Shiga Like Toxin 1
Description
Structure and Functional Mechanism
Shiga-like toxin 1 consists of two structural subunits:
-
A subunit: A 293-amino acid enzymatic chain (32 kDa) responsible for ribosomal RNA cleavage via its RNA-glycohydrolase activity. It is cleaved by trypsin into A1 (catalytic domain) and A2 (anchoring peptide) linked by a disulfide bond .
-
B subunit pentamer: Five identical 69-amino acid monomers (7.7 kDa each) that bind the glycolipid receptor globotriaosylceramide (Gb3) on host endothelial cells .
The toxin follows retrograde trafficking from endosomes to the Golgi and endoplasmic reticulum, where the A1 fragment translocates into the cytosol to inhibit protein synthesis . Structural studies reveal three Gb3-binding sites per B monomer, enabling high-affinity interactions with host cells .
Subtypes and Genetic Variants
Stx1 is categorized into subtypes based on sequence divergence and functional differences:
Stx1 subtypes are encoded by lysogenic bacteriophages integrated into the E. coli genome, enabling horizontal gene transfer .
Clinical and Epidemiological Significance
-
Severity: Strains producing Stx1a are more frequently associated with bloody diarrhea and HUS compared to Stx1c . For example, 41% of patients infected with Stx1a-positive STEC developed bloody diarrhea, whereas no cases were reported for Stx1c .
-
Prevalence: Stx1c accounts for ~14% of human STEC isolates, often linked to non-O157 serotypes (e.g., O91, O113) .
-
Detection Challenges: Commercial reverse passive latex agglutination (RPLA) assays show reduced sensitivity for Stx1c due to antigenic divergence . Advanced methods like recombinase polymerase amplification (RPA) improve subtype-specific detection .
Comparative Toxicity with Stx2
While Stx1 and Stx2 share a similar mechanism, key differences include:
Diagnostic and Therapeutic Considerations
Product Specs
Purified by affinity chromatographic technique.
Q&A
Here’s a structured collection of FAQs tailored for academic researchers studying Shiga-like Toxin 1 (SLT-1), incorporating experimental design considerations, methodological guidance, and data-driven insights from peer-reviewed sources:
What experimental approaches reliably confirm SLT-1 presence in bacterial isolates?
Methodological Answer:
-
PCR amplification: Target the stx1 gene using primers specific to conserved regions (e.g., stx1A catalytic domain). Include controls for stx2 to avoid cross-reactivity .
-
Immunoassays: Use monoclonal antibodies (mAbs) against SLT-1 B-subunit (e.g., clone FabC11:Stx1) in ELISAs. Note: Some commercial assays show weak reactivity with SLT-1c variants, requiring validation with in-house antibodies .
-
Functional assays: Measure Vero cell cytotoxicity; compare with neutralization using SLT-1-specific mAbs to confirm toxin activity .
How do I design an in vitro model for SLT-1 cytotoxicity studies?
Methodological Answer:
-
Cell lines: Use Gb3 receptor-rich cells (e.g., Vero or HCT-8). Validate receptor expression via flow cytometry with anti-Gb3 antibodies .
-
Dose optimization: Titrate SLT-1 (0.1–10 ng/mL) and monitor apoptosis via caspase-3 activation at 24–72 hrs .
-
Controls: Include SLT-1 B-subunit-only treatments to isolate receptor-binding effects from catalytic A-subunit activity .
How can contradictory data on SLT-1 receptor specificity be resolved?
Experimental Design Framework:
-
Glycan microarray screening: Profile SLT-1 binding against 500+ glycans to identify non-Gb3 receptors (e.g., Gb4) .
-
Mutational analysis: Engineer SLT-1 B-subunit residues (e.g., Gln-64, Asp-17) implicated in Gb3 binding and compare binding affinity via SPR .
-
In vivo validation: Use transgenic mice expressing human Gb3 vs. wild-type to assess tissue-specific toxicity .
What strategies improve SLT-1 neutralization in preclinical models?
Data-Driven Solutions:
-
Bispecific antibodies: Combine mAbs targeting A-subunit (e.g., 1C10) and B-subunit (e.g., 2F10) for synergistic neutralization .
-
Recombinant decoy receptors: Express soluble Gb3 analogs (e.g., STARFISH) to competitively inhibit toxin binding .
-
Vaccine development: Test fusion proteins like Stx2B-Stx1B, which elicit cross-neutralizing antibodies in murine models (survival rates: 90% vs. 40% in controls) .
How do I address variability in SLT-1 production across bacterial strains?
Troubleshooting Guide:
What biophysical techniques characterize SLT-1 holotoxin stability?
Advanced Methodologies:
-
Crystallography: Resolve SLT-1 B-pentamer structure (PDB: 1R4Q) to identify Gb3-binding pockets .
-
Hydrogen-deuterium exchange MS: Map conformational changes in A-subunit during endosomal trafficking .
-
Surface plasmon resonance (SPR): Quantify binding kinetics (KD) between SLT-1 and Gb3 analogs (e.g., Pk trisaccharide) .
How are SLT-1/Stx2 hybrid toxins detected and characterized?
Integrated Workflow:
Product Science Overview
Shiga-like toxins (SLTs), also known as verotoxins, are a family of related protein toxins produced by certain strains of bacteria, notably Shigella dysenteriae and Shiga-toxin-producing Escherichia coli (STEC) such as E. coli O157:H7. These toxins are responsible for causing severe gastrointestinal diseases, including bloody diarrhea and hemorrhagic colitis, which can sometimes lead to fatal systemic complications .
Shiga-like toxins consist of two main components: the A subunit and the B subunit. The A subunit has RNA N-glycosidase activity, which inhibits eukaryotic protein synthesis, leading to cell death. The B subunit, on the other hand, forms a pentamer that binds to the functional cell-surface receptor globotriaosylceramide (Gb3). This binding is crucial for the toxin’s entry into the host cell .
The recombinant B subunit of Shiga-like Toxin-1 (SLT-1B) is a 7 kDa protein containing 69 amino acid residues. It is expressed in Escherichia coli and is used in various research applications due to its ability to bind specifically to Gb3 receptors. This specificity makes it a valuable tool for studying cell-specific vectorization, labeling, and imaging purposes .
The recombinant SLT-1B subunit has been utilized in several scientific studies and applications:
- Detection of Carbohydrate Ligands: The B subunit can be labeled with digoxigenin and used as a probe to detect carbohydrate ligands in immunochemical and flow cytometric applications. This allows for the measurement of carbohydrate binding activity in a simple and quantitative manner .
- Therapeutic and Vaccine Research: The specificity of SLT-1B for Gb3 receptors has been explored for its potential in therapeutic and vaccine research. By targeting Gb3-expressing cells, researchers aim to develop targeted therapies and vaccines that can neutralize the effects of Shiga-like toxins .
- Cell-Specific Vectorization: The ability of SLT-1B to bind specifically to Gb3 receptors overexpressed in tumor cells makes it a promising candidate for cell-specific vectorization. This can be used for targeted drug delivery and imaging in cancer research .
