Genome-wide RNA interference of the nhr gene family in barber's pole worm identified members crucial for larval viability in vitro.

Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans, a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode Clades I (10-13), III (16-42), IV (33-35) and V (25-64). Using an in vitro biosystem, we demonstrated that 40 nhr genes in a blood-feeding nematode Haemonchus contortus (clade V; barber's pole worm) were responsive to host serum and one nhr gene (i.e., nhr-64) was consistently stimulated by anthelmintics (i.e., ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e., nhr-105) and development (i.e., nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets.

Proteomic differences between extracellular vesicles and extracellular vesicle-depleted excretory/secretory products of barber's pole worm.

BACKGROUND: Components of excretory/secretory products (ESPs) of helminths have been proposed as vaccine targets and shown to play a role in modulating host immune responses for decades. Such research interest is further increased by the discovery of extracellular vesicles (EVs) in the ESPs of parasitic worms. Although efforts have been made to reveal the cargos of EVs, little is known about the proteomic differences between EVs and canonical ESPs released by parasitic worms from animals. METHODS: The total ESPs of Haemonchus contortus (barber's pole worm) were obtained by short-term in vitro culturing of young adult worms, and small EVs were isolated from ESPs using an ultracentrifugation method. Data-dependent acquisition (DDA) label-free Nano-LC-MS/MS was used to quantify the proteomic difference between small EVs and EV-depleted ESPs of H. contortus. Functional annotation and enrichment of the differential proteins were performed regarding cellular components, molecular functions, pathways, and/or biological processes. RESULTS: A total of 1697 proteins were identified in small EVs and EV-depleted ESPs of H. contortus adult worms, with 706 unique proteins detected in the former and 597 unique proteins in the latter. It was revealed that proteins in small EVs are dominantly cytoplasmic, whereas proteins in EV-depleted ESPs are mainly extracellular; canonical ESPs such as proteases and small GTPases were abundantly detected in small EVs, and SCP/TAP-, DUF-, and GLOBIN domain-containing proteins were mainly found in EV-depleted ESPs. Compared with well-characterised proteins in small EVs, about 50% of the proteins detected in EV-depleted ESPs were poorly characterised. CONCLUSIONS: There are remarkable differences between small EVs and EV-depleted ESPs of H. contortus in terms of protein composition. Immune modulatory effects caused by nematode ESPs are possibly contributed mainly by the proteins in small EVs.

A secreted BPTI/Kunitz inhibitor domain-containing protein of barber's pole worm interacts with host NLRP3 inflammasome activation-associated G protein subunit to inhibit IL-1ß and IL-18 maturation in vitro.

Protease inhibitors are major components of excretory/secretory products released by parasitic nematodes and have been proposed to play roles in host-parasite interactions. Haemonchus contortus (the barber's pole worm) encodes for several serine protease inhibitors, and in a previous study we identified a trypsin inhibitor-like serine protease inhibitor of this blood-feeding nematode, SPI-I8, as necessary for anticoagulation. Here, we demonstrated that a bovine pancreatic trypsin inhibitor/Kunitz-type serine protease inhibitor (BPTI/Kunitz) domain-containing protein highly expressed in parasitic stages, HCON_00133150, is involved in suppressing proinflammatory cytokine production in mammalian cells. Fluorescent labelling of HCON_00133150 revealed a punctate localisation at the inner hypodermal membrane of H. contortus, an organ closely related to the excretory column. Yeast two-hybrid screening and immunoprecipitation-mass spectrometry identified that the recombinant HCON_00133150 physically interacted with a range of host proteins including the G protein subunit beta 1 of sheep (Ovis aries; OaGNB1), a negative regulator of NLRP3 inflammasome activation. Interestingly, heterologous expression of HCON_00133150 enhanced the inhibitory effect of OaGNB1 on NLRP3 inflammasome and the maturation of proinflammatory cytokines IL-1ß and IL-18 in transfected cells. 1-to-1 orthologues (n = 33) of BPTI/Kunitz inhibitor domain-containing proteins were predicted in clades III, IV and V (but not clade I) parasitic nematodes. Structural (tandem BPTI/Kunitz inhibitor domains inverted into the globular reticulation) and functional (a GNB1 enhancer) characterisation of HCON_00133150 and its orthologues elucidated that these molecules might contribute to immune suppression by parasitic nematodes in animals and humans.

Fatty acid- and retinol-binding protein 6 does not control worm fatty acid content in Caenorhabditis elegans but might play a role in Haemonchus contortus parasitism.

BACKGROUND: Nematodes have lost the ability to synthesise necessary lipids de novo and have complementally evolved the capacity to acquire fatty acids and their derivatives from a diet or host animal. Nematode-specific fatty acid- and retinol-binding protein (FAR) family is one approach that facilitates lipid acquisition, representing an Achilles heel and potential target against roundworms of socioeconomic significance. However, little is known about their detailed functional roles in either free-living or parasitic nematodes. METHODS: A genome-wide identification and curation were performed to screen the FAR family members of Haemonchus contortus. Their transcription patterns in worms were also analysed to identify the targets. Ligand binding assay and molecular docking were conducted to verify the fatty acid binding activities of FAR proteins of interest. RNA interference (RNAi) and heterologous expression (rescuing) experiments were designed to explore the potential roles of the selected FAR protein in nematodes. Localisation of the protein was shown in sections of paraffin-embedded worms after an immunohistochemistry (IHC) assay. RESULTS: Here, an orthologue of far-6 in the model organism Caenorhabditis elegans (Ce-far-6) was functionally characterised in a parasitic nematode, H. contortus (Hc-far-6). It is demonstrated that knockdown of Ce-far-6 gene did not affect worm fat content, reproduction, or lifespan, but decreased worm body length at an early life stage of C. elegans. In particular, the Ce-far-6 mutant associated phenotype was completely rescued by Hc-far-6, suggesting a conserved functional role. Surprisingly, there were distinct tissue expression patterns of FAR-6 in the free-living C. elegans and parasitic H. contortus. High transcriptional level of Hc-far-6 and dominant expression of FAR-6 in the intestine of the parasitic stage of H. contortus link this gene/protein to nematode parasitism. CONCLUSIONS: These findings substantially enhance our understanding of far genes and the associated lipid biology of this important parasitic nematode at a molecular level, and the approaches established are readily applicable to the studies of far genes in a broad range of parasites.

PP2Acα-B'/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites.

Here, we report that the inhibition of the PP2A subfamily by okadaic acid results in an accumulation of polysaccharides in the acute infection stage (tachyzoites) of Toxoplasma gondii, which is a protozoan of global zoonotic importance and a model for the apicomplexan parasites. The loss of the catalytic subunit α of PP2A (ΔPP2Acα) in RHΔku80 leads to the polysaccharide accumulation phenotype in the base of tachyzoites as well as residual bodies and significantly compromises the intracellular growth in vitro and the virulence in vivo. A metabolomic analysis revealed that the accumulated polysaccharides in ΔPP2Acα are derived from interrupted glucose metabolism, which affects the production of ATP and energy homeostasis in the T. gondii knockout. The assembly of the PP2Acα holoenzyme complex involved in the amylopectin metabolism in tachyzoites is possibly not regulated by LCMT1 or PME1, and this finding contributes to the identification of the regulatory B subunit (B'/PR61). The loss of B'/PR61 results in the accumulation of polysaccharide granules in the tachyzoites as well as reduced plaque formation ability, exactly the same as ΔPP2Acα. Taken together, we have identified a PP2Acα-B'/PR61 holoenzyme complex that plays a crucial role in the carbohydrate metabolism and viability in T. gondii, and its deficiency in function remarkably suppresses the growth and virulence of this important zoonotic parasite both in vitro and in vivo. Hence, rendering the PP2Acα-B'/PR61 holoenzyme functionless should be a promising strategy for the intervention of Toxoplasma acute infection and toxoplasmosis. IMPORTANCE Toxoplasma gondii switches back and forth between acute and chronic infections, mainly in response to host immunologic status, which is characterized by flexible but specific energy metabolism. Polysaccharide granules are accumulated in the acute infection stage of T. gondii that have been exposed to a chemical inhibitor of the PP2A subfamily. The genetic depletion of the catalytic subunit α of PP2A leads to this phenotype and significantly affects the cell metabolism, energy production, and viability. Further, a regulatory B subunit PR61 is necessary for the PP2A holoenzyme to function in glucose metabolism and in the intracellular growth of T. gondii tachyzoites. A deficiency of this PP2A holoenzyme complex (PP2Acα-B'/PR61) in T. gondii knockouts results in the abnormal accumulation of polysaccharides and the disruption of energy metabolism, suppressing their growth and virulence. These findings provide novel insights into cell metabolism and identify a potential target for an intervention against a T. gondii acute infection.

The non-glycosylated protein of Toxocara canis MUC-1 interacts with proteins of murine macrophages.

Toxocariasis is a neglected parasitic disease caused predominantly by larvae of Toxocara canis. While this zoonotic disease is of major importance in humans and canids, it can also affect a range of other mammalian hosts. It is known that mucins secreted by larvae play key roles in immune recognition and evasion, but very little is understood about the molecular interactions between host cells and T. canis. Here, using an integrative approach (affinity pull-down, mass spectrometry, co-immunoprecipitation and bioinformatics), we identified 219 proteins expressed by a murine macrophage cell line (RAW264.7) that interact with prokaryotically-expressed recombinant protein (rTc-MUC-1) representing the mucin Tc-MUC-1 present in the surface coat of infective larvae of T. canis. Protein-protein interactions between rTc-MUC-1 and an actin binding protein CFL1 as well as the fatty acid binding protein FABP5 of RAW264.7 macrophages were also demonstrated in a human embryonic kidney cell line (HEK 293T). By combing predicted structural information on the protein-protein interaction and functional knowledge of the related protein association networks, we inferred roles for Tc-MUC-1 protein in the regulation of actin cytoskeletal remodelling, and the migration and phagosome formation of macrophage cells. These molecular interactions now require verification in vivo. The experimental approach taken here should be readily applicable to comparative studies of other ascaridoid nematodes (e.g. T. cati, Anisakis simplex, Ascaris suum and Baylisascaris procyonis) whose larvae undergo tissue migration in accidental hosts, including humans.

Mycophenolate mofetil preconditioning protects mouse liver against ischemia/reperfusion injury in wild type and toll-like receptor 4 knockout mice.

BACKGROUND: Mycophenolate mofetil (MMF), an immunosuppressive drug, exerts anti-inflammatory effects on organs during ischemia/reperfusion (I/R) injury. However, the exact function of MMF in hepatic I/R injury remains largely unknown. The purpose of this study was to explore the role and potential mechanism of MMF protection in hepatic I/R injury. METHODS: Male wild type (WT) and TLR4 knockout (KO) mice were injected intraperitoneally with MMF or normal saline. Animals underwent 90 min of partial hepatic ischemia, followed by 1, 6, or 24 h of reperfusion. Hepatic histology, serum amiotransferase, inflammatory cytokines, hepatocyte apoptosis, and hepatocyte autophagy were examined to assess liver injury. RESULTS: Treatment with MMF significantly decreased hepatic I/R injury as indicated by a reduction in serum aminotransferase levels, Suzuki scores, and the overall degree of necrosis. MMF treatment inhibited TLR4 activation dramatically. MMF administration also significantly inhibited the activation of the NF-κB pathway and the expression of pro-inflammatory cytokines. In TLR4 KO mice, MMF still exerted protection from hepatic I/R injury. MMF treatment inhibited hepatocyte apoptosis, as indicated by reduced TUNEL staining, and reduced the accumulation of cleaved caspase-3. In addition, MMF may induce autophagy and increase autophagic flux before and after hepatic reperfusion by augmenting the expression of LC3-II, P62, and Beclin-1. The induction of autophagy by MMF treatment may be related to TLR4 activation. CONCLUSIONS: Our results indicate that MMF treatment ameliorates hepatic I/R injury. The mechanism of action likely involves the ability of MMF to decrease apoptosis and the inflammatory response while inducing autophagy.