Secreted cathepsin L-like peptidases are involved in the degradation of trapped antibodies on the surface of Echinostoma caproni.

Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.

The transcriptome of Echinostoma caproni adults: further characterization of the secretome and identification of new potential drug targets.

Echinostomes are cosmopolitan parasites that infect a large number of different warm-blooded hosts, both in nature and in the laboratory. They also constitute an important group of food-borne trematodes of public health importance mainly in Southeast Asia and the Far East. In addition, echinostomes are an ideal model to study several aspects of intestinal helminth biology, since they present a number of advantages. For example, echinostomes are large worms whose life cycle is relatively easy to maintain in the laboratory. Recently, several studies documented their great value in the study of intestinal helminth-vertebrate host relationship. Detailed knowledge of their genome, transcriptome and proteome is likely to have an important impact on the development of control strategies for intestinal helminths. We present the first transcriptome of the adult stage of Echinostoma caproni using 454 sequencing coupled to a semi-automated bioinformatic analyses. 557,236 raw sequence reads were assembled into 28,577 contiguous sequences using iAssembler. 23,296 putative proteins were characterized based on homology, gene ontology and/or biochemical pathways. Comparisons of the transcriptome of E. caproni with those of other trematodes revealed similarities in the transcription pattern of molecules inferred to have key roles in parasite-host interactions. Enzymatic proteins like kinases and peptidases were abundant. Of the 3415 predicted excretory/secretory proteins compiled (including non-classical secretory proteins), 180 different proteins were confirmed by proteomic analysis. Potential drug targets were also identified. BIOLOGICAL SIGNIFICANCE: In this study the first transcriptome of the adult stage of E. caproni is presented and compared to those of other trematodes revealing similarities in transcription for molecules inferred to have key roles in parasite-host interactions. 3415 predicted excretory/secretory proteins were compiled, being 180 different proteins confirmed by proteomic analysis. The current transcriptome data increased by nine times the number of previous protein identifications. In addition, potential drug targets for this parasite were identified. The present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of E. caproni, as well as a basis for applied outcomes, such as the development of novel methods of intervention against this model organism and related parasites.

Metabolic profiling of Echinostoma caproni and Schistosoma mansoni in their definitive and intermediate hosts.

This review examines metabolic profiling of Schistosoma mansoni and Echinostoma caproni in their definitive and intermediate hosts. The earlier coverage of the literature on metabolic profiling was reviewed by Wang et al. 2010, Advances in Parasitology, 73, 373-404 and covered mainly studies using proton nuclear magnetic resonance spectroscopy. The methods focused upon in our review are mainly chromatographic. In the studies reviewed, various metabolites were analyzed in hosts infected with either E. caproni or S. mansoni and compared to the uninfected controls.

Screening trematodes for novel intervention targets: a proteomic and immunological comparison of Schistosoma haematobium, Schistosoma bovis and Echinostoma caproni.

With the current paucity of vaccine targets for parasitic diseases, particularly those in childhood, the aim of this study was to compare protein expression and immune cross-reactivity between the trematodes Schistosoma haematobium, S. bovis and Echinostoma caproni in the hope of identifying novel intervention targets. Native adult parasite proteins were separated by 2-dimensional gel electrophoresis and identified through electrospray ionisation tandem mass spectrometry to produce a reference gel. Proteins from differential gel electrophoresis analyses of the three parasite proteomes were compared and screened against sera from hamsters infected with S. haematobium and E. caproni following 2-dimensional Western blotting. Differential protein expression between the three species was observed with circa 5% of proteins from S. haematobium showing expression up-regulation compared to the other two species. There was 91% similarity between the proteomes of the two Schistosoma species and 81% and 78·6% similarity between S. haematobium and S. bovis versus E. caproni, respectively. Although there were some common cross-species antigens, species-species targets were revealed which, despite evolutionary homology, could be due to phenotypic plasticity arising from different host-parasite relationships. Nevertheless, this approach helps to identify novel intervention targets which could be used as broad-spectrum candidates for future use in human and veterinary vaccines.

Excretory/secretory proteome of the adult stage of Echinostoma caproni.

The excretory/secretory proteome of Echinostoma caproni (Trematoda: Echinostomatidae) adults collected from experimentally infected mice was investigated using a proteomic approach. We performed a shot-gun liquid chromatography/tandem mass spectrometry for the separation and identification of tryptic peptides from the excretory/secretory products of E. caproni adult worms. Database search was performed using MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems). A total of 39 parasite proteins were accurately identified. Strikingly, metabolic enzymes, and particularly glycolytic enzymes, constituted the largest protein family in the excretory/secretory proteome of E. caproni adult worms. Moreover, representative proteins involved in parasite structure, response against stress, chaperones, calcium-binding, and signal transduction were also identified. This work extends our knowledge of host-parasite relationships in the E. caproni-rodent model that is extensively used to analyze the factors determining the intestinal helminth rejection. Consequently, information on many proteins may be useful to better understand the molecular basis that determines the survival of this parasite in the definitive host.

Panorganismal metabolic response modeling of an experimental Echinostoma caproni infection in the mouse.

Metabolic profiling of host tissues and biofluids during parasitic infections can reveal new biomarker information and aid the elucidation of mechanisms of disease. The multicompartmental metabolic effects of an experimental Echinostoma caproni infection have been characterized in 12 outbred female mice infected orally with 30 E. caproni metacercariae each, using a further 12 uninfected animals as a control group. Mice were killed 36 days postinfection and brain, intestine (colon, ileum, jejeunum), kidney, liver, and spleen were removed. Metabolic profiles of tissue samples were measured using high-resolution magic angle spinning (1)H NMR spectroscopy and biofluids measured by applying conventional (1)H NMR spectroscopy. Spectral data were analyzed via principal component analysis, partial least-squares-derived methods and hierarchical projection analyses. Infection-induced metabolic changes in the tissues were correlated with altered metabolite concentrations in the biofluids (urine, plasma, fecal water) using hierarchical modeling and correlation analyses. Metabolic descriptors of infection were identified in liver, renal cortex, intestinal tissues but not in spleen, brain or renal medulla. The main physiological change observed in the mouse was malabsorption in the small intestine, which was evidenced by decreased levels of various amino acids in the ileum, for example, alanine, taurine, glutamine, and branched chain amino acids. Furthermore, altered gut microbial activity or composition was reflected by increased levels of trimethylamine in the colon. Our modeling approach facilitated in-depth appraisal of the covariation of the metabolic profiles of different biological matrices and found that urine and plasma most closely reflected changes in ileal compartments. In conclusion, an E. caproni infection not only results in direct localized (ileum and jejenum) effects, but also causes remote metabolic changes (colon and several peripheral organs), and therefore describes the panorganismal metabolic response of the infection.

Molecular cloning and characterization of Echinostoma caproni heat shock protein-70 and differential expression in the parasite derived from low- and high-compatible hosts.

We cloned and expressed Echinostoma caproni HSP70 in Escherichia coli. This molecule presents an open reading frame (ORF) of 655 amino acids, and a theoretical molecular weight of 71 kDa. E. caproni HSP70 protein showed a high homology to other helminth molecules, major differences being located in the C-terminal region of the molecule, with a hydrophobic portion. Studies of protein and messenger RNA (mRNA) expression revealed a distinct pattern, depending on the host (low- or high-compatible). Specific polyclonal antisera raised against the recombinant protein expressed in Escherichia coli demonstrated its selective presence in excretory/secretory products (ESP) of adult parasites obtained from high-compatible hosts. Immunological studies showed clearly the association of HSP70 with the parasite surface and other structures, including eggs.

Excretory-secretory proteome of larval Schistosoma mansoni and Echinostoma caproni, two parasites of Biomphalaria glabrata.

Schistosoma mansoni and Echinostoma caproni are two trematode species that use different strategies (mimicry and immunosuppression, respectively) to interfere with the snail innate immune system. Parasites excretory-secretory (ES) products have been shown to play a key role in these host-parasite immune interactions. However, they remain largely uncharacterized in larval trematodes. We developed a global proteomic approach to characterize the ES proteome of S. mansoni and E. caproni primary sporocysts. In ES products of both parasites, we found proteins involved in reactive oxygen species scavenging, glycolysis, signalling or calcium binding (superoxide dismutase Cu/Zn; glutathione S-transferase; aldo-keto-reductase; triose-phosphate isomerase; glyceraldehyde-3-phosphate dehydrogenase; aldolase, enolase, MICAL-like, calreticulin). According to their predicted functions, we propose a model in which these proteins (i) are involved in antioxidant activity, (ii) prevent hemocyte encapsulation process or (iii) favor invasion and migration of sporocysts in host tissues. These results suggest that S. mansoni and E. caproni sporocysts develope a strong immune protection during the first hours of infection giving them enough time to build up a long lasting immune evasion strategy relying on molecular mimicry or immunosuppression, respectively.

Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections.

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.

Hemozoin formation in Echinostoma trivolvis rediae.

Rediae of the trematode Echinostoma trivolvis, from naturally infected Helisoma trivolvis snails, form a black pigment while inside the snail host. Here we examine the black pigment to show that the insolubility characteristics in detergent and weak base solution are identical to Plasmodium falciparum hemozoin. Laser desorption mass spectrometry of the purified pigment demonstrates the presence of heme. Examination of purified pigment under polarized light microscopy illuminates ordered birefringent crystals. Field emission in lens scanning electron microscopy reveals irregular ovoid crystals of 200-300 nm in diameter. The purified pigment crystals seeded extension of monomeric heme onto the crystal which by Fourier Transform Infrared analysis is beta-hematin. Rediae of a second echinostome parasite, Echinostoma caproni, from experimentally infected Biomphalaria glabrata, do not produce measurable or recoverable heme crystals. These observations are consistent with heme crystal formation by a hematophagous parasite within a non-vertebrate intermediate host.

Specific tyrosine phosphorylation in response to bile in Fasciola hepatica and Echinostoma friedi.

Protein tyrosine phosphorylation (PY) is a well-known signalling mechanism which is also involved in host-parasite interactions. Despite its transcendence, PY has been poorly studied in parasitic helminths. The aim of this study is to examine the effect of bile salts on the PY pattern in parasitic trematodes. Two distinct adult models were analysed: Echinostoma friedi, of intestinal habitat, and Fasciola hepatica, naturally inhabitant of host biliary channels. Our results show that bile salts induce specific and distinct protein PY in both trematode species, indicating that this signalling process seems to be also involved in host-trematode relationships.

Effects of tonicity on the release of neutral lipids in Echinostoma caproni adults and observations on lipids in excysted metacercariae.

High performance thin layer chromatography was used to analyze neutral lipids in worm incubates isotonic, hypotonic, and hypertonic to the intestinal habitat of adult Echinostoma caproni. Qualitative analysis revealed the presence of free sterols, free fatty acids, triacylglycerols, and a steryl ester/hydrocarbon fraction in all incubate samples. The most abundant neutral lipid fraction released into the incubation medium was the triacylglycerol fraction. This fraction was quantified after worms were maintained for 2 h at 37.5 degrees C in hypertonic (Locke's 2x solution), isotonic (Locke's 0.5x solution) and hypotonic (deionized water) media. Percentages of triacylglycerols on a wet-weight basis found in Locke's 2x, 0.5x, and deionized water were 0.369, 3.23, and 0.242, respectively, suggesting that the optimal medium to obtain maximal excretory-secretory products is the Locke's 0.5x solution. Histochemical staining of whole excysted metacercariae with oil red O did not detect neutral lipids. Analysis of 500 excysted metacercariae incubated for 2 h at 37.5 degrees C revealed that free sterols, free fatty acids, and triacylglycerols were released in amounts of 16.2, 1.59, and 5.34 ng/organism, respectively. Our results were compared with previous studies on neutral lipids in excysted metacercariae and adults of E. trivolvis. Variations in the results of our study compared with others reflect intrinsic differences in the species of echinostome used.

Schistosoma mansoni and Echinostoma caproni excretory-secretory products differentially affect gene expression in Biomphalaria glabrata embryonic cells.

Biomphalaria glabrata embryonic (Bge) cells have been shown to be a valuable in vitro cellular model for the study of snail host-parasite interactions. They both promote the growth and differentiation of various trematode species including Schistosoma mansoni, and Echinostoma caproni and share some morphological and functional features with circulating haemocytes. As an approach to investigate snail genes potentially regulated following exposure to trematode excretory-secretory (ES) products, we compared gene expression profiles of Bge cells exposed to saline solution, or saline solution containing ES products from S. mansoni or E. caproni, two trematode species parasitizing B. glabrata. Following differential display RT-PCR analysis we characterized 23 differentially displayed cDNAs and we focussed on the 5 cDNAs showing sequence similarity to known genes for expression validation. Using RT-PCR, we confirmed that ES products from S. mansoni and E. caproni differentially affect the expression levels of 4 out of the 5 transcripts. These partial transcripts corresponded to novel B. glabrata sequences, and showed significant sequence similarity to genes coding for (i) cytochrome C, (ii) methyl-binding proteins, (iii) glutamine synthetases, and (iv) protease inhibitors from the Kunitz family. The possible significance of these gene expression changes in host-parasite molecular interactions is discussed.

Differences in adult excretory-secretory products between geographical isolates of Echinostoma caproni.

Because of the potentially important role of parasite excretory-secretory (ES) products in regulating intermate relationships and host-parasite interactions, we investigated intraspecific variability of adult ES products from a trematode species. Adults from 3 geographical isolates of Echinostoma caproni were collected and maintained under in vitro conditions. ES products were collected at 4, 8, and 22 hr of in vitro maintenance. In order to test for interspecific variability, ES products from a different echinostome species (Echinostoma sp.) were collected in a similar way. Major ES polypeptides were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and silver staining. The polypeptide patterns of total ES products appeared very stable in time for each isolate. ES products from Echinostoma sp. showed a distinct polypeptide profile with none of the major bands being shared with the E. caproni isolates. Although polypeptide patterns from the 3 E. caproni isolates shared most major bands, isolate-specific bands could be observed. Two isolates exhibited a band at 85 and 119 kDa, respectively, whereas the third isolate was characterized by the absence of both bands. These results, together with the previously reported role of ES products in molecular signaling raise the question of the importance of intraspecific ES product differences in evolutionary processes such as assortative mating or local host adaptation.

Effects of benzimidazole anthelmintics as microtubule-active drugs on the synthesis and transport of surface glycoconjugates in Hymenolepis microstoma, Echinostoma caproni, and Schistosoma mansoni.

Hymenolepis microstoma (Cestoda), Echinostoma caproni, and Schistosoma mansoni (Digenea) were exposed to benzimidazoles to determine the influence of the drugs on the secretion of glycoconjugates that protect the worms' surface. Worms were obtained from mice treated with mebendazole or albendazole, and the glycoconjugates were localized in the parasite tissues by cytochemistry using lectin-gold conjugates. Events leading to the death of H. microstoma and E. caproni extended over a medication period for at least 2-3 days, and the following interrelated phases were discernible. Upon depolymerization of the microtubules the tegumentary cytons continued to synthesize glycoconjugates for up to about 24 h. Vesicles containing the glycans accumulated in the cytons, but their microtubule-based transport to the distal tegument was inhibited. At about 1 day the Golgi complex became fragmented and the production of glycans sharply declined. As a consequence of this and an ongoing turnover of the surface coat the contents of glycoconjugates in the distal tegument decreased. Similar effects were produced by vinblastine and colchicine in vitro. In contrast, benzimidazole treatment of S. mansoni, which is reportedly inefficacious, did not alter the replenishment of the surface glycoconjugates. Diminution of the coating with glycoconjugates of the surface of drug-sensitive species constitutes a secondary effect of benzimidazoles that might, synergistically with immune mechanisms of the host, enhance the expulsion of the worms.

Histochemical glycogen and neutral lipid in Echinostoma trivolvis cercariae and effects of exogenous glucose on cercarial longevity.

Histochemical glycogen and neutral lipid studies were conducted on Echinostoma trivolvis cercariae maintained in artificial spring water (ASW) at 24-25 degrees C for up to 24 h after emergence from host snails. Treatment of whole cercariae by the periodic acid Schiff (PAS) reagent with or without 1% malt diastase showed that cercariae depleted glycogen mainly from the tail by 6 to 24 h postemergence. The posterior tip of the tail remained PAS positive and diastase fast suggesting the presence of mucopolysaccharides there. Fresh cercariae or those stained up to 24 h postemergence with Oil Red O showed the presence of neutral lipid droplets in the excretory system. There was no discernible difference in the size, abundance, or distribution of these droplets in fresh or aged cercariae. Cercariae maintained in ASW plus 1% glucose for 12 or 23 h showed no evidence of resynthesizing glycogen. Nevertheless, cercariae survived longer in 1% glucose than in either 0.0, 0.1 or 0.5% glucose; but only at 23 h were any differences statistically greater (one way ANOVA, P < 0.05).

Tubulin polyglycylation in Platyhelminthes: diversity among stable microtubule networks and very late occurrence during spermiogenesis.

The distribution of glycylated tubulin has been analyzed in different populations of stable microtubules in a digenean flatworm, Echinostoma caproni (Platyhelminthes). Two cellular types, spermatozoa and ciliated excretory cells, have been analyzed by means of immunofluorescence, immunogold, and immunoblotting techniques using two monoclonal antibodies (mAbs), AXO 49, and TAP 952, specifically directed against differently glycylated isoforms of tubulin. The presence of glycylated tubulin in the two cell types was shown. However, the differential reactivities of TAP 952 and AXO 49 mAbs with the two axoneme types suggest a difference in their glycylation level. In addition, within a single cell, the spermatozoon, cortical microtubules underlying the flagellar membrane, and axonemal microtubules were shown to comprise different tubulin isoforms, the latter ones only being labelled with one of the antiglycylated tubulin mAbs, TAP 952. Similarly, the antiacetylated (6-11B-1) and polyglutamylated (GT335) tubulin mAbs decorated the two types of axonemal microtubules, but not the cortical ones. From these data, a subcellular sorting of posttranslationally modified tubulin isoforms within spermatozoa, on the one hand, and a cellular sorting of glycylated isoforms inside the whole organism, on the other hand, is demonstrated in the flatworm E. caproni. Last, a sequential occurrence of tubulin posttranslational modifications was observed in the course of spermiogenesis. Acetylation appears first, followed shortly by glutamylation; glycylation takes place at the extreme end of spermiogenesis and, specifically, in a proximo-distal process. Thus in agreement with, and extending other studies [Bré et al., 1996], glycylation appears to close the sequence of posttranslational events occurring in axonemal microtubules during spermiogenesis.

Spermiogenesis and spermatozoon of Echinostoma caproni (Platyhelminthes, Digenea): transmission and scanning electron microscopy, and tubulin immunocytochemistry.

Spermiogenesis and the spermatozoon of Echinostoma caproni (from experimentally infested laboratory mice) were investigated by several methods. Transmission electron microscopy shows that spermiogenesis consists of proximo-distal fusion of three processes followed by elongation of the spermatid. Scanning electron microscopy shows that the spermatozoon is a filiform cell, 235 microns in length, with a cylindrical anterior extremity and a broader posterior extremity. Epifluorescence microscopy, including immunocytochemistry of tubulin and labelling of nucleus with specific dyes, has provided valuable additional information. Migration of the nuclei from the common cytoplasmic mass of spermatids to the distal part of the elongating spermatids is visualized, and centrioles demonstrated in the proximal, anterior region, and the nucleus in the distal, posterior region of the spermatozoon. One axoneme has a distal extremity which in the mature spermatozoon extends 30 microns more distally than the other, with the result that the posterior part of the spermatozoon contains a single axoneme and nucleus. Immunocytochemistry experiments show that a region, 15 microns in length, not labelled by the anti-tubulin antibodies with certain fixation-permeabilization procedures, corresponds to a region which, by transmission electron microscopy, shows external ornamentation on the membrane. This region has a bilaterally asymmetric pattern (in TEM), forms angles or coils according to the fixation used, and marks the boundary between two distinct patterns of movement. Spermiogenesis and the spermatozoon in E. caproni correspond to the general pattern found in the digeneans, with the exception of this asymmetric region. It is emphasized that the use of various methods provides a better understanding of sperm structure than transmission electron microscopy alone, particularly in the case of long, filiform spermatozoa.

Effects of diet on the lipid composition of Echinostoma caproni (Trematoda) in ICR mice.

High-performance thin-layer chromatography (HPTLC) was used to determine neutral lipids and phospholipids in the intestinal trematode Echinostoma caproni from experimentally infected ICR mice fed a high-fat diet (hen's egg yolk) as compared with worms from mice fed a standard laboratory diet. Worms were removed from the hosts at 2, 3, and 4 weeks postinfection (p.i.). Analysis by TLC-densitometry showed significantly greater amounts of triacylglycerols and free sterols at 2, 3, and 4 weeks p.i. in worms from mice on the high-fat diet as compared with worms from mice on the standard laboratory diet. Significantly greater amounts of phosphatidylcholine and phosphatidylethanolamine were found in worms from mice on the high-fat diet as compared with worms from those on the standard diet at 2 weeks p.i. but not at 3 and 4 weeks p.i. The results of this study suggest that the host diet influences the lipid content of E. caproni adults.

In vitro studies on intraspecific and interspecific chemical attraction in daughter rediae of Echinostoma trivolvis and E. caproni.

In vitro pairing and aggregation studies on daughter rediae of Echinostoma trivolvis and E. caproni were done at 22 degrees C in a Petri dish bioassay containing an agar substratum and a Locke's solution overlay. Pairing or aggregation was considered positive when rediae were in contact or within 1 mm of each other. Intraspecific and interspecific pairing or aggregation occurred in the bioassay when rediae were initially placed 5 or 10 mm apart. Movement of a single redia in the bioassay to a dialysis sac containing 1-10 rediae showed that intraspecific and interspecific pairing occurred in the absence of redial tactile stimulation. Movements of single rediae in the bioassay to agar plugs impregnated with redial excretory/secretory (ES) products occurred. The lipophilic fraction of the ES products was significantly more attractive than the hydrophilic fraction. The significance of redial chemical communication is not clear.