关键词: 蜡样芽孢杆菌, 致吐毒素cereulide, 生物合成, 非核糖体肽合成酶, 毒性

Abstract:

Bacillus cereus (B. cereus) is a gram-positive facultative anaerobe that can produce spores to survive adverse environments. And it is widely present in soil, water, air and a variety of foods. Pathogenic B. cereus is one of the most common food-borne pathogens, and the toxins produced by B. cereus are the main cause of food poisoning. Cereulide is a major toxin produced by pathogenic B. cereus, which is a small molecule lipophilic cyclic dodecadepsipeptide with stable structural properties. Cereulide can cause mild food poisoning with emetic symptoms, such as nausea and vomiting, and it may induce severe fatal diseases such as hepatic encephalopathy or acute liver failure. Current researches believed that cereulide caused vomiting by stimulating the vagus nerve, and induced the loss of mitochondrial membrane potential by acting as a potassium ionophore, which ultimately led to cell death. However, the toxic mechanism of hepatic encephalopathy or acute liver failure caused by cereulide remains unclear. Cereulide is encoded by the cereulide synthetase gene cluster (ces) and is synthesized by the non-ribosomal peptide synthetase (NRPS) system. Cereulide is composed by two hydroxy acids and two amino acid residues [-D-HIC-D-Ala-L-HIV-L-Val-], which forms a trimer phenolphthalein after three iterations and shows a structural specificity and representativeness. However, isocereulides may be produced due to the flexibility of the NRPS system. Therefore, the toxicity of cereulide is closely related to its biosynthesis process. Based on previous studies, this review summarized and proposed the biosynthesis mechanism of cereulide. Firstly, the CesA and CesB domained in ces recognize D-α-ketocarboxylic acid, L-alanine, L-α-ketoisovalerate and L-valine, respectively, which formed the main synthetic unit dipeptide of cereulide by covalent bonding. Secondly, a tetrapeptide was synthesized by repeating the above process. Thirdly, the second tetrapeptide was synthesized through repeated reactions, and the two tetrapeptides formed an octapeptide through esterification. Fourthly, the above reaction was repeated to form a ternary complex product peptide. Lastly, because the surface structure of the active center of the thioesterase domain in ces-NRPS prevented external water molecules from entering, it induced an internal nucleophilic attack reaction and finally released a circular cereulide. The risk of food poisoning caused by cereulide producing B. cereus was underestimated. In addition, our previous studies have found that some probiotic Bacillus products were contaminated with cereulide-producing B. cereus strains. This posed a potential risk to food safety and public health. This review briefly summarized the characteristics and toxic mechanisms of cereulide, which would provide a scientific basis for the prevention of cereulide. This review also summarized and proposed the biosynthesis process of cereulide. Functions of two domains in the synthesis process need to be focused. The main function of ketoreductase (KR) domain was that it could catalyze the formation of esters of keto acid at the beginning of the biosynthesis processes. The important role of thioesterase (TE) domain was to form repeating units and the cyclic peptide in the last link of synthesis. These could serve as a model for other cyclic peptides synthesized by the non-ribosomal peptide synthetase system.

Key words: Bacillus cereus, cereulide, biosynthesis, non-ribosomal peptide synthetase, toxicity