Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula migrating to final localization. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far concerning the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled distinguishing PA and CA glands for microdissection. Using 7.5 × 106 μm3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least 2 of 3 replicates in either gland type (PA = 455, 40 exclusives; CA = 421, 6 exclusives; 60 proteins differed significantly in their abundance between the glands). Peptidases of 5 catalytic types accounted for ca. 8 % and 6 % of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. CA glands were rich in venom allergen-like proteins. The assembled total cercarial body proteome included 1631 identified proteins and revealed additional interesting factors possibly related to tissue invasion.HighlightsProteomes of two penetration gland types in schistosome cercariae greatly differPostacetabular glands possess 40 unique proteins and are abundant in hydrolasesCircumacetabular glands posses 6 unique proteins and are rich in VAL proteinsPeptidases make up 8 % of postacetabular and 6 % of circumacetabular gland proteinsCercarial elastase is unique to circumacetabular glands of Trichobilharzia szidatiNote: Supplementary data associated with this article All supplementary data files can be accessed from the following link: http://www.helminthology.cz/supplementary_files.html

The Transcriptome of Schistosoma mansoni Developing Eggs Reveals Key Mediators in Pathogenesis and Life Cycle Propagation

Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by parasite eggs becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species—Schistosoma mansoni, S. japonicum and S. haematobium—the transcriptomic profiles of developing eggs remain under unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis, days 1-3 and 3-6 post-oviposition, respectively. Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq datasets from S. mansoni eggs collected from the livers of rodent hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidial development and life cycle progression of schistosomes.

Genomic reconstruction of the introduction and diversification of golden potato cyst nematode populations in Indonesia

Potato cyst nematodes (PCN), the umbrella term for Globodera rostochiensis and G. pallida, co-evolved with their Solanaceous hosts in the Andeans. From there, PCN proliferated worldwide to virtually all potato production areas. PCN is a major factor limiting the potato production in Indonesia. In our survey, only G. rostochiensis was found. Fourteen field populations were collected on Java and Sumatra, and unique variants were called by mapping re-sequencing data on a G. rostochiensis reference genome. A phylogenetic tree based on 1.4 million unique variants showed a genotypic separation between the outgroup, a Scottish Ro1 population, and all Indonesian populations. This separation was comparable in size to the genotypic distinction between the Javanese and the Sumatran PCN populations. Next, variants within PCN effector gene families SPRYSEC, 1106, 4D06, and venom allergen-like protein (VAL) that all interfere with the host innate immune system were compared. Distinct selective pressures acted on these effector families; while SPRYSECs (4,341 SNPs/indels) behaved like neutral genes, the phylogenetic trees of 1106, 4D06 and VAL proteins (respectively 235, 790 and 150 SNPs/indels) showed deviating topologies. Our data suggest that PCN was introduced on Java not too long after the introduction of potato in the middle of the 18th century. Soon thereafter, the pathogen established on Sumatra, and started to diversify independently. This scenario was corroborated by diversification patterns of the effector families 1106, 4D06 and VAL. Our data demonstrate how genome re-sequencing data from a non-indigenous pathogen can be used to reconstruct the introduction and diversification process.

The transcriptome of Schistosoma mansoni developing eggs reveals key mediators in pathogenesis and life cycle propagation.

Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by the eggs laid by the parasites and becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species, i.e. Schistosoma mansoni, S. japonicum and S. haematobium, the transcriptomic profiles of developing eggs remain largely unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis (days 1-3 and 3-6 post-oviposition, respectively). Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq dataset from S. mansoni eggs collected from the livers of murine hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were found enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidia development and life cycle progression of schistosomes.

A Venom Allergen-Like Protein, RsVAP, the First Discovered Effector Protein of Radopholus similis That Inhibits Plant Defense and Facilitates Parasitism

Radopholus similis is a migratory endoparasitic nematode that is extremely harmful to host plants. Venom allergen-like proteins (VAPs) are members of the cysteine-rich secretory protein family that are widely present in plants and animals. In this study, we cloned a VAP gene from R. similis, designated as RsVAP. RsVAP contains an open reading frame of 1089 bp encoding 362 amino acids. RsVAP is specifically expressed in the esophageal gland, and the expression levels of RsVAP are significantly higher in juveniles than in other life stages of R. similis. This expression pattern of RsVAP was consistent with the biological characteristics of juveniles of R. similis, which have the ability of infection and are the main infection stages of R. similis. The pathogenicity and reproduction rate of R. similis in tomato was significantly attenuated after RsVAP was silenced. In tobacco leaves transiently expressing RsVAP, the pathogen-associated molecular pattern-triggered immunity (PTI) induced by a bacterial flagellin fragment (flg22) was inhibited, while the cell death induced by two sets of immune elicitors (BAX and Gpa2/RBP-1) was repressed. The RsVAP-interacting, ras-related protein RABA1d (LeRabA1d) was identified in tomato hosts by yeast two-hybrid and co-immunoprecipitation assays. RsVAP may interact with LeRabA1d to affect the host defense response, which in turn facilitates nematode infection. This study provides the first evidence for the inhibition of plant defense response by a VAP from migratory plant-parasitic nematodes, and, for the first time, the target protein of R. similis in its host was identified.

Bioinformatics evaluation of the homologues of Schistosoma mansoni biomarker proteins of bladder cancer in other Schistosoma species

Schistosomiasis remains a public health problem in developing countries. An ideal diagnostic test capable of detecting parasites as early as possible after the onset of infection is therefore highly desired. The identification of biomarker proteins associated with active infection and immune response may constitute the basis for the development of a successful diagnostic test. The aim of this study is to contribute to the development of protein biomarkers for schistosomiasis using a bioinformatics approach. The homologues of 36 previously identified urine biomarker proteins from a Schistosoma mansoni database, were identified in other Schistosoma species using SMARTBLAST and analyzed for similarities and differences using multiple sequence alignment. Of the 36 S. mansoni biomarker proteins, 29 had homologues with both S. haematobium and S. japonicum or either of S. haematobium and S. japonicum. Most of the 29 S. mansoni biomarker proteins aligned with their homologues with many conserved regions. However, some vital biomarker proteins like venom allergen-like proteins, which had been proposed as a putative drug and vaccine target, showed more semi conserved regions in which the amino acids had similar shape but weakly similar properties. The predictions of 12 markers found in all three species also show that treatment of infections may possibly benefit from the investigational drug Artenimol and specific nutraceuticals or supplements. Experimental evaluation is needed to confirm the potential of the proteins as biomarkers for early diagnosis of schistosomiasis and associated bladder cancer.

Quantitative Proteomics Comparison of Total Expressed Proteomes of Anisakis simplex Sensu Stricto, A. pegreffii, and Their Hybrid Genotype

The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes’ biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomics.