13 research outputs found
Microexon gene transcriptional profiles and evolution provide insights into blood processing by the <i>Schistosoma japonicum</i> esophagus
<div><p>Background</p><p>Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a <i>Schistosoma japonicum</i> infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of <i>S</i>. <i>japonicum</i> esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process.</p><p>Methodology/Principal findings</p><p>The heads and a small portion of the rear end of male and female <i>S</i>. <i>japonicum</i> worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled <i>de novo</i> using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome <i>Trichobilharzia regenti</i>, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood.</p><p>Significance/Conclusions</p><p>The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.</p></div
Frame analysis of the feeding process in <i>S. mansoni</i>.
<p>Single frame from movies of active male worms feeding in vitro on a dilute suspension of erythrocytes. (A) Blood accumulating in the lumen of the anterior esophagus to form a bulge (AOB). (B) The deflated anterior esophagus is replaced by a bulge in the posterior (POB), as blood transits. (C) Ingested blood (IB, arrowed) entering the lumen of the esophageal gland (EG, outlined, based on >40 consecutive images) flows as dark line around the plug (P) of material as it passes to the transverse gut (TG). A and B filmed at ×10 magnification, C at ×40. Scale bars: A & B, 100 µm; C, 25 µm.</p
Gel shift of SmMEG-4.1 and its glycosylation.
<p>(A) 2D electrophoretic separation of head proteins after extraction. Arrows indicate actual position of Sm-MEG-4.1 protein while red square showed its predicted location. (B) Western blot of 2D gel probed with anti- SmMEG-4.1 antibody. Arrows indicate the position of the MEG protein. (C) Western blot of 2D gel probed with peanut agglutinin (PNA) reveals O-glycosylation of SmMEG-4.1 (arrowed). (D) Permeabilized whole worm reacted with FITC-labelled PNA showing reactivity of the esophageal gland (EG), nephridial canals (N) and flame cells (arrowed). Scale bar: 50 µm.</p
Three different views of esophageal cellular morphology.
<p>A–C, anterior esophagus; D–F, posterioresophageal gland. A and D show the whole region in optical section; B, C and E, F show only the respective esophageal linings at the much greater resolution provided by electron microscopy. (A) Confocal image of <i>S. japonicum</i> adult male stained with Langeron's carmine, showing arrangement of densely packed tegument cell bodies around the anterior esophageal lining; the musculature (M) is located as the dark line between the two. (L, Lumen; G, Central ganglion; TCB, tegument cell bodies). (B) SEM, anterior esophageal lining of <i>S. mansoni</i> showing its highly corrugated surface. (C) TEM, anterior esophageal lining of <i>S. mansoni</i> showing longitudinal orientation of the corrugations containing discoid bodies (arrowed and inset), typical of the tegument. (D) Confocal image of <i>S. japonicum</i> adult male stained with Langeron's carmine showing arrangement of cell bodies that comprise the esophageal gland around the posterior esophageal lining. (EGCB, esophageal gland cell bodies; PL, Plates; L, Lumen; M, Musculature; DJ, Desmosome junction between esophageal lining and gastrodermal epithelium.) (E) SEM of posterior esophageal lining of <i>S. mansoni</i> showing the luminal surface greatly extended to form thin plates (arrowed). (L, Esophageal lumen; LE, Leucocyte.) (F) TEM of the thin plates; a central double line (white arrows) is evident in each plate and discoid bodies (black arrows) are numerous. A single crystalloid vesicle (starred) is located close to a potential docking site. Scale bars: A, 50 µm; B, 1 µm; C, 500 nm; D, 50 µm; inset, 100 nm; E, 5 µm; F, 500 nm.</p
Host antibodies target antigens in the esophageal lumen of <i>S. mansoni</i>.
<p>Permeabilized female worms from mouse (A and B) and male worms from hamster (C and D) incubated with fluorescent-labelled antibodies (green) and counterstained with phalloidin (orange) to highlight the musculature. (A) Test with anti-mouse IgG. (B) Control with anti-hamster IgG. (C) Control with anti-mouse IgG. (D) Test with anti-hamster IgG. Strong specific antibody binding was only detected in the esophageal lumen (arrowed) of the relevant parasite, plus the gonopore region (G) of the female from mouse. Scale bars: A & B, 20 µm; C & D, 50 µm.</p
Erythrocyte processing in <i>S. mansoni</i>.
<p>(A) Confocal image of a feeding female worm fixed in vitro and stained with Langeron's carmine. Intact erythrocytes are visible within the oral cavity (OC) and the lumen (L) of the anterior esophagus. The erythrocytes can only be seen when the laser intensity is greatly amplified as they do not react well with the stain. (B) Material lying between esophageal plates (pl) of a male worm fixed in vivo, which typically has two distinct forms, small very electron dense granules (g) and looser striated bodies (sb). (C) High magnification TEM of the inter-plate aggregates showing the alternating light and dark striated material; the dark striations comprise two closely apposed layers (arrowed). (D) TEM of the posterior esophageal lumen from a male worm fixed in vivo. Three ghosts (gh) are present, showing different stages of haemoglobin leakage, plus two intact erythrocytes (ie). Scale bars: A, 10 µm; B, 500 nm; C, 100 nm; D, 5 µm.</p
Layout of the esophageal region and its musculature.
<p>(A) To-scale confocal images of <i>S. japonicum</i> adult male (left) and female (right) stained with Langeron's carmine, to illustrate the large discrepancy in size of their esophageal glands. (B) A longitudinal side view of a female <i>S. mansoni</i>, stained with phalloidin to show only the distribution of F-actin in muscles. The minute inner circular (ICM) and outer longitudinal muscle fibers (OLM) that invest the syncytial esophageal lining appear as a fine meshwork. In comparison the larger circular and longitudinal fibers of the body wall (BW) and oral sucker (S) are intensely stained. An oral sphincter (OS, in side view) comprising a stronger circular fiber is visible at the junction between oral cavity and esophagus; a posterior sphincter (PS) is present at the junction between the esophagus and the transverse gut (TG) (inset, en face view). Scale bars: A, 50 µm; B and inset, 10 µm.</p
Localisation of transcripts and proteins in the esophageal gland of whole permeabilised adult worms.
<p>A–D, males; F–I, females. (A and F) Low magnification images of SmMEG-4.1 localization revealed by WISH, to show the absolute specificity of gene expression in the esophageal gland (EG). (B and G) SmMEG-14 expression solely in the esophageal gland; (C and H) SmMEG-4.2 is expressed more strongly in the female than the male esophageal gland. (D and I) Localization of <i>S. japonicum</i> MEG-4.1 protein (green) and nuclei (blue) by immunocytochemistry, in the esophageal gland. (E) High magnification of esophageal gland cell bodies reveals the abundant sites of active SjMEG-4.1 synthesis and packaging (i.e. endoplasmic reticulum and Golgi; green). The small 0.3 µm dots (inset, arrowed) at the limit of resolution are probably individual crystalloid vesicles. Scale bars: A–D, F, 100 µm; G–I, 25 µm; E, 10 µm; Inset, 2 µm.</p
Head-enriched genes in female worms.
<p><b>A)</b> Scatter plot of differential gene expression in female heads and tails, based on the Trinity assembly of raw reads from HiSeq. Those genes differentially expressed >16-fold in the heads with a FPKM of >16 were considered for analysis. B) Differentially expressed genes encoding secreted or membrane proteins classified by category. C) A comparison of the expression level of selected genes in male and female heads. The correlation coefficient r between the two parameters = 0.83.</p
Putative functions of head-enriched genes.
<p>Differentially expressed head genes in the box in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006235#pntd.0006235.g001" target="_blank">Fig 1</a>, classified by category.</p