Polyethylene Glycol Conjugation Enhances Mosquito-Larvicidal Activity of Lysinibacillus Sphaericus BinA Protein

Abstract

Mosquitoes are known to spread human diseases like West Nile fever, dengue, malaria, zika etc. accounting for millions of deaths worldwide. Lysinibacillus sphaericus, a gram positive, spore producing commensal soil bacterium, has been used worldwide for controlling mosquito population like Culex and Anopheles, and is regarded as safe against non-target organisms. Binary toxin, composed of BinA (41.9 kDa) and BinB (51.4 kDa) component proteins, is responsible for the high larvicidal activity of several L. sphaericus strains. The two proteins exert high toxicity when administered together. BinA alone displays larvicidal activity, in the absence of BinB, albeit at reduced levels. But instability, shorter half-lives and rapid proteolytic digestion can limit their use as an effective insecticide. We for the first time demonstrate the beneficial effect of PEGylation (covalent attachment of polyethylene glycol) on mosquito-larvicidal activity of BinA protein. PEG-protein conjugates were synthesized using PEG-isocyanate polymer. The resulting bio-conjugates were purified to homogeneity by column chromatography methods. These were characterized by various biophysical methods like MALDI-TOF, DLS, DSF and CD. Two different isoforms of PEG-BinA conjugates are expected from biophysical analysis, which appear to be mono-PEGylated but may differ in the site of PEG attachment to BinA protein. The PEGylated proteins displayed preservation of protein's native structure and exhibited improved thermal stability by about 3-5 oC. The PEGylated proteins were also checked for stability against complex proteolytic environment. Regardless of the site of modification, the two isoforms showed a remarkable 7-fold improvement in the larvicidal activity of BinA protein against 3rd instar Culex larvae, compared to the unmodified protein. The PEGylation of recombinantly produced BinA can be achieved easily. It promises a judicious approach towards mosquito population control.