Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains.
Biophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ββ′σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ββ′σ subunits of polymerase.
To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of this α-proteobacteria and in deciphering the role of Wol GreA in filarial development.
Diethylcarbamazine and ivermectin, the conventional antifilarial drugs, exert poor activity on adult filarial worms. Brugia malayi, a causative agent of human lymphatic filariasis, harbors an obligate alpha-proteobacterium, Wolbachia. Because the endosymbiont is required for the development, viability, and fertility of the parasite, wolbachial proteins seem to be a potential target for antifilarial drugs. Transcription elongation factor is one of the bacterial factors that plays an essential role in efficient transcription by facilitating the movement of RNA polymerase on DNA template. The factor is also necessary for bacterial viability because deletion of greA leads to hypersensitivity to environmental assaults such as ionic detergents, elevated temperature or osmotic shock. Functional characterization of Wol GreA with its domains was undertaken to ascertain the molecular trappings underlying Wolbachia maintenance within the filarial parasite and gain further insight into the mutualism between the bacteria and the nematode host. In the present study, GreA of Wolbachia revealed phylogenetic divergence from other bacteria. Wol GreA, a member of the Gre family, authenticates its essentiality by the presence of evolutionarily conserved N and C terminal domains, where dimeric CTD exhibited chemical cross-linking and direct binding to α2ββ′σ subunits of Wol RNAP.