The Food Poisoning Toxins of Bacillus cereus

Abstract

Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease.

Keywords: Bacillus cereus; cereulide; cytotoxicity; cytotoxin K; food poisoning; hemolysin BL; non-hemolytic enterotoxin; pore formation.

Figure 4

Genetic organization of the operons encoding the tripartite enterotoxins of B. cereus. (A) The hblCDAB operon of strain F837/76, encoding the proteins Hbl L2, L1, B and B’ [198,199]. Upstream and downstream of hblB, a stem loop was identified [204]. The promoter region, as well as the transcriptional start TAA is shown [201]. 236 bp upstream of the promoter, a PlcR-box is located [202]. (B) The nhe operon of strain NVH 0075-95, encoding NheA, B and C [91,210]. Two PlcR-boxes as well as two possible transcriptional start sites exist [201,209,210]. Upstream and downstream of nheC, a stem loop was identified [207,210].

Figure 6

Properties of the CytK enterotoxin from B. cereus. (A) The cytK-1 and cytK-2 genetic regions according to Böhm et al. [210]. The promoter regions including (putative) transcriptional regulator binding sites are shown. (B) Putative mode of action of CytK during pore formation according to related β-barrel pore-forming toxins [419,423,424]. Step 1: Soluble monomers in solution. Step 2: Target membrane binding due to putative interaction with liposomes. Step 3: Oligomerization into heptamers. Step 4: Conformational changes and pore formation.

Figure 1

Structure of the depsipeptide toxin cereulide, the causative agent for the emetic type of B. cereus food-borne intoxications.

Figure 2

Genetic organization of the ces locus encoding the genetic determinants essential for non-ribosomal assembly of the cereulide toxin, located on the pX01-like mega-plasmid pCER270. For detailed description of the ces locus see text and [154,155,161,162]. Promoters are indicated by arrows. The main promoter P₁ (indicated in red) drives the polycistronic transcription of the ces operon. A hairpin indicates the terminator. Abbreviation: cesH, a hydrolase/esterase; cesP, a phosphopantetheinyl transferase; cesT, a type II thioesterase; cesA and cesB, structural cereulide synthetase genes; cesC and cesD, ABC transporter.

Figure 3

Toolbox for research and diagnostics of emetic B. cereus and the cereulide toxin. Several tools have been developed, which allow the identification of emetic B. cereus by means of molecular methods, such as conventional polymerase chain reaction (PCR) and real time PCR, mass spectrometry and spectroscopy, such as Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS) and artificial neural network assisted Fourier transform infrared spectroscopy (ANN-assisted FTIR). Furthermore, tools have been developed to monitor the ces-NRPS (Non-Ribosomal Peptide Synthetase) expression on a transcriptional and translational level, such as a lux-promotor transcription assay and an immunoassay targeting the cereulide synthetase. In addition, mass spectrometry methods for quantitation of the cereulide toxin and isocereulides and cell culture-based assays for cytotoxicity studies are available, such as stable isotope dilution assay liquid chromatography mass spectrometry (SIDA LC-MS) and Hep-2 cell culture assays. Details of the respective methods are provided in Section 3.4. Images for the composite figure are based on the following publications: [7,36,48,162,165,183,184,185].

Figure 5

Comparative depiction of the pore forming mechanism of the three-component enterotoxin complexes Hbl and Nhe. (A) Step 1: Complex formation in solution as well as presence of free Hbl and Nhe components [301,308,324,327,329,330]. (B) Step 2: Membrane attachment as Hbl B-L1 complexes [204,301], or as NheB-C complexes resulting in small, permeable “pro-pores” [327,330]. (C) Step 3: Attachment of Hbl L2 or NheA [301,324,329]. (D) Step 4: Conformational changes and completion of the full pore [301,324,329].