Recombinant Bacillus cereus Enterotoxin, partial
Description
Introduction to Recombinant Bacillus cereus Enterotoxin, Partial
Recombinant Bacillus cereus Enterotoxin, partial (Product Code: CSB-EP302168BQJ) is a bioengineered protein fragment derived from the enterotoxin complex of B. cereus, a bacterium responsible for diarrheal food poisoning. This recombinant product represents a truncated version of the native toxin, engineered for research purposes to study toxin-subunit interactions, epitope mapping, or structural biology. The partial designation indicates that it represents a specific region of the full-length enterotoxin, enabling precise experimental focus on functional domains .
Epitope Mapping and Antibody Development
The partial enterotoxin serves as a tool for studying antigenic regions. For example:
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NheC C-Terminal Peptide: A synthetic peptide representing the C-terminal 15 amino acids of NheC (native toxin) was used to generate polyclonal antibodies for immunoassays .
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Recombinant Subunits: Partial fragments, like this product, enable targeted detection of specific toxin domains, aiding in the development of diagnostic assays .
Toxin-Subunit Interaction Studies
Native B. cereus enterotoxins (e.g., NheA, NheB, NheC) require precise stoichiometry and binding order (NheC → NheB → NheA) to form cytotoxic pores . The recombinant partial enterotoxin could be used to:
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Disrupt Assembly: By blocking critical binding sites, researchers can study how food components or inhibitors interfere with toxin function .
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Neutralization Assays: Antibodies against subunits (e.g., NheB) reduce cytotoxicity by 98% , highlighting the utility of recombinant fragments in toxin neutralization studies.
Comparative Analysis with Native Enterotoxins
Below is a comparison of the recombinant partial enterotoxin with native B. cereus enterotoxin components:
| Feature | Recombinant Partial Enterotoxin | Native Nhe Complex (NheA/B/C) | Native Hbl Complex (L1/L2/B) |
|---|---|---|---|
| Molecular Weight | 18.3 kDa | NheA: 41.0 kDa; NheB: 39.8 kDa; NheC: 36.5 kDa | L1: ~38 kDa; L2: ~35 kDa; B: ~38 kDa |
| Toxicity | Non-toxic (fragment) | Cytotoxic (pore-forming) | Hemolytic and cytotoxic |
| Genetic Organization | N/A (recombinant) | Operon (nheABC) | Operon (hblCDA) |
| Binding Order | N/A (fragment) | NheC → NheB → NheA | B → L2 → L1 |
| Key Applications | Epitope mapping, structural studies | Food poisoning, cytotoxicity assays | Hemolysis, pore formation studies |
Product Specs
Target Background
Q&A
What are the primary enterotoxins produced by Bacillus cereus implicated in foodborne illness?
B. cereus produces two main enterotoxin complexes associated with the diarrheal form of gastroenteritis: hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE). HBL consists of three components: a binding component B and two lytic components L1 and L2, working together to form a membrane-attacking complex. NHE is also a tripartite toxin with proteins of varying molecular weights. Additionally, B. cereus produces alveolysin, a pore-forming exotoxin that disrupts intestinal epithelial barriers, and cereulide, an emetic toxin with distinct pathogenic mechanisms .
How is the genetic organization of enterotoxin operons structured in B. cereus?
Northern blot analysis of B. cereus RNA revealed a large 5.1-kb transcript hybridizing with a 500-bp probe internal to the B-component coding sequence. This suggests that the hblA gene (encoding the B component) may be transcribed as part of a polycistronic message, potentially including the structural genes for the two lytic components (L1 and L2). This polycistronic organization likely facilitates coordinated expression of all components necessary for functional toxin assembly .
What expression vectors and host systems are most effective for recombinant production of B. cereus enterotoxin components?
Escherichia coli remains the predominant expression host for recombinant B. cereus toxin components. Research indicates successful expression using temperature-induced vectors such as pIET98 with E. coli BL21 strain, which employs a runaway replication mechanism. For the hblA gene, expression has been achieved in E. coli using the native B. cereus promoter. For Fnr protein expression, two different tagging approaches have been documented: C-terminal His-tagged and N-terminal Strep-tagged constructs, each produced in E. coli BL21 CodonPlus(DE3)-RIL under IPTG induction .
What are the optimal conditions for maximizing recombinant enterotoxin component yield?
Fed-batch cultivation using semi-synthetic high-cell density medium with exponentially increasing nutrient supply has been shown to achieve optimal yields. Cultivation parameters that significantly impact yield include:
The scalability of this approach has been demonstrated from 5L to 200L without loss of productivity .
How can researchers overcome challenges in maintaining proper folding and functionality of recombinant enterotoxin components?
The choice of tagging strategy significantly impacts protein folding and oligomerization state. Research shows that C-terminal His-tagged Fnr predominantly exists as monomers, while N-terminal Strep-tagged Fnr exists mainly as oligomers. The oligomeric state of apo-StrepFnr is dithiothreitol-sensitive, indicating the importance of disulfide bridges for oligomerization. Importantly, only monomeric forms of both recombinant apoFnr proteins bind to target DNA sequences, suggesting that maintaining the proper quaternary structure is critical for functionality .
What purification strategies yield the highest purity and functional recovery for recombinant enterotoxin components?
A systematic multi-step purification approach has been successfully employed for B. cereus recombinant proteins:
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Heat denaturation: Exploits the relative thermostability of target proteins
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Liquid-liquid extraction: Separates based on phase partitioning
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Gel filtration: Removes aggregates and separates by molecular size
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Anion-exchange chromatography: Final polishing step for homogeneity
This approach yields highly pure enzyme with approximately 65% recovery of activity. The integrity of purified recombinant proteins can be verified by molecular weight determination and N-terminal amino acid sequencing .
How can researchers verify that recombinant enterotoxin components retain native biological activity?
Functional verification of recombinant enterotoxin components requires demonstration of characteristic biological activities. For the HBL complex, combining recombinant B component with purified L components should produce a ring-shaped zone of hemolysis, which is the typical reaction of hemolysin BL. For DNA-binding proteins like Fnr, verification involves electrophoretic mobility shift assays (EMSAs) to confirm binding to specific promoter regions. Target promoters include those of enterotoxin regulators fnr, resDE, and plcR, as well as structural genes hbl and nhe .
How does oxygen tension modulate the expression of B. cereus enterotoxin genes at the molecular level?
B. cereus enterotoxin gene expression is regulated by oxygen availability through the transcription factor Fnr. Unlike B. subtilis Fnr, B. cereus Fnr exhibits the unusual property of remaining active under aerobic conditions. Under aerobiosis, apoFnr (without FeS cluster) binds as a monomer to specific sequences in promoter regions of both regulatory genes (fnr, resDE, plcR) and structural enterotoxin genes (hbl, nhe). The oligomeric state of apoFnr appears to be redox-regulated through disulfide bridge formation, potentially serving as a redox-sensing mechanism that fine-tunes enterotoxin expression in response to environmental oxygen levels .
What is the role of the two-component ResDE system in B. cereus virulence gene regulation?
The ResDE two-component regulatory system plays a critical role in B. cereus virulence gene expression. ResDE influences growth performance, glucose metabolism, and the expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe). Studies with resDE and resE mutants under various oxygenation conditions have revealed that this system mediates responses to changes in extracellular oxidoreduction potential. The ResDE system likely functions in concert with Fnr, as Fnr has been shown to bind to the resDE promoter region, suggesting hierarchical regulation of enterotoxin expression .
What is the structural basis for Fnr-mediated regulation of enterotoxin genes?
Analysis of Fnr binding sites in B. cereus enterotoxin promoters reveals a complex regulatory architecture:
| Promoter Region | Classification | Binding Site Locations | Potential Regulatory Mode |
|---|---|---|---|
| fnr | Class I activating | Upstream of transcription start | Activation |
| resDE, plcR | Class II | At or near -35 region | Activation/Repression |
| nhe, hbl | Class III | Multiple sites including -10 region and downstream of +1 | Complex activation/repression |
The presence of Crp/Fnr boxes both upstream and downstream of transcription start sites suggests an interplay of activation and repression mechanisms. Additionally, both hbl and nhe promoters have long untranslated regions (UTRs), suggesting potential post-transcriptional regulation mechanisms that may involve interaction between transcriptional regulators and ribosomal proteins .
What molecular mechanisms underlie the intestinal barrier disruption caused by B. cereus alveolysin?
Alveolysin disrupts intestinal epithelial barriers through a complex cellular mechanism:
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The toxin increases production of membrane-anchored protein CD59 and cilia- and flagella-associated protein 100 (CFAP100) in intestinal epithelial cells
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CFAP100 interacts with microtubules and promotes microtubule polymerization
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Elevated CFAP100 levels stabilize microtubules, leading to disorganization of the microtubule network
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This disorganization perturbs tight and adherens junctions between epithelial cells
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The disruption of cell junctions depends on CFAP100 upregulation, which in turn depends on CD59 and activation of PI3K-AKT signaling
This mechanism explains how alveolysin permeabilizes the intestinal epithelium in a manner consistent with intestinal symptoms and potentially facilitates bacterial escape from the intestine to cause systemic infections .
How do enterotoxin expression profiles differ among B. cereus strains from various sources?
Enterotoxin production varies considerably among B. cereus strains. Individual strains may produce hemolysin BL (HBL), nonhemolytic enterotoxin (NHE), or both toxin complexes. Some strains have the capability to produce both enterotoxins and emetic toxin, particularly those capable of starch digestion. In a Norwegian study of 85 B. cereus isolates from milk products, approximately 59% were found to produce enterotoxin, demonstrating substantial variation in virulence potential among environmental isolates .
What are the most sensitive and specific methods for detecting recombinant B. cereus enterotoxin components?
Multiple detection approaches have been developed for B. cereus enterotoxins, each with specific applications:
| Detection Method | Target Component | Commercial Kit | Sensitivity/Specificity Considerations |
|---|---|---|---|
| Immunological assay | L2 component of HBL | BCET-RPLA (Oxoid) | Detects specific protein component |
| Immunological assay | 45-kDa protein of NHE | BDE (Tecra) | Main protein detected from NHE complex |
| PCR | Gene sequences (hblA, etc.) | Various | Detects genetic potential but not expression |
| Bioassays | Functional toxicity | None commercial | Required for emetic toxin which has low antigenicity |
Notably, the emetic toxin (cereulide) has proven difficult to detect immunochemically due to its low antigenicity, necessitating the development of bioassays for its detection .
How can researchers differentiate between the contributions of different enterotoxin components in pathogenesis studies?
Molecular genetic approaches using homologous recombination have proven effective for studying the specific contributions of individual enterotoxin components. Recombinant plasmids can be introduced into B. cereus by electroporation, with homologous recombination resulting in single crossover events that generate specific mutants. Creating mutants deficient in specific enterotoxin components allows researchers to assess their individual contributions to virulence. Additionally, complementation studies using recombinant enterotoxin components can restore function in mutant strains, confirming the role of specific components .
What potential therapeutic targets have been identified based on B. cereus enterotoxin research?
Recent research has identified alveolysin and CFAP100 as potential therapeutic targets for preventing B. cereus-associated intestinal diseases and systemic infections. By targeting alveolysin directly or disrupting the CFAP100-dependent pathway, it may be possible to prevent intestinal barrier disruption and subsequent bacterial translocation into deeper tissues. This approach could be particularly valuable for preventing the progression from self-limiting gastrointestinal illness to more severe systemic infections, especially in immunocompromised patients .
What are the future directions for recombinant B. cereus enterotoxin research?
Future research directions should focus on several key areas:
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Elucidating the complex interplay between transcriptional activation and repression by Fnr and other regulators in enterotoxin expression
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Investigating the potential post-transcriptional regulation mechanisms involving the long UTRs of hbl and nhe transcripts
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Developing improved expression systems for difficult-to-express enterotoxin components
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Creating engineered toxoid variants for vaccine development
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Exploring the potential of enterotoxin components as targeted delivery vehicles for therapeutic agents
Additionally, further investigation of the mechanisms employed by B. cereus to optimize virulence gene expression in response to environmental oxygen tension changes, such as those encountered during infection in a human host, will provide valuable insights into pathogenesis .
