Proteomic Analysis of Adult Ascaris suum Fluid Compartments and Secretory Products

Background

Strategies employed by parasites to establish infections are poorly understood. The host-parasite interface is maintained through a molecular dialog that, among other roles, protects parasites from host immune responses. Parasite excretory/secretory products (ESP) play major roles in this process. Understanding the biology of protein secretion by parasites and their associated functional processes will enhance our understanding of the roles of ESP in host-parasite interactions.

Methodology/Principal Findings

ESP was collected after culturing 10 adult female Ascaris suum. Perienteric fluid (PE) and uterine fluid (UF) were collected directly from adult females by dissection. Using SDS-PAGE coupled with LC-MS/MS, we identified 175, 308 and 274 proteins in ESP, PE and UF, respectively. Although many proteins were shared among the samples, the protein composition of ESP was distinct from PE and UF, whereas PE and UF were highly similar. The distribution of gene ontology (GO) terms for proteins in ESP, PE and UF supports this claim. Comparison of ESP composition in A. suum, Brugia malayi and Heligmosoides polygyrus showed that proteins found in UF were also secreted by males and by larval stages of other species, suggesting that multiple routes of secretion may be used for homologous proteins. ESP composition of nematodes is both phylogeny- and niche-dependent.

Conclusions/Significance

Analysis of the protein composition of A. suum ESP and UF leads to the conclusion that the excretory-secretory apparatus and uterus are separate routes for protein release. Proteins detected in ESP have distinct patterns of biological functions compared to those in UF. PE is likely to serve as the source of the majority of proteins in UF. This analysis expands our knowledge of the biology of protein secretion from nematodes and will inform new studies on the function of secreted proteins in the orchestration of host-parasite interactions.     

Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of Ascaris reveals a novel fold and two discrete lipid-binding sites

Background

Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.

Methodology/Principal Findings

The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.

Conclusions/Significance

This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.     

Phylogeographical Studies of Ascaris spp. Based on Ribosomal and Mitochondrial DNA Sequences

Background

The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the world''s most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples.

Methodology

151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions.

Principal Findings

No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum.

Conclusions/Significance

These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This conclusion has implications on the systematics, transmission and control programs relating to ascariasis.     

Signatures of selection in sheep bred for resistance or susceptibility to gastrointestinal nematodes

Background

Gastrointestinal nematodes are one of the most serious causes of disease in domestic ruminants worldwide. There is considerable variation in resistance to gastrointestinal nematodes within and between sheep breeds, which appears to be due to underlying genetic diversity. Through selection of resistant animals, rapid genetic progress has been demonstrated in both research and commercial flocks. Recent advances in genome sequencing and genomic technologies provide new opportunities to understand the ovine host response to gastrointestinal nematodes at the molecular level, and to identify polymorphisms conferring nematode resistance.

Results

Divergent lines of Romney and Perendale sheep, selectively bred for high and low faecal nematode egg count, were genotyped using the Illumina® Ovine SNP50 BeadChip. The resulting genome-wide SNP data were analysed for selective sweeps on loci associated with resistance or susceptibility to gastrointestinal nematode infection. Population differentiation using F_ST and Peddrift revealed sixteen regions, which included candidate genes involved in chitinase activity and the cytokine response. Two of the sixteen regions identified were contained within previously identified QTLs associated with nematode resistance.

Conclusions

In this study we identified fourteen novel regions associated with resistance or susceptibility to gastrointestinal nematodes. Results from this study support the hypothesis that host resistance to internal nematode parasites is likely to be controlled by a number of loci of moderate to small effects.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-637) contains supplementary material, which is available to authorized users.     

Peptidases Compartmentalized to the Ascaris suum Intestinal Lumen and Apical Intestinal Membrane

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.     

Tissue-specific direct targets of Caenorhabditis elegans Rb/E2F dictate distinct somatic and germline programs

Background

The tumor suppressor Rb/E2F regulates gene expression to control differentiation in multiple tissues during development, although how it directs tissue-specific gene regulation in vivo is poorly understood.

Results

We determined the genome-wide binding profiles for Caenorhabditis elegans Rb/E2F-like components in the germline, in the intestine and broadly throughout the soma, and uncovered highly tissue-specific binding patterns and target genes. Chromatin association by LIN-35, the C. elegans ortholog of Rb, is impaired in the germline but robust in the soma, a characteristic that might govern differential effects on gene expression in the two cell types. In the intestine, LIN-35 and the heterochromatin protein HPL-2, the ortholog of Hp1, coordinately bind at many sites lacking E2F. Finally, selected direct target genes contribute to the soma-to-germline transformation of lin-35 mutants, including mes-4, a soma-specific target that promotes H3K36 methylation, and csr-1, a germline-specific target that functions in a 22G small RNA pathway.

Conclusions

In sum, identification of tissue-specific binding profiles and effector target genes reveals important insights into the mechanisms by which Rb/E2F controls distinct cell fates in vivo.     

Nematode Homologue of PQBP1, a Mental Retardation Causative Gene,Is Involved in Lipid Metabolism

Background

PQBP1 is a causative gene for X-linked mental retardation (MR) whose patients frequently show lean body. C. elegans has a strictly conserved homologue gene of PQBP1, T21D12.3.

Methodology and Principal Findings

We generated Venus-transgenic and T21D12.3-mutant nematodes to analyze developmental expression patterns and in vivo functions of the nematode PQBP1 homologue protein (pqbp-1.1). During development, pqbp-1.1 is expressed from cell proliferation stage to larva stage. In larva, intestinal cells show the highest expression of pqbp-1.1, while it decreases in adult worms. The mutants of pqbp-1.1 show a decrease of the lipid content in intestinal cells. Especially, incorporation of fatty acid into triglyceride is impaired. ShRNA-mediated repression of PQBP1 also leads to reduction of lipid content in mammalian primary white adipocytes.

Conclusion/ Significance

These results suggest that pqbp-1.1 is involved in lipid metabolism of intestinal cells. Dysfunction of lipid metabolism might underlie lean body, one of the most frequent symptoms associating with PQBP1-linked MR patients.     

The New Anthelmintic Tribendimidine is an L-type (Levamisole and Pyrantel) Nicotinic Acetylcholine Receptor Agonist

Background

Intestinal parasitic nematodes such as hookworms, Ascaris lumbricoides, and Trichuris trichiura are amongst most prevalent tropical parasites in the world today. Although these parasites cause a tremendous disease burden, we have very few anthelmintic drugs with which to treat them. In the past three decades only one new anthelmintic, tribendimidine, has been developed and taken into human clinical trials. Studies show that tribendimidine is safe and has good clinical activity against Ascaris and hookworms. However, little is known about its mechanism of action and potential resistance pathway(s). Such information is important for preventing, detecting, and managing resistance, for safety considerations, and for knowing how to combine tribendimidine with other anthelmintics.

Methodology/Principal Findings

To investigate how tribendimidine works and how resistance to it might develop, we turned to the genetically tractable nematode, Caenorhabditis elegans. When exposed to tribendimidine, C. elegans hermaphrodites undergo a near immediate loss of motility; longer exposure results in extensive body damage, developmental arrest, reductions in fecundity, and/or death. We performed a forward genetic screen for tribendimidine-resistant mutants and obtained ten resistant alleles that fall into four complementation groups. Intoxication assays, complementation tests, genetic mapping experiments, and sequencing of nucleic acids indicate tribendimidine-resistant mutants are resistant also to levamisole and pyrantel and alter the same genes that mutate to levamisole resistance. Furthermore, we demonstrate that eleven C. elegans mutants isolated based on their ability to resist levamisole are also resistant to tribendimidine.

Conclusions/Significance

Our results demonstrate that the mechanism of action of tribendimidine against nematodes is the same as levamisole and pyrantel, namely, tribendimidine is an L-subtype nAChR agonist. Thus, tribendimidine may not be a viable anthelmintic where resistance to levamisole or pyrantel already exists but could productively be used where resistance to benzimidazoles exists or could be combined with this class of anthelmintics.     

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma

Background

Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed.

Results

Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101.

Conclusions

Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.     

Helminth and intestinal protozoa infections, multiparasitism and risk factors in Champasack province, Lao People's Democratic Republic

Background

Detailed investigations of multiparasitism are scarce in the Mekong River basin. We assessed helminth (trematode, nematode, and cestode), and intestinal protozoa infections, and multiparasitism in random population samples from three different eco-epidemiological settings in Champasack province, southern Lao People''s Democratic Republic (Lao PDR), and determined underlying risk factors.

Methodology

Two stool samples were collected from 669 individuals aged ≥6 months over consecutive days and examined for helminth infections using the Kato-Katz method. Additionally, one stool sample per person was subjected to a formalin-ethyl acetate concentration technique for diagnosis of helminth and intestinal protozoa infections. Questionnaires were administered to obtain individual and household-level data pertaining to behavior, demography and socioeconomic status. Risk factors for hepato-biliary and intestinal parasitic infections and multiparasitism were determined using multiple logistic regressions analyses.

Principal Findings

Multiple species intestinal parasite infections were common: 86.6% of the study participants harbored at least two and up to seven different parasites concurrently. Regarding nematode infections, hookworm was the most prevalent species (76.8%), followed by Ascaris lumbricoides (31.7%) and Trichuris trichiura (25.0%). Regarding trematodes, Opisthorchis viverrini and Schistosoma mekongi infections were found in 64.3% and 24.2% of the participants, respectively. Infections with intestinal protozoa were rare.

Conclusions/Significance

There is a pressing need to intensify and sustain helminth control interventions in the southern part of Lao PDR. Given the high prevalence with nematode and trematode infections and the extent of multiparasitism, preventive chemotherapy is warranted. This intervention should be coupled with health education and improved access to clean water and adequate sanitation to consolidate morbidity control and enhance sustainability.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.