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Background Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusion Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana. Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing. We detected 3423 proteins, where 1543 and 113 proteins showed significant changes in abundance in N and P treatments, respectively. The analysis includes the global correlation between proteomic and transcriptomic data, the regulation of subcellular proteomes in different compartments, gene/protein functional groups, and metabolic pathways. The results show that triacylglycerol (TAG) accumulation under nitrogen deprivation was associated with substantial downregulation of protein synthesis and photosynthetic activity. Oil accumulation was also accompanied by a diverse set of responses including the upregulation of diacylglycerol acyltransferase (DGAT), lipase, and lipid body associated proteins. Deprivation of phosphorus had comparatively fewer, weaker effects, some of which were linked to the remodeling of respiratory metabolism.

The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion.

Peptides present in the seminal fluid of Drosophila melanogaster can function as antimicrobial agents, enzyme inhibitors and as pheromones that elicit physiological and behavioural responses in the post-mated female. Understanding the molecular interactions by which these peptides influence reproduction requires detailed knowledge of their molecular structures. However, this information is often lacking and cannot be gleaned from just gene sequences and standard proteomic data. We now report the native structures of four seminal fluid peptides (andropin, CG42782, Met75C and Acp54A1) from the ejaculatory duct of male D. melanogaster. The mature CG42782, Met75C and Acp54A1 peptides each have a cyclic structure formed by a disulfide bond, which will reduce conformational freedom and enhance metabolic stability. In addition, the presence of a penultimate Pro in CG42782 and Met75C will help prevent degradation by carboxypeptidases. Met75C has undergone more extensive post-translational modifications with the formation of an N-terminal pyroglutamyl residue and the attachment of a mucin-like O-glycan to the side chain of Thr4. Both of these modifications are expected to further enhance the stability of the secreted peptide. The glycan has a rare zwitterionic structure comprising an O-linked N-acetyl hexosamine, a hexose and, unusually, phosphoethanolamine. A survey of various genomes showed that andropin, CG42782, and Acp54A1 are relatively recent genes and are restricted to the melanogaster subgroup. Met75C, however, was also found in members of the obscura species groups and in Scaptodrosophila lebanonensis. Andropin is related to the cecropin gene family and probably arose by tandem gene duplication, whereas CG42782, Met75C and Acp54A1 possibly emerged de novo. We speculate that the post-translational modifications that we report for these gene products will be important not only for a biological function, but also for metabolic stability and might also facilitate transport across tissue barriers, such as the blood-brain barrier of the female insect. BIOLOGICAL SIGNIFICANCE: Seminal fluid peptides of D. melanogaster function as antimicrobials, enzyme inhibitors and as pheromones, eliciting physiological and behavioural responses in the post-mated female. A fuller understanding of how these peptides influence reproduction requires knowledge not only of their primary structure, but also of their post-translational modification. However, this information is often lacking and difficult to glean from standard proteomic data. The reported modifications, including the unusual glycosylation, adds much to our knowledge of this important class of peptides in this model organism, par excellence.

The production of Parma dry-cured ham involves the steps of salting, drying, and ripening. Although sea salt is the only preserving agent, there are strategies being developed with the goal of reducing salt content in order to decrease its negative impact on consumer health. A 24 h pressure treatment was applied before salting to reduce thickness and inequalities in shape. To evaluate the potential impact of the pressure step on the process outcome, differential proteomic analyses by complementary 2D-PAGE and LC-MS/MS were carried out on exudates collected at day 1, 5, and 18 of the salting phase for hams treated or untreated with pressure. Specific proteins were found differentially abundant in exudates from pressed vs unpressed hams and as a function of time. These changes include glycolytic enzymes and several myofibrillar proteins. These findings indicate that pressure causes a faster loosening of the myofibrillar structure with the release of specific groups of proteins.

Diagnostic classification accuracy is critical in expression proteomics to ensure that as many true differences as possible are identified with acceptable false-positive rates. We present a comparison of the diagnostic accuracy of iTRAQ with three label-free methods, peak area, spectral counting, and emPAI, for relative quantification using a spiked proteome standard. We provide the first validation of emPAI for intersample relative quantification and find clear differences among the four quantification approaches that could be considered when designing an experiment. Spectral counting was observed to perform surprisingly well in all regards. Peak area performed best for smaller fold differences and was shown to be capable of discerning a 1.1-fold difference with acceptable specificity and sensitivity. The performance of iTRAQ was dramatically worse than the label-free methods with low abundance proteins. Using the iTRAQ data set for validation, we also demonstrate a novel iTRAQ analysis regime that avoids the use of ratios in significance testing and outperforms a common commercial alternative.

The secretome of a parasite in its definitive host can be considered to be its genome in trans, to the extent that secreted products encoded by the parasite fulfill their function in the host milieu. The 'extended phenotype' of the filarial parasite, Brugia malayi, is of particular interest because of the evidence that infection results in potent down-modulation of the host immune response. We collected B. malayi 'excretory-secretory' (BES) proteins from adult parasites and using a combination of shotgun LC-MS/MS and 2D gel electrophoresis, identified 80 B. malayi and two host proteins in BES, of which 31 (38%) were detectable in whole worm extract (BmA). Products which were enriched in BES relative to BmA included phosphatidylethanolamine-binding protein (PEB), leucyl aminopeptidase (LAP, homologue of ES-62 from the related filaria Acanthocheilonema viteae), N-acetylglucosaminyltransferase (GlcNAcT) and galectin-1, in addition to the previously described major surface glycoprotein, glutathione peroxidase (gp29, GPX-1) and the cytokine homologue macrophage migration inhibitory factor (MIF-1). One of the most abundant released proteins was triose phosphate isomerase (TPI), yet many other glycolytic enzymes (such as aldolase and GAPDH) were found only in the somatic extract. Among the more prominent novel products identified in BES were a set of 11 small transthyretin-like proteins, and three glutamine-rich-repeat mucin-like proteins. Notably, no evidence was found of any secreted protein corresponding to the genome of the Wolbachia endosymbiont present in B. malayi. Western blotting with anti-phosphorylcholine (PC) monoclonal antibody identified that GlcNAcT, and not the ES-62 homologue, is the major PC-bearing protein in BES, while probing with human filariasis sera showed preferential reactivity to galectin-1 and to processed forms of myotactin. Overall, this analysis demonstrates selective release of a suite of newly identified proteins not previously suspected to be involved at the host-parasite interface, and provides important new perspectives on the biology of the filarial parasite.

Dendritic cells (DCs) play a pivotal role in polarising Th lymphocyte subsets but it is unclear what molecular events occur when DCs generate Th2-type responses. Here, we analysed plasma membrane-enriched fractions from immature, pro-Th1 and pro-Th2 DCs and used a combination of iTRAQ labelling and LC–MS/MS to quantify changes in the proteomes. Analysis was performed on triplicate biological samples and changes verified by flow cytometry. MHC class II molecules and CD29 were up-regulated in pro-Th1 DCs whilst CD18 and CD44 were up-regulated in pro-Th2 DCs. One of the most down-regulated molecules in pro-Th1 DCs was YM-1 whilst the greatest decrease in pro-Th2 DCs was NAP-22. Other molecules up-regulated in pro-Th2 DC compared to pro-Th1 DCs included some potentially involved in protein folding during antigen processing (clathrin and Rab-7), whilst other non-membrane proteins such as enzymes/transporters related to cell metabolism (malate dehydrogenase, pyruvate kinase, and ATPase Na+/K+) were also recorded. This suggests that pro-Th2 DCs are more metabolically active while pro-Th1 DCs have a mature ‘end state’. Overall, although several molecules were preferentially expressed on pro-Th2 DCs, our proteomics data support the view of a ‘limited maturation’ of pro-Th2 DCs compared to pro-Th1 DCs., Graphical abstract Highlights ► Analysis of plasma membrane-enriched proteomes of differentially matured DCs. ► Relative expression determined by iTRAQ labelling combined with LC–MS/MS. ► Differential expression of immune associated proteins by pro-Th2 versus pro-Th1 DCs. ► Data support ‘limited maturation’ but not ‘unique’ phenotype of pro-Th2 DCs.

The intestinal helminth parasite, Heligmosomoides polygyrus bakeri offers a tractable experimental model for human hookworm infections such as Ancylostoma duodenale and veterinary parasites such as Haemonchus contortus. Parasite excretory-secretory (ES) products represent the major focus for immunological and biochemical analyses, and contain immunomodulatory molecules responsible for nematode immune evasion. In a proteomic analysis of adult H. polygyrus secretions (termed HES) matched to an extensive transcriptomic dataset, we identified 374 HES proteins by LC-MS/MS, which were distinct from those in somatic extract HEx, comprising 446 identified proteins, confirming selective export of ES proteins. The predominant secreted protein families were proteases (astacins and other metalloproteases, aspartic, cysteine and serine-type proteases), lysozymes, apyrases and acetylcholinesterases. The most abundant products were members of the highly divergent venom allergen-like (VAL) family, related to Ancylostoma secreted protein (ASP); 25 homologues were identified, with VAL-1 and -2 also shown to be associated with the parasite surface. The dominance of VAL proteins is similar to profiles reported for Ancylostoma and Haemonchus ES products. Overall, this study shows that the secretions of H. polygyrus closely parallel those of clinically important GI nematodes, confirming the value of this parasite as a model of helminth infection.

Background The membrane-associated and membrane-spanning constituents of the Schistosoma mansoni tegument surface, the parasite's principal interface with the host bloodstream, have recently been characterized using proteomic techniques. Biotinylation of live worms using membrane-impermeant probes revealed that only a small subset of the proteins was accessible to the reagents. Their position within the multilayered architecture of the surface has not been ascertained. Methodology/Principal Findings An enzymatic shaving approach on live worms has now been used to release the most accessible components, for analysis by MS/MS. Treatment with trypsin, or phosphatidylinositol-specific phospholipase C (PiPLC), only minimally impaired membrane integrity. PiPLC-enriched proteins were distinguished from those released in parasite vomitus or by handling damage, using isobaric tagging. Trypsin released five membrane proteins, Sm200, Sm25 and three annexins, plus host CD44 and the complement factors C3 and C4. Nutrient transporters and ion channels were absent from the trypsin fraction, suggesting a deeper location in the surface complex; surprisingly, two BAR-domain containing proteins were released. Seven parasite and two host proteins were enriched by PiPLC treatment, the vaccine candidate Sm29 being the most prominent along with two orthologues of human CD59, potentially inhibitors of complement fixation. The enzymes carbonic anhydrase and APD-ribosyl cyclase were also enriched, plus Sm200 and alkaline phosphatase. Host GPI-anchored proteins CD48 and CD90, suggest ‘surface painting’ during worm peregrination in the portal system. Conclusions/Significance Our findings suggest that the membranocalyx secreted over the tegument surface is not the inert barrier previously proposed, some tegument proteins being externally accessible to enzymes and thus potentially located within it. Furthermore, the detection of C3 and C4 indicates that the complement cascade is initiated, while two CD59 orthologues suggest a potential mechanism for its inhibition. The detection of several host proteins is a testimonial to the acquisitive properties of the tegument surface. The exposed parasite proteins could represent novel vaccine candidates for combating this neglected disease., Author Summary Adult schistosome parasites can reside in the host bloodstream for decades surrounded by components of the immune system. It was originally proposed that their survival depended on the secretion of an inert bilayer, the membranocalyx, to protect the underlying plasma membrane from attack. We have investigated whether any proteins were exposed on the surface of live worms using incubation with selected hydrolases, in combination with mass spectrometry to identify released proteins. We show that a small number of parasite proteins are accessible to the enzymes and so could represent constituents of the membranocalyx. We also identified several proteins acquired by the parasite on contact with host cells. In addition, components of the cytolytic complement pathway were detected, but these appeared not to harm the worm, indicating that some of its own surface proteins could inhibit the lytic pathway. We suggest that, collectively, the ‘superficial’ parasite proteins may provide good candidates for a schistosome vaccine.

The schistosome tegument provides a major interface with the host blood stream in which it resides. Our recent proteomic studies have identified a range of proteins present in the complex tegument structure, and two models of protective immunity have implicated surface proteins as mediating antigens. We have used the QconCAT technique to evaluate the relative and absolute amounts of tegument proteins identified previously. A concatamer comprising R- or K-terminated peptides was generated with [(13)C(6)] lysine/arginine amino acids. Two tegument surface preparations were each spiked with the purified SmQconCAT as a standard, trypsin digested, and subjected to MALDI ToF-MS. The absolute amounts of protein in the biological samples were determined by comparing the areas under the pairs of peaks, separated by 6m/z units, representing the light and heavy peptides derived from the biological sample and SmQconCAT, respectively. We report that aquaporin is the most abundant transmembrane protein, followed by two phosphohydrolases. Tetraspanin Tsp-2 and Annexin-2 are also abundant but transporters are scarce. Sm200 surface protein comprised the bulk of the GPI-anchored fraction and likely resides in the secreted membranocalyx. Two host IgGs were identified but in amounts much lower than their targets. The findings are interpreted in relation to the models of protective immunity.

The surface tegument of the liver fluke Fasciola hepatica is a syncytial cytoplasmic layer bounded externally by a plasma membrane and covered by a glycocalyx, which constitutes the interface between the parasite and its ruminant host. The tegument’s interaction with the immune system during the fluke’s protracted migration from the gut lumen through the peritoneal cavity and liver parenchyma to the lumen of the bile duct, plays a key role in the fluke’s establishment or elimination. However, little is known about proteins of the tegument surface or its secretions. We applied techniques developed for the blood fluke, Schistosoma mansoni, to enrich a tegument surface membrane preparation and analyse its composition by tandem mass spectrometry using new transcript databases for F. hepatica. We increased the membrane and secretory pathway components of the final preparation to ∼30%, whilst eliminating contaminating proteases. We identified a series of proteins or transcripts shared with the schistosome tegument including annexins, a tetraspanin, carbonic anhydrase and an orthologue of a host protein (CD59) that inhibits complement fixation. Unique to F. hepatica, we also found proteins with lectin, cubulin and von Willebrand factor domains plus 10 proteins with leader sequences or transmembrane helices. Many of these surface proteins are potential vaccine candidates. We were hampered in collecting tegument secretions by the propensity of liver flukes, unlike blood flukes, to vomit their gut contents. We analysed both the ‘vomitus’ and a second supernatant released from haematin-depleted flukes. We identified many proteases, some novel, as well as a second protein with a von Willebrand factor domain. This study demonstrates that components of the tegumental surface of F. hepatica can be defined using proteomic approaches, but also indicates the need to prevent vomiting if tegument secretions are to be characterised.

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity., Author Summary Intestinal helminth parasites are highly prevalent in humans and animals, and survive for long periods by deviating the host immune system. No vaccines are currently available to control these infections. Many helminths invade through barrier surfaces (such as the skin or the digestive tract) and develop through tissue larval stages before reaching their final niche such as the intestinal lumen. We studied the tissue larval stage of a mouse parasite, Heligmosomoides polygyrus, to test whether proteins released by this stage could elicit protective immunity, and found that they indeed constitute very effective vaccine antigens. Proteomic analysis to identify the individual proteins released by the larvae demonstrated that while many products are shared between tissue-dwelling larvae and adults occupying the intestinal lumen, larvae express higher levels of two gene families linked to immunomodulation, namely the Sushi protein family and the ShK toxin family. Antibody analysis of serum from vaccinated mice identified two major antigens recognised by the protective immune response as VAL-1 and ACE-1, which are respectively members of the venom allergen and acetylcholinesterase families. This work establishes that tissue larvae are the source of protective antigens for future vaccines, and highlights their production of two potentially immunomodulatory gene families.

Due to genetic selection towards reduced subcutaneous fat, the amount of intramuscular fat (IMF) in commercial pigs has been reduced (

Antimicrobial silver (Ag+) coatings on orthopaedic implants may reduce infection rates, but should not be to the detriment of regenerative cell populations, primarily mesenchymal stem/stromal cells (MSCs). We determined intramedullary silver release profiles in vivo, which were used to test relevant Ag+ concentrations on MSC function in vitro. We measured a rapid elution of Ag+ from intramedullary pins in a rat femoral implantation model, delivering a maximum potential concentration of 7.8 µM, which was below toxic levels determined for MSCs in vitro (EC50, 33 µM). Additionally, we present in vitro data of the reduced colonisation of implants by Staphylococcus aureus. MSCs exposed to Ag+ prior to/during osteogenic differentiation were not statistically affected. Notably, at clonal density, the colony-forming capacity of MSCs was significantly reduced in the presence of 10 µM Ag+, suggesting that a subpopulation of clonal MSCs was sensitive to Ag+ exposure. At a molecular level, surviving colony-forming MSCs treated with Ag+ demonstrated a significant upregulation of components of the peroxiredoxin/thioredoxin pathway and processes involved in glutathione metabolism compared to untreated controls. Inhibition of glutathione synthesis using l-buthionine sulfoxamine eliminated MSC clonogenicity in the presence of Ag+, which was rescued by exogenous glutathione.