Solution Structure,Membrane Interactions,and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.     

Excretory/Secretory Proteome of the Adult Developmental Stage of Human Blood Fluke, Schistosoma japonicum

Schistosomes are the causative agents of schistosomiasis, one of the most prevalent and serious of the parasitic diseases that currently infects ∼200 million people worldwide. Schistosome excretory/secretory (ES) proteins have been shown to play important roles in modulating mammalian host immune systems. In our current study, we performed a global proteomics identification of the ES proteins from adult worms of Schistosoma japonicum, one of the three major schistosome species. Our results unambiguously identified 101 proteins, including 53 putatively secreted proteins. By quantitative analysis, we revealed fatty acid-binding protein as a major constituent of the in vitro ES proteome. Strikingly the heat shock proteins HSP70s, HSP90, and HSP97 constituted the largest protein family in the ES proteome, implying a central role for these proteins in immunomodulation in the host-parasite relationship. Other important S. japonicum ES proteins included actins, 14-3-3, aminopeptidase, enolase, and glyceraldehyde-3-phosphate dehydrogenase, some of which have been considered as viable vaccine candidates and therapeutic targets. A comparison with previous studies suggests that 48.5% of S. japonicum ES proteins are common to other parasite ES products, indicating that the molecular mechanisms involved in evading the host immune response may be conserved across different parasites. Interestingly seven host proteins, including antimicrobial protein CAP18, immunoglobulins, and a complement component, were identified among in vitro S. japonicum ES products likely originating from the schistosome tegument or gut, indicating that host innate and acquired immune systems could defend against schistosome invasion. Our present study represents the first attempt at profiling S. japonicum ES proteins, provides an insight into host-parasite interactions, and establishes a resource for the development of diagnostic agents and vaccines for the control of schistosomiasis.Schistosomes, or blood flukes, are water-borne parasites that are the causative agents of schistosomiasis. An estimated 200 million people worldwide are infected with schistosomes with an additional 650 million people at risk of infection (1). One of the major species of schistosomes, Schistosoma japonicum, is a mammalian parasite endemic in East Asia, especially in China and the Philippines. Schistosomes have complex life cycles. Larval schistosome worms (cercariae) are released by freshwater snails and subsequently invade their definite hosts, human or other mammals, via skin penetration. Once in a host animal, cercariae develop into schistosomula and adult worms, which reside in the portal mesenteric system of the host. When the females lay eggs, some eggs leave the host body and hatch in bodies of water as miracidia. The miracidia seek out and penetrate intermediate host snails, completing the schistosome life cycle.Schistosome strategies for evasion of the host immune system, which permit extended survival in mammalian hosts, are not well understood. These dominant evasion strategies have been described as a system of mimicry capable of producing antigens that are similar to endogenous host components (2–4), antigen disguise through acquisition of host molecules to cover the outer worm surface (5, 6), and immunological modulation through interference with host immune systems (7–10). Among these strategies, schistosome excretory/secretory (ES)¹ products have been shown to elicit host immunological modulation functions (7, 11). Schistosome ES proteins are released or secreted from epithelial surfaces of the gut and/or tegument as well as other specialized ES organs throughout almost all life stages. Schistosoma mansoni primary sporocysts have been reported to synthesize and secrete a wide variety of glycoproteins when cultured in vitro (12, 13). These glycoproteins were shown to have antioxidant activities against potential oxidative killing by mollusk defense systems (14). Similarly ES molecules from schistosome cercariae were also reported to down-regulate host immune responses (7). The anti-inflammatory activity of S. mansoni schistosomula ES products (ESPs) was found to be dominantly associated with Sm16.8 protein (15). In addition, ES proteins from S. mansoni adult worms (16), eggs (17), and miracidia (18) have also been investigated.Identification of all ES complex components is important for understanding how schistosomes regulate host immune systems to establish chronic infections and also other aspects of parasite-host interaction. Importantly this information can be expected to facilitate the discovery of vaccines and new therapeutic drug targets as well as new diagnostic reagents for schistosomiasis control. Proteomics approaches encompass the most efficient and powerful tools for identification of protein complexes and have been widely used to decipher the ES components of the filarial parasite Brugia malayi (19), Leishmania (Trypanosomatidae) (20), nematodes (21–26), and Trematoda (27–37). For the genus Schistosoma, the ES compositions of S. mansoni have been identified in many developmental life stages, including sporocyst (34), cercaria (35, 36), and egg (37) but have not been characterized in the adult worm.Characterization of the S. japonicum ES proteome has not been reported. S. japonicum is significantly different from S. mansoni and Schistosoma hematobium in skin invasion, skin migration, and its developmental patterns of swift migration and maturation (38–40). As such, S. japonicum represents a distinct and valuable model for the study of blood fluke immune evasion strategies. Our research group recently generated and reported a large number of S. japonicum protein-coding genes and expressed sequence tags (ESTs) (41, 42). This preliminary work provides important translated protein sequence data resources for mass spectrum data searching. The present study characterized the in vitro ES proteome of adult worms of S. japonicum (43) using a high throughput LC-MS/MS screening. This life stage spans the longest time frame of parasitic interaction with the host that is distinct from previous reports on proteomics identification of ES compositions in other species. Finally the present study confidently identified 101 S. japonicum ES proteins. This information represents substantial progress toward deciphering the worm ES proteome. These new data provide the basis for further investigations into the molecular basis of schistosome modulation of host immunity, increase the possibility of identifying vaccine candidates and new drug targets, and may aid the development of protein probes for selective and sensitive diagnosis of schistosomiasis.     

SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

Background

Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins.

Methodology/Principal Findings

We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms.

Conclusions/Significance

SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable ‘marker sequences’ for inclusion in future phylogenetic analyses.     

MicroRNAs Are Involved in the Regulation of Ovary Development in the Pathogenic Blood Fluke Schistosoma japonicum

Schistosomes, blood flukes, are an important global public health concern. Paired adult female schistosomes produce large numbers of eggs that are primarily responsible for the disease pathology and critical for dissemination. Consequently, understanding schistosome sexual maturation and egg production may open novel perspectives for intervening with these processes to prevent clinical symptoms and to interrupt the life-cycle of these blood-flukes. microRNAs (miRNAs) are key regulators of many biological processes including development, cell proliferation, metabolism, and signal transduction. Here, we report on the identification of Schistosoma japonicum miRNAs using small RNA deep sequencing in the key stages of male-female pairing, gametogenesis, and egg production. We identified 38 miRNAs, including 10 previously unknown miRNAs. Eighteen of the miRNAs were differentially expressed between male and female schistosomes and during different stages of sexual maturation. We identified 30 potential target genes for 16 of the S. japonicum miRNAs using antibody-based pull-down assays and bioinformatic analyses. We further validated some of these target genes using either in vitro luciferase assays or in vivo miRNA suppression experiments. Notably, suppression of the female enriched miRNAs bantam and miR-31 led to morphological alteration of ovaries in female schistosomes. These findings uncover key roles for specific miRNAs in schistosome sexual maturation and egg production.     

Uncovering Biological Application of Brazilian Green Propolis: A Phenotypic Screening against Schistosoma mansoni

The chemotherapy of schistosomiasis remains centered in the use of praziquantel, however, there has been growing resistant parasites to this drug. Thus, the aim of this work was to evaluate in vitro schistosomicidal activity of the hexanes/dichloromethane 1 : 1 extract of Brazilian green propolis (Pex), as well as its major isolated compounds artepillin C, caffeic acid, coumaric acid and drupanin against Schistosoma mansoni. The Pex was active by displaying an IC₅₀ value of 36.60 (26.26–51.13) μg mL^?1 at 72 h against adult worms of S. mansoni. The major isolated compounds were inactive with IC₅₀ values >100 μM, however, the combination of the isolated compounds (CM) in the same range found in the extract was active with an IC₅₀ value of 41.17 (39.89–42.46) μg mL^?1 at 72 h. Pex and CM induced alteration in the tegument of S. mansoni, and caffeic acid caused alteration in egg's maturation. Pex displayed in vitro activity against adult worms’ and eggs’ viability of S. mansoni, which opens new perspectives to better understand the synergistic and/or additive effects promoted by both Pex extract and CM against schistosomiasis features.     

Analysis of Schistosoma mansoni genes using the expressed sequence tag approach

Expressed sequence tags (ESTs) are partial cDNA sequences read from both ends of random expressed gene fragments used for discovering new genes. DNA libraries from four different developmental stages of Schistosoma mansoni used in this study generated 141 ESTs representing about 2.5% of S. mansoni sequences in dbEST. Sequencing was done by the dideoxy chain termination method. The sequences were submitted to GenBank for homology searching in nonredundant databases using Basic Local Alignment Search Tool for DNA (BLASTN) alignment and for protein (BLASTX) alignment at the National Center for Biotechnology Information (NCBI). Among submitted ESTs, 29 were derived from lambdagt11 sporocyst library, 70 from lambdaZap adult worm library, 31 from lambdaZap cercarial library, and 11 from lambdaZap female B worm library. Homology search revealed that eight (5.6%) ESTs shared homology to previously identified S.mansoni genes in dbEST, 15 (10.6%) are homologous to known genes in other organisms, 116 (81.7%) showed no significant sequence homology in the databases, and the remaining sequences (2.1%) showed low homologies to rRNA or mitochondrial DNA sequences. Thus, among the 141 ESTs studied, 116 sequences are derived from noval, uncharactarized S. mansoni genes. Those 116 ESTs are important for identification of coding regions in the sequences, helping in mapping of schistosome genome, and identifying genes of immunological and pharmacological significance.     

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.     

RNA干扰MDCht9基因的家蝇幼虫的转录组分析

家蝇(Musca domestica)属于双翅目昆虫,广泛分布于世界各地,目前家蝇抗药现象较为严重.为了筛选及挖掘与防治有害医学昆虫相关的分子靶标,本研究以家蝇幼虫为实验对象,采用RNAi方法沉默家蝇几丁质酶MDCht9基因后,分析幼虫mRNA转录组表达变化,初步探讨MDCht9的功能.研究结果表明,在注射dsRNA ...     

Underestimation of Schistosoma mansoni prevalences

Field methods used for detecting Schistosoma mansoni infection miss a certain proportion of the infections. Prevalences of infection appear to be far under-estimated by faecal screening, with important consequences for control and research. Sake de Vlos and Bruno Gryseels investigate how the number of undetected infections can be statistically inferred from population surveys.     

Fingerprint of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia, using RAPD-PCR

In the time schistosomisis control programs are implemented in many countries, schistosomiasis continues to spread throughout the world. Among these control strategies is the vector control. Within this context, analysis of the genetic variability of the intermediate host snails is important because it allows identification of specific sequences of the genome of this mollusk related to determine their fingerprint. We investigated Biomphalaria arabica, which is found in Saudi Arabia, the intermediate host of Schistosoma mansoni infection. Genetic fingerprint was studied by RAPD-PCR using our own different random primers as well as published primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers of B. arabica. This information will be helpful in the identification of the snails and demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for establishment of genetic barcode development.