Abstract
Objectives 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.Methodology/Principal FindingsBiophysical 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.Conclusions/SignificanceTo 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.
Highlights
Lymphatic filariasis (LF), recognized as the world’s most disabling and disfiguring disease, is a huge and costly problem in developing nations
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
Summary
Lymphatic filariasis (LF), recognized as the world’s most disabling and disfiguring disease, is a huge and costly problem in developing nations. LF caused by the mosquito-borne, thread-like nematodes belonging to the genera Wuchereria and Brugia affects around 120 million people worldwide. The only reliable chemotherapeutic control measures against this devastating parasitic disease are community-wide distributions of diethylcarbamazine or ivermectin in combination with albendazole [3]. These antifilarial drugs are principally microfilaricidal, and exhibit only limited macrofilaricidal efficacy, allowing adult worms to survive in human hosts for up to decades. Repeated treatments are required over many years to interrupt the transmission This raises the possibility of parasites becoming resistant to conventional drugs. A recent report indicates that the depletion of Wolbachia by anti-
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