Recombinant Listeria innocua serovar 6a Protein psiE homolog (psiE)

What is the Recombinant Listeria innocua serovar 6a Protein psiE homolog (psiE)?

Recombinant Listeria innocua serovar 6a Protein psiE homolog is a full-length protein (137 amino acids) expressed in E. coli systems, typically with an N-terminal His-tag for purification purposes . It belongs to a class of proteins found in Listeria species that may be involved in stress response pathways. The protein is encoded by the psiE gene (lin0828) in Listeria innocua strain CLIP 11262 . While its exact biological function remains under investigation, structural and sequence analysis suggests potential involvement in bacterial stress response mechanisms.

How does psiE in Listeria innocua compare to its homolog in pathogenic Listeria species?

The psiE protein in Listeria innocua serovar 6a shows remarkable sequence similarity to its homolog in pathogenic Listeria monocytogenes serotype 4b. Comparing their amino acid sequences reveals nearly identical composition with only one amino acid difference at position 135, where Listeria innocua has leucine (L) while Listeria monocytogenes has methionine (M) :

This high degree of conservation suggests important functional constraints on this protein across Listeria species, despite their differential pathogenicity . This makes L. innocua psiE an excellent model for studying conserved stress response mechanisms without the biosafety concerns associated with pathogenic L. monocytogenes.

What expression systems are recommended for producing recombinant psiE protein?

For recombinant production of Listeria innocua psiE protein, E. coli expression systems have been successfully employed . The methodological approach involves:

• Gene synthesis or PCR amplification of the psiE gene (lin0828)

• Cloning into an expression vector with an N-terminal His-tag

• Transformation into competent E. coli cells

• Induction of protein expression (typically using IPTG for T7 promoter-based systems)

• Cell lysis and protein extraction

• Purification using nickel affinity chromatography

For optimal expression, consider:

• Using BL21(DE3) or similar E. coli strains designed for recombinant protein expression

• Employing codon optimization if expression yields are low

• Testing various induction conditions (temperature, IPTG concentration, induction time)

• Including protease inhibitors during extraction to prevent degradation

The recombinant protein has been successfully produced with greater than 90% purity as determined by SDS-PAGE .

Purification:

The His-tagged psiE protein can be purified using standard immobilized metal affinity chromatography (IMAC) protocols. For optimal results:

• Use equilibrated Ni-NTA or similar resin

• Apply cleared lysate containing the His-tagged protein

• Wash with buffer containing low imidazole concentration (10-20 mM) to remove non-specific binding

• Elute with buffer containing higher imidazole concentration (250-500 mM)

• Perform buffer exchange to remove imidazole via dialysis or gel filtration

Storage:

For optimal stability, the following storage conditions are recommended:

• Short-term storage (up to one week): 4°C in appropriate buffer

• Long-term storage: -20°C/-80°C with 50% glycerol as cryoprotectant

• Storage buffer: Tris/PBS-based buffer, pH 8.0, with 6% trehalose

• Aliquot before freezing to avoid repeated freeze-thaw cycles, which can significantly reduce protein activity

• When reconstituting lyophilized protein, brief centrifugation prior to opening is recommended to bring contents to the bottom of the vial

The recommended reconstitution protocol involves using deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, followed by addition of glycerol to a final concentration of 50% for long-term storage .

What functional assays can be used to study psiE protein activity?

Based on its potential role in stress response mechanisms, several functional assays can be employed to investigate psiE protein activity:

• Acid Stress Response Assays:

  • Expose bacterial cultures with and without psiE to acidic conditions (pH 4.0-5.5)

  • Monitor survival rates at different time points

  • Compare growth kinetics under various acidic conditions

• Protein-Protein Interaction Studies:

  • Pull-down assays using His-tagged psiE as bait

  • Yeast two-hybrid screening to identify interaction partners

  • Co-immunoprecipitation with suspected partner proteins

• Expression Analysis:

  • qRT-PCR to monitor psiE expression under various stress conditions

  • Western blot analysis using antibodies against the recombinant protein

  • Reporter gene assays using psiE promoter regions

• Structural Analysis:

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to evaluate protein stability under different conditions

  • ATP binding assays if the protein contains the ATP-binding motif mentioned for other USPs

When designing these assays, consider including appropriate positive and negative controls, and validate results using multiple methodological approaches.

What is the relationship between psiE and stress response in Listeria species?

While the specific role of psiE in Listeria innocua has not been fully characterized, research on Universal Stress Proteins (USPs) in Listeria provides valuable context. USPs are upregulated under stress conditions, particularly acid stress, and play crucial roles in bacterial adaptation and survival .

In a study of Listeria innocua ATCC 33090, a USP was found to be upregulated not only during stationary phase but also during exponential growth phase under acid stress conditions . This USP was predicted to have a homo-tetrameric quaternary structure with each monomer showing a Rossmann-like α/β-fold architecture consisting of five parallel β-strands and four α-helices . The protein contained a conserved ATP-binding motif G-2X-G-9X-G(S/T-N), suggesting ATP might play a role in stabilizing the tetrameric assembly .

Although psiE is not explicitly identified as this USP in the research, its classification as a stress-response protein in Listeria suggests it may function in similar stress adaptation pathways. Further investigation is needed to determine whether psiE interacts with other stress response proteins or contributes to specific stress adaptation mechanisms in Listeria innocua.

How does psiE potentially contribute to Listeria persistence in food processing environments?

Listeria species, including L. innocua, demonstrate remarkable persistence in food processing environments (FPEs). While the specific contribution of psiE to this persistence has not been directly established, several stress survival mechanisms have been identified in Listeria that may involve proteins like psiE:

• Stress Survival Islets (SSIs): Listeria species possess genetic elements known as stress survival islets (SSI-1 and SSI-2) that confer advantages for growth and survival under suboptimal conditions such as low pH, alkaline pH, or oxidative stress . These islets are typically overrepresented among food isolates and implicated in the persistence of clonally related groups .

• Acid Adaptation: Listeria species can adapt to acidic environments, a trait particularly important in dairy processing facilities where acidification is common. Proteins involved in acid stress response, potentially including psiE, may contribute to this adaptation mechanism .

• Biofilm Formation: Persistent strains often demonstrate enhanced biofilm formation capabilities. Stress response proteins may play roles in regulating biofilm development under adverse conditions.

Research has shown that L. innocua strains isolated from dairy processing plants can persist despite cleaning procedures, with 17 L. innocua strains isolated from dairy processing facilities showing resistance to multiple antibiotics . While these studies don't specifically implicate psiE, they highlight the importance of understanding stress response proteins in addressing Listeria persistence in food environments.

What evolutionary relationships exist between psiE and similar proteins across Listeria species?

Phylogenetic analysis of stress response proteins in Listeria reveals interesting evolutionary relationships. USPs from Listeria species are distinct from USPs of other bacteria like Pseudomonas spp., Escherichia coli, and Salmonella spp., and cluster in a separate and heterogeneous class that includes USPs from both Listeria spp. and Lactobacillus spp. .

The high sequence similarity between psiE in L. innocua (Q92DI5) and L. monocytogenes (Q721Y1) suggests a recent common ancestor before the divergence of these species . Both proteins are 137 amino acids in length with only a single amino acid difference (position 135: L vs M), indicating strong evolutionary conservation.

This conservation is particularly interesting considering that L. innocua lacks many of the virulence factors present in L. monocytogenes:

• L. innocua is generally considered non-pathogenic, lacking the prfA-virulence gene cluster present in L. monocytogenes

• L. innocua comprises multiple subgroups, with atypical subgroup D serving as an evolutionary link between L. monocytogenes and L. innocua

• Some atypical hemolytic L. innocua strains carry pathogenic islands similar to L. monocytogenes (LIPI-1 and LIPI-3)

The high conservation of psiE across pathogenic and non-pathogenic Listeria species suggests it plays a fundamental role in bacterial physiology independent of virulence functions, possibly related to environmental stress adaptation .

How might psiE interact with ATP, and what experimental approaches can verify this interaction?

Based on research on Universal Stress Proteins (USPs) in Listeria, there is evidence for an ATP-binding motif G-2X-G-9X-G(S/T-N) that suggests ATP may play a role in protein function and/or stabilization of quaternary structure . If psiE contains similar motifs, it might interact with ATP in comparable ways.

To investigate potential ATP interactions with psiE, the following experimental approaches could be employed:

• ATP Binding Assays:

  • Fluorescence-based assays using fluorescent ATP analogs

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics

  • Surface plasmon resonance (SPR) to assess binding kinetics

• Structural Analysis:

  • X-ray crystallography of psiE with and without ATP

  • NMR studies to identify ATP binding sites

  • In silico molecular docking and molecular dynamics simulations

• Functional Impact:

  • ATPase activity assays to determine if psiE hydrolyzes ATP

  • Mutagenesis of predicted ATP-binding residues followed by functional assays

  • Crosslinking studies to assess if ATP affects protein oligomerization

• Comparative Analysis:

  • Alignment of psiE sequence with known ATP-binding USPs

  • Homology modeling based on USPs with resolved ATP-binding structures

  • Phylogenetic analysis of ATP-binding motifs across related proteins

A comprehensive approach combining multiple methods would provide the strongest evidence for ATP binding and its functional significance in psiE activity.

How does L. innocua psiE research inform our understanding of pathogenic Listeria species?

Although Listeria innocua is generally considered non-pathogenic, studying its proteins like psiE provides valuable insights into the biology of pathogenic Listeria species, particularly L. monocytogenes:

• Model System Advantages: L. innocua serves as a safer model organism for studying conserved Listeria proteins without the biosafety concerns associated with pathogenic species . The extremely high sequence similarity between L. innocua psiE and L. monocytogenes psiE (differing by only one amino acid) suggests functional conservation .

• Evolutionary Insights: Comparative studies between L. innocua and L. monocytogenes proteins reveal which genes/proteins are conserved across both pathogenic and non-pathogenic species (like psiE) versus those unique to pathogenic strains .

• Stress Response Mechanisms: Research on stress response proteins in L. innocua, including potential roles of psiE, can illuminate how Listeria species adapt to environmental stresses encountered during infection processes or in food production environments .

• Emerging Pathogenicity Concerns: Recent research has identified atypical hemolytic L. innocua strains carrying pathogenicity islands similar to L. monocytogenes (LIPI-1, LIPI-3), challenging the traditional view of L. innocua as universally non-pathogenic . A case report documented L. innocua meningoencephalitis in a three-year-old child, suggesting some strains may cause serious disease .

This research underscores the importance of studying conserved proteins like psiE across Listeria species to understand both common physiological mechanisms and the specific adaptations that contribute to pathogenicity.

What methodological approaches can differentiate the functional roles of psiE in different Listeria species?

To investigate potential functional differences in psiE between Listeria species, researchers can employ several comparative methodological approaches:

• Comparative Gene Expression Analysis:

  • RNA-Seq or qRT-PCR to compare psiE expression patterns in L. innocua versus L. monocytogenes under various stress conditions

  • Promoter analysis to identify differences in regulatory elements controlling psiE expression

  • Reporter gene assays using psiE promoters from different species

• Cross-Species Complementation Studies:

  • Gene knockout studies of psiE in both species

  • Complementation of knockout strains with psiE from the other species

  • Phenotypic comparison of wild-type, knockout, and complemented strains

• Protein-Protein Interaction Networks:

  • Interactome mapping using techniques like co-immunoprecipitation or bacterial two-hybrid systems

  • Comparative analysis of psiE interaction partners across species

  • Identification of species-specific versus conserved interactions

• Structural Biology Approaches:

  • Comparative structural analysis of psiE from different species

  • Investigation of how the L→M substitution at position 135 affects structure and function

  • Binding studies with potential ligands or interaction partners

• Stress Response Profiling:

  • Challenge experiments exposing both species to various stressors (acid, oxidative, osmotic)

  • Monitoring psiE expression and protein levels under these conditions

  • Correlation of psiE activity with stress tolerance phenotypes

These approaches would provide insights into whether psiE functions identically across Listeria species or has acquired species-specific roles related to pathogenicity or environmental adaptation.

What is the potential role of psiE in the recently documented cases of L. innocua pathogenicity?

Recent research has challenged the traditional view of Listeria innocua as non-pathogenic, with documented cases of human infection, including a severe case of meningoencephalitis in a three-year-old boy . While the specific involvement of psiE in these cases remains unexplored, several considerations merit investigation:

• Atypical Hemolytic Strains: Some L. innocua isolates have been found to carry pathogenic islands similar to L. monocytogenes (LIPI-1 and LIPI-3) and can invade human cells . These atypical strains might utilize stress response proteins like psiE differently than typical non-pathogenic strains.

• Stress Adaptation During Infection: If psiE functions in acid stress response as suggested for other USPs , it might contribute to survival in the acidic environment of the stomach or within phagolysosomes during infection.

• Host-Pathogen Interactions: The highly conserved nature of psiE across Listeria species suggests it may play a role in basic physiological processes encountered during host-pathogen interactions.

• Virulence Regulation: In L. monocytogenes, the expression of virulence factors is controlled by the PrfA regulator protein . Research has identified PrfA-dependent proteins in L. monocytogenes not found in L. innocua . While psiE hasn't been specifically identified as PrfA-regulated, investigating its potential regulation in the context of virulence factor expression networks could provide insights into atypical pathogenic L. innocua strains.

Experimental approaches to investigate these questions could include comparative genomic analysis of pathogenic versus non-pathogenic L. innocua strains, transcriptomic profiling during infection models, and protein interaction studies to identify potential virulence-associated partners of psiE.

What are common challenges in expressing and purifying recombinant psiE protein?

Researchers working with recombinant Listeria innocua psiE protein may encounter several challenges during expression and purification processes:

• Protein Solubility Issues:

  • Challenge: The amino acid sequence of psiE contains hydrophobic regions that may affect solubility

  • Solution: Optimize expression conditions (lower temperature, reduced IPTG concentration), use solubility-enhancing fusion tags (SUMO, MBP), or employ detergent-based extraction methods

• Protein Stability Concerns:

  • Challenge: Maintaining protein stability during purification and storage

  • Solution: Include glycerol (50%) and trehalose (6%) in storage buffers , avoid repeated freeze-thaw cycles, and store working aliquots at 4°C for up to one week

• Contaminating Proteins:

  • Challenge: Achieving high purity (>90%) for functional studies

  • Solution: Implement two-step purification protocols combining IMAC with size exclusion or ion exchange chromatography

• Protein Activity Verification:

  • Challenge: Confirming that recombinant psiE retains native functionality

  • Solution: Develop functional assays based on predicted stress response roles, such as ATP binding assays or acid stress protection experiments

• Batch-to-Batch Variability:

  • Challenge: Ensuring consistent protein quality across production batches

  • Solution: Standardize expression and purification protocols, implement quality control metrics (SDS-PAGE, activity assays), and prepare larger single batches for long-term studies

When troubleshooting expression issues, systematic optimization of expression parameters (host strain, media composition, induction conditions) is recommended, along with careful attention to buffer composition and pH during purification to maximize protein stability and functionality.

How can researchers verify the structural integrity of purified recombinant psiE?

Verifying the structural integrity of purified recombinant psiE is crucial for ensuring experimental reliability. Several complementary methods can be employed:

• Biophysical Characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure components

  • Thermal shift assays to evaluate protein stability and proper folding

  • Dynamic light scattering (DLS) to confirm homogeneity and detect aggregation

  • Limited proteolysis to verify compact, folded structure resistant to digestion

• Structural Analysis:

  • Small-angle X-ray scattering (SAXS) for low-resolution structural information

  • Nuclear magnetic resonance (NMR) for solution structure determination

  • X-ray crystallography for high-resolution structural analysis

  • Homology modeling compared with experimentally determined structures of similar USPs

• Functional Verification:

  • ATP binding assays if the protein contains ATP-binding motifs similar to other USPs

  • Oligomerization state analysis (size exclusion chromatography, analytical ultracentrifugation)

  • Thermal stability comparisons under different conditions (pH, salt concentration)

• Computational Analysis:

  • Molecular dynamics simulations to assess structural stability

  • In silico prediction of disorder regions and comparison with experimental data

  • Analysis of surface electrostatics to identify potential interaction interfaces

For recombinant psiE specifically, comparison with the predicted homo-tetrameric quaternary structure described for Listeria USPs (with Rossmann-like α/β-fold containing five parallel β-strands and four α-helices) would provide validation of proper folding .