Molecular & Biochemical Characterization of a Putative Chemotherapeutic Target of Filarial

Abstract

Lymphatic filariasis, an infectious disease caused by Wuchereria bancrofti, Brugia malayi and Brugia timori (lymph-dwelling nematode), is transmitted by mosquitoes. About 1.3 billion people living in tropical and subtropical areas of more than 83 countries worldwide are affected by this disease. The Global Programme to Eliminate Lymphatic Filariasis was established in 1999 with the objective of interrupting transmission of the parasites in all endemic countries by 2020. The mainstay of filarial disease control for several decades has been a limited number of drugs viz., Diethylcarbamazine, albendazole and ivermectin. These drugs are not effective against all stages of the parasite and have many possible side effects. Furthermore, signs of emerging drug resistance are becoming increasingly apparent. Therefore novel chemotherapeutic drugs effective against adult parasites are urgently needed. As complete genome sequence of B. malayi is available, comparative genomics would undoubtedly lead to a greater understanding of filarial parasite evolution, host parasite interaction and biochemical processes of filarial parasite, especially related to those that are crucial in drug and immunological response. Current filariasis control strategies are not entirely successful and filarial infections are on the rise. In the absence of availability of antifilarial vaccines, chemotherapy remains the mainstay for treatment of the diseases caused by filarial parasites. However, the precise primary effects of different chemical classes of compounds currently used as antifilarials are still unclear. A clear understanding of the mode of action of antifilarials awaits greater knowledge of the biochemical pathways operating in filarial parasites. Interfering with parasite specific metabolic pathways could lead to a new range of antifilarial drugs whose structure and mode of action are different from those currently in use. G6PD, a rate-limiting enzyme of PPP is responsible for the generation of NADPH and ribose 5-phosphate. This pathway is an alternative route to glycolysis for degradation of glucose. The tightly controlled concentrations of ROS and fluctuations in redox potential are important mediators of signaling processes, stress response, development and aging. G6PD, a critical cytosolic antioxidant enzyme maintains the NADPH/NADP+, GSH/GSSG ratio and plays a crucial role in the protection from redox stress induced apoptosis and in cell growth. Glycolysis serves as a major pathway for hexose utilization in filarial parasites. Inhibition of G6PD activity causes accumulation of 6-phosphogluconate, which inhibits glycolysis by competitively inhibiting the glucose-6-phospho isomerase. Therefore decrease in G6PD activity and as a result NADPH level will impair the entire antioxidant system. The differences between host and parasitic G6PD can be fruitfully utilized for designing of selective inhibitors with antifilarial activity. G6PD from Brugia malayi was successfully cloned and expressed. RNA from Brugia malayi was isolated and cDNA was synthesized. G6PD gene was PCR amplified from cDNA and cloned in cloning vector. The positive clones were confirmed by restriction digestion and sequencing of the clones. These clones were sub cloned in the expression vector. Recombinant G6PD clone was transformed in E. coli cells for expression of protein. Recombinant protein was purified by affinity column and purified protein was biochemically characterized. The optimum conditions for enzyme activity were evaluated. The effect of various G6PD inhibitors, SH inhibitors, metal ions, antifilarials and CDRI compounds on the enzyme activity were studied. Various spectroscopic techniques viz., CD and fluorescence were used to study the tertiary and secondary structure of BmG6PD and also the effect of cofactor on tryptophan accessibility, stability and folding of the purified recombinant BmG6PD. The structural characterization of holoenzyme and apoenzyme of BmG6PD was carried out by fluorescence spectroscopy to determine dissociation constant, the influence of the bound cofactor on the protein structure, the effect of chemical denaturants such as urea and GdmCl on the protein structure and stability. The oligomeric state of BmG6PD was studied by size-exclusion chromatography and glutaraldehyde crosslinking. The expression and localization of BmG6PD was studied in microfilariae and adult stage of parasite by polyclonal antibody raised against the recombinant BmG6PD protein. Western immunoblotting and immunofluoresence microscopic studies using polyclonal antibodies have been presented to illustrate the localization of G6PD in B. malayi parasites. The homology model of BmG6PD was constructed and refined BmG6PD model was used for docking studies.