Unraveling flp-11/flp-32 dichotomy in nematodes

AbstractFMRFamide-like peptide (FLP) signalling systems are core to nematode neuromuscular function. Novel drug discovery efforts associated with nematode FLP/FLP receptor biology are advanced through the accumulation of basic biological data that can reveal subtle complexities within the neuropeptidergic system. This study reports the characterisation of FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32), two distinct flp genes which encode the analogous peptide, AMRN(A/S)LVRFamide, in multiple nematode species – the only known example of this phenomenon within the FLPergic system of nematodes. Using bioinformatics, in situ hybridisation, immunocytochemistry and behavioural assays we show that: (i) flp-11 and -32 are distinct flp genes expressed individually or in tandem across multiple nematode species, where they encode a highly similar peptide; (ii) flp-11 does not appear to be the most widely expressed flp in Caenorhabditis elegans; (iii) in species expressing both flp-11 and flp-32, flp-11 displays a conserved, restricted expression pattern across nematode clades and lifestyles; (iv) in species expressing both flp-11 and flp-32, flp-32 expression is more widespread and less conserved than flp-11; (v) in species expressing only flp-11, the flp-11 expression profile is more similar to the flp-32 profile observed in species expressing both; and (vi) FLP-11 peptides inhibit motor function in multiple nematode species. The biological significance and evolutionary origin of flp-11 and -32 peptide duplication remains unclear despite attempts to identify a common ancestor; this may become clearer as the availability of genomic data improves. This work provides insight into the complexity of the neuropeptidergic system in nematodes, and begins to examine how nematodes may compensate for structural neuronal simplicity. From a parasite control standpoint, this work underscores the importance of basic biological data, and has wider implications for the utility of C. elegans as a model for parasite neurobiology

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologs have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicates a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localisation throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localise to mammalian motile cilia and we demonstrate here that ULK4 also localises to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/STK36 in a pathway required for stable assembly of motile cilia

The Structure of Hydrogenase-2 from <i>Escherichia coli</i>:Implications for H₂ -Driven Proton Pumping

Under anaerobic conditions Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from H2 oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly-bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein, HybB. To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In this paper we describe a new over-expression system that has facilitated determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex

RNA interference in adult Ascaris suum - an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

AbstractThe sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance

Recognition and killing of Brugia malayi microfilariae by human immune cells is dependent on the parasite sample and is not altered by ivermectin treatment

Mass administration of macrocyclic lactones targets the transmission of the causative agents of lymphatic filariasis to their insect vectors by rapidly clearing microfilariae (Mf) from the circulation. It has been proposed that the anti-filarial action of these drugs may be mediated through the host immune system. We recently developed an in vitro assay for monitoring the attachment to and killing of B. malayi Mf by human neutrophils (PMNs) and monocytes (PBMCs), however, the levels of both cell to worm attachment and leukocyte mediated Mf killing varied greatly between individual experiments. To determine whether differences in an individual's immune cells or the Mf themselves might account for the variability in survival, PMNs and PBMCs were isolated from 12 donors every week for 4 weeks and the cells used for survival assays with a different batch of Mf, thereby keeping donors constant but varying the Mf sample. Results from these experiments indicate that, overall, killing is Mf-rather than donor-dependent. To assess whether ivermectin (IVM) or diethylcarbamazine (DEC) increase killing, Mf were incubated either alone or with immune cells in the presence of IVM or DEC. Neither drug induced a significant difference in the survival of Mf whether cultured with or without cells, with the exception of DEC at 2 h post incubation. In addition, human PBMCs and PMNs were incubated with IVM or DEC for 1 h or 16 h prior to RNA extraction and Illumina sequencing. Although donor-to-donor variation may mask subtle differences in gene expression, principle component analysis of the RNASeq data indicates that there is no significant change in the expression of any genes from the treated cells versus controls. Together these data suggest that IVM and DEC have little direct effect on immune cells involved in the rapid clearance of Mf from the circulation. Keywords: Brugia malayi, Ivermectin, Microfilariae, Neutrophils, Peripheral blood mononuclear cell

In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes

Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the lead targets. In order to successfully validate NP-GPCRs for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identification of receptor activating ligand(s) via deorphanisation. Such efforts require the identification of all neuropeptide ligands within parasites. Here we mined the genomes of nine therapeutically relevant pathogenic nematodes to characterise the neuropeptide-like protein complements and demonstrate that: (i) parasitic nematodes possess a reduced complement of neuropeptide-like protein-encoding genes relative to Caenorhabditis elegans; (ii) parasite neuropeptide-like protein profiles are broadly conserved between nematode clades; (iii) five Ce-nlps are completely conserved across the nematode species examined; (iv) the extent and position of neuropeptide-like protein-motif conservation is variable; (v) novel RPamide-encoding genes are present in parasitic nematodes; (vi) novel Allatostatin-C-like peptide encoding genes are present in both C. elegans and parasitic nematodes; (vii) novel neuropeptide-like protein families are absent in C. elegans; and (viii) highly conserved nematode neuropeptide-like proteins are bioactive. These data highlight the complexity of nematode neuropeptide-like proteins and reveal the need for nomenclature revision in this diverse neuropeptide family. The identification of neuropeptide-like protein ligands, and characterisation of those with functional relevance, advance our understanding of neuropeptide signalling to support exploitation of the neuropeptidergic system as an anthelmintic target

Human Leukocytes Kill <i>Brugia malayi</i> Microfilariae Independently of DNA-Based Extracellular Trap Release

<div><p>Background</p><p><i>Wuchereria bancrofti</i>, <i>Brugia malayi</i> and <i>Brugia timori</i> infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases.</p><p>Principal findings</p><p><i>In vitro</i>, PMNs expel large amounts of NETs that capture but do not kill <i>B</i>. <i>malayi</i> Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill <i>B</i>. <i>malayi</i> Mf <i>in vitro</i> and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf <i>in vitro</i>, which may reflect the pathological heterogeneity associated with filarial parasitic infections.</p><p>Conclusions/Significance</p><p>Human innate immune cells are able to recognize, attach to and kill <i>B</i>. <i>malayi</i> microfilariae in an <i>in vitro</i> system. This suggests that, <i>in vivo</i>, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.</p></div