain the tegument architecture in <i>S</i>. <i>japonicum</i>.

<p>(A) qRT-PCR and Western blot analysis to investigate the effect of <i>SjTeg-20</i> silencing on the expression of SjIAP. qRT-PCR data are representative results and shown as the mean ± standard error from triplicate experiments. * <i>P</i> ≤ 0.05. (B) qRT-PCR and Western blot analysis of the effect of SjIAP silencing on the expression of SjTeg-20. qRT-PCR data are representative results and shown as the mean ± standard error from triplicate experiments. * <i>P</i> ≤ 0.05. (C) Co-silencing of SjIAP and SjTeg-20 significantly elevated caspase 3/7 activity determined from worm protein lysates. Data are representative results and shown as the mean ± standard error from triplicate experiments. ** <i>P</i> ≤ 0.01. (D) Effect of co-transfection of recombinant plasmids for expressing SjIAP and SjTeg-20 on the caspase activity in mammalian cells. HEK293T cells were treated with Cyclosporin A for 12 h and then transfected with recombinant plasmids expressing SjIAP and SjTeg-20 (2 μg). At 12 h post transfection, cells were collected for determining caspase 3/7 activity. Data are representative results and show the mean ± standard error from triplicate experiments. ** <i>P</i> ≤ 0.01. (E) Effect of co-transfection of the recombinant plasmids expressing SjIAP and SjTeg-20 on cell apoptosis in mammalian cells. HEK293T cells were treated with Cyclosporin A for 12 h, transfected with recombinant plasmids expressing SjIAP and SjTeg-20 (2 μg), stained with Annexin V-FITC kit, and subjected to flow cytometry. Data are representative results from triplicate experiments. (F) Quantitation of apoptosis cells transfected with recombinant plasmids expressing SjIAP and SjTeg-20 in HEK293T cells using flow cytometry. Data are representative result and shown the mean ± standard error from triplicate experiments. ** <i>P</i> ≤ 0.01. (G) TEM of morphological alterations in the tegument of <i>S</i>. <i>japonicum</i> treated with <i>SjIAP</i> siRNA and/or <i>SjTeg-20</i> siRNA. Data are representative results from at least 15 worms investigated in three independent experiments. Scale bars: 2 μM. (H) Quantification of tegument changes due to co-silencing SjIAP and SjTeg-20 shown in (G). The tegument defects were analyzed using Image J based on the ratio of the area occupied by the tegument cytoplasm to the total area of the tegument. Data show the mean and standard errors derived from four randomly selected parasites. ** <i>P</i> ≤ 0.01. Abbreviations: Mus-muscles; teg-surface layer of the tegument. (I) Co-silencing SjIAP and SjTeg-20 resulted in significantly increased worm mortality. At 4 days of post treatment, worms were stained with Hoechst 33258 dye, and the mortality was determined by microscopy. Data are representative results and shown as the mean ± standard error from triplicate experiments. ** <i>P</i> ≤ 0.01.</p

Effect of SjIAP silencing on caspase activity and worm survival.

<p>(A) Incubation of adult worms with <i>SjIAP</i> siRNA-951 <i>in vitro</i> led to a significant decrease of <i>SjIAP</i> expression at the transcript level. At 4 days of post-treatment, the RNA was isolated from the worms. Transcript levels of <i>SjIAP</i> relative to the reference gene <i>SjNADH</i> were determined in parasites transfected with <i>SjIAP</i> siRNA and control siRNA using qRT-PCR. The data illustrate representative results and show mean±SEM derived from triplicate experiments. ** <i>P</i> ≤ 0.01. (B) Western blot analysis of the effect of SjIAP silencing at the protein level. The densitometry results (arbitrary units [AU]) of the Western blot analyzed by Image J were shown as a bar graph. Each bar represents mean±SEM from triplicate experiments. Representative Western blot result was also shown. ** <i>P</i> ≤ 0.01. (C) SjIAP silencing resulted in increased caspase 3/7 activity. At 4 days of post-treatment, the protein lysates of worms were prepared and used to determine caspase 3/7 activity. Data are representative result and shown the mean and standard errors from triplicate experiments. ** <i>P</i> ≤ 0.01. (D) qRT-PCR analysis of the transcript levels of different caspases (caspase 2, caspase 3, and caspase 7) in SjIAP-silenced and control schistosomes determined relative to <i>SjNADH</i> at 4 days post-treatment. (E) SjIAP silencing led to an increased number of TUNEL⁺ cells in the tegument (arrows) of schistosomes at 4 days post-treatment. Data are representative results from at least 20 worms investigated in at least three independent experiments. (F) SjIAP silencing resulted in increased worm mortality. At 4 days post-treatment, the worms were stained with Hoechst 33258 dye, and the mortality was determined by microscopy. Data are representative and show the mean and standard errors from three separate experiments. ** <i>P</i> ≤ 0.01.</p

Silencing of SjIAP and SjTeg-20 decreased worm burden and egg deposition in mice <i>in vivo</i>.

<p>(A) Schematic showing the schedule for siRNA injection in mice. Each treatment included 6 mice (n = 6) (B) qRT-PCR analysis of <i>SjIAP</i> mRNA from surviving schistosomes isolated from mice injected with <i>SjIAP</i> siRNA by hydrodynamic tail vein injection, determined relative to <i>SjNADH</i>. Data are shown as the mean ± standard error and are representative results from triplicate experiments. ** <i>P</i> ≤ 0.01. (C) Effect of <i>SjIAP</i> siRNA injection on the worm burden in mice. At 14 days post infection, mice were administrated with <i>SjIAP</i> siRNA five times with one day interval. At 28 days of post infection, worms were perfused from mice, and the number of worms was counted. ** <i>P</i> ≤ 0.01. (D) Effect of <i>SjIAP</i> siRNA injection on egg deposition in the liver of mice. ** <i>P</i> ≤ 0.01. (E) qRT-PCR analysis of <i>SjIAP</i> mRNA levels from schistosomes isolated from mice injected with <i>SjIAP</i>/<i>SjTeg-20</i> siRNA by hydrodynamic tail vein injection in the biological replicates. Levels of <i>SjIAP</i> mRNA relative to <i>SjNADH</i> were analyzed by qRT-PCR in surviving worms used for RNA isolation. Data are shown as the mean ± standard error and representative results from triplicate experiments. * <i>P</i> ≤ 0.05 and ** <i>P</i> ≤ 0.01. (F) qRT-PCR analysis of <i>SjTeg-20</i> mRNA level from schistosomes isolated from mice injected with <i>SjIAP</i>/<i>SjTeg-20</i> siRNAs by hydrodynamic tail vein injection in the biological replicates. Levels of <i>SjTeg-20</i> mRNA relative to <i>SjNADH</i> were analyzed by qRT-PCR in surviving worms used for RNA isolation. Data are shown as mean ± standard error and representative results from triplicate experiments. ** <i>P</i> ≤ 0.01. (G) Effect of <i>SjIAP</i>/<i>SjTeg-20</i> siRNA injection on the worm burden in mice. At 14 days post infection, mice were administered with <i>SjIAP</i>/<i>SjTeg-20</i> siRNA as indicated above. At 28 days of post infection, worms were perfused from mice, and the number of worms was counted. * <i>P</i> ≤ 0.05 and ** <i>P</i> ≤ 0.01. (H) Effect of <i>SjIAP</i>/<i>SjTeg-20</i> siRNA injection on egg deposition in the liver of mice. * <i>P</i> ≤ 0.05 and ** <i>P</i> ≤ 0.01.</p

Effects of SjTeg-20 silencing on caspase activity, tegument destruction, and worm mortality.

<p>(A) Screening of the best siRNA duplex for inhibiting SjTeg-20. Three siRNA duplexes were electroporated into <i>in vitro</i> cultured schistosomes, respectively, and <i>SjTeg-20</i> mRNA levels were determined relative to <i>SjNADH</i> in <i>SjTeg-20</i> siRNA and control siRNA-treated worms at 4 days post electroporation by qRT-PCR. Data illustrate representative results and show the mean and standard errors derived from triplicate experiments. ** <i>P</i> ≤ 0.01. (B) Validation of the <i>SjTeg-20</i> siRNA-132 duplex for silencing SjTeg-20 using western blot analysis. The densitometry results (arbitrary units [AU]) of the Western blot analyzed by Image J were shown as a bar graph. Each bar represents mean±SEM from triplicate experiments. Representative Western blot result was also shown. ** <i>P</i> ≤ 0.01. (C) SjTeg-20 silencing resulted in morphological alterations in the tegument of <i>S</i>. <i>japonicum</i> as determined by SEM. Data are representative results from at least 15 worms investigated in three independent experiments. (D) SjTeg-20 silencing resulted in increased caspase 3/7 activity of cultured worms (28 d) <i>in vitro</i> which were electroporated with <i>SjTeg-20</i> siRNA-132. (E) SjTeg-20 silencing increased worm mortality based on Hoechst 33258 dye staining and microscopic observations. Data are representative results and shown as the mean ± standard error from triplicate experiments. ** <i>P</i> ≤ 0.01 (Student’s t test, <i>SjTeg-20</i> siRNA vs control siRNA).</p

SjIAP inhibition by siRNA led to morphological alterations in the tegument of <i>S</i>. <i>japonicum</i>.

<p>(A) Scanning electronic microscopy (SEM) of the tegument of <i>S</i>. <i>japonicum</i> following treatment with <i>SjIAP</i> siRNA. Tegument ridges and sensory structures were observed in the mid part of the worm body. Data are representative results from at least 20 worms investigated in three independent experiments. (B) Transmission electronic microscopy (TEM) of the tegument of <i>S</i>. <i>japonicum</i> following treatment with <i>SjIAP</i> siRNA. Data are representative results from at least 15 worms investigated in three independent experiments. Scale bars: 2 μM. (C) Quantification of tegument changes due to SjIAP silencing shown in (B). Tegument defects were analyzed using Image J software based on the ratio of the area occupied by the tegument cytoplasm to the total area of the tegument. Data show the mean and standard errors derived from four randomly selected parasites. ** <i>P</i> ≤ 0.01 (Student’s t test, <i>SjIAP</i> siRNA vs control siRNA).</p

Developmental expression and localization of SjIAP in <i>S</i>. <i>japonicum</i>.

<p>(A) Expression of <i>SjIAP</i> at different stages of the <i>S</i>. <i>japonicum</i> life cycles. Eggs were isolated from liver of rabbits infected with <i>S</i>. <i>japonicum</i> cercariae, cercariae were collected from infected <i>Oncomelania hupensis</i> snails, and the parasites were collected from infected rabbits at 7, 14, 21, 28, and 35 days post-infection, respectively. The mRNA expression levels of <i>SjIAP</i> relative to <i>SjNADH</i> were analyzed by qRT-PCR. Data illustrate representative results and show the mean and standard errors derived from triplicate experiments. (B) Immunohistochemistry analysis of SjIAP in adult schistosomes using an antiserum against SjIAP [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006654#pntd.0006654.ref021" target="_blank">21</a>]. Arrows indicate the schistosome tegument. Scale bars: 50 μm.</p

Performance of the SMWM.

<p>Performance of the SMWM.</p