Abstract
ABSTRACTClostridium perfringens epsilon toxin (Etx) is categorized as the third most lethal bioterrorism agent by the Centers for Disease Control and Prevention (CDC), with no therapeutic counter measures available for humans. Here, we have developed a high-affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse glycosides (numbered 1-12). SAR of glycoside-Etx heptamers revealed exceptionally strong H-bond interactions of glycoside-4 with a druggable pocket in the oligomerization and β-hairpin region of Etx. Analysis of its structure suggested that glycoside-4 might self-aggregate to form a robust micelle-like supra-molecular complex due to its linear side-chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in turn leads to blockage of pore formation. Downstream evaluation revealed that glycoside-4 effectively blocked cell death of Etx-treated cultured primary cells and maintained cellular homeostasis via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing the mitochondrial membrane and impairing high mobility group box 1 (HMGB1) translocation from nucleus to cytoplasm. Furthermore, a single dosage of glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work reports for the first time a potent, nontoxic glycoside with strong ability to occlude toxin lethality, representing it as a bio-arm therapeutic against Etx-based biological threat.
Highlights
IntroductionOut of five strains (A-E) of C. perfringens, epsilon toxin is secreted by toxinotypes B and D
The findings showed that glycoside-4 could decrease Ca2+ influx in epsilon toxin (Etx)-treated Madin-Darby canine kidney (MDCK) cells, as FACS analysis depicted a drastic reduction of Fluo-4 AM intensity, which was similar to the untreated control (Fig. 2F)
According to the Centers for Disease Control and Prevention (CDC) list of potent bio-weapons, C. perfringens Etx has been categorized as a class B type of lethal neurotoxin that poses potential threat to domesticized animals and humans (CDC, 2000)
Summary
Out of five strains (A-E) of C. perfringens, epsilon toxin is secreted by toxinotypes B and D It causes fatal enterotoxaemia, called the kidney pulp disease, in domestic ruminants, resulting in heavy economic losses (Songer, 1996; Titball, 2009). As per previous reports (Knapp et al, 2009), domain II is known for playing crucial roles in oligomerization and pore formation, and amino acid mutation at this domain has been implicated in altered cytotoxicity Based on this information, we hypothesize that accessing the druggability of domain II in Etx might lead to the development of a promising antidote against epsilon toxin. None of these therapeutic measures are effective in counteracting the effects of the toxin
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