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

Inhibition of sporozoite invasion <i>in vitro</i> by antibodies specific to rEtAMA1.

<p>(A) Representative flow cytometry charts used to determine the inhibitory activity of the antibody on labeled sporozoites of <i>E. tenella</i>. Left panel, non-infected cells; Middle panel, infected cells as a negative control; right panel: infection with sporozoites preincubated with inhibitory antibodies of rEtAMA1 at a concentration of 400 µg/mL. (B) Dose-dependent inhibition of sporozoite invasion by antibodies specific to rEtAMA1 and GST. The results are representative of three individual experiments, and the error bars indicate standard deviations. (*) According to the Student’s t-test, the differences between the treatment with anti-GST antibodies and anti-rEtAMA1 antibodies at the same IgG concentration were significant (P<0.05).</p

EtAMA1 mRNA expression levels in different developmental stages of <i>E. tenella</i> as revealed by SYBR Green quantitative real-time PCR.

<p>EtAMA1 mRNA levels in sporulated oocysts (SO), unsporulated oocysts (UO), sporozoites (SZ) and second-generation merozoites (MZ) were normalized to the 18S rRNA level of the corresponding stage. *P<0.05, n = 3.</p

Characterization of the EtAMA1 gene.

<p>(A) Multiple-sequence alignment of AMA1 proteins from <i>E. tenella</i> (Et), <i>E. maxima</i> (Em), <i>N. caninum</i> (Nc), <i>T. gondii</i> (Tg) and <i>P. falciparum</i> (Pf). Strictly conserved residues are indicated with a black background, and five different cysteine residues with species of <i>Plasmodium</i> in DIII are indicated with a gray arrowhead. The cleavage site of the EtAMA1 putative signal peptide is indicated by an arrowhead, and the transmembrane region is indicated by ( ); cysteine residues that formed disulfide bonds in EtAMA1 are indicated by domain (I, II and III) and bond (a, b and c) designations according to TgAMA1. (B) Phylogenetic tree of AMA1 proteins from <i>E. maxima</i> (Em), <i>E. tenella</i> (Et), <i>N. caninum</i> (Nc), <i>T. gondii</i> (Tg), <i>P. falciparum</i> (Pf), <i>P. vivax</i> (Pv), <i>B. bovis</i> (Bbo), <i>B. gibsoni</i> (Bg),and <i>P. bigemina</i> (Pbi).</p

Western Blotting analysis of EtAMA1 in different life stages of <i>E. tenella</i> probed with anti-rEtAMA1 antibodies.

<p>(A) The cytosol proteins of parasites. (B) The membrane proteins of parasites. Lanes: UO, unsporulated oocysts; SO, sporulated oocysts; SZ, sporozoites; MZ, second-generation merozoites.</p

EtAMA1 localization in infected ceca as visualized by immunohistochemistry analysis.

<p>Details of parasites immuno-stained with anti-rEtAMA1 antibodies, visualized with FITC (green) and counter-stained with DAPI (blue), while the normal ceca collected at the same time point as controls. (A) 24-h p.i., sporozoites (SZ); (B) 48-h p.i., first-generation schizonts (fSC); (C) 72-h p.i., immature second-generation schizonts (isSC); (D) 96-h p.i., second-generation schizonts (sSC); (E) 24-h p.i., normal ceca; (F) 48-h p.i., normal ceca; (G) 72-h p.i., normal ceca; (H) 96-h p.i., normal ceca; (I) 120-h p.i., third-generation schizonts (or gametocytes) (tSC or GC); (J) 144-h p.i., microgametocytes (miGC) and macrogametocytes (mrGC); (K) 144-h p.i., zygotes (ZG); (L) 168-h p.i., unsporulated oocysts (UO); (M) 120-h p.i., normal ceca; (N) 144-h p.i., normal ceca; (O) 168-h p.i., normal ceca; (P) 110-h p.i., second-generation merozoites purified from ceca (sMZ).</p

Differential Expression of microRNAs in the Non-Permissive Schistosome Host <i>Microtus fortis</i> under Schistosome Infection

<div><p>The reed vole <i>Microtus fortis</i> is the only mammal known in China in which the growth, development and maturation of schistosomes (<i>Schistosoma japonicum</i>) is prevented. It might be that the anti-schistosomiasis mechanisms of <i>M. fortis</i> associate with microRNA-mediated gene expression, given that the latter has been found to be involved in gene regulation in eukaryotes. In the present study, the difference between pathological changes in tissues of <i>M. fortis</i> and of mice (<i>Mus musculus</i>) post-schistosome infection were observed by using hematoxylin-eosin staining. In addition, microarray technique was applied to identify differentially expressed miRNAs in the same tissues before and post-infection to analyze the potential roles of miRNAs in schistosome infection in these two different types of host. Histological analyses showed that <i>S. japonicum</i> infection in <i>M. fortis</i> resulted in a more intensive inflammatory response and pathological change than in mice. The microarray analysis revealed that 162 miRNAs were expressed in both species, with 12 in liver, 32 in spleen and 34 in lung being differentially expressed in <i>M. fortis</i>. The functions of the differentially expressed miRNAs were mainly revolved in nutrient metabolism, immune regulation, etc. Further analysis revealed that important signaling pathways were triggered after infection by <i>S. japonicum</i> in <i>M. fortis</i> but not in the mice. These results provide new insights into the general mechanisms of regulation in the non-permissive schistosome host <i>M. fortis</i> that exploits potential miRNA regulatory networks. Such information will help improve current understanding of schistosome development and host–parasite interactions.</p> </div

Immunolocalization of SjGALE from adult male and female <i>S. japonicum</i>.

<p>Polyclonal anti-rSjGALE antibody was used as primary antibody and serum from naive mice was used as a negative control. Alexa Flour 488-conjugated (green) anti-mouse IgG was used as a second antibody. DAPI was used to stain nuclei (blue). (A): male adult worm. (C): male and female paring worms. (E): negative control. (B, D and F): DIC images of A, C, E, separately. (G): Detail of the image from panel (C) for detection of SjGALE (60× magnification).</p

Expression profiling of miRNAs in different tissues of <i>M. fortis</i> and mice following <i>S. japonicum</i> infection.

<p>A:Comparison of observed miRNA in liver, spleen and lung of <i>M. fortis</i> and mice; B-D: miRNA expression profile in different tissue of <i>M.fortis</i> and mice following <i>S.japonicum</i> infection. The left and right panel shows a heat-map of selected miRNAs that showed changes in expression in different tissue in <i>M. fortis</i> and mice following <i>S. japonicum</i> infection. B: differentially expressed miRNA in liver, C: differentially expressed miRNA in spleen, D: differentially expressed miRNA in lung. Clustering of the microarray showing the stastically significant(*p<0.05) miRNAs in different tissues of <i>M. fortis</i> compared with mice. Three replicates from each tissue cluster together. Both up-regulated (the red color ) and down-regulated (the green color) miRNAs from the mean were identified. Then Columns and rows represent samples and particular miRNAs.</p