Proteins exposed at the adult schistosome surface revealed by biotinylation

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.     

Internalization of macromolecules from the medium in Suctoria

Takophrya infusionum like all other Suctoria lacks an oral cavity. Its feeding apparatus consists of tentacles, long narrow tubes through which the contents of the living prey are ingested. For normal growth, reproduction, and longevity of clones, Tokophrya needs supplements deriving from the medium in addition to living prey. Since Tokophrya lacks a mouth, these supplements can reach the cytoplasm only through the complex structure of the cortex, which is composed of a three- membraned pellicle and a dense epiplasm. In addition, external to the cortex, an extraneous coat covers the whole organism. Only the outer pellicular plasma membrane is continuous; the other two and the epiplasm are interrupted by the outer plasma membrane which invaginates at intervals forming the so-called pits. The invaginated plasma membrane dips down into the cytoplasm where it extends to form a saccule. Experiments with cationized ferritin and Thorotrast provide evidence that internalization of these macromolecules takes place through the pits by pinocytosis. The membrane of the saccules of the pits forms invaginations which pinch off giving rise to small, flattened vesicles containing the tracers. The tracers were never found free in the cytoplasm but exclusively in the flat vesicles. These vesicles are thus the vehicles transporting macromolecules from the medium to the cytoplasm. The saccules of the pits are the natural loci of pinocytosis and together with the flattened vesicles perform an important function in Suctoria, supplying the organisms with macromolecules from the medium.     

Detection of localization and internalization of membrane DNA in the Tetrahymena by the lactoferrin--colloidal gold technique

It is known that lactoferrin binds to DNA. In the present study a lactoferrin-gold complex bound initially to the ciliary membrane, then appeared in coated pits and intracytoplasmic vesicles. On treatment for 1 h, the gold particles could be detected near to the nucleus. The lactoferrin-gold complex neither bound to the surface, nor appeared in the cytoplasm of those cells which had been exposed to DNAse for pretreatment.     

Ultrastructural observations on the redial tegument of Paramphistomum epiclitum from the planorbid snail, Indoplanorbis exustus.

The morphology of the tegument in the redia of Paramphistomum epiclitum (Digenea: Paramphistomidae) resembles that shown by most larval and adult digeneans; an outer surface syncytium is in continuity with the cytoplasm of in-sunken, nucleated cytons. Although tegumental cytons usually contain a single nucleus, some display up to six nuclei. The tegumental syncytium lining the pharynx of P. epiclitum rediae lack underlying cytons. The apical membrane of the tegument is elaborated by folds and microvilli, which presumably facilitate uptake of nutrients and/or exchange of ions involved in osmoregulation. A single type of secretory body, resulting from the fusion of smaller vesicles produced at Golgi complexes in the cytons, occurs throughout the tegument. Uniciliate sensory receptors occur in the surface syncytium particularly around the oral opening.     

Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics

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.     

Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis: a rational approach to select vaccine candidates

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.     

Hymenolepis diminuta: ultrastructural observations on complement-mediated tegumental lysis and destrobilation of 4-day-old worms in vitro

Changes in the ultrastructure of the tegument and subtegumental cells of 4-day-old Hymenolepis diminuta were studied in vitro in 50% fresh normal rat serum over a 5-h period and compared with heat-inactivated serum and saline controls. First, membrane-bound vesicles accumulate above the microthrix-border. After 30–40 min large vacuoles, which may contain membranous elements, appear in the tegument at a time when the surface of the young strobila is virtually denuded of the microthrix-border. With prolonged incubations there are subtegumental secretory inclusions with dark, enveloping cytoplasm in the tegument and finally the apical plasma membrane, together with the majority of the matrix, is lost. The disrupted portion of the worm is abruptly demarcated from the comparatively intact scolex/anterior neck region by a constriction. Even after 5 h incubation there is no evidence of loss of tegumental matrix components from regions anterior to the constriction but the neck region shows a significant denudation of the microthrix layer and the tegument contains numerous inclusions. The scolex tegument only showed little evidence of loss of membrane from the surface. Possible mechanisms for the avoidance of complement-mediated lysis in the anterior region are discussed.     

Topography and ultrastructure of the tegument of Bucephalus anguillae (Digenea: Bucephalidae), a parasite of the European eel Anguilla anguilla (Osteichthyen: Anguillidae)

The tegumental ultrastructure of the intestinal fluke Bucephalus anguillae was studied with the use of scanning and transmission electron microscopy. The surface of the tegument is covered by transverse ridges from which protrude numerous closely packed, digitated, and claw-shaped spines. Cobblestone-like units of the tegument were observed on the crescent-shaped formation of the rhynchus and at the posterior part of the body. Three types of sensory structures were examined, i.e., 2 uniciliated receptors and 1 without cilia. As anterior-posterior differences were observed, particular attention was given to spines and sensory receptors. Spine insertion zones and average cilia length are variable between anterior and posterior tegument areas. Ultrastructural study revealed that the tegument of B. anguillae has a typical syncytial organization with a distal cytoplasm lying over a basal matrix and cytons below. Cytoplasmic bridges allowed transit of secretory vesicles and granules. Diagrams of spines and sensory receptors were made to help in understanding the nature of these structures.     

In rat dorsal root ganglion neurons,herpes simplex virus type 1 tegument forms in the cytoplasm of the cell body

The herpes simplex virus type 1 (HSV-1) tegument is the least understood component of the virion, and the mechanism of tegument assembly and incorporation into virions during viral egress has not yet been elucidated. In the present study, the addition of tegument proteins (VP13/14, VP16, VP22, and US9) and envelope glycoproteins (gD and gH) to herpes simplex virions in the cell body of rat dorsal root ganglion neurons was examined by immunoelectron microscopy. All tegument proteins were detected diffusely spread in the nucleus within 10 to 12 h and, at these times, nucleocapsids were observed budding from the nucleus. The majority (96%) of these nucleocapsids had no detectable label for tegument and glycoproteins despite the presence of tegument proteins in the nucleus and glycoproteins adjacent to the nuclear membrane. Immunolabeling for tegument proteins and glycoproteins was found abundantly in the cytoplasm of the cell body in multiple discrete vesicular areas: on unenveloped, enveloped, or partially enveloped capsids adjacent to these vesicles and in extracellular virions. These vesicles and intracytoplasmic and extracellular virions also labeled with Golgi markers, giantin, mannosidase II, and TGN38. Treatment with brefeldin A from 2 to 24 h postinfection markedly inhibited incorporation into virions of VP22 and US9 but to a lesser degree with VP16 and VP13/14. These results suggest that, in the cell body of neurons, most tegument proteins are incorporated into unenveloped nucleocapsids prior to envelopment in the Golgi and the trans-Golgi network. These findings give further support to the deenvelopment-reenvelopment hypothesis for viral egress. Finally, the addition of tegument proteins to unenveloped nucleocapsids in the cell body allows access to these unenveloped nucleocapsids to one of two pathways: egress through the cell body or transport into the axon.     

鸭胚成纤维细胞中鸭瘟强毒的增殖特性研究

通过细胞培养物光学显微观察、细胞超薄切片研究、定量PCR检测技术对鸭胚成纤维细胞中鸭瘟强毒的增殖特性进行了研究.结果表明:鸭瘟强毒接种鸭胚成纤维细胞后42 h,可使细胞培养物出现大量明显的蚀斑病变.细胞培养物的超薄切片电镜观察研究表明,病毒核酸在胞核内常集中分布,呈直径35～45 nm的圆形颗粒状;核衣壳在胞核和胞浆内都有分布,呈直径90～100 nm的网形颗粒状;成熟病毒位于胞浆空泡内,呈直径150～300 nm的圆形,有囊膜和皮层结构;病毒通过囊膜与胞膜融合入侵细胞,在核内生成核酸、装配核衣壳,在胞浆中得到皮层,出芽到胞浆空泡内获得囊膜,通过胞吐作用释放到胞外.定量PCR研究表明:鸭瘟强毒接种细胞后10 h开始明显复制,接毒后30 h时含量趋于稳定,接毒后22 h时开始向胞外释放,50 h时达最大值,细胞和上清中病毒含量的增幅均为103左右,病毒主要存在于细胞中,其含量为上清的102～103倍.     

An electron microscope study of the tegument of Hunterella nodulosa Mackiewicz and McCrae, 1962 (cestoidea: Caryophyllidea)

Ultrastructural observations using transmission and scanning electron microscopy reveal the tegument to be basically similar to that of other cestodes. The syncytial distal cytoplasm is devoid of organelles except for rod-shaped bodies, believed to be secretory vesicles, and lamellated bodies which probably contribute the raw material for new microtriches. There is evidence that these vesicles originate from the Golgi found in the sub-cuticular cells.Three types of microtriches are described: typical ones with well-developed spines, ones with short filaments instead of spines, and ones with no spines. Microtriches with spines are found only on the anterior part of the worm and may serve to anchor the worm. Microtriches on the posterior have no spines and are believed to be primarily involved in the absorption of nutrients. Between these two regions there is a transitional zone where all three types of microtriches can be found. In general the microtriches are quite uniformly distributed throughout the surface of the worm. The presence of cestodarian-like microtriches raises interesting evolutionary questions.Histochemical tests localized acid and alkaline phosphatase activity on various parts of the tegument, as well as on host intestine, while acian blue tests showed that acid mucopolysaccharide levels correspond with the concentration of the tegument vesicles.     

Uptake of ferritin by the mosaic egg surface of Brachydanio

The internalization of membrane from the mosaic egg surface of the zebra fish, Brachydanio, was investigated using anionic ferritin and transmission electron microscopy. The cortical cytoplasm of the 5-min activated egg showed numerous membrane-bound vesicles not found in the unactivated egg cortex. Two types of vesicles were identified: uncoated (smooth) and coated. Coated vesicles measured about 0.7 to 0.9 micrometer in diameter. Coated pits, considered to be precursors to the formation of coated vesicles, were frequently observed at the base of membrane-lined cortical granule crypts. Anionic ferritin was localized over coated pits and in both smooth and coated vesicles. The absence of any morphological evidence of a surface origin for smooth vesicles suggested these ferritin-labeled organelles might be formed by coated vesicle fusion. Our results indicate that the plasma membrane redundancy created by the exocytosis of cortical granules in Brachydanio appears to be resolved in part by the internalization of membrane through endocytosis.     

河鲈锚首吸虫体壁的超微结构观察

寄生鳜鳃部的河鲈锚首吸虫的体壁由表皮合胞体、基板、环肌、纵肌和表皮细胞核周体所组成。合胞体顶部质膜起伏形成表皮的嵴纹,基部质膜折叠形成指状突起伸入到合胞体中。合胞体表面覆盖着一层糖萼。河鲈锚首吸虫的表皮中含有四类分泌体,即电子致密的分泌颗粒、中等电子致密的分泌颗粒、有膜包围的电子稀疏的分泌体和多囊体．可见分泌体和合胞体基质通过胞吐作用排到体外,未见吞饮小泡,推测表皮的主要功能在于分泌和渗透压调节而非营养吸收。在外侧头瓣的乳突状结构所在处,合胞层较薄,基板平滑,在实质组织中的一腔体样结构中可见囊状体、电子致密度各异的颗粒体、泡状体和电子致密的基质团,神经突起分布于腔体周围,这类乳突可能代表一类新的非纤毛感受器类型。     

Synthesis of macromolecules by the epithelial surfaces of Schistosoma mansoni: an autoradiographic study.

The use of tritiated leucine as a marker for protein synthesis and of tritiated glucosamine as a marker for polysaccharide/glycoprotein synthesis, is described. Adult worms were pulse-labelled by incubation in medium containing the substrate. Labelled worms were then incubated in chase medium, without labelled substrate, for varying lengths of time before fixation. The distribution of label which had been incorporated into macromolecules in the worm tissues, was examined by light and electron microscope autoradiography. It was estimated that the tegument and tegument cell bodies were the source of 67--80%, and the gut epithelium of 20--30%, of exportable leucine-containing protein. Conversely, the gut epithelium was the source of 72%, and the tegument cells 28%, of exportable glucosamine-containing polysaccharide. The specific activity of labelled protein reached a peak in the tegument cytoplasm after 1.5 h of chase incubation. Half of the labelled protein was secreted into the worm's environment by 3 h of chase incubation. The half-life of secretory protein in gut cells appears to be around 2 h. Labelled protein disappears from the gut lumen relatively rapidly but labelled polysaccharide remains in the lumen at high specific activity for at least 24 h. The major carbohydrate labelled may be the glycocalyx on the luminal surface of the gut epithelial cells. The results suggest that the bulk of worm secretions have a rapid turnover with a half-life of a few hours. Against this background of rapid mass secretion a slower process of membrane turnover would be difficult to detect and quantitatively small.     

Endocytosis in cultures of Blastocystis hominis

A study of the function of the electron-dense pits in the vacuolar and granular forms of Blastocystis hominis was undertaken. Immuno-electron microscopy using anti-clathrin antibody and colloidal gold demonstrated clathrin to be associated with all forms of the pits and some cytoplasmic vesicles. Cationized ferritin traced the pathway of endocytosis from the surface of the coated pits through internalization via electron-dense coated vesicles and uncoated vesicles and tubules in the cytoplasm. The cationized ferritin particles accumulated in the central vacuole, suggesting a metabolic or storage role for this structure. Differences in the accumulation of cationized ferritin particles were noted between vacuolar and granular forms. The hydrolytic enzyme acid phosphatase was not detected within the central vacuole suggesting that this structure does not act as a lysosome.     

Induction cues for tegument formation during the transformation of Schistosoma mansoni cercariae

Adult schistosomes are parasitic blood flukes that have a continuous double lipid bilayered membrane surrounding the entire worm. This tegumental membrane is synthesised during invasion of the vertebrate host by free-swimming infectious forms called cercariae. As cercariae invade their final hosts they lose their tails and encounter a changing environment that includes altered temperature, sugar concentration and osmolarity. We have identified a glucose transporter protein designated SGTP4 that is found exclusively in the outer adult tegument and on membranous vesicles within the tegumental cytoplasm. By using immunofluorescence analysis to monitor the appearance and distribution of SGTP4 we can track the process of new tegumental membrane formation and examine the cues that trigger this developmental pathway. Cercariae in water do not transform their tegument while those incubated in rich medium do so rapidly. We have examined which of the many constituents of rich medium are responsible for triggering this transformation. Incubation in a solution of moderate osmolarity (120 mOsM PBS) is sufficient by itself to trigger tegument transformation, albeit at a slower rate relative to incubation in rich medium. Adding either glucose (to 100 mM) to the solution or increasing the temperature of incubation (from 22 degrees C to 37 degrees C) further increased the rate of tegument biogenesis. The introduction of glucose together with an increase in the incubation temperature further accelerated the process, suggesting that these factors act synergistically to promote transformation rates. The critical nature of osmolarity in inducing the process is highlighted by the fact that transformation proceeds as efficiently in 360 mOsM alone as it does in rich medium. While the fatty acids linolenic acid (cis-9, cis-12, cis-15-octadecatrienoic acid at 1 mM) and capric acid (Decanoic acid, at 0.1 mM) have both been proposed to stimulate tegumental transformation, we show that neither promotes the morphogenesis of a normal schistosomulum tegument. The schistosomicide praziquantel (to 1 mM) has no detectable effect on new tegument formation.     

Cytoskeletal features of the syncytial epidermis of the tapeworm Hymenolepis diminuta

A hallmark feature of parasitic platyhelminths is a cytoarchitecturally unusual syncytial epidermis composed of a peripheral layer of continuous cytoplasm (the ectocytoplasm) connected to underlying nucleated cell bodies by small cytoplasmic bridges. The helminth epidermis, or tegument, plays important roles in protection and nutrient acquisition; cestodes, in fact, completely lack a gastrointestinal tract and absorb all nutritive material through the tegument. Perhaps not surprisingly, the cestode tegument bears certain resemblances to the mucosal epithelium of the vertebrate small intestine, including the possession of a microvillous brush border upon the surface of the ectocytoplasm. In contrast to the intestinal epithelial cell, however, very little is known concerning the nature and organization of the cytoskeleton within the helminth epidermis. Therefore, a number of different microscopical preparative techniques were used to examine the tegument of the tapeworm Hymenolepis diminuta for the presence and distribution of microfilaments, intermediate filaments, and microtubules. It was found that both actin-containing microfilaments and intermediate-sized filaments are present but are restricted to specific locations along the plasmalemmae of the ectocytoplasm. In contrast, microtubules are found throughout the tegument, and are concentrated in the supranuclear regions of the perikarya and in the cytoplasmic bridges interconnecting the perikarya and ectocytoplasm. Unlike brush borders of most other epithelia, the cestode epidermal brush border lacks a filamentous terminal web and is instead associated with microtubules. A network of fine filaments, 5-8 nm in diameter but distinct from actin-containing microfilaments, runs throughout the ectocytoplasm and appears to interlink tegumental vesicles. These fine filaments may represent the primary "skeletal" system responsible for maintaining the structure of the tegumental cytoplasm.     

Morphometric analysis of coated pits and vesicles in the proximal and distal caput epididymidis

A morphometric analysis of coated and uncoated structures found in the apical portion of principal cells from both the proximal and distal caput epididymidis has been carried out. Almost all endocytic, coated vesicles are found within 1 micron of the luminal surface of principal cells and the volume fraction of these and of uncoated vesicles is much greater in the proximal caput epididymidis. A serial section analysis indicated that many coated "vesicles" are tangentially sectioned coated pits and that a complex network of interconnected vesicular and tubular structures exists in the apical cytoplasm. Efferent duct ligation has no effect on the number of size of large coated and uncoated vesicles in either the proximal or distal caput epididymidis, indicating that substances delivered to principal cells from the lumen are not required to maintain the endocytic machinery. However, this treatment does result in a considerable increase in the number of large coated vesicles associated with the basal surface of principal cells from the proximal but not the distal caput epididymidis. The volume fraction of small, presumably exocytic, coated vesicles is significantly greater in the apical cytoplasm of cells from the distal caput epididymidis in control animals. Efferent duct ligation results in a significant increase in the volume fraction of these vesicles in the proximal but not distal caput epididymidis. These results show that there are marked differences in structure among principal cells from these two regions of the epididymis and that this may reflect differences in control and function.     

Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).