Exploring the neurotransmitter labyrinth in nematodes

Nematodes include both free-living species such as Caenorhabditis elegans and major parasites of humans, livestock and plants. The apparent simplicity and uniformity of their nervous system belies a rich diversity of putative signalling molecules,particularly neuropeptides. This new appreciation stems largely from the genome-sequencing project with C. elegans, which is due to be completed by the end of 1998. The project has provided additional insights into other aspects of nematode neurobiology, as have studies on the mechanism of action of anthelmintics. Here, progress on the identification, localization, synthesis and physiological actions of transmitters identified in nematodes is explored.

Post-mortem examination of a wild muntjac from Northern Ireland.

1. Deer are of major concern with regards to impacts on biodiversity, forestry and agriculture as well as human health. The invasive Reeves’ muntjac deer Muntiacus reevesi, native to Asia but established in Great Britain, has recently appeared in the wild in Ireland.

2. The first verified record in the wild in Northern Ireland was confirmed during June 2009 as a result of a road traffic accident near Newtownards, Co. Down. The second record was a culled animal shot in the grounds of Mount Stewart, Co. Down during June 2011.

3. The current report aimed to perform a detailed investigation of the most recently obtained animal to establish a) its age, b) its genetic relationship with the first animal and c) the threat it might pose in terms of carrying endo- or ecto-parasites or other micro-pathogens, principally viruses and bacteria.

4. Analysis of its dentition suggested the culled animal was 56 weeks old (range 55-57 weeks).

5. Genetic analysis indicated that there was no possibility of a father-son relationship with the buck killed in 2009. There was a 6.25% probability of both individuals being full-sibs and a 25% probability of a half-sib relationship.

6. The deer was free from all the major pathogens. Most significantly, a novel species of Gammaherpesvirus was detected most closely matching type 2 ruminant rhadinovirus (Gammaherpesvirinae) from mule deer. Further sequencing is required to provide a definitive classification. There is no evidence suggesting this virus is pathogenic but its detection is nonetheless of concern.

7. During late 2009 (after the first animal was recovered) and early 2010, there were a number of sightings of muntjac within the vicinity of Mount Stewart. Our results indicate that the animal shot in June 2011 could not have been this same animal, as the shot animal must have been born in spring 2010. Moreover, genetic analysis indicates that the two recovered individuals were highly unlikely to share the same mother and father, suggesting they were the offspring of a minimum of three breeding adults (two fathers and one mother or two mothers and one father). This brings the total number of known individuals to 5 including the two offspring. The location of the breeding adults is unknown and may be either in captivity with subsequent escapees, or deliberate releases, or be free living in the wild. However, a further sighting during early 2012 may suggest a wild origin.

LOCALIZATION OF DIPLOPTERA-PUNCTATA ALLATOSTATIN-LIKE IMMUNOREACTIVITY IN HELMINTHS - AN IMMUNOCYTOCHEMICAL STUDY

The nervous systems of helminths are predominantly peptidergic in nature, although it is likely that the full range of regulatory peptides used by these organisms has yet to be elucidated. Attempts to identify novel helminth neuropeptides are being made using immunocytochemistry with antisera raised against peptides isolated originally from insects. One of these antisera was raised against allatostatin III, a peptide isolated originally from the cockroach, Diploptera punctata, and a member of a family of related peptides found in insects. Allatostatin immunoreactivity was found throughout the nervous systems of Mesocestoides corti tetrathyridia, and adult Moniezia expansa, Diclidophora merlangi, Fasciola hepatica, Schistosoma mansoni, Ascaris suum and Panagrellus redivivus. Immunostaining was observed in the nerve cords and anterior ganglia of all the helminths. It was also apparent in the subtegumental nerves and around the reproductive apparatus of the flatworms, in neurones in the pharynx of D. merlangi, F. hepatica, A. suum and P. redivivus, and in fibres innervating the anterior sense organs in the nematodes. Immunostaining in all species was both reproducible and specific in that it could be abolished by pre-absorption of the antiserum with allatostatins I-IV. These results suggest that molecules related to the D. punctata allatostatins are important components in the nervous systems of a number of helminth parasites, and a free-living nematode. Their distribution within the nervous system suggests they function as neurotransmitters/ neuromodulators with roles in locomotion, feeding, reproduction and sensory perception.

NEUROPEPTIDE-F (MONIEZIA-EXPANSA) - LOCALIZATION AND CHARACTERIZATION USING SPECIFIC ANTISERA

Immunocytochemical techniques used in conjunction with confocal scanning laser microscopy (CSLM) and electron microscopy have been used to demonstrate, for the first time, the distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF) in the cestode, Moniezia expansa. Antisera were raised to intact NPF(1-39) and to the C-terminal decapeptide of NPF(30-39). These antisera were characterized and validated for use in both immunocytochemistry and radioimmunoassay (RIA). NPF immunoreactivity (IR) was detected using both antisera throughout all of the major components of the central and peripheral nervous systems of the worm. The pattern of NPF-IR was found to mirror the IR obtained using a C-terminally directed pancreatic polypeptide (PP) antiserum and FMRFamide antisera; blocking studies using these antisera revealed that FMRFamide and PP antisera cross-react with NPF(M. expansa). RIA of acid-alcohol extracts of the worm measured 114 ng/g using the C-terminal NPF antiserum and 56 ng/g using the whole-molecule-directed antiserum. While the C-terminally-directed NPF antiserum cross-reacts with NPF-related peptides from other invertebrates, the whole-molecule-directed NPF antiserum is specific for NPF(M. expansa). The C-terminal NPF antiserum has potential for use in the identification and purification of NPF analogues from other platyhelminth parasites.

A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase

We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.-Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase.

Defense peptides secreted by helminth pathogens: antimicrobial and/or immunomodulator molecules?

Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections.

The phylogeny, structure and function of trematode cysteine proteases, with particular emphasis on the Fasciola hepatica cathepsin L family

Helminth parasites (nematodes, flatworms and cestodes) infect over 1 billion of the world's population causing high morbidity and mortality. The large tissue-dwelling worms express papain-like cysteine peptidases, termed cathepsins that play important roles in virulence including host entry, tissue migration and the suppression of host immune responses. Much of our knowledge of helminth cathepsins comes from studies using flatworms or trematode (fluke) parasites. The developmentally-regulated expression of these proteases correlates with the passage of parasites through host tissues and their encounters with different host macromolecules. Recent phylogenetic, biochemical and structural studies indicate that trematode cathepsins exhibit overlapping but distinct substrate specificities due to divergence within the protease active site. Here we provide an overview of the evolution, biochemistry and structure of these important enzymes and highlight how recent advances in proteomics and gene silencing techniques are allowing researchers to probe their biological functions. We focus mainly on members of the cathepsin L gene family of the animal and human pathogen, Fasciola hepatica, because of our deep understanding of their function, biochemistry and structure.

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125V in the presence of 50 µg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.

Zoonotic helminth infections with particular emphasis on fasciolosis and other trematodiases

Zoonotic infections are among the most common on earth and are responsible for >60 per cent of all human infectious diseases. Some of the most important and well-known human zoonoses are caused by worm or helminth parasites, including species of nematodes (trichinellosis), cestodes (cysticercosis, echinococcosis) and trematodes (schistosomiasis). However, along with social, epidemiological and environmental changes, together with improvements in our ability to diagnose helminth infections, several neglected parasite species are now fast-becoming recognized as important zoonotic diseases of humans, e.g. anasakiasis, several fish-borne trematodiasis and fasciolosis. In the present review, we discuss the current disease status of these primary helminth zoonotic infections with particular emphasis on their diagnosis and control. Advances in molecular biology, proteomics and the release of helminth genome-sequencing project data are revolutionizing parasitology research. The use of these powerful experimental approaches, and their potential benefits to helminth biology are also discussed in relation to the future control of helminth infections of animals and humans.

An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: Proteins associated with invasion and infection of the mammalian host

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum:importance of active site metal ions in the binding of substrates and inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene

Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1.