The genomes of four tapeworm species reveal adaptations to parasitism.

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

Functional characterization of a novel family of acetylcholine-gated chloride channels in Schistosoma mansoni.

Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels.

flp-32 Ligand/receptor silencing phenocopy faster plant pathogenic nematodes.

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R-silenced worms also display an increase in migration rate. This work demonstrates that Gp-flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR.

The genome of the blood fluke Schistosoma mansoni.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.

The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea.

BACKGROUND: G protein-coupled receptors (GPCRs) constitute one of the largest groupings of eukaryotic proteins, and represent a particularly lucrative set of pharmaceutical targets. They play an important role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing major pathogens as well as established model organisms. The recent availability of genomic data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first comprehensive effort to identify and analyze GPCRs in this important phylum. RESULTS: Application of a novel transmembrane-oriented approach to receptor mining led to the discovery of 117 S. mansoni GPCRs, representing all of the major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin and 5 Frizzled. Similarly, 418 Rhodopsin, 9 Glutamate, 21 Adhesion, 1 Secretin and 11 Frizzled S. mediterranea receptors were identified. Among these, we report the identification of novel receptor groupings, including a large and highly-diverged Platyhelminth-specific Rhodopsin subfamily, a planarian-specific Adhesion-like family, and atypical Glutamate-like receptors. Phylogenetic analysis was carried out following extensive gene curation. Support vector machines (SVMs) were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification. CONCLUSIONS: Genome-wide investigation of GPCRs in two platyhelminth genomes reveals an extensive and complex receptor signaling repertoire with many unique features. This work provides important sequence and functional leads for understanding basic flatworm receptor biology, and sheds light on a lucrative set of anthelmintic drug targets.

Identification of multiresistant Salmonella isolates capable of subsisting on antibiotics.

This study assessed the ability of Salmonella (572 isolates) to subsist on 12 different antibiotics. The majority (11/12) of the antibiotics enabled subsistence for at least 1 of 140 isolates. Furthermore, 40 isolates were able to subsist on more than one antibiotic. Antibiotic resistance and antibiotic subsistence do not appear to be equivalent.

Neuropeptide physiology in helminths.

Parasitic worms come from two distinct, distant phyla, Nematoda (roundworms) and Platyhelminthes (flatworms). The nervous systems of worms from both phyla are replete with neuropeptides and there is ample physiological evidence that these neuropeptides control vital aspects of worm biology. In each phyla, the physiological evidence for critical roles for helminth neuropeptides is derived from both parasitic and free-living members. In the nematodes, the intestinal parasite Ascaris suum and the free-living Caenorhabditis elegans have yielded most of the data; in the platyhelminths, the most physiological data has come from the blood fluke Schistosoma mansoni. FMRFamide-like peptides (FLPs) have many varied effects (excitation, relaxation, or a combination) on somatic musculature, reproductive musculature, the pharynx and motor neurons in nematodes. Insulin-like peptides (INSs) play an essential role in nematode dauer formation and other developmental processes. There is also some evidence for a role in somatic muscle control for the somewhat heterogeneous grouping ofpeptides known as neuropeptide-like proteins (NLPs). In platyhelminths, as in nematodes, FLPs have a central role in somatic muscle function. Reports of FLP physiological action in platyhelminths are limited to a potent excitation of the somatic musculature. Platyhelminths are also abundantly endowed with neuropeptide Fs (NPFs), which appear absent from nematodes. There is not yet any data linking platyhelminth NPF to any particular physiological outcome, but this neuropeptide does potently and specifically inhibit cAMP accumulation in schistosomes. In nematodes and platyhelminths, there is an abundance of physiological evidence demonstrating that neuropeptides play critical roles in the biology of both free-living and parasitic helminths. While it is certainly true that there remains a great deal to learn about the biology of neuropeptides in both phyla, physiological evidence presently available points to neuropeptidergic signaling as a very promising field from which to harvest future drug targets.

Evaluation of the pathogenicity and virulence of three strains of Salmonella organisms in calves and pigs.

OBJECTIVE: To assess in pigs the pathogenicity and virulence of 3 strains of Salmonella spp capable of causing atypical salmonellosis in cattle. ANIMALS: 36 Holstein calves and 72 pigs experimentally infected with Salmonella spp. PROCEDURES: Representative Salmonella strains associated with 3 new disease phenotypes (protozoa-mediated hypervirulence, multisystemic cytopathicity, and encephalopathy) that have been characterized in cattle during the past 10 years were orally inoculated into pigs. Clinical manifestations were compared with those observed in cattle. Samples were collected from various tissues, and the presence of Salmonella organisms was assessed qualitatively and quantitatively by use of Salmonella-selective media. RESULTS: Of the 3 unique Salmonella disease phenotypes observed in cattle, only protozoa-mediated hypervirulence was observed in pigs. Hypervirulence was related to a more rapid onset of disease and higher pathogen burden in pigs than in cattle. This phenotype was observed in pigs inoculated with multiresistant Salmonella enterica serotypes Typhimurium or Choleraesuis bearing the Salmonella genomic island 1 (SGI1) integron. CONCLUSIONS AND CLINICAL RELEVANCE: Salmonella hypervirulence was identified in pigs noculated with SGI1-bearing strains exposed to free-living protozoa. Additionally, an SGI1-bearing strain of Salmonella Choleraesuis was detected that resulted in augmented virulence in pigs. Therefore, it appeared that protozoa-associated salmonellosis was analogous in pigs and cattle. Salmonella-mediated encephalopathy and multisystemic cytopathicity did not appear to be relevant diseases in pigs.

The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.

Probing protein function in parasitic flatworms is hampered by the difficulties associated with the development of transgenic approaches. Although RNA interference (RNAi) in schistosomes shows much promise, it has not been reported in other trematodes. Here, we show the successful silencing of the cysteine proteases cathepsin B and L in the infective stage of Fasciola hepatica newly excysted juveniles (NEJs). Silencing resulted in marked reductions in target transcript levels and significant diminution in the encoded proteins in the gut. RNAi of either enzyme in NEJs induced transient, abnormal locomotory phenotypes, and significantly reduced penetration of the rat intestinal wall.

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 (> or = 18 h) and concentration (> or = 0.1 microg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomotory 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.

Praziquantel Interaction with Mammalian Targets in the Spotlight.

Chan et al. recently demonstrated that the antischistosomal drug praziquantel has a potent and specific interaction with human 5-HT2B receptors, and that the drug also elicits contraction of mouse mesenteric vasculature apparently mediated by the same receptor subtype We consider what this might mean about the drug's molecular therapeutic targets in both the worm and the host.

Identification of a platyhelminth neuropeptide receptor.

We report the characterisation of the first neuropeptide receptor from the phylum Platyhelminthes, an early-diverging phylum which includes a number of important human and veterinary parasites. The G protein-coupled receptor (GPCR) was identified from the model flatworm Girardia tigrina (Tricladida: Dugesiidae) based on the presence of motifs widely conserved amongst GPCRs. In two different assays utilising heterologous expression in Chinese hamster ovary cells, the Girardia GPCR was most potently activated by neuropeptides from the FMRFamide-like peptide class. The most potent platyhelminth neuropeptide in both assays was GYIRFamide, a FMRFamide-like peptide known to be present in G. tigrina. There was no activation by neuropeptide Fs, another class of flatworm neuropeptides. Also active were FMRFamide-like peptides derived from other phyla but not known to be present in any platyhelminth. Most potent among these were nematode neuropeptides encoded by the Caenorhabditis elegans flp-1 gene which share a PNFLRFamide carboxy terminal motif. The ability of nematode peptides to stimulate a platyhelminth receptor demonstrates a degree of structural conservation between FMRFamide-like peptide receptors from these two distinct, distant phyla which contain parasitic worms.

Interaction of plant essential oil terpenoids with the southern cattle tick tyramine receptor: A potential biopesticide target.

An outbreak of the southern cattle tick, Rhipicephalus (Boophilus) microplus, (Canestrini), in the United States would have devastating consequences on the cattle industry. Tick populations have developed resistance to current acaricides, highlighting the need to identify new biochemical targets along with new chemistry. Furthermore, acaricide resistance could further hamper control of tick populations during an outbreak. Botanically-based compounds may provide a safe alternative for efficacious control of the southern cattle tick. We have developed a heterologous expression system that stably expresses the cattle tick's tyramine receptor with a G-protein chimera, producing a system that is amenable to high-throughput screening. Screening an in-house terpenoid library, at two screening concentrations (10 µM and 100 µM), has identified four terpenoids (piperonyl alcohol, 1,4-cineole, carvacrol and isoeugenol) that we believe are positive modulators of the southern cattle tick's tyramine receptor.

Intestinal Enterobacteriaceae that Protect Nematodes from the Effects of Benzimidazoles.

The objective of this study was to investigate an interaction between nematodes and gut Enterobacteriaceae that use benzimidazoles as a carbon source. By addressing this objective, we identified an anthelmintic resistance-like mechanism for gastrointestinal nematodes. We isolated 30 gut bacteria (family Enterobacteriaceae) that subsist on and putatively catabolize benzimidazole-class anthelmintics. C. elegans was protected from the effects of benzimidazoles when co-incubated with these Enterobacteriaceae that also protect adult ascarids from the effects of albendazole. This bacterial phenotype represents a novel mechanism by which gastrointestinal nematodes are potentially spared from the effects of benzimidazoles, without any apparent fitness cost to the parasite.

Current status of sensitivity to praziquantel in a focus of potential drug resistance in Egypt.

A decade ago, a study revealed that praziquantel (PZQ) failed to cure 1.6% of those with intestinal schistomiasis in five villages of the Nile delta region. The recommended dosage of PZQ is a single 40 mg/kg oral dose, and each of these patients continued to pass viable Schistosoma mansoni eggs despite three successive doses at or above this level. The eggs passed by these uncured villagers produced adult worms that were, in most cases, significantly less responsive to PZQ in vitro. This report investigates the current sensitivity of S. mansoni infections to PZQ after 10 years of therapeutic pressure in the same villages, testing the hypothesis that the number of drug failures would have increased as continued drug pressure selected for worms with diminished sensitivity to PZQ. The data show that these villages have experienced a significant decrease in the prevalence and intensity of S. mansoni infections, with present infection rate of 10.9%, compared with 25.1% in 1994. The first treatment resulted in normal range of cure rates, between 73.8 and 92.3% in each of the five villages in the study. After three successive doses (40, 40, and 60 mg/kg, the same treatment protocol applied a decade ago) there were no uncured patients remaining in the study. This shows that there has not been an increase of drug failure, despite 10 years of therapeutic pressure in these villages where there had been resistant infections and worms with decreased response to PZQ.

A functionally atypical amidating enzyme from the human parasite Schistosoma mansoni.

Many neuropeptide transmitters require the presence of a carboxy-terminal alpha-amide group for biological activity. Amidation requires conversion of a glycine-extended peptide intermediate into a C-terminally amidated product. This post-translational modification depends on the sequential action of two enzymes (peptidylglycine alpha-hydroxylating monooxygenase or PHM, and peptidyl-alpha-hydroxyglycine alpha-amidating lyase or PAL) that in most eukaryotes are expressed as separate domains of a single protein (peptidylglycine alpha-amidating monooxygenase or PAM). We identified a cDNA encoding PHM in the human parasite Schistosoma mansoni. Transient expression of schistosome PHM (smPHM) revealed functional properties that are different from other PHM proteins; smPHM displays a lower pH-optimum and, when expressed in mammalian cells, is heavily N-glycosylated. In adult worms, PHM is found in the trans-Golgi network and secretory vesicles of both central and peripheral nerves. The widespread occurrence of PHM in the nervous system confirms the important role of amidated neuropeptides in these parasitic flatworms. The differences between schistosome and mammalian PHM suggest that it could be a target for new chemotherapeutics.

A constitutively active G protein-coupled acetylcholine receptor regulates motility of larval Schistosoma mansoni.

The neuromuscular system of helminths controls a variety of essential biological processes and therefore represents a good source of novel drug targets. The neuroactive substance, acetylcholine controls movement of Schistosoma mansoni but the mode of action is poorly understood. Here, we present first evidence of a functional G protein-coupled acetylcholine receptor in S. mansoni, which we have named SmGAR. A bioinformatics analysis indicated that SmGAR belongs to a clade of invertebrate GAR-like receptors and is related to vertebrate muscarinic acetylcholine receptors. Functional expression studies in yeast showed that SmGAR is constitutively active but can be further activated by acetylcholine and, to a lesser extent, the cholinergic agonist, carbachol. Anti-cholinergic drugs, atropine and promethazine, were found to have inverse agonist activity towards SmGAR, causing a significant decrease in the receptor's basal activity. An RNAi phenotypic assay revealed that suppression of SmGAR activity in early-stage larval schistosomulae leads to a drastic reduction in larval motility. In sum, our results provide the first molecular evidence that cholinergic GAR-like receptors are present in schistosomes and are required for proper motor control in the larvae. The results further identify SmGAR as a possible candidate for antiparasitic drug targeting.

Functional analysis of Girardia tigrina transcriptome seeds pipeline for anthelmintic target discovery.

BACKGROUND: Neglected diseases caused by helminth infections impose a massive hindrance to progress in the developing world. While basic research on parasitic flatworms (platyhelminths) continues to expand, researchers have yet to broadly adopt a free-living model to complement the study of these important parasites. METHODS: We report the high-coverage sequencing (RNA-Seq) and assembly of the transcriptome of the planarian Girardia tigrina across a set of dynamic conditions. The assembly was annotated and extensive orthology analysis was used to seed a pipeline for the rational prioritization and validation of putative anthelmintic targets. A small number of targets conserved between parasitic and free-living flatworms were comparatively interrogated. RESULTS: 240 million paired-end reads were assembled de novo to produce a strictly filtered predicted proteome consisting of over 22,000 proteins. Gene Ontology annotations were extended to 16,467 proteins. 2,693 sequences were identified in orthology groups spanning flukes, tapeworms and planaria, with 441 highlighted as belonging to druggable protein families. Chemical inhibitors were used on three targets in pharmacological screens using both planaria and schistosomula, revealing distinct motility phenotypes that were shown to correlate with planarian RNAi phenotypes. CONCLUSIONS: This work provides the first comprehensive and annotated sequence resource for the model planarian G. tigrina, alongside a prioritized list of candidate drug targets conserved among parasitic and free-living flatworms. As proof of principle, we show that a simple RNAi and pharmacology pipeline in the more convenient planarian model system can inform parasite biology and serve as an efficient screening tool for the identification of lucrative anthelmintic targets.