In Vitro Silencing of Brugia malayi Trehalose-6-Phosphate Phosphatase Impairs Embryogenesis and In Vivo Development of Infective Larvae in Jirds

Abstract

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.

An improved method for the large scale preparation of α,α′-trehalose-6-phosphate

“…1,2 T6PP functions both in the synthesis of a,a 0 -trehalose, which is required for survival and/or virulence, and in the removal of T6PP that is necessary to avoid cell toxicity. [1][2][3][4][5] Because a,a 0 -trehalose synthesis is restricted to plants and in particular to lower organisms, including parasitic nematodes, opportunistic fungi and pathogenic bacteria, T6PP has been identified as an attractive platform for the development of small molecule inhibitors to serve as antihelminthics, antifungals and antibacterials. 2,6,7 To this end, large quantities of homogenous T6P are required to support high-throughput inhibitor screening, evaluation and SAR analyses.…”

The gene expression and the activity of enzyme synthesis of trehalose during development ofAscaris suum(Nematoda) eggs

“…Kormish and McGhee (2005) developed the lethal gob-1 phenotype to demonstrate that T6P accumulation leads to nematode death. Kushwaha et al (2012) also demonstrated that tpp gene silencing was lethal for Brugia malayi larvae and that the observed lethal effects impaired in vivo development.…”

Establishing RNAi in a Non-Model Organism: The Antarctic Nematode Panagrolaimus sp. DAW1

“…A recent screening of the PaDAW1 genome did not confirm the presence of these proteins, possibly explaining the limited efficiency of RNAi [10]. However, Kushwaha et al [37] reported that B . malayi genes can be efficiently silenced by RNAi despite the lack of SID-1 and SID-2 in this species, suggesting the involvement of other genes.…”