BackgroundThe trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP) is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes.Methodology and Principal FindingsIn the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds.Conclusions/SignificanceThe present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.

In vitro studies have shown unequivocally that bilirubin is an antioxidant. We hypothesized that bilirubin serves a physiological role of an antioxidant in vivo. To investigate the probable protective role of bilirubin in vivo, term babies with clinical jaundice were grouped into four categories-serum total bilirubin (STB) <160 mg/l, 160-200 mg/l, >200 mg/l, and kernicterus. Serum bilirubin, serum albumin, plasma glucose-6-phosphate dehydrogenase (G6PD), lipid peroxidation in blood cells, and reduced glutathione (GSH) content in whole blood were investigated. We also measured superoxide dismutase (SOD) and catalase in hemolysate and total plasma antioxidant capacity (TAC). Lipid peroxidation and antioxidant enzymes were significantly lower in babies with STB <200 mg/l compared to controls. TAC had a positive and MDA had a negative correlation with STB till 200 mg/l. However, TAC had a negative and MDA had a positive correlation with bilirubin >200 mg/l and in babies with bilirubin encephalopathy. Elevated levels of MDA, SOD, and catalase and significantly decreased levels of reduced glutathione and total antioxidant capacity were observed in STB >200 mg/l group. Antioxidant enzymes were also significantly inhibited in bilirubin encephalopathy babies. Post phototherapy, MDA production and antioxidant levels were significantly increased whilst total antioxidant capacity and reduced glutathione were significantly decreased compared to pre-phototherapy values. Exchange transfusion resulted in reduced oxidative stress in subjects with encephalopathy, whereas no significant difference was observed in other babies with STB >200 mg/l. Taken together, the present study propounds that bilirubin acts as a physiological antioxidant till 200 mg/l concentration in full-term normal neonates. It is conjectured that beyond 200 mg/l, it can no longer be considered physiologic. However, the cause of pathological jaundice needs to be identified and treated. The present data documents that phototherapy also induces oxidative stress.

BackgroundThe phosphoglycerate mutase (PGM) enzyme catalyzes the interconversion of 2- and 3-phosphoglycerate in the glycolytic /gluconeogenic pathways that are present in the majority of cellular organisms. They can be classified as cofactor-dependent PGM (dPGM) or cofactor-independent PGM (iPGM). Vertebrates, yeasts, and many bacteria have only dPGM, while higher plants, nematodes, archaea, and many other bacteria have only iPGM. A small number of bacteria, including Escherichia coli and certain archaea and protozoa, contain both forms. The silencing of ipgm in Caenorhabditis elegans (C. elegans) has demonstrated the importance of this enzyme in parasite viability and, therefore, its potential as an anthelmintic drug target. In this study, the role of the Brugia malayi (B. malayi) ipgm in parasite viability, microfilaria release, embryogenesis, and in vivo development of infective larvae post-gene silencing was explored by applying ribonucleic acid (RNA) interference studies.ResultsThe in vitro ipgm gene silencing by small interfering RNA (siRNA) leads to severe phenotypic deformities in the intrauterine developmental stages of female worms with a drastic reduction (~90%) in the motility of adult parasites and a significantly reduced (80%) release of microfilariae (mf) by female worms in vitro. Almost half of the in vitro-treated infective L3 displayed sluggish movement. The in vivo survival and development of siRNA-treated infective larvae (L3) was investigated in the peritoneal cavity of jirds where a ~45% reduction in adult worm establishment was observed.ConclusionThe findings clearly suggest that iPGM is essential for both larval and adult stages of B. malayi parasite and that it plays a pivotal role in female worm embryogenesis. The results thus validate the Bm-iPGM as a putative anti-filarial drug target.

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (K m) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.