LLO PEST free
Description
Mechanism of Action
The PEST-like sequence in wild-type LLO prevents cytotoxicity by:
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Promoting endocytosis: Interaction with Ap2a2 mediates removal of LLO from the plasma membrane .
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Limiting membrane damage: Restricts pore-forming activity to phagosomes (pH 5.5) rather than the cytosol (pH 7.0) .
In LLO PEST-free, deletion of this sequence results in:
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Accumulation on host membranes: Increased association with the plasma membrane, leading to cell detachment and death .
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Uncontrolled pore formation: Sustained cytolytic activity across pH ranges, causing rapid host cell lysis .
3.1. Virulence Studies
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Mouse models: LLO PEST-free strains exhibit 10,000-fold reduced virulence compared to wild-type L. monocytogenes due to excessive host cell death .
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Intracellular replication: Loss of the PEST sequence prevents bacterial growth in the cytosol, as gentamicin enters lysed cells and kills bacteria .
3.2. Functional Complementation
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The PEST-like sequence from human GPCRs (e.g., calcium-sensing receptor) can partially restore intracellular replication when engineered into LLO PEST-free, confirming the modular role of PEST motifs in toxin regulation .
3.3. ERK1/2 Signaling Activation
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LLO PEST-free retains the ability to activate ERK1/2 phosphorylation in Caco-2 cells, demonstrating that pore-forming activity—not the PEST sequence—drives this immune response .
Implications for Pathogenesis
The PEST sequence is critical for L. monocytogenes to balance virulence and cytotoxicity:
Product Specs
Q&A
Basic Research Questions
What experimental models are optimal for studying LLO PEST-free mutants in intracellular pathogenesis?
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Primary models:
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Key parameters:
How does the PEST-like sequence influence LLO stability in host cells?
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Mechanism: The PEST-like sequence targets LLO for AP-2-mediated endocytosis, limiting its accumulation on the plasma membrane ( ).
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Methodological validation:
Advanced Research Questions
How do contradictory findings about the PEST sequence’s role in proteasomal degradation reconcile?
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Resolution: Discrepancies arise from genetic copy number (plasmid vs. chromosomal) and assay sensitivity. Prioritize chromosomal mutants for physiological relevance ( ).
What advanced techniques elucidate AP-2’s role in mitigating LLO cytotoxicity?
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Yeast two-hybrid (Y2H) screening: Identified Ap2a2 as a binding partner of the LLO PEST motif ( ).
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Functional complementation:
How can researchers design LLO PEST-free mutants to study host-pathogen adaptation?
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Strain construction:
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Phenotypic validation:
Methodological Best Practices
What controls are critical when analyzing LLO PEST-free mutants?
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Essential controls:
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Data normalization:
How to resolve conflicting results in PEST sequence studies?
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Troubleshooting steps:
Key Research Gaps and Future Directions
Product Science Overview
Listeriolysin O (LLO) is a cholesterol-dependent cytolysin produced by the pathogenic bacterium Listeria monocytogenes. This protein plays a crucial role in the pathogenicity of L. monocytogenes, enabling the bacterium to escape from the phagosomal compartment of host cells and proliferate within the cytoplasm . The PEST sequence, a proline, glutamic acid, serine, and threonine-rich region, is known to regulate the cytotoxicity of LLO. The PEST-free recombinant version of LLO is engineered to lack this sequence, thereby altering its regulatory properties.
Listeriolysin O is synthesized as a precursor protein composed of 529 amino acid residues. It possesses a typical signal sequence at its N-terminus, which is cleaved to produce the mature protein . The mature LLO is secreted as a monomer and functions by forming pores in the host cell membrane, facilitating the escape of L. monocytogenes from the phagosome into the cytoplasm .
The recombinant LLO, including the PEST-free variant, is typically produced by expressing the hlyA gene (encoding LLO) in Escherichia coli. The purification process involves a one-step method that yields a significant amount of functional LLO. This method ensures the stability and activity of the protein, which can be retained at 4°C for over a year . The recombinant LLO can be tagged with either an N-terminus or C-terminus His tag to facilitate purification .
The synthetic route for producing recombinant LLO involves cloning the hlyA gene into an expression vector suitable for E. coli. The expression of the gene is induced, and the protein is subsequently purified using affinity chromatography. The PEST-free variant is engineered by deleting the PEST sequence from the hlyA gene before cloning .
Listeriolysin O’s pore-forming activity is essential for its function in facilitating the escape of L. monocytogenes from the phagosome. The protein interacts with cholesterol in the host cell membrane, leading to pore formation. This activity is crucial for the bacterium’s intracellular survival and proliferation . The absence of the PEST sequence in the recombinant variant does not significantly affect its pore-forming ability but may alter its regulatory properties .
The recombinant LLO, particularly the PEST-free variant, has several applications in research and biotechnology. It is used as a tool to study the intracellular lifecycle of L. monocytogenes and the host-pathogen interactions. Additionally, it has potential applications in vaccine development and as an adjuvant in immunotherapy .
