Navegando por Autor "Wilson, R. Alan"

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Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics.
(2011) Borges, William de Castro; Simpson, Deborah M.; Dowle, Adam A.; Curwen, Rachel S.; Oates, Jane Thomas; Beynon, Robert J.; Wilson, R. Alan
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 [13C6] 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 6 m/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.
Characterization of phosphodiesterase-5 as a surface protein in the tegument of Schistosoma mansoni.
(2009) Rofatto, Henrique Krambeck; Tararam, Cibele Aparecida; Borges, William de Castro; Wilson, R. Alan; Leite, Luciana Cesar de Cerqueira; Farias, Leonardo Paiva
Schistosoma mansoni is a major causative agent of schistosomiasis, an important parasitic disease that constitutes a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection and the development of an effective vaccine still remains the most desirable means of control for this disease. In thisworkwe describe the cloning and characterization of a S. mansoni nucleotidepyrophosphatase/phosphosdiesterase type 5(SmNPP-5), previously identifiedinthe tegument by proteomic studies. In silico analysis predicts an N-terminal signal peptide, three N-glycosylation sites and a C-terminal transmembrane domain similar to that described for mammalian isoforms. Real-time quantitative RT-PCR andWestern blot analyses determined that SmNPP-5 is significantly upregulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages; additionally, the native protein was demonstrated to be N-glycosylated. Immunolocalization experiments and tegument surface membrane preparations confirm the protein as a tegument surface protein. Furthermore, the ectolocalization of this enzyme was corroborated through the hydrolysis of the phosphodiesterase specific substrate (_-Nph-5_-TMP) by living adult and 21-day-old worms. Interestingly, pre-incubation of adult and 21-day-old worms with anti-rSmNPP-5 antibody was able to reduce by 50–60% the enzyme activity. These results suggest that SmNPP-5 is closely associated with the new tegument surface generation after cercarial penetration, and being located at the host–parasite interface, is a potential target for immune intervention.
Dissection of schistosome tissues under LC–MS compatible preservative conditions for quantitative proteomics.
(2023) Neves, Leandro Xavier; Wilson, R. Alan; Brownridge, Philip; Holman, Stephen W.; Harman, Victoria Margaret Elizabeth; Eyers, Claire E.; Beynon, Robert J.; Borges, William de Castro
Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography– mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.
Do schistosome vaccine trials in mice have an intrinsic flaw that generates spurious protection data?
(2016) Wilson, R. Alan; Li, Xiao Hong; Borges, William de Castro
The laboratory mouse has been widely used to test the efficacy of schistosome vaccines and a long list of candidates has emerged from this work, many of them abundant internal proteins. These antigens do not have an additive effect when co-administered, or delivered as SWAP homogenate, a quarter of which comprises multiple candidates; the observed protection has an apparent ceiling of 40–50 %. We contend that the low level of maturation of penetrating cercariae (~32 % for Schistosoma mansoni) is a major limitation of the model since 68/100 parasites fail to mature in naïve mice due to natural causes. The pulmonary capillary bed is the obstacle encountered by schistosomula en route to the portal system. The fragility of pulmonary capillaries and their susceptibility to a cytokine-induced vascular leak syndrome have been documented. During lung transit schistosomula burst into the alveolar spaces, and possess only a limited capacity to re-enter tissues. The acquired immunity elicited by the radiation attenuated (RA) cercarial vaccine relies on a pulmonary inflammatory response, involving cytokines such as IFNγ and TNFα, to deflect additional parasites into the alveoli. A principal difference between antigen vaccine protocols and the RA vaccine is the short interval between the last antigen boost and cercarial challenge of mice (often two weeks). Thus, after antigen vaccination, challenge parasites will reach the lungs when both activated T cells and cytokine levels are maximal in the circulation. We propose that “protection” in this situation is the result of physiological effects on the pulmonary blood vessels, increasing the proportion of parasites that enter the alveoli. This hypothesis will explain why internal antigens, which are unlikely to interact with the immune response in a living schistosomulum, plus a variety of heterologous proteins, can reduce the level of maturation in a non-antigen-specific way. These proteins are “successful” precisely because they have not been selected for immunological silence. The same arguments apply to vaccine experiments with S. japonicum in the mouse model; this schistosome species seems a more robust parasite, even harder to eliminate by acquired immune responses. We propose a number of ways in which our conclusions may be tested.
Elimination of Schistosoma mansoni adult worms by rhesus macaques : basis for a therapeutic vaccine?
(2008) Wilson, R. Alan; Langermans, Jan A. M.; Dam, Govert J. van; Vervenne, Richard A.; Hall, Stephanie L.; Borges, William de Castro; Dillon, Gary P.; Thomas, Alan W.; Coulson, Patricia S.
Background: Among animal models of schistosomiasis, the rhesus macaque is unique in that an infection establishes but egg excretion rapidly diminishes, potentially due to loss of adult worms from the portal system via shunts or death by immune attack. Principal Findings: To investigate this, six rhesus macaques were exposed to Schistosoma mansoni cercariae and the infection monitored until portal perfusion at 18 weeks. Despite a wide variation in worm numbers recovered, fecal egg output and circulating antigen levels indicated that a substantial population had established in all animals. Half the macaques had portal hypertension but only one had portacaval shunts, ruling out translocation to the lungs as the reason for loss of adult burden. Many worms had a shrunken and pallid appearance, with degenerative changes in intestines and reproductive organs. Tegument, gut epithelia and muscles appeared cytologically intact but the parenchyma was virtually devoid of content. An early and intense IgG production correlated with low worm burden at perfusion, and blood-feeding worms cultured in the presence of serum from these animals had stunted growth. Using immunoproteomics, gut digestive enzymes, tegument surface hydrolases and antioxidant enzymes were identified as targets of IgG in the high responder animals. Significance: It appears that worms starve to death after cessation of blood feeding, as a result of antibody-mediated processes. We suggest that proteins in the three categories above, formulated to trigger the appropriate mechanisms operating in rhesus macaques, would have both prophylactic and therapeutic potential as a human vaccine.
Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis : a rational approach to select vaccine candidates.
(2011) Borges, William de Castro; Dowle, Adam A.; Curwen, Rachel S.; Oates, Jane Thomas; Wilson, R. Alan
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 ofc omplement 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.
Epitope mapping of exposed tegument and alimentary tract proteins identifies putative antigenic targets of the attenuated schistosome vaccine.
(2020) Farias, Leonardo Paiva; Vance, Gillian M.; Coulson, Patricia S.; Souza, Juliana Vitoriano de; Silva Neto, Almiro Pires da; Wangwiwatsin, Arporn; Neves, Leandro Xavier; Borges, William de Castro; McNicholas, Stuart; Wilson, Keith Sanderson; Leite, Luciana Cezar de Cerqueira; Wilson, R. Alan
The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.
Exploring the Fasciola hepatica tegument proteome.
(2011) Wilson, R. Alan; Wright, Janelle M.; Borges, William de Castro; Manuel, Sophie J. Parker; Dowle, Adam A.; Ashton, Peter D.; Young, Neil D.; Gasser, Robin B.; Spithill, Terry W.
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.
Mapping the epitopes of Schistosoma japonicum esophageal gland proteins for incorporation into vaccine constructs.
(2020) Li, Xiao Hong; Vance, Gillian M.; Cartwright, Jared; Cao, Jian Ping; Wilson, R. Alan; Borges, William de Castro
Background The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. Methodology/Principal findings We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection.Conclusions/Significance It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.
Microexon gene transcriptional profiles and evolution provide insights into blood processing by the Schistosoma japonicum esophagus.
(2018) Li, Xiao Hong; DeMarco, Ricardo; Neves, Leandro Xavier; James, Sally R.; Newling, Katherine; Ashton, Peter D.; Cao, Jian Ping; Wilson, R. Alan; Borges, William de Castro
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 Schistosoma japonicum 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 S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process.
Proteômica quantitativa baseada em espectrometria de massas para estudo do trato alimentar de Schistosoma mansoni com ênfase na glândula esofagiana e suas secreções.
(2018) Neves, Leandro Xavier; Borges, William de Castro; Wilson, R. Alan; Borges, William de Castro; Palmisano, Giuseppe; Castro, Ieso de Miranda; Cota, Renata Guerra de Sá; Marco, Ricardo de
A esquistossomose é uma doença tropical negligenciada que afeta mais de 240 milhões de pessoas em 78 países. Apesar dos esforços para erradicação da doença, fatores como baixa cobertura dos programas de tratamento quimioterápico, métodos diagnóstico pouco sensíveis e reinfecções em regiões endêmicas, constituem desafios para o controle da transmissão. O desenvolvimento de vacinas contra schistosomas constitui uma ferramenta alternativa de combate à doença, porém, a identificação de antígenos protetores tem sido um grande desafio. Neste sentido, destaca-se o modelo de auto cura da infecção em macaco Rhesus (Macaca mulatta), no qual observa-se eliminação de parasitos após uma resposta humoral direcionada às interfaces do parasito, como tegumento e secreções da glândula esofagiana. Neste contexto, o presente trabalho teve como objetivo geral a caracterização proteômica em larga escala de produtos da glândula esofagiana e demais secreções do trato alimentar de S. mansoni para proposição de novos alvos vacinais. Uma abordagem quantitativa avaliou a presença de proteínas de interface em homogenato solúvel de vermes adultos, revelando uma baixa representatividade de moléculas de interesse nessa fração. A partir disso, um método de enriquecimento tecidual foi utilizado para a análise detalhada do esôfago de vermes machos adultos. As análises shotgun quantitativas apontaram 628 grupos de proteínas enriquecidos na região esofagiana, incluindo Microexon genes (MEG), hidrolases, glicosil transferases e proteínas para transporte vesicular. Ademais, essa abordagem permitiu a detecção de proteoformas de MEGs geradas por substituição e/ou deleção de resíduos de aminoácidos. Nove proteínas expressas na glândula esofagiana foram selecionadas para quantificação absoluta por QconCAT. A MEG-12 apresentou a maior concentração entre os alvos quantificados. A estimativa do número de cópias por célula das proteínas secretadas sugere que tais moléculas constituem alvos abundantes neste subproteoma. Por último, a caracterização proteômica do sobrenadante de cultura de S. mansoni resultou na detecção de um repertório expandido de proteínas do trato alimentar, incluindo MEGs esofagianas, reforçando a eventual exposição das secreções nessa interface parasito-hospedeiro. Em conjunto, as abordagens proteômicas de caracterização da região esofagiana e trato alimentar de S. mansoni resultaram na identificação de moléculas com potencial aplicação biotecnológica no desenvolvimento de vacinas, bem como alvos terapêuticos.
Quantitative proteomics of enriched esophageal and gut tissues from the human blood fluke Schistosoma mansoni pinpoints secreted proteins for vaccine development.
(2020) Neves, Leandro Xavier; Wilson, R. Alan; Brownridge, Philip; Harman, Victoria Margaret Elizabeth; Holman, Stephen W.; Beynon, Robert J.; Eyers, Claire E.; DeMarco, Ricardo; Borges, William de Castro
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.
Schistosome feeding and regurgitation.
(2014) Skelly, Patrick J.; Da'dara, Akram A.; Li, Xiao Hong; Borges, William de Castro; Wilson, R. Alan
Schistosomes are parasitic flatworms that infect .200 million people worldwide, causing the chronic, debilitating disease schistosomiasis. Unusual among parasitic helminths, the long-lived adult worms, continuously bathed in blood, take up nutrients directly across the body surface and also by ingestion of blood into the gut. Recent proteomic analyses of the body surface revealed the presence of hydrolytic enzymes, solute, and ion transporters, thus emphasising its metabolic credentials. Furthermore, definition of the molecular mechanisms for the uptake of selected metabolites (glucose, certain amino acids, and water) establishes it as a vital site of nutrient acquisition. Nevertheless, the amount of blood ingested into the gut per day is considerable: for males ,100 nl; for the more actively feeding females ,900 nl, .4 times body volume. Ingested erythrocytes are lysed as they pass through the specialized esophagus, while leucocytes become tethered and disabled there. Proteomics and transcriptomics have revealed, in addition to gut proteases, an amino acid transporter in gut tissue and other hydrolases, ion, and lipid transporters in the lumen, implicating the gut as the site for acquisition of essential lipids and inorganic ions. The surface is the principal entry route for glucose, whereas the gut dominates amino acid acquisition, especially in females. Heme, a potentially toxic hemoglobin degradation product, accumulates in the gut and, since schistosomes lack an anus, must be expelled by the poorly understood process of regurgitation. Here we place the new observations on the proteome of body surface and gut, and the entry of different nutrient classes into schistosomes, into the context of older studies on worm composition and metabolism. We suggest that the balance between surface and gut in nutrition is determined by the constraints of solute diffusion imposed by differences in male and female worm morphology. Our conclusions have major implications for worm survival under immunological or pharmacological pressure.
The 20S proteasome of Schistosoma mansoni : a proteomic analysis.
(2007) Borges, William de Castro; Cartwright, Jared; Ashton, Peter D.; Braschi, Simon; Cota, Renata Guerra de Sá; Rodrigues, Vanderlei; Wilson, R. Alan; Curwen, Rachel S.
Proteasomes are molecular machines found in virtually all cells that provide one of the mechanisms for protein turnover. We have analysed the 20S proteasome of Schistosoma mansoni, the first multimeric complex isolated from this helminth parasite. Three chromatographic steps were employed to yield a highly homogeneous preparation. 2-DE of the purified complex revealed 58 spots, of which 46 could be assigned either an a or a b proteasome signature by MS. Most of the 14 transcripts (7a and 7b) encoded by the parasite genome were represented by multiple spots and we suggest that this diversity is due to PTMs of subunits. For most of the isoforms, variations in pI predominated although alterations in mass were also observed. 2-DE separations of extracts from infective cercariae and blood-dwelling adult worms probed by Western blotting, using a human anti-a subunit antibody, revealed different patterns of reactivity, most probably in a3 and a6 subunits, on the basis of sequence conservation. This difference was rapidly lost following transformation of the cercaria to the skin schistosomulum stage, suggesting that changes in the proteasome structure, likely caused by the introduction of a new set of PTMs, precede remodelling of the parasite body prior to intravascular migration.
The proteasome-ubiquitin pathway in the Schistosoma mansoni egg has development - and morphology - specific characteristics.
(2011) Mathieson, William; Borges, William de Castro; Wilson, R. Alan
Schistosoma mansoni eggs, consisting of an ovum surrounded by nutritive vitelline cells packaged in a tanned protein shell, are produced by paired worms residing in the mesenteric veins of the human host. The vitelline cells are degraded as the larval miracidium matures, the fully developed egg either crossing the gut wall to escape the host or becoming lodged in the host’s tissues where it dies and disintegrates, inducing a potentially pathological immune response. Thus, the egg is central to both the transmission of the parasite and the aetiology of the disease. Herewepresent the first study investigating protein turnover in the egg. We establish that the ubiquitin-proteasome pathway (UPP) changes with egg development and furthermore, that the morphological components of the fully developed egg (the miracidium and the subshell envelope) also exhibit different proteasome subunit expression profiles. We conclude that the UPP is responsible not only for degrading the vitelline cells but is also more highly developed in the envelope than in the miracidium. The envelope is involved in the defence of the miracidium and produces the proteins that the egg secretes, presumably to facilitate its escape from the host, so the UPP probably has a multi-faceted role in the egg’s biology.
The schistosome eesophageal gland : initiator of blood processing.
(2013) Li, Xiao Hong; Borges, William de Castro; Manuel, Sophie J. Parker; Vance, Gillian M.; DeMarco, Ricardo; Neves, Leandro Xavier; Evans, Garet J. O.; Wilson, R. Alan
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.
Vaccination with enzimatically cleaved GPI-anchored proteins from Schistosoma mansoni induced protection against challenge infection.
(2012) Martins, Vicente de Paulo; Pinheiro, Carina da Silva; Figueiredo, Bárbara de Castro Pimentel; Assis, Natan Raimundo Gonçalves de; Morais, Suellen Batistoni de; Caliari, Marcelo Vidigal; Azevedo, Vasco Ariston de Carvalho; Borges, William de Castro; Wilson, R. Alan; Oliveira, Sergio Costa
The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-termstrategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γ and TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from the S. mansoni tegument as vaccine candidate.
What’s in SWAP? Abundance of the principal constituents in a soluble extract of Schistosoma mansoni revealed by shotgun proteomics.
(2015) Neves, Leandro Xavier; Sanson, Ananda Lima; Wilson, R. Alan; Borges, William de Castro
Background: The soluble antigen preparation of adult schistosomes (SWAP) has often been used to probe host responses against these blood-dwelling parasites. Despite its long established use there is limited knowledge about its composition. The information we provide here on the molecular composition of SWAP may contribute as a guide for a rational selection of antigenic targets. Methods: Label-free quantitative shotgun proteomics was employed to determine the composition and abundance of SWAP constituents. Briefly, paired adult Schistosoma mansoni worms were sonicated in PBS pH 7.2 and ultracentrifuged for recovery of the soluble supernatant. An aliquot was subjected to trypsin digestion. Resulting peptides were separated under ultra-high performance liquid chromatography and analysed using an orbitrap mass spectrometer. Spectral data were interrogated using SequestHT against an in-house S. mansoni database. Proteins were quantified by recording the mean area under curve obtained for up to three most intense detected peptides. Proteins within the 90th percentile of the total SWAP mass were categorized according to their sub-cellular/tissue location. Results: In this work we expanded significantly the list of known SWAP constituents. Through application of stringent criteria, a total of 633 proteins were quantitatively identified. Only 18 proteins account to 50 % of the total SWAP mass as revealed by their cumulative abundance. Among them, none is predicted as a secreted molecule reinforcing the point that SWAP is dominated by cytosolic and cytoskeletal proteins. In contrast, only 3 % of the mass comprised proteins proposed to function at the host-parasite interfaces (tegument and gut), which could conceivably represent vulnerable targets of a protective immune response. Paradoxically, at least 11 SWAP proteins, comprising ~25 % of its mass, have been tested as vaccine candidates. Conclusions: Our data suggest that use of SWAP to probe host responses has greatest value for diagnostic purposes or assessing intensity of infection. However, the preparation is of limited utility as an antigen source for investigating host responses to proteins expressed at or secreted from worm-host interfaces. The data also pose the question as to why vaccination with SWAP, containing so many proposed vaccine candidates, has no additive or even synergistic effects on the induction of protection.