Pfarr KM et al. (2009) Parasite Immunol "Filariasis and lymphoedema."

Pfarr KM, Specht S, Debrah AY, Hoerauf A

[Parasite Immunol, 2009]

Among the causes of lymphoedema (LE), secondary LE due to filariasis is the most prevalent. It affects only a minority of the 120 million people infected with the causative organisms of lymphatic filariasis (LF), Wuchereria bancrofti and Brugia malayi/timori, but is clustered in families, indicating a genetic basis for development of this pathology. The majority of infected individuals develop filarial-specific immunosuppression that starts even before birth in cases where mothers are infected and is characterized by regulatory T-cell responses and high levels of IgG4, thus tolerating high parasite loads and microfilaraemia. In contrast, individuals with this pathology show stronger immune reactions biased towards Th1, Th2 and probably also Th17. Importantly, as for the aberrant lymph vessel development, innate immune responses that are triggered by the filarial antigen ultimately result in the activation of vascular endothelial growth factors (VEGF), thus promoting lymph vessel hyperplasia as a first step to lymphoedema development. Wolbachia endosymbionts are major inducers of these responses in vitro, and their depletion by doxycycline in LF patients reduces plasma VEGF and soluble VEGF-receptor-3 levels to those seen in endemic normals preceding pathology improvement. The search for the immunogenetic basis for LE could lead to the identification of risk factors and thus, to prevention; and has so far led to the identification of single-nucleotide polymorphisms (SNP) with potential functional relevance to VEGF, cytokine and toll-like receptor (TLR) genes. Hydrocele, a pathology with some similarity to LE in which both lymph vessel dilation and lymph extravasation are shared sequelae, has been found to be strongly associated with a VEGF-A SNP known for upregulation of this (lymph-)angiogenesis factor.

Pfarr KM et al. (2000) Exp Parasitol "Brugia malayi: localization of nitric oxide synthase in a lymphatic filariid."

Pfarr KM, Fuhrman JA

[Exp Parasitol, 2000]

Nitric oxide synthase converts L-arginine to citrulline and nitric oxide, a gaseous signaling molecule critical to multiple physiological responses. Nitric oxide synthase was detected by Western blot analysis of Brugia malayi extracts using an antibody raised against a peptide from murine brain nitric oxide synthase. Using NADPH diaphorase staining and immunohistochemistry, nitric oxide synthase was localized in the parasitic nematode B. malayi. As in Ascaris suum, nitric oxide synthase was detected in the body wall muscles of adult B. malayi. This localization pattern is in agreement with the role of nitric oxide in the control of muscle tone in other invertebrates and in vertebrates. A novel finding was the localization of nitric oxide synthase in the oocytes, in developing embryos, and in spermatozoa. B. malayi nitric oxide synthase may play a role in developmental signaling, as has been suggested for Drosophila and Ilyanassa, a marine mud snail.

Pfarr KM et al. (2008) Parasitology "The mitochondrial heat shock protein 60 (HSP60) is up-regulated in Onchocerca volvulus after the depletion of Wolbachia."

Hoerauf A, Schmetz C, Buttner DW, Pfarr KM, Heider U

[Parasitology, 2008]

Wolbachia, a genus of endosymbiotic bacteria of filarial worms, represent novel targets for anti-filarial therapy. The efficacy of compounds against Wolbachia has been evaluated using antiserum raised against the 60 kDa heat shock protein (HSP60) which binds specifically to this protein in both Wolbachia and mitochondria. It has been shown that Wolbachia stains (using such specific probes) stronger than the mitochondria in untreated Onchocerca volvulus, whereas after the depletion of Wolbachia (with drugs) staining of the mitochondria is increased. Herein, immunogold electron microscopy showed that specific anti-HSP60 serum specifically labelled Wolbachia and filarial mitochondria, and that both have distinct localization patterns, thus allowing them to be differentiated. Immunohistochemistry of O. volvulus showed that HSP60 staining is increased in the mitochondria after Wolbachia depletion in the hypodermis, epithelia, muscles, oocytes, embryos, and developing spermatozoa. This could have been the result of the antiserum preferentially binding to the Wolbachia when they are present or due to increased expression of the protein in the absence of the bacteria. To address this, mRNA levels of filarial hsp60 in O. volvulus were measured. After the depletion of Wolbachia, the transcription of hsp60 was significantly greater (7.7 fold) compared with untreated worms. We hypothesize that the increased expression of HSP60 in the absence of Wolbachia is due to a disruption of the homeostasis of the endosymbiosis.

Arumugam S et al. (2014) Exp Parasitol "Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria."

Pfarr KM, Arumugam S, Hoerauf A

[Exp Parasitol, 2014]

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

Puah SM et al. (2014) ScientificWorldJournal "Molecular characterization of putative virulence determinants in Burkholderia pseudomallei."

Puthucheary SD, Chua KH, Wang JT, Pan YJ, Puah SM

[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.

Heider U et al. (2006) Int J Med Microbiol "Differential display of genes expressed in the filarial nematode Litomosoides sigmodontis reveals a putative phosphate permease up-regulated after depletion of Wolbachia endobacteria."

Blaxter M, Heider U, Hoerauf A, Pfarr KM

[Int J Med Microbiol, 2006]

Mutualist symbiotic Wolbachia endobacteria are found in most filarial nematodes. Wolbachia are essential for embryogenesis and for larval development into adults, and thus represent a new target for anti-filarial drug development. Tetracycline antibiotics deplete Wolbachia in animal model filaria Litomosoides sigmodontis and Brugia pahangi, as well as in the human parasites Brugia malayi, Onchocerca volvulus and Wuchereria bancrofti. Very little is known about the molecular details of the symbiotic interaction between Wolbachia and filarial nematodes. Nematode genes that respond to anti-Wolbachia antibiotic treatment may play important roles in the symbiosis. Differential display PCR was used to detect several candidate genes that are up-regulated after 3, 6, 15, 30 and 36 days of tetracycline treatment. One of these genes, Ls-ppe-1, was similar to a family of phosphate permeases, and had putative orthologues in O. volvulus and B. malayi. Ls-ppe-1 steady-state mRNA levels were elevated by day 3-6 of treatment, and remained elevated through to 70 days post-treatment. In Caenorhabditis elegans, the knockdown of a homologous phosphate permease results in embryonic lethality, with the production of degenerating embryos, a phenotype also seen in filarial nematodes after depletion of Wolbachia with tetracycline. The potential role of Ls-ppe-1 in the nematode-bacterial symbiosis is discussed.

Arata, Yukinobu et al. (2020) MicroPubl Biol

Sako, Yasushi, Kimura, Hiroshi, Arata, Yukinobu, Ikeda, Yusaku, Oshima, Taku

[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.

Luo X et al. (2016) Chemosphere "A novel method for assessing the toxicity of silver nanoparticles in Caenorhabditis elegans."

Luo X, Yang Y, Wu L, Zhang Y, Chen S, Wang S, Xu S, Xu A

[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.

Silimon, Ruth L. et al. (2013) International Worm Meeting "Overexpression, purification and characterization of C. elegans glyceraldehyde-3-phosphate dehydrogenase isozymes."

Walstrom, Katherine M., Spengler, Justin W., Valbuena, Valeria S. M., Gautier, Megan K., Silimon, Ruth L., Schwabe-Warf, Derek

[International Worm Meeting, 2013]

Glyceraldehyde-3-phosphate (G3P) dehydrogenase (GPD) is the glycolytic enzyme that adds inorganic phosphate to its substrate so that net ATP production is possible. C. elegans has 4 different GPD isozymes. Embryonic GPD-1 and GPD-4 are nearly identical, while the homologous GPD-2 and GPD-3 are expressed in postembryonic worms (Yarbrough and Hetch, JBC 259, 14711, 1984; Huang et al., 1989, JMB 206, 411). GPD-3 is involved in protection from anoxia (Mendenhall et al., 2006, Genetics 174, 1173) and upregulated in dauers and daf-2 mutants (McElwee et al. 2006, Mech. Age. Dev. 127, 922). We subcloned, overexpressed, and purified both GPD-1/4 and GPD-3 and performed enzyme kinetics studies with all three substrates under the optimal reaction conditions at pH 8.0-8.5. Both isozymes exhibited substrate inhibition at high concentrations of phosphate and NAD+, but the embryonic form was inhibited at lower concentrations of phosphate or NAD+ than GPD-3. GPD-1/4 appeared to have a higher specific activity than GPD-3. We determined that the KM values for NAD+, phosphate, and G3P for GPD-3 were 0.3 mM, 0.4 mM, and 2.6 mM, respectively. The KM values for NAD+ and phosphate for GPD-1/4 were 1 mM and ~0.6 mM. The KM values from a partially purified GPD preparation from a mixed population of C. elegans had NAD+ and G3P KM values of 1 mM and 0.3 mM, respectively. Based on the results of Yarbrough and Hetch, we expected the enzyme activity of the adult GPD-2 and GPD-3 enzymes to predominate in the endogenous GPD mixture purified from the worms. Based on our current kinetics results, the endogenous protein mixture had a KM for NAD+ more similar to the GPD-1/4 enzyme. We are continuing to compare the activities of the C. elegans GPD enzymes to determine which isozyme(s) are active in the endogenous GPD mixture.

Yakushiji Y et al. (2004) FEBS Lett "Identification of a cysteine residue important for the ATPase activity of C. elegans fidgetin homologue."

Yamanaka K, Ogura T, Yakushiji Y

[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.