Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation

Abstract

The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

Highly Effective Broad Spectrum Chimeric Larvicide That Targets Vector Mosquitoes Using a Lipophilic Protein

“…Another problem with a report that BinA and BinB prepared separately and then mixed together formed a tetramer 28 is that in a subsequent study it was shown the Ls protein complex studied 29 by the former group was a spore coat protein complex, not the Bin toxin. In other studies it has been suggested reassociation of BinA and BinB may be required for important conformational changes essential to both molecules so that activated Bin can bind receptors, interact with membrane lipids for additional structural alterations, and induce its internalization 18 , 20 , 27 . We do not question these results under the conditions tested, but our in vivo results reported here provide strong evidence that BinA once activated is highly toxic without requiring BinB for conformational changes, nor does it appear to require interactions with microvillar membrane lipids for toxicity.…”

“…Whereas Bin’s intoxication has been well described cytologically 5 , 16 – 18 , its mode of action at the molecular level remains unknown. In many studies over the past decade it appears to be assumed that BinA and BinB cystallize separately in Ls, dissolve after ingestion in the midgut, are activated, and then associate to form an activated dimer or tetramer 20 – 22 , 27 (2BinA + 2BinB; see Fig. 6) 20 .…”

Cytotoxic Effects and Intracellular Localization of Bin Toxin from Lysinibacillus sphaericus in Human Liver Cancer Cell Line

“…The decrease in the cytotoxic effect on HepG2 cells caused by the combination of Bin subunits in comparison to individual Bin toxin subunits observed in the present study was in concordance with a previous report by Chankamngoen et al [ 11 ]. In fact, Surya et al [ 16 ] indicated that change in conformational state of Bin toxin monomeric subunits to heterodimeric form occurs when present together in an equimolar ratio in an aqueous solution. The formation of heterodimers in BinA + BinB might affect the interaction of Bin toxin with membrane or with the targeted receptor in susceptible cancer cell lines, resulting in the difference in cytotoxic effects.…”

Membrane insertion of α-xenorhabdolysin in near-atomic detail

“…Interestingly, the transmembrane helices of XaxA that fortify the inner ring of helices of XaxB, do not completely span the membrane ( Figure S10). The arrangement of the components clearly shows that XaxAB is not a binary toxin as suggested [14,15], but rather a bi-component toxin, such as BinAB from Lysinibacillus sphaericus [26,27] and leukocidin A & B (LukGH and SF) from Staphylococcus aureus [28,29] where both proteins contribute to building the pore.…”