Abstract:
The endosymbiotic Wolbachia of Brugia malayi are an attractive antifilarial drug target. These bacteria possess an mutualistic relationship with the filarial parasite and impair filarial growth and survival if treated with tetracycline, doxycycline etc. thereby presenting Wolbachia as an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from Wolbachia endosymbiont of Brugia malayi its biophysical properties, specific inhibitors assay and immunologic response in BALB/c.
The gene was found to be expressed in all the major life-stages of B. malayi. The solubilised GST-fusion enzyme expressed in E. coli was found to be in monomer form in native state having Mr ~ 62 kDa. The specific inhibitors were used against the enzyme on B. malayi adult and microfilariae for 7 days in vitro at various concentrations and NSC-659390 proved to be the most potent compound (IC50 0.32 µM) followed by NSC-658343 (4.13 µM) and NSC-657589 (7.5 µM). On intraperitoneal administration in adult B. malayi I.P. transplanted jirds at 5 mg/kg x 7 days, all the compounds killed a significant proportion of implanted worms. The subcutaneous administration of the recombinant protein in BALB/c mice generated no specific immune response; rather there was downregulation of BALB/c immunity. The docking studies of enzyme and inhibitors and in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity.
An understanding of the functional mechanism of rRNA methylation in Wolbachia would certainly pave the way for future research on this enzyme. Findings suggest evaluation of compounds on other bacterial pathogens possessing methyltransferase with DPPY motif and design and synthesis of analogous inhibitors.
