Schistosomes are blood-dwelling helminth parasites causing a debilitating disease in the tropics. Major challenges to control the disease persist and vaccines would provide an additional tool, but their development has been problematic. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target schistosome proteins from the tegument, gut and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted comparative proteomics of the schistosome esophagus and gut for detection of secreted antigens. Shotgun proteome analysis of the male schistosomes esophagus identified 13 proteins encoded by microexon genes (MEG), eleven of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for absolute quantification of selected targets. MEGs 12, 4.2, 4.1 and Venom allergen-like protein 7 were the mostly abundant, spanning over 245-6 million copies per cell, while aspartyl protease, palmitoyl thioesterase and a galactosyl transferase were present at <1 million cpc. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialised machinery for protein glycosylation in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.

Based on state of the art QconCAT technology, a construct was designed by concatenating proteotypic peptides to obtain an artificial protein that served as the internal standard for simultaneous quantification of multiple antigens produced by the Schistosoma mansoni esophageal glands. Moreover, sequences consisting of three amino acids found naturally flanking the proteotypic peptides in the endogenous target proteins were preserved in the EsoCAT construct to provide conditions for both analyte and internal standard to be evenly digested. Four replicates with 20 µg of the esophageal (ESO) homogenate were spiked in with the purified EsoCAT protein, followed by trypsinolysis. In parallel, 20 μg digests containing only the ESO proteome were produced and used for isotope dilution by factors of 1:10 and 1:100. This ensured the EsoCAT internal standard spanned a concentration range of two orders of magnitude (~0.15-150 fmol/μg of ESO proteome). Absolute quantification was performed on a nanoACQUITY UPLC™ system coupled to a Xevo TQS triple quadrupole mass spectrometer (Waters) set to Selected Reaction Monitoring (SRM) acquisition mode, with Q1 and Q3 operating at unit mass resolution. Peptides were resolved on the analytical column HSS T3 nanoACQUITY C18 (1.8 µm, 75 µm x 150 mm; Waters) over a 90 min linear gradient of 3-40% (v/v) MeCN in 0.1% (v/v) formic acid, at a constant flow of 300 nl∙min-1, at 35 °C. SRM acquisition was scheduled with 4 min windows and dwell times adjusted to achieve ≥12 sampling points per peak. Quantification of endogenous and surrogate peptides was performed using Skyline v4.2 by integrating extracted ion chromatograms for 3-4 transitions, monitored in light and heavy channels, respectively . An equivalent number of transitions with random mass shifts (decoys) was also monitored allowing peak validation and FDR calculation by mProphet. Analyte quantification was finally achieved using the light to heavy ratios observed in the isotopic dilution that fulfilled the following criteria: (i) closest 1:1 ratio (never exceeding 10x fold) between the internal standard and endogenous analyte that exhibited a (ii) mProphet’s FDR ≤ 0.05 score in at least three out of four replicates.

Adult S. mansoni worms were obtained by portal perfusion of 45 day-infected mice using 10 mM HEPES buffered RPMI-1640 medium, pH 7.4, containing 4 IU/ml of heparin. After extensive washes in pre-warmed medium (37 °C), parasites were instantly fixed in RNAlater® solution and stored at 4 °C. Adult male worms immersed in ice-cold RNAlater® were carefully held and dissected using fine curved tweezers (Ideal Tek) and Vannas scissors (John Weiss), under a stereomicroscope at x35 magnification. First, the oral sucker was removed by an incision at its posterior, at the very beginning of the esophagus. Next, a second cut along the line of the transverse gut released the esophageal fragment (ESO). The detailed and illustrated dissection procedure is uploaded at protocols.io repository (https://www.protocols.io/) and accessible via DOI dx.doi.org/10.17504/protocols.io.tq2emye. ESO fragments were washed twice in 1 mL of ice-cold PBS, pH 7.4, to remove RNAlater. Samples were sonicated in an ice bath for 5 cycles of 10 second pulses at 30% amplitude , with 50 seconds intervals. Total protein concentration was determined using a Coomassie Plus (Bradford) assay kit (Thermo Scientific) by interpolation from a BSA standard curve, according to the manufacturer’s instructions. Sample aliquots equivalent to 20 µg of protein were transferred to LoBind tubes (Eppendorf) and made up to 100 µL by the addition of 25 mM ammonium bicarbonate (AMBIC). Protein denaturation was induced by the addition of 20 µL of RapiGest SF 1% w/v (Waters) and heating at 80 °C for 10 min. Next, sample volume was adjusted to 180 µL with AMBIC before reduction (addition of 10 µL 60 mM dithiothreitol, 60 °C for 10 min) and alkylation (addition of 10 µL 180 mM iodoacetamide and incubation at room temperature for 30 min). MS-grade trypsin (Promega) was added at an enzyme:substrate ratio of 1:50 and digestion allowed to occur at 37 °C for 16 h. Trypsinolysis was terminated by addition of 1.5 µL trifluoroacetic acid. Acidified samples (pH<2) were then incubated at 37 °C for 45 min to precipitate RapiGest SF, which was later removed by centrifugation at 13,000g for 15 min at 7 °C. Supernatant was recovered for LC-MS analysis.