Date Published: December 7, 2015
Publisher: Public Library of Science
Author(s): R. Alan Wilson, Xiao Hong Li, Sandy MacDonald, Leandro Xavier Neves, Juliana Vitoriano-Souza, Luciana C. C. Leite, Leonardo P. Farias, Sally James, Peter D. Ashton, Ricardo DeMarco, William Castro Borges, José M. C. Ribeiro. http://doi.org/10.1371/journal.pntd.0004272
Abstract: BackgroundThe schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes.Methodology/Principal FindingsWe have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands.ConclusionsA proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets.
Partial Text: Adult schistosome worms reside in the host vascular system actively feeding on blood that contains antibodies, complement factors and effector leucocytes, yet they are apparently unaffected by this ‘toxic’ diet. Indeed, their attested longevity in the hepatic portal system (Schistosoma mansoni and S. japonicum) or the venous plexuses around the bladder (S. haematobium) illustrates the sophisticated yet poorly understood mechanisms they must deploy to evade the host immune response in such a hostile environment . The schistosome alimentary tract comprises an oral sucker around the mouth, a short esophagus and an extended gut caecum that runs to the extreme posterior . The caecum comprises a syncytial gastrodermis that is both secretory and absorptive, and an associated network of muscle fibres responsible for peristalsis. It occupies a larger proportion of body cross section in females (16%) than males (6%) , reflecting the disparate balance between nutrient uptake across the body surface and gut in the two sexes . The proteolytic enzymes responsible for breakdown of ingested proteins in the acidic environment of the gut lumen have been well researched [reviewed in 2]. In addition, a proteomic analysis of the vomitus released by worms in short term culture  has revealed the presence of other hydrolases, as well as ‘transport’ proteins capable of binding lipids (e.g. saposins) and inorganic ions (ferritin, calumenin). In vitro feeding experiments with labelled dextran have demonstrated the occurrence of endocytosis at the gastrodermal surface , while laser capture microdissection has been used to identify genes encoding transporters putatively expressed on the luminal surface of the gastrodermis .
Our aim in this study was to obtain an insight into those genes expressed in the distinctive tissues of the schistosome esophagus that encode the proteins involved in the initial processing of ingested blood. The difficulties in characterising patterns of gene expression that occur in the discrete organ systems of an acoelomate metazoan with a solid body plan should not be underestimated. Laser capture microdissection [5, 32, 33] has been applied but the amount of tissue obtained and the precision needed to excise the organ of choice without contamination, are major limitations. Moreover, the studies to date have used microarray analysis to detect differences in gene expression between tissues, a technique which has inherent limitations. The fixed design of the array, especially if coverage is partial , leaves gaps in the repertoire and furthermore does not permit new genes to be identified. The dynamic range of detection is also limited (typically a maximum of 200-fold), due to high background levels, cross hybridisation and saturation of signals .