Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus

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.

The Food Poisoning Toxins of Bacillus cereus

“…However, also for Nhe strain-specific variations exist [5]. All three recombinant Nhe protein components were successfully overexpressed in B. anthracis [204] and, just recently, in a eukaryotic cell-free protein expression system [309]. The Nhe and Hbl proteins share sequence identities, both between the three components of each complex and between the two enterotoxin complexes.…”

The Bacillus cereus Food Infection as Multifactorial Process

“…It has been shown that the B. cereus enterotoxins affect target cells of a great variety of different tissue, origin and species [388,391,[408][409][410][411][412][413]. Furthermore, they form pores on planar lipid bilayers [393,414,415], which favored the assumption of rather unspecific cell binding for several years. Nevertheless, just recently, LPS-induced TNF-α factor (LITAF) was determined as the main and its related protein CDIP1 as alternative receptor for Hbl, while a specific binding site for Nhe has still not been discovered [410].…”

Characterization of Bacillus cereus in Dairy Products in China

“…Therefore, the diversity and perfection of detection techniques of B. cereus and its virulence factors are necessary to improve the safety of dairy products in the future. Beyond that, the toxicity mechanisms of Bacillus virulence factors deserve to be illustrated, as host cells have frequently interacted with B. cereus infection [1,31,33,136,137]. Thus, comprehending the cytotoxicity of B. cereus is greatly beneficial in providing the therapeutic strategy for related illnesses caused by intaking Bacillus-contaminated dairy products.…”