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Mol Biochem Parasitol,
2008]
An unusual feature of nematodes is the covalent attachment of immunomodulatory phosphorylcholine (PC) moieties to N-type glycans. Our previous work on the filarial nematode glycoprotein ES-62 has enabled us to predict the identity of enzymes necessary for PC-N-glycan biosynthesis. Here, we addressed these predictions using gene knockout technology applied to C. elegans and present two pieces of confirmatory data. Employing a triple null mutant worm lacking all three genes that encode active UDP-N-acetyl-d-glucosamine: alpha-3-d-mannoside beta1, 2-N-acetylglucosaminyltransferase I (GnT I) we have confirmed our earlier prediction that a crucial step in the generation of the substrate for PC transfer is addition of terminal GlcNAc to the alpha1-3-linked mannose residue of the glycan by GnT I. Second, by silencing genes responsible for expressing enzymes of the Kennedy pathway of phosphatidylcholine biosynthesis by RNA interference (RNAi), we have confirmed our belief for a role for diacylglycerol: choline phosphotransferase (CPT) in PC-N-glycan biosynthesis.
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ScientificWorldJournal,
2014]
The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.
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MicroPubl Biol,
2020]
OP50 is an Escherichia coli strain conventionally used as a bacterial food in the laboratory maintenance of Caenorhabditis elegans on agar plates. It has also been used to feed C. elegans in longitudinal cultures within microfluidic devices (MFDs) (Hulme et al., 2010; Li et al., 2015), where it has been subject to killing by ultraviolet irradiation or pasteurization performed to suppress clogging due to biofilm formation and aggregation (Li et al., 2015; Zhuo et al., 2017). However, the killed bacterial food can change C. elegans aging dynamics, likely due to influences on C. elegans physiology (Saul et al., 2009; Gruber et al.;, 2007; Garigan et al., 2002). Further development of longitudinal culturing systems for C. elegans in MFDs requires elucidation of the mechanisms that underlie food bacteria clogging and delineation of culture conditions in which living bacterial food can be incorporated without clogging. Bacteria switch from planktonic growth to aggregated growth under conditions of environmental stress, in the presence of toxins (e.g. antibiotics), and when there is a lack of nutrients (Trunk et al., 2018). Biofilms, such as dental plaque, are bacterial communities that are organized in a film-like form in which they are embedded in a self-produced polymeric matrix on biotic or abiotic surfaces; pellicles are floating biofilms that form at liquid-air interfaces. Meanwhile, autoaggregations are aggregated communities of bacteria suspended in solution, such as bacterial flocs formed in activated sludge. Biofilms and autoaggregations are formed by both shared and independent genetic and physico-chemical mechanisms (Trunk et al., 2018; Berne et al., 2018; Berne et al., 2015). In this study, we examined OP50 biofilm formation.Biofilm formation is mediated by flagellin proteins (e.g. FliC), which form flagella, and the adhesion protein FimH, which is located at the tips of type I pili (Berne et al., 2018, Jones et al., 1995; Pratt and Kolter, 1998; Friedlander et al., 2013). We compared the biofilm formation ability of OP50 with that of the biofilm-forming (Wood et al., 2006) wild-type BW251113 E. coli strain as well as that of two BW251113-derived knockouts produced with a kanamycin (Km) cassette characterized as biofilm formation defective mutants: JW4283: BW25113 fimH::Km (a fimH knockout) and JW1908: BW25113 fliC::Km (a fliC knockout) (Baba et al., 2006). Compared to the original BW251113 strain, BW251113 fliC::Km had a significantly reduced ability to form biofilm on glass and polystyrene (Fig. 1A and 1B, p < 0.05) and BW25113 fimH::Km had a significantly reduced ability to form biofilm on glass (Fig. 1A, p < 0.05; biofilm formation on polystyrene showed a near-significant reduction trend Fig. 1B, p = 0.0574). Compared with the original BW251113 strain, we found that OP50 had a significantly reduced biofilm formation ability on polystyrene (Fig. 1B, p < 0.05; biofilm formation on glass showed a near-significant reduction trend, Fig. 1A, p = 0.0507). The biofilm formation ability of OP50 was as low as that seen with the BW251113 biofilm formation defective mutants, and similar to that of OP50 fliC::Km and OP50 fimH::Km mutants (Fig. 1A and 1B), which were constructed by transferring fliC::Km and fimH::Km alleles to OP50 by P1 transduction (Fig. 1C and 1D). Therefore, we conclude that the original OP50 strain is itself a biofilm formation defective mutant.
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Chemosphere,
2016]
At present, nanotechnology has been producing nanoscale materials with unprecedented speed. Nanomaterials could be inevitably released into the environment owing to their widespread use, and their potential toxicity has caused a great concern. With regard to assessment of nanomaterial toxicity, many studies probably don't truly reflect their toxicity, because the nanoparticles were not stable and uniformly dispersed in the medium. In the present study, the semi-fluid nematode growth gelrite medium (NGG) was used to achieve better distribution of silver nanoparticles (AgNPs). We aimed to evaluate the toxicity of AgNPs in three different culture methods, such as the NGG, nematode growth medium (NGM) and K-medium (KM). Our transmission electron microscopy, hydrodynamic diameter, and inductively coupled plasma-atomic emission spectrometry results demonstrated that AgNPs homogeneously and stably dispersed in NGG compared to that in liquid KM. Furthermore, the conventional toxicity end points, such as body length, fecundity, lifespan, population growth, germline cell apoptosis, reactive oxygen species, and mitochondrial membrane potential were used to assess the toxicity of AgNPs to Caenorhabditis elegans (C.elegans) in NGG, NGM and KM. Our results showed that the toxicity of AgNPs obtained in the NGG test medium was much higher than that in the standard NGM and KM. In addition to the improved dispersion of nanoparticles, NGG also offered advantages for long-term studies and likely provided a convenient nematode toxicity testing method. These results revealed that the NGG test medium was a suitable and sensitive culture method for the evaluation of AgNPs toxicity using C.elegans.
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FEBS Lett,
2004]
Based on the amino acid alignment, Caenorhabditis elegans F32D1.1 was identified to be a homologue of the mammalian fidgetin. We produced and purified the F32D1.1 protein by using a baculovirus-expression system. F32D1.1 has an ATPase activity, which is sensitive to N-ethylmaleimide. Km and Vmax for the ATPase activity of F32D1.1 were estimated to be 0.44 mM and 225 nmol/mg/min, respectively. When the cysteine at the position of 368 was mutated to alanine, the ATPase activity was greatly decreased; Vmax was decreased to one-sixth, while Km remained similar. These results suggest that the unique position of cysteine 368, located immediately downstream of the Walker A motif, plays an important role in the ATP hydrolysis process of C. elegans F32D1.1 protein.
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Southeast Asian J Trop Med Public Health,
1977]
A survey was carried out among persons residing in 8 villages in the Province of West Kalimantan, Indonesia to determine the prevalence of filariasis. Finger tip blood smears were obtained at night from over 3,000 people and microfilariae of Brugia malayi were found in 108 (3.5%) and Wuchereria bancrofti in 10 (0.3%). Most B. malayi (96 carriers) was found in Kakap, a village near the coast, 20 km from the provincial capital of Pontianak. Nine of 10 cases of W. bancrofti were located in Pahauman, a village 130 km northeast of the provincial capital. Periodicity studies indicate the strain of B. malayi to be subperiodic. In Kakap 18% of 226 persons examined had a clinical history of filariasis and elephantiasis was seen in 13%. This is the first report of rural bancroftian filariasis in the area. A few Mansonia species of mosquitoes were examined but none were infected with filarial larvae.
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J Biol Chem,
1999]
N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.
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J Neurochem,
1985]
We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.
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Biochim Biophys Acta Proteins Proteom,
2021]
Malate dehydrogenase (MDH) catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate in the citric acid cycle. Eukaryotes have one MDH isozyme that is imported into the mitochondria and one in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 and mitochondrial MDH-2 in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we measured KM values for oxaloacetate of 54 and 52 uM and KM values for NADH of 61 and 107 uM for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH-1 and MDH-2 from a mixed population of worms and separated them using anion exchange chromatography. Both endogenous enzymes had a KM for oxaloacetate similar to that of the corresponding recombinant enzyme. Recombinant MDH-1 and MDH-2 had maximum activity at 40 degrees C and 35 degrees C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Protein homology modeling predicted that MDH-1 had more intersubunit salt-bridges than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer intersubunit ionic interactions compared to mammalian MDH2 enzymes. These results suggest that the increased stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.
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Exp Parasitol,
2015]
Full length cDNAs encoding phosphofructokinase (PFK) were cloned from Teladorsagia circumcincta (TcPFK) and Haemonchus contortus (HcPFK). TcPFK (2361bp) and HcPFK (2367bp) cDNA encoded 787 and 789 amino acid proteins respectively. The predicted amino acid sequences showed 98% similarity with each other and 70% with a Caenorhabditis elegans PFK. Substrate binding sites were completely conserved in both proteins. Soluble N-terminal His-tagged PFK proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH7.0, Km for fructose 6-phosphate was 0.50+/-0.01 and 0.55+/-0.01mM respectively when higher (inhibiting concentration, 0.3mM) ATP concentration was used and the curve was sigmoidal. The Vmax for TcPFK and HcPFK were 1110+/-16 and 910+/-10 nM min(-1)mg(-1) protein respectively. Lower ATP concentration (non-inhibiting, 0.01mM) did not change the Vmax for TcPFK and HcPFK (890+/-10 and 860+/-12 nM min(-1)mg(-1) protein) but the substrate affinity doubled and Km for fructose 6-phosphate were 0.20+/-0.05 and 0.25+/-0.01mM respectively. Recognition of TcPFK and HcPFK by mucosal and serum antibodies in nematode exposed animals demonstrates antigenicity and suggests involvement in the host response to nematode infection.