The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the β-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.

The 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. We 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. A 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. Schistosomes feed on blood and we have previously shown that its processing begins in the esophagus, which does not act simply as a conduit. It comprises anterior and posterior compartments, each surrounded by glands that secrete proteins into the lumen. Erythrocytes are ruptured as they pass through the compartments and leucocytes are tethered and killed but blood fails to clot. We wanted to identify the proteins secreted from these glands by sequencing the transcriptomes of head and tail preparations to pinpoint those messenger RNAs predominantly or exclusively present only in the heads. We found approximately 50 such proteins, the largest group of 27 being encoded by microexon genes. A second group comprised hydrolytic enzymes that operate at an acid pH. We showed by hybridisation experiments that expression of these genes is indeed localised to either the anterior or the posterior gland. We have suggested that this complex mixture of secreted proteins act together to perform the biological processes that occur in the lumen or, in the case of O-glycosylated membrane proteins, form a protective lining coat. We now want to discover which of them can serve as immune targets in infected animal hosts.

The Schistosoma mansoni Venom-Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in the host-pathogen interaction. Some of these molecules were suggested by us and others as potential immunomodulators and vaccine candidates, due to their functional classification, expression profile and predicted localization. From a vaccine perspective, one of the concerns is the potential allergic effect of these molecules. Herein, we characterized the putative secreted proteins SmVAL4 and SmVAL26 and explored the mouse model of airway inflammation to investigate their potential allergenic properties. The respective recombinant proteins were obtained in the Pichia pastoris system and the purified proteins used to produce specific antibodies. SmVAL4 protein was revealed to be present only in the cercarial stage, increasing from 0–6 h in the secretions of newly transformed schistosomulum. SmVAL26 was identified only in the egg stage, mainly in the hatched eggs' fluid and also in the secretions of cultured eggs. Concerning the investigation of the allergic properties of these proteins in the mouse model of airway inflammation, SmVAL4 induced a significant increase in total cells in the bronchoalveolar lavage fluid, mostly due to an increase in eosinophils and macrophages, which correlated with increases in IgG1, IgE and IL-5, characterizing a typical allergic airway inflammation response. High titers of anaphylactic IgG1 were revealed by the Passive Cutaneous Anaphylactic (PCA) hypersensitivity assay. Additionally, in a more conventional protocol of immunization for vaccine trials, rSmVAL4 still induced high levels of IgG1 and IgE. Our results suggest that members of the SmVAL family do present allergic properties; however, this varies significantly and therefore should be considered in the design of a schistosomiasis vaccine. Additionally, the murine model of airway inflammation proved to be useful in the investigation of allergic properties of potential vaccine candidates. The Schistosoma mansoni Venom Allergen Like proteins (SmVALs) have been identified in the Transcriptome and Post-Genomic studies as targets for immune interventions. Two secreted members of the family were obtained as recombinant proteins in the native conformation. Antibodies produced against them showed that SmVAL4 was present mostly in cercarial secretions and SmVAL26 in egg secretions and that only the native SmVAL4 contained carbohydrate moieties. Due to concerns with potential allergic characteristics of this class of molecules, we have explored the mouse model of airway inflammation in order to investigate these properties in a more confined system. Sensitization and challenge with rSmVAL4, but not rSmVAL26, induced extensive migration of cells to the lungs, mostly eosinophils and macrophages; moreover, immunological parameters were also characteristic of an allergic inflammatory response. Our results showed that the allergic potential of this class of proteins can be variable and that the vaccine candidates should be characterized; the mouse model of airway inflammation can be useful to evaluate these properties.