Tegument surface membranes of adult Schistosoma mansoni: Development of a method for their isolation

Roberts, S.M.; MacGregor, A.; Vojvodic, Maria; Wells, Edward; Crabtree, Jean E.; Wilson, R. A.

doi:10.1016/0166-6851(83)90104-4

Cited by 70 publications

(68 citation statements)

References 36 publications

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“…Methods were devised more than 30 years ago to detach the tegument surface complex for compositional analysis (7)(8)(9). However, despite separation of the protein constituents by electrophoresis (10,11), little progress was made in obtaining their identities.…”

Section: Molecular and Cellular Proteomics 5:347-356 2006mentioning

confidence: 99%

Proteins Exposed at the Adult Schistosome Surface Revealed by Biotinylation

Braschi

Wilson

2006

Molecular & Cellular Proteomics

236

309

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The human blood-dwelling parasite Schistosoma mansoni can survive in the hostile host environment for decades and must therefore display effective strategies to evade the host immune responses. The surface of the adult worm is covered by a living syncytial layer, the tegument, bounded by a complex multilaminate surface. This comprises a normal plasma membrane overlain by a secreted bilayer, the membranocalyx. Recent proteomic studies have identified constituents of the tegument, but their relative locations remain to be established. We labeled the most exposed surface proteins using two impermeant biotinylation reagents that differed only in length. We anticipated that the two reagents would display distinct powers of penetration, thereby producing a differential labeling pattern. The labeled proteins were recovered by streptavidin affinity and identified by tandem mass spectrometry. A total of 28 proteins was identified, 13 labeled by a long form reagent and the same 13 plus a further 15 labeled by a short form reagent. The parasite proteins included membrane enzymes, transporters, and structural proteins. The short form reagent additionally labeled some cytosolic and cytoskeletal proteins, the latter being constituents of the intracellular spines. Only a single secreted protein was labeled, implying a location between the plasma membrane and the membranocalyx or as part of the latter. Four host proteins, three immunoglobulin heavy chains and C3c/C3dg, a fragment of complement C3, were labeled by both reagents indicating their exposed situation. The presence of the degraded complement C3 implicates inhibition of the classical pathway as a major element of the immune evasion strategy, whereas the recovery of only one truly secreted protein points to the membranocalyx acting primarily as an inert protective barrier between the immune system and the tegument plasma membrane. Collectively the labeled parasite proteins merit investigation as potential vaccine candidates. Molecular & Cellular Proteomics 5:347-356, 2006.The trematode Schistosoma mansoni is a long lived parasite of the human hepatic portal system, infecting millions of people in Africa and South and Central America (1). Infection of the mammalian host occurs by direct cercarial penetration through the skin followed by transformation into schistosomula. These then undertake a protracted intravascular migration to the hepatic portal vein where they begin to feed on blood, mature, and pair. The male carries the female up the mesenteric blood vessels to the gut wall where she begins to deposit eggs. A proportion of these pass through the tissue and reach the gut lumen to continue the life cycle, whereas others are washed downstream to the liver where they initiate the granulomatous inflammation that characterizes the disease. That schistosomes can survive for 30 -40 years in humans (2) attests to their possession of an effective immune evasion strategy.The mature worm is covered by a syncytial cytoplasmic layer, the tegument, attached to underlying cell bo...

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Section: Molecular and Cellular Proteomics 5:347-356 2006mentioning

confidence: 99%

Proteins Exposed at the Adult Schistosome Surface Revealed by Biotinylation

Braschi

Wilson

2006

Molecular & Cellular Proteomics

236

309

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“…Tegument membranes were removed from adult worms by a freeze/thaw method and surface membranes enriched by sucrose-gradient centrifugation as previously described (Roberts et al 1983). They were pelleted by centrifugation at 100g for 30 min at 4°C, resuspended in 0.5 mL of 10 mM Tris, and enriched on a continuous 20%-70% sucrose gradient, with centrifugation at 100,000g for 40 min.…”

Section: Cloning and Sequencing Of Genomic Regions Of Meg-3mentioning

confidence: 99%

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

DeMarco¹,

Mathieson²,

Manuel³

et al. 2010

Genome Res.

162

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Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (#36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.

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“…The first proteomic analysis of the S. mansoni tegument utilised the freeze-thaw method developed by Roberts et al (1983) to detach the tegument syncytium from the underlying worm body (van Balkom et al 2005). The proteins in the whole tegument and the body fractions were separated by 1-D electrophoresis, the gel sectioned and each piece treated with trypsin.…”

Section: Ms-based Study Of the Tegument Proteomementioning

confidence: 99%

“…The tegument was detached from approximately 2000 worm pairs using the freeze-thaw method, and the surface membranes were enriched by density gradient centrifugation (Roberts et al 1983) prior to further analysis. Furthermore, we incorporated a differential extraction procedure on the membrane fraction with reagents of increasing solubilising strength, to remove sequentially the cytosolic and cytoskeletal proteins, yielding an insoluble pellet highly enriched in membranes.…”

Section: Proteomics Of the Tegument Surface Membranesmentioning

confidence: 99%

“…Methods were developed more than 20 years ago to detach the tegument using detergents (Kusel 1970, Oaks et al 1981, McDiarmid et al 1983, chemicals (Bennett & Seed 1977) or physical shearing (Roberts et al 1983), but until recently, little was progressed beyond the identification of few proteins, using antibody or histochemical studies. With the advent of mass spectrometry (MS)-based proteomics, together with the recent sequencing of the S. mansoni genome (www.schistoDB.org) and transcriptome (Verjovski-Almeida et al 2003), it has become possible to compile a much more complete inventory of the protein constituents of the tegument and its surface.…”

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confidence: 99%

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