Aaron G. Maule

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall–degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

NEUROPEPTIDE-F - A NOVEL PARASITIC FLATWORM REGULATORY PEPTIDE FROM MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

Using a C-terminally directed pancreatic polypeptide (PP) antiserum and immunocytochemical methods, PP-immuno-reactivity (IR) was localized throughout the central (CNS) and peripheral nervous systems (PNS) of the cestode, Moniezia expansa . In the CNS, immunostaining was evident in the paired cerebral ganglia (primitive brain), connecting commissure, and the paired longitudinal nerve cords that are cross-linked by numerous regular transverse connectives. The PNS was seen to consist of a fine anastomosing nerve-net of immunoreactive fibres, many of which were closely associated with reproductive structures. Radioimmunoassay of this peptide IR in acid-alcohol extracts of the worm measured 192·8 ng/g of PP–IR. HPLC analyses of the M. expansa PP–IR identified a single molecular form which was purified to homogeneity. Plasma desorption mass spectrometry (PDMS) of purified parasite peptide resolved a single peptide with a molecular mass of 4599±10 Da. Automated gas-phase Edman degradation identified a 39-amino acid peptide with a C-terminal phenyl-alaninamide. Examination of its primary structure shows that it displays significant sequence homology with the vertebrate neuropeptide Y superfamily, suggesting that this platyhelminth-derived peptide is the phylogenetic precursor. Neuropeptide F ( M. expansa ) is the first regulatory peptide to be fully sequenced from the phylum Platyhelminthes and may represent a member of an important new class of invertebrate neuropeptide.

Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Physiology

Part I: Phylogeny, Genetics and Transcriptomes * The Evolution of Parasitism in Flatworms, D T J Littlewood, Natural History Museum, UK * Genomes and Genomics of Parasitic Flatworms, D A Johnston, Natural History Museum, UK * Genetic Discrimination of Echinococcus Species and Strains, D P McManus, University of Queensland, Australia * Ribosomal DNA Variation in Parasitic Flatworms, D Blair, James Cook University, Australia * Genetic Studies on Monogeneas with Emphasis on Gyrodactylus, C O Cunningham and I Matejusovo, FRS Marine Laboratory, UK * The Schistosome Transcriptome, S Verjovski-Almeida and R DeMarco, Universidade de Sao Paulo, Brazil * Transgenic Flatworms, C G Grevelding, Justus-Liebig-University Giessen, Germany Part II: Immunobiology, Host-Parasite Interaction and Control * Immunobiology of Schistosomes, S G Forrester, University of Ontario, Canada and E J Pearce, University of Pennsylvania, USA * Cestode Infection: Immunological Considerations From Host and Tapeworm Perspectives, D M McKay and R A Webb, York University, Canada * Parasitic Flatworms: Signal Transduction at the Host-Parasite Interface, T P Yoshino, J J Vermeire and J Humphries, University of Wisconsin-Madison, USA * Parasite Effects on the Snail Host Transcriptome, M Knight and N Raghavan, Biomedical Research Institute, USA * Developments in the chemotherapy of parasitic flatworms, G C Coles, University of Bristol, UK * Drug Resistance in Schistosomes, T A Day, Iowa State University, USA and S Botros, Theodor Bilharz Research Institute, Egypt * Praziquantel: Mechanism of Action, R M Greenberg, Marine Biological Laboratory, USA * Cestode Vaccine Development, M W Lightowlers, University of Melbourne, Australia * The Development of a Schistosome Vaccine, R A Wilson and P S Coulson, University of York, UK Part III: Protein Function, Metabolism and Physiology * Flatworm Parasite Proteomics, R M Morphew, J Barrett and P M Brophy, University of Wales, UK * Proteases in Trematode Biology, J P Dalton, University of Technology, Australia, C R Caffrey, M Sajid, University of California, USA, C Stack, S Donnelly, University of Technology, Australia, A Loukas, T Don, Queensland Institute of Medical Research, J McKerrow, University of California, USA, D W Halton, Queens University Belfast, UK and P J Brindley, Tulane University Health Sciences Center, USA * Signalling Molecules and Nerve-Muscle Function, A G Maule, N J Marks and T A Day * Unusual Aspects of Metabolism in Flatworm Parasites, A G M Tielens and J J van Hellemond, Utrecht University, Netherlands * Glycoconjugate Structures, M Wuhrer, Leiden University Medical Center, Netherlands and R Geyer, University of Giessen, Germany * Gene Silencing in Flatworms using RNA interference, P J Skelly, Tufts University School of Veterinary Medicine, USA.

flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference

The potato cyst nematode Globodera pallida is a serious pest of potato crops. Nematode FMRFamide‐like peptides (FLPs) are one of the most diverse neuropeptide families known, and modulate sensory and motor functions. As neuromuscular function is a well‐established target for parasite control, parasitic nematode FLP signaling has significant potential in novel control strategies. In the absence of transgenic parasitic nematodes and the reported ineffectiveness of neuronal gene RNAi in Caenorhabditis elegans, nothing is known about flp function in nematode parasites. In attempts to evaluate flp function in G. pallida, we have discovered that, unlike in C. elegans, these genes are readily susceptible to RNAi. Silencing any of the five characterized G. pallida flp genes (Gp‐flp‐1, ‐6, ‐12, ‐14, or ‐18) incurred distinct aberrant behavioral phenotypes consistent with key roles in motor function. Further delineation of these effects revealed that double‐stranded RNA exposure time (≥18 h) and concentration (≥0.1 μg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomo‐tory activities, do not display redundancy, and are susceptible to RNAi, paving the way for the investigation of RNAi‐mediated flp gene silencing as a novel plant parasite control strategy.—Kimber, M. J., McKinney, S., McMaster, S., Day, T. A., Fleming, C. C., Maule, A. G. flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference. FASEB J. 21, 1233–1243 (2007)

RNAi effector diversity in nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.

Parasitic peptides! The structure and function of neuropeptides in parasitic worms.

Parasitic worms come from two very different phyla-Platyhelminthes (flatworms) and Nematoda (roundworms). Although both phyla possess nervous systems with highly developed peptidergic components, there are key differences in the structure and action of native neuropeptides in the two groups. For example, the most abundant neuropeptide known in platyhelminths is the pancreatic polypeptide-like neuropeptide F, whereas the most prevalent neuropeptides in nematodes are FMRFamide-related peptides (FaRPs), which are also present in platyhelminths. With respect to neuropeptide diversity, platyhelminth species possess only one or two distinct FaRPs, whereas nematodes have upwards of 50 unique FaRPs. FaRP bioactivity in platyhelminths appears to be restricted to myoexcitation, whereas both excitatory and inhibitory effects have been reported in nematodes. Recently interest has focused on the peptidergic signaling systems of both phyla because elucidation of these systems will do much to clarify the basic biology of the worms and because the peptidergic systems hold the promise of yielding novel targets for a new generation of antiparasitic drugs.

REGULATORY PEPTIDES IN HELMINTH-PARASITES

Publisher Summary This chapter focuses on the regulatory peptides in helminth parasites. Regulatory peptides are a diverse group of biologically active peptides, usually of between 1–5 kDa molecular weight and 2–50 amino acid residues in length, that operate as neurohormonal mediators in intercellular communication. In vertebrates, regulatory peptides are synthesized by the endocrine and nervous systems and, indeed, are also expressed by the cells of the immune system; in invertebrates, they are largely elaborated by the nervous system and function as neurotransmitters, neuromodulators, and as trophic agents. Once a regulatory peptide has been identified within a helminth parasite, either through its activity in a specific bioassay system or as a result of its cross-reactivity with antisera under immunocytochemical or immunoassay conditions, the next logical step is its quantification, isolation, and structural characterization. Regulatory peptides have been quantified most commonly using immunofluorescence detection systems, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) techniques. These systems generally work well when peptides of known structure are being quantified. Peptide quantification in the platyhelminths has been carried out using a range of peptide antisera, most commonly raised against vertebrate regulatory peptides in radioimmunoassay methodologies. The quantification and characterization of regulatory peptides in nematodes presents a situation somewhat different from that in the platyhelminths. The structural characterization of parasitic helminth regulatory peptides has progressed only slowly, following their discovery in the nervous system of D. dendriticum.

Platyhelminth FMRFamide-related peptides (FaRPs) contract Schistosoma mansoni (Trematoda: Digenea) muscle fibres in vitro

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10(-9) and 10(-7) M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10(-8)-10(-6) M and 10(-7)-10(-5) M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 microM FMR-D-Famide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0.5 microM EGTA was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high K+-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.