Quantitative Proteomics of Enriched Esophageal and Gut Tissues from the Human Blood Fluke Schistosoma mansoni Pinpoints Secreted Proteins for Vaccine Development

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can selfcure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion 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

The schistosome oesophageal gland: initiator of blood processing

Background: Although the ultrastructure of the schistosome esophageal gland was described .35 years ago, its role in the processing of ingested blood has never been established. The current study was prompted by our identification of MEG-4.1 expression in the gland and the observation of erythrocyte uncoating in the posterior esophagus. Methodology/Principal Findings: The salient feature of the posterior esophagus, characterized by confocal and electron microscopy, is the enormous increase in membrane surface area provided by the plate-like extensions and basal invaginations of the lining syncytium, with unique crystalloid vesicles releasing their contents between the plates. The feeding process was shown by video microscopy to be divided into two phases, blood first accumulating in the anterior lumen before passing as a bolus to the posterior. There it streamed around a plug of material revealed by confocal microscopy as tethered leucocytes. These were present in far larger numbers than predicted from the volume of the lumen, and in varying states of damage and destruction. Intact erythrocytes were detected in the anterior esophagus but not observed thereafter, implying that their lysis occurred rapidly as they enter the posterior. Two further genes, MEGs 4.2 and 14, were shown to be expressed exclusively in the esophageal gland. Bioinformatics predicted that MEGs 4.1 and 4.2 possessed a common hydrophobic region with a shared motif, while antibodies to SjMEG-4.1 showed it was bound to leucocytes in the esophageal lumen. It was also predicted that MEGs 4.1 and 14 were heavily O-glycosylated and this was confirmed for the former by 2D-electrophoresis and Western blotting. Conclusions/Significance: The esophageal gland and its products play a central role in the processing of ingested blood. The binding of host antibodies in the esophageal lumen shows that some constituents are antibody targets and could provide a new source of vaccine candidates.FAPEMIGCAPESShanghai Health Bureau - Overseas Public Health Training ProgrammeChina CDC - Young Scholar Scientific Research Foundatio

A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus

<p>Abstract</p> <p>Background</p> <p>Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of <it>Eucalyptus </it>using Single Feature Polymorphism (SFP) markers.</p> <p>Results</p> <p>SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. <it>In silico </it>validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the <it>Eucalyptus grandis </it>genome.</p> <p>Conclusions</p> <p>The <it>Eucalyptus </it>1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on <it>Eucalyptus </it>maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species.</p

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

Atmosfera modificada e 1-metilciclopropeno na conservação pós-colheita de kiwis cv. Bruno Modified atmosphere and 1-methylciclopropene on postharvest conservation of kiwis cv. Bruno

Neste trabalho avaliou-se, em kiwis da cv Bruno, a ação do 1-Metilciclopropeno (1-MCP), na concentração de 625 ppb, associado ou não à atmosfera modificada gerada com o emprego de embalagem de polietileno de baixa densidade (PEBD), de 22µm. Os tratamentos testados foram: T1, controle (sem embalagem e 1-MCP); T2, sem embalagem com 1-MCP; T3, com embalagem de PEBD sem 1-MCP; T4, embalagem de PEBD mais o 1-MCP, sendo após armazenados em câmara fria a - 0,5 ± 0,5 ºC e 95 ± 5% de umidade relativa (U.R.). As avaliações foram realizadas após o pré-resfriamento, aos 45 dias, 45 + 5 dias (22 ± 3ºC e 75 ± 5% de U.R.), 90 dias e 90 + 5 dias (22 ± 3ºC e 75 ± 5% de U.R.). As variáveis analisadas foram: firmeza de polpa (FP), sólidos totais (ST), acidez titulável (AT), concentração de etileno e de CO2 e análise sensorial (somente ao final do experimento). A maior firmeza de polpa e acidez titulável, o menor conteúdo de sólidos totais, as menores concentrações de etileno e CO2 e a melhor aceitabilidade pelos julgadores foi obtida com os frutos acondicionadas em PEBD de 22 µm e tratadas com 1-MCP.<br>In this work was evaluated, for kiwis cv. Bruno, the Methylcyclopropene action (1-MCP), in concentration of 625 ppb, associated or not with modified atmosphere (MA) using low density polyethylene bags (LDPE) of 22µm. The following treatments were tested: T1: control (no bags and 1-MCP); T2 no bags with 1-MCP; T3 LDPE bags no 1-MCP; T4 LDPE bags and 1-MCP, then they were stored in cold chamber at - 0,5 ± 0,5 ºC and 95 ± 5% of RH for 45 or 90 days. The fruits were evaluated after pre-cooling, 45 days, 45 + 5 days (22 ± 3ºC and 75 ± 5% of RH.), 90 days and 90 + 5 days (22 ± 3ºC and 75 ± 5% of RH.). The parameters analyzed were: pulp firmness (PF), total solids (TS), titritable acidity (TA), ethylene and CO2 concentration, and sensorial analyzes (only in the end of the experiment). The largest pulp firmness and titritable acidity, the smaller content of total soluble, the smaller concentration of ethylene and CO2 and the better acceptability for the taste panel was obtained with the fruits stored in LDPE of 22 µm and treated with 1-MCP

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

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