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

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

Genes differentially expressed in male heads encoding secretory proteins.

<p>Genes differentially expressed in male heads encoding secretory proteins.</p

Head-enriched genes in male worms.

<p>Scatter plot of differential gene expression in heads and tails based on the Trinity assembly of raw reads by MiSeq. Difference is defined as the ratio of head FPKM/tail FPKM and abundance as the FPKM for each gene. The box delimits the genes that are differentially expressed >8-fold in the heads with a FPKM of >16. Genes with an FPKM <8 are omitted from the dot plot.</p

Evolution of MEG-8/-9 family.

<p><b>A)</b> Multiple alignment of the residues of the sequence from MEG-8 and MEG-9 family members that displayed detectable similarity at the amino acid level. <b>B)</b> Phylogenetic tree of MEG-8 family members based on alignment of the conserved region. The tree was constructed using Bayesian inference implemented on MrBayes. Numbers next to each node indicate posterior probabilities. Nodes with posterior probability lower than 50% were collapsed and the tree was rooted at midpoint. <b>C)</b> Schematic representation of gene structure of a representative gene member of the MEG-8 and MEG-9 families. Exon boxes are proportional to their lengths in bp. Lines represent introns shown with a length not proportional to their size. Boxes in light and dark grey indicate exons coding for residues conserved in MEG-8 or in MEG-8 and MEG-9 families, respectively. The nucleotide and protein sequence at the 5’ boundary of the last exon and the adjacent intron is shown, with homologous residues aligned. The white box in the nucleotide sequence highlights the canonical splicing site.</p