Abstract
Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins.
Highlights
Foodborne diseases caused by bacterial pathogens play an important role in the worldwide health-care system
As the coexpression of all three subunits simultaneously in a 10:10:1 molar plasmid ratio led to the active tripartite toxin, we wanted to determine whether the addition of NheA and NheB plasmid at higher ratios than 2:2:1 led to hemolytic activity
The autoradiograph clearly showed defined protein bands (Supplementary Fig. 2c) and all reactions showed hemolytic activity (Supplementary Fig. 2d), which suggests that NheA and NheB generally have to be present in excess over NheC
Summary
Foodborne diseases caused by bacterial pathogens play an important role in the worldwide health-care system. Previous studies have demonstrated the importance of the three subunits NheA, NheB and NheC for complex and pore-formation. To display maximum functional activity and cytotoxicity, Nhe subunits must interact at a 10:10:1 NheA:NheB:NheC molar ratio[6]. New methods for the characterization of bacterial toxins are required in order to develop diagnostic tools for their detection In this context, cell-free protein synthesis (CFPS) has emerged as a rapid and efficient method for the synthesis and functional characterization of toxins[10,11,12]. We demonstrate the cell-free synthesis of the functional tripartite Nhe toxin. We show that eukaryotic cell-free protein synthesis systems offer a way to synthesize functionally active, multicomponent toxins in a fast and efficient manner
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