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SUMMARYNematodes represent one of the most abundant and species-rich groups of animals on the planet, with parasitic species causing chronic, debilitating infections in both livestock and humans worldwide. The prevalence and success of the nematodes is a direct consequence of the exceptionally protective properties of their cuticle. The synthesis of this cuticle is a complex multi-step process, which is repeated 4 times from hatchling to adult and has been investigated in detail in the free-living nematode, Caenorhabditis elegans. This process is known as moulting and involves numerous enzymes in the synthesis and degradation of the collagenous matrix. The nas-36 and nas-37 genes in C. elegans encode functionally conserved enzymes of the astacin metalloprotease family which, when mutated, result in a phenotype associated with the late-stage moulting defects, namely the inability to remove the preceding cuticle. Extensive genome searches in the gastrointestinal nematode of sheep, Haemonchus contortus, and in the filarial nematode of humans, Brugia malayi, identified NAS-36 but not NAS-37 homologues†. Significantly, the nas-36 gene from B. malayi could successfully complement the moult defects associated with C. elegans nas-36, nas-37 and nas-36/nas-37 double mutants, suggesting a conserved function for NAS-36 between these diverse nematode species. This conservation between species was further indicated when the recombinant enzymes demonstrated a similar range of inhibitable metalloprotease activities.

Heat shock protein 90 (Hsp-90) is a highly conserved essential protein in eukaryotes. Here we describe the molecular characterisation of hsp-90 from three nematodes, the free-living Caenorhabditis elegans (Ce) and the parasitic worms Brugia pahangi (Bp) and Haemonchus contortus (Hc). These molecules were functionally characterised by rescue of a Ce-daf-21 (hsp-90) null mutant. Our results show a gradient of rescue: the C. elegans endogenous gene provided full rescue of the daf-21 mutant, while Hc-hsp-90 provided partial rescue. In contrast, no rescue could be obtained using a variety of Bp-hsp-90 constructs, despite the fact that Bp-hsp-90 was transcribed and translated in the mutant worms. daf-21 RNA interference (RNAi) experiments were carried out to determine whether knock-down of the endogenous daf-21 mRNA in N2 worms could be complemented by expression of either parasite gene. However neither parasite gene could rescue the daf-21 (RNAi) phenotypes. These results indicate that factors other than the level of sequence identity are important for determining whether parasite genes can functionally complement in C. elegans.

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

The FK506-binding proteins (FKBs) represent ubiquitous enzymes that catalyze the rate-limiting peptidyl prolyl cis-trans isomerization step in protein folding. The nematode Caenorhabditis elegans has eight FKBs, three of which (FKB-3, -4, and -5) have dual peptidyl prolyl cis-trans isomerase (PPIase) domains, signal peptides and ER retention signals. PPIase activity has been detected for recombinant FKB-3. Both FKB-3 and -5 are expressed in the exoskeleton-synthesizing hypodermis with transcript peaks that correspond to the molting and collagen synthesis cycles. FKB-4 is expressed at a low level throughout development. No phenotypes were observed in deletion mutants in each of the secretory pathway FKBs. Combined triple and fkb-4, -5 double deletion mutants were however found to arrest at 12 degrees C, but developed normally at 15-25 degrees C. This cold-sensitive larval lethal effect was not maternally derived, occurred during embryogenesis, and could be rescued following the transgenic introduction of a wild type copy of either fkb-4 or fkb-5. The temperature-sensitive defects also affected molting, cuticle collagen expression, hypodermal seam cell morphology, and the structural integrity of the cuticular extracellular matrix. This study establishes that the secretory pathway FK506-binding PPIase enzymes are essential for normal nematode development, collagen biogenesis, and the formation of an intact exoskeleton under adverse physiological conditions.

Graphical abstract, Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several μM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect ‘Dumpy’ (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals.

Background Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event. Results microRNAs differentially expressed during the early post-infective stages of Brugia pahangi L3 were identified by microarray analysis. One of these, bpa-miR-5364, was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade III nematodes, and is a novel member of the let-7 family, which are known to have key developmental functions in the free-living nematode Caenorhabditis elegans. Predicted mRNA targets of bpa-miR-5364 were identified using bioinformatics and comparative genomics approaches that relied on the conservation of miR-5364 binding sites in the orthologous mRNAs of other filarial nematodes. Finally, we confirmed the interaction between bpa-miR-5364 and three of its predicted targets using a dual luciferase assay. Conclusions These data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. This study is the first to identify parasitic nematode mRNAs that are verified targets of specific microRNAs and demonstrates that post-transcriptional control of gene expression via stage-specific expression of microRNAs may be important in the success of filarial infection. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1536-y) contains supplementary material, which is available to authorized users.

Graphical abstract, Highlights • Astacin metalloprotease, DPY-31, is conserved throughout the nematode phylum. • DPY-31 is crucial to Teladorsagia circumcincta cuticle formation. • Matrix metalloprotease inhibitors are efficacious against recombinant DPY-31. • Novel hydroxamate inhibitors caused Dumpy and Moult defects in nematodes. • DPY-31 is a potential target for future nematode control., Parasitic nematodes cause chronic, debilitating infections in both livestock and humans worldwide, and many have developed multiple resistance to the currently available anthelmintics. The protective collagenous cuticle of these parasites is required for nematode survival and its synthesis has been studied extensively in the free-living nematode, Caenorhabditis elegans. The collagen synthesis pathway is a complex, multi-step process involving numerous key enzymes, including the astacin metalloproteases. Nematode astacinsare crucial for C. elegans development, having specific roles in hatching, moulting and cuticle synthesis. NAS-35 (also called DPY-31) is a homologue of a vertebrate procollagen C-proteinase and performs a central role in cuticle formation of C. elegans as its mutation causes temperature-sensitive lethality and cuticle defects. The characterisation of DPY-31 from the ovine gastrointestinal nematode Teladorsagia circumcincta and its ability to rescue the C. elegans mutant is described. Compounds with a hydroxamate functional group have previously been shown to be potent inhibitors of procollagen C-proteinases and were therefore examined for inhibitory activity against the T. circumcincta enzyme. Phenotypic screening against T. circumcincta, Haemonchus contortus and C. elegans larval stages identified compounds that caused body morphology phenotypes consistent with the inhibition of proteases involved in cuticle collagen synthesis. These compounds correspondingly inhibited the activity of recombinant T. circumcincta DPY-31, supporting the hypothesis that this enzyme may represent a potentially novel anthelmintic drug target.

The integral role that collagens play in the morphogenesis of the nematode exoskeleton or cuticle makes them a useful marker in the examination of the collagen synthesizing machinery. In this study, a green fluorescent protein-collagen fusion has been constructed by using the Caenorhabditis elegans adult-specific, hypodermally synthesized collagen COL-19. In wild-type nematodes, this collagen marker localized to the circumferential annular rings and the lateral trilaminar alae of the cuticle. Crosses carried out between a COL-19::GFP integrated strain and several morphologically mutant strains, including blister, dumpy, long, small, squat, and roller revealed significant COL-19 disruption that was predominantly strain-specific and provided a structural basis for the associated phenotypes. Disruption was most notable in the cuticle overlying the lateral seam cell syncytium, and confirmed the presence of two distinct forms of hypodermis, namely the circumferentially contracting lateral seam cells and the laterally contracting ventral-dorsal hypodermis. The effect of a single aberrant collagen being sufficient to mediate widespread collagen disruption was exemplified by the collagen mutant strain dpy-5 and its disrupted COL-19::GFP and DPY-7 collagen expression patterns. Through the disrupted pattern of COL-19 and DPY-7 in a thioredoxin mutant, dpy-11, and through RNA interference of a dual oxidase enzyme and a vesicular transport protein, we also show the efficacy of the COL-19::GFP strain as a marker for aberrant cuticle collagen synthesis and, thus, for the identification of factors involved in the construction of collagenous extracellular matrices.

Background: Collagen prolyl 4-hydroxylases (C-P4H) are involved in the formation of extracellular matrices. Results: The full complement of C-P4H enzymes from the human infective parasite Brugia malayi have been bioinformatically, biochemically, and functionally characterized. Conclusion: C-P4H enzymes are essential for development in B. malayi. Significance: Unique features of these essential enzymes may be exploited in future control mechanisms., Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.

The exoskeleton or cuticle performs many key roles in the development and survival of all nematodes. This structure is predominantly collagenous in nature and requires numerous enzymes to properly fold, modify, process and cross-link these essential structural proteins. The cuticle structure and its collagen components are conserved throughout the nematode phylum but differ from the collagenous matrices found in vertebrates. This structure, its formation and the enzymology of nematode cuticle collagen biogenesis have been elucidated in the free-living nematode Caenorhabditis elegans. The dpy-31 gene in C. elegans encodes a procollagen C-terminal processing enzyme of the astacin metalloprotease or bone morphogenetic protein class that, when mutated, results in a temperature-sensitive lethal phenotype associated with cuticle defects. In this study, orthologues of this essential gene have been identified in the phylogenetically diverse parasitic nematodes Haemonchus contortus and Brugia malayi. The DPY-31 protein is expressed in the gut and secretory system of C. elegans, a location also confirmed when a B. malayi transcriptional dpy-31 promoter-reporter gene fusion was expressed in C. elegans. Functional conservation between the nematode enzymes was supported by the fact that heterologous expression of the H. contortus dpy-31 orthologue in a C. elegans dpy-31 mutant resulted in the full rescue of the mutant body form. This interspecies conservation was further established when the recombinant nematode enzymes were found to have a similar range of inhibitable protease activities. In addition, the recombinant DPY-31 enzymes from both H. contortus and B. malayi were shown to efficiently process the C. elegans cuticle collagen SQT-3 at the correct C-terminal procollagen processing site.

The cuticle of parasitic nematodes performs many critical functions and is essential for proper development and for protection from the host immune response. The biosynthesis, assembly, modification and turnover of this exoskeleton have been most extensively studied in the free-living nematode, Caenorhabditis elegans, where it represents a complex multi-step process involving a whole suite of enzymes. The biosynthesis of the cuticle has an additional level of complexity, as many of the enzymes also require additional proteins to aid their activation and selective inhibition. Blister-5 (BLI-5) represents a protein with a kunitz-type serine protease interacting domain and is involved in cuticle collagen biosynthesis in C. elegans, through its interaction with subtilisin-like processing enzymes (such as BLI-4). Mutation of the bli-5 gene causes blistering of the collagenous adult cuticle. Homologues of BLI-5 have been identified in several parasitic species that span different nematode clades. In this study, we molecularly and biochemically characterize BLI-5 homologues from the clade V nematodes C. elegans and Haemonchus contortus and from the clade III filarial nematode Brugia malayi. The nematode BLI-5 orthologues possess a shared domain structure and perform similar in vitro and in vivo functions, performing important proteolytic enzyme functions. The results demonstrate that the bli-5 genes from these diverse parasitic nematodes are able to complement a C. elegansbli-5 mutant and thereby support the use of the C. elegans model system to examine gene function in the experimentally less-amenable parasitic species.

We have rigorously tested the hypothesis that Trypanosoma brucei gambiense Type 1 is composed of genetically homogenous populations by examining the parasite population present in Human African Trypanosomiasis (HAT) patients from the Democratic Republic of Congo (DRC) and Cameroon (CAM). We amplified eight microsatellite markers by PCR directly from blood spots on FTA filters, thereby avoiding the significant parasite selection inherent in the traditional isolation techniques of rodent inoculation or in vitro culture. All microsatellite markers were polymorphic, although for four markers there was only polymorphism between the DRC and CAM populations, not within populations, suggesting very limited genetic exchange. Within the largest population from the DRC, Hardy–Weinberg equilibrium is not evident at any loci. This evidence suggests a clonal population. However, there was significant sub-structuring between the DRC and CAM samples ( F ST = 0.32), indicating that Trypanosoma brucei gambiense Type 1 has genetically distinct clades. The data combine to indicate that genetic exchange plays a very limited role. The finding of distinct clades in different places suggests the possibility that samples from humans with clinical signs represent clonal expansions from an underlying population that requires identifying and characterising.

The collagen prolyl 4-hydroxylases (P4Hs) are\ud essential for proper extracellular matrix\ud formation in multicellular organisms. The\ud vertebrate enzymes are α2β2 tetramers, in\ud which the β subunits are identical to protein\ud disulfide isomerase (PDI). Unique P4H forms\ud have been shown to assemble from the\ud Caenorhabditis elegans catalytic α subunit\ud isoforms PHY-1 and PHY-2 and the β subunit\ud PDI-2. A mixed PHY-1/PHY-2/(PDI-2)2\ud tetramer is the major form, while PHY-1/PDI-\ud 2 and PHY-2/PDI-2 dimers are also assembled\ud but less efficiently. Cloning and\ud characterization of the orthologous subunits\ud from the closely related nematode\ud Caenorhabditis briggsae revealed distinct\ud differences in the assembly of active P4H\ud forms in spite of the extremely high amino\ud acid sequence identity (92-97%) between the\ud C. briggsae and C. elegans subunits. In\ud addition to a PHY-1/PHY-2(PDI-2)2 tetramer\ud and a PHY-1/PDI-2 dimer, an active (PHY-\ud 2)2(PDI-2)2 tetramer was formed in C.\ud briggsae instead of a PHY-2/PDI-2 dimer.\ud Site-directed mutagenesis studies and\ud generation of inter-species hybrid polypeptides\ud showed that the N-terminal halves of the\ud Caenorhabditis PHY-2 polypeptides\ud determine their assembly properties. Genetic\ud disruption of C. briggsae phy-1 (Cb-dpy-18)\ud via a Mos1 insertion resulted a small (short)\ud phenotype that is less severe than the dumpy\ud (short and fat) phenotype of the corresponding\ud C. elegans mutants (Ce-dpy-18). C. briggsae\ud phy-2 RNA interference produced no visible\ud phenotype in the wild type nematodes but\ud produced a severe dumpy phenotype and larval\ud arrest in phy-1 mutants. Genetic\ud complementation of the C. briggsae and C.\ud elegans phy-1 mutants was achieved by\ud injection of a wild type phy-1 gene from either\ud species.

Caenorhabditis elegans represents an excellent model in which to dissect the biosynthesis and assembly of the nematode cuticle. A sequenced genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of cuticle components. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. For many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterised bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed.

Protein disulfide isomerase (PDI) is a multifunctional protein required for many aspects of protein folding and transit through the endoplasmic reticulum. A conserved\ud family of three PDIs have been functionally analysed using genetic mutants of the model organism Caenorhabditis elegans. PDI-1 and PDI-3 are individually nonessential,\ud whereas PDI-2 is required for normal post-embryonic development. In combination, all three genes are synergistically essential for embryonic development\ud in this nematode. Mutations in pdi-2 result in severe body morphology defects, uncoordinated movement, adult sterility, abnormal molting and aberrant collagen\ud deposition. Many of these phenotypes are consistent with a role in collagen biogenesis and extracellular matrix formation. PDI-2 is required for the normal function of prolyl 4-hydroxylase, a key collagen-modifying enzyme. Site-directed mutagenesis indicates that the independent catalytic activity of PDI-2 may also perform an essential\ud developmental function. PDI-2 therefore performs two critical roles during morphogenesis. The role of PDI-2 in collagen biogenesis can be partially restored following complementation of the mutant with human PDI.

Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterised by low parasitaemias. Multiple Displacement Amplification (MDA) amplifies DNA with a simple in vitro step, and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a twenty-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multi-copy ribosomal ITS region or 177 bp repeats, and a twenty-fold increase in sensitivity over nested PCR against a single copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses.