Recombinant Yersinia pseudotuberculosis serotype O:3 Protein AaeX (aaeX)

What is the genomic characterization of the aaeX gene in Yersinia pseudotuberculosis serotype O:3?

The aaeX gene in Y. pseudotuberculosis is a conserved sequence located on the chromosome that encodes a small membrane protein. While specific data for serotype O:3 is limited in current literature, multilocus sequence typing (MLST) approaches have been instrumental in characterizing the genetic structure of Y. pseudotuberculosis strains. MLST analysis typically involves the sequencing of seven housekeeping genes: mdh, recA, sucA, fumC, aroC, pgi, and gyrB . For proper genomic characterization of aaeX in serotype O:3 specifically, researchers should:

• Extract bacterial DNA using QIAamp DNA kits or similar protocols with minor modifications for optimal yield

• Design primers targeting the aaeX gene and flanking regions

• Perform PCR amplification using high-fidelity polymerase

• Sequence the amplified products and analyze using bioinformatics tools

• Compare sequences with reference genomes using BLAST or similar tools

For Y. pseudotuberculosis characterization, DNA extraction should follow established protocols involving homogenization of samples followed by incubation and supernatant processing .

How does the function of AaeX protein in Y. pseudotuberculosis serotype O:3 compare to other Yersinia species?

The AaeX protein shows functional conservation across Yersinia species, though with species-specific variations that reflect evolutionary adaptations. In Y. enterocolitica, AaeX functions as a membrane protein potentially involved in environmental adaptation mechanisms . For Y. pseudotuberculosis serotype O:3, researchers should consider:

• Comparative sequence analysis with homologs from Y. enterocolitica strain 8081 AaeX protein

• Structural prediction using bioinformatics tools to identify conserved domains

• Expression analysis under various environmental conditions to determine regulation patterns

• Knockout studies to assess phenotypic changes

• Complementation experiments to confirm functional roles

The pathogenicity of Y. pseudotuberculosis is determined by several virulence factors, including the virulence plasmid pYV, the high-pathogenicity island (HPI), and the Y. pseudotuberculosis-derived mitogen (YPM) . Understanding how AaeX interacts with these established virulence determinants provides context for its functional significance.

What are the optimal conditions for recombinant expression of Y. pseudotuberculosis serotype O:3 AaeX protein?

For optimal recombinant expression of Y. pseudotuberculosis serotype O:3 AaeX protein, researchers should consider the following parameters:

When designing expression constructs, consider:

• Codon optimization for the host organism

• Addition of purification tags (His6, GST, etc.) at N or C terminus

• Inclusion of appropriate signal sequences if membrane localization is desired

• Selection of vector with suitable promoter strength

• Optimization of solubilization conditions if AaeX forms inclusion bodies

For purification, a staged approach involving initial capture by affinity chromatography followed by polishing steps (ion exchange, size exclusion) typically yields the purest protein preparations .

How does the expression of AaeX protein impact the virulence mechanisms of engineered Y. pseudotuberculosis vaccine strains?

The impact of AaeX protein expression on virulence mechanisms in engineered Y. pseudotuberculosis vaccine strains represents a complex interplay of factors. When evaluating this relationship, researchers should:

• Establish baseline virulence using wild-type strains versus modified strains with controlled AaeX expression

• Quantify interactions between AaeX and known virulence factors (particularly those in the Type III Secretion System)

• Evaluate translocation efficiency of effector proteins in different AaeX expression backgrounds

• Assess immune response profiles with varied AaeX levels

In recombinant attenuated Y. pseudotuberculosis vaccine development, strains like χ10069 (with Δ yopK Δ yopJ Δ asd triple mutations) have demonstrated effectiveness in delivering Y. pestis fusion proteins as protective antigens . This approach induces potent humoral and cell-mediated immune responses, leading to protection against both subcutaneous and intranasal challenges with virulent Y. pestis and oral challenge with Y. enterocolitica WA and Y. pseudotuberculosis PB1+ .

The modulatory effects of AaeX expression should be evaluated within this context of attenuated strain development. Researchers should particularly assess how AaeX expression affects:

• Colonization efficiency in mucosal tissues

• Retention time in lymphoid tissues

• Stimulation of dendritic cells and T-cell responses

• Balance between attenuation and immunogenicity

What methodologies are most effective for analyzing AaeX protein interactions with host immune components?

To effectively analyze AaeX protein interactions with host immune components, researchers should employ multiple complementary approaches:

In vitro methods:

• Surface Plasmon Resonance (SPR) - For quantitative binding kinetics between purified AaeX and immune molecules

• Co-immunoprecipitation - To identify protein-protein interactions in cell lysates

• ELISA-based assays - For screening potential binding partners

• Flow cytometry - To detect binding to immune cell surfaces

Ex vivo methods:

• Immune cell stimulation assays with:

  • Dendritic cells - Measure activation markers (CD80/86, MHC-II, cytokine production)

  • Macrophages - Assess phagocytosis efficiency, cytokine profiles

  • T cells - Evaluate proliferation, cytokine production

• Confocal microscopy to visualize:

  • Protein localization

  • Co-localization with immune receptors

  • Internalization dynamics

In vivo methods:

• Transgenic mouse models expressing tagged versions of AaeX

• Adoptive transfer experiments with labeled immune cells

• Cytokine/chemokine profiling in tissues following exposure

When assessing immune responses to recombinant Y. pseudotuberculosis strains, researchers have demonstrated that vaccination with attenuated strains delivering Y. pestis antigens can induce significant protection against challenge with virulent strains . This suggests that AaeX may play a role in modulating host immune interactions, which should be systematically investigated using the methodologies outlined above.

What are the challenges in resolving crystal structures of membrane-associated proteins like AaeX, and what alternative structural biology approaches can be employed?

Resolving crystal structures of membrane-associated proteins like AaeX presents several significant challenges:

• Expression and purification obstacles:

  • Low natural expression levels necessitate optimization of recombinant systems

  • Hydrophobic regions cause aggregation and misfolding

  • Detergent selection critically impacts structural integrity

• Crystallization barriers:

  • Detergent micelles create heterogeneous samples

  • Limited polar surfaces reduce crystal contact points

  • Conformational flexibility hinders crystal packing

Alternative structural biology approaches include:

For AaeX specifically, a hybrid approach is recommended:

What serotyping methods are most reliable for confirming Y. pseudotuberculosis serotype O:3 identification?

For reliable serotyping of Y. pseudotuberculosis serotype O:3, researchers should employ a multi-method approach:

• Slide agglutination with specific antisera:

  • Use commercial Y. pseudotuberculosis O:1–O:6 antisera (such as those from Denka Seiken)

  • Perform tests on clean cultures grown under standardized conditions

  • Include positive and negative controls to ensure specificity

• O-genotyping using multiplex PCR:

  • Apply the Bogdanovich et al. method for molecular confirmation

  • Design primers targeting serotype-specific regions of the O-antigen gene cluster

  • Run alongside reference strains representing different serotypes

• Whole genome sequencing approach:

  • Sequence the entire O-antigen gene cluster

  • Analyze wzz gene variations, which are crucial for O-antigen chain length determination

  • Compare with reference database sequences for conclusive identification

• Mass spectrometry:

  • Analyze lipopolysaccharide profiles using MALDI-TOF

  • Compare spectral patterns with reference serotypes

  • Use for rapid screening before confirmation with other methods

For isolation prior to serotyping, selective media should be used:

• CIN agar showing "bull's-eye" colonies with transparent areas

• CHROMagar yielding mauve colonies

• Followed by incubation in PBS with heat treatment (10 min at 100°C)

• Centrifugation to obtain supernatant for PCR confirmation

The combined approach provides the most reliable serotyping, as molecular methods complement traditional serological approaches, reducing potential cross-reactivity issues.

How can researchers optimize DNA extraction protocols specifically for Y. pseudotuberculosis serotype O:3 genomic analysis?

Optimizing DNA extraction protocols for Y. pseudotuberculosis serotype O:3 requires attention to several critical factors:

• Sample preparation optimization:

  • Homogenize samples thoroughly in Lab-Blender 80 or equivalent until complete homogeneity

  • For environmental or clinical samples, employ selective enrichment at 4°C for 14 days prior to extraction

  • Use 200 μL of supernatant as template for subsequent PCR applications

• Extraction method refinements:

  • Utilize QIAamp DNA Blood Mini Kit with specific modifications for Y. pseudotuberculosis

  • Increase lysis time to account for robust cell wall characteristics

  • Adjust elution volume based on expected DNA concentration

  • Consider multiple elution steps to improve yield

• Quality control parameters:

  • Measure DNA concentration using NanoDrop spectrophotometry (aim for 150-200 ng for optimal PCR results)

  • Assess A260/A280 ratio (1.8-2.0 indicates high purity)

  • Verify integrity using gel electrophoresis (should show minimal degradation)

  • Include extraction controls to monitor potential contamination

• PCR inhibitor management:

  • Add additional washing steps if samples contain PCR inhibitors

  • Consider using BSA (bovine serum albumin) or specialized PCR enhancers

  • Perform dilution series to identify optimal template concentration

For subsequent gene detection, the TaqMan rt-PCR assay has been successfully employed for Yersinia detection, with specific primers and probes targeting the ail gene and wzz gene to distinguish Y. pseudotuberculosis serotypes . Researchers should always validate extraction methods by:

• Testing recovery efficiency with spiked samples

• Comparing multiple extraction methods on identical samples

• Assessing reproducibility across technical replicates

What are the key considerations when designing immunization protocols using recombinant Y. pseudotuberculosis-based vaccines?

When designing immunization protocols using recombinant Y. pseudotuberculosis-based vaccines, researchers must address several critical considerations:

• Attenuation strategy selection:

  • Triple mutations (Δ yopK Δ yopJ Δ asd) have demonstrated effective attenuation while maintaining immunogenicity

  • Balance safety profile with adequate colonization for immune stimulation

  • Consider auxotrophic mutations (like Δ asd) for biological containment

• Antigen delivery design:

  • The Type III Secretion System (T3SS) provides efficient antigen delivery

  • YopE amino acid 1 to 138 fusion constructs show effective translocation

  • Plasmid stability must be maintained without antibiotic selection in vivo

• Administration route optimization:

  • Oral administration mimics natural infection route and stimulates mucosal immunity

  • Intranasal administration targets respiratory immunity

  • Dose titration studies should determine minimal effective dose

• Immunization schedule parameters:

  Parameter	Recommendation	Rationale
  Priming dose	10⁹-10¹⁰ CFU	Establishes initial immune response
  Booster timing	21-28 days after prime	Allows memory cell development
  Booster dose	Equal to or less than prime	Prevents excessive reactogenicity
  Route consistency	Same as prime	Targets same immune compartments
  Adjuvant need	Generally unnecessary	Bacterial components provide adjuvant effect

• Immune response monitoring:

  • Humoral immunity: Measure serum IgG and mucosal IgA antibodies

  • Cell-mediated immunity: Assess T-cell proliferation and cytokine production

  • Challenge studies: Evaluate protection against different routes of infection

• Safety assessment protocols:

  • Monitor bacterial shedding and persistence

  • Evaluate potential for reversion to virulence

  • Assess reactogenicity and adverse events

The recombinant attenuated Y. pseudotuberculosis PB1+ strain (χ10069) with Δ yopK Δ yopJ Δ asd triple mutations has demonstrated effectiveness in delivering Y. pestis fusion proteins as protective antigens . Mice vaccinated orally with this strain show potent humoral and cell-mediated immune responses, providing effective protection against both subcutaneous and intranasal challenges with virulent Y. pestis and oral challenge with related Yersinia species .

How should researchers analyze evolutionary relationships between Y. pseudotuberculosis serotype O:3 AaeX and homologous proteins in other pathogens?

To analyze evolutionary relationships between Y. pseudotuberculosis serotype O:3 AaeX and homologous proteins in other pathogens, researchers should implement a systematic approach:

• Sequence acquisition and preparation:

  • Retrieve AaeX sequences from Y. pseudotuberculosis serotype O:3 and related species

  • Include homologs from other Yersinia species and more distant Enterobacteriaceae

  • Ensure proper annotation and sequence validation

  • Create multiple sequence alignments using MUSCLE, MAFFT, or similar algorithms

• Phylogenetic analysis workflow:

  • Select appropriate evolutionary models (JTT, WAG, or LG for proteins)

  • Construct trees using multiple methods:

    • Maximum Likelihood (RAxML, PhyML)

    • Bayesian Inference (MrBayes, BEAST)

    • Distance-based methods (Neighbor-Joining)

  • Implement bootstrap analysis (>1000 replicates) or posterior probability assessment

  • Root trees using distant homologs as outgroups

• Molecular evolution analysis:

  • Calculate dN/dS ratios to identify selection pressure

  • Apply branch-site models to detect episodic selection

  • Identify conserved domains indicating functional importance

  • Map sequence variations to structural models when available

• Contextual analysis:

  • Correlate evolutionary patterns with pathogenicity differences

  • Examine gene synteny and genomic context across species

  • Investigate horizontal gene transfer evidence

  • Consider ecological niches and host interactions

When analyzing AaeX specifically, researchers should place findings within this broader evolutionary context, noting whether AaeX evolution follows similar patterns to housekeeping genes or shows evidence of different selective pressures.

What statistical approaches are most appropriate for analyzing immune response data following vaccination with Y. pseudotuberculosis-based vaccines?

When analyzing immune response data following vaccination with Y. pseudotuberculosis-based vaccines, researchers should select statistical approaches appropriate for the experimental design and data characteristics:

• For comparing antibody titers or cell counts between groups:

  • For normally distributed data: Student's t-test (two groups) or ANOVA with post-hoc tests (multiple groups)

  • For non-normally distributed data: Mann-Whitney U test (two groups) or Kruskal-Wallis with Dunn's post-hoc test (multiple groups)

  • For repeated measures: Paired t-test or repeated measures ANOVA with appropriate post-hoc tests

• For survival analysis following challenge:

  • Kaplan-Meier survival curves for visualization

  • Log-rank (Mantel-Cox) test for comparing survival between groups

  • Cox proportional hazards regression for adjusting for covariates

• For correlating immune parameters with protection:

  • Pearson or Spearman correlation coefficients based on data distribution

  • Logistic regression to identify predictors of protection

  • Receiver Operating Characteristic (ROC) analysis to determine predictive thresholds

• For multivariate analysis of complex immune responses:

  • Principal Component Analysis (PCA) to identify major sources of variation

  • Hierarchical clustering to identify patterns of immune response

  • Partial Least Squares Discriminant Analysis (PLS-DA) to identify components most associated with protection

• For longitudinal data analysis:

  • Linear mixed-effects models to account for within-subject correlation

  • Generalized Estimating Equations (GEE) for population-average effects

  • Area Under the Curve (AUC) analysis for cumulative responses

When reporting results, include:

• Effect sizes with confidence intervals, not just p-values

• Clear statements about multiple testing correction methods

• Appropriate visualizations (box plots, scatter plots with regression lines)

• Sample size justification and power calculations

In studies with Y. pseudotuberculosis vaccines, strain χ10069(pYA5199) demonstrated potent humoral and cell-mediated immune responses that provided effective protection against both subcutaneous and intranasal challenges with virulent pathogens . Such protection data should be analyzed with survival analysis methods, while the underlying immune correlates should be examined with the appropriate parametric or non-parametric tests based on data distribution characteristics.

How can researchers effectively analyze and interpret proteomics data to understand AaeX protein interactions within bacterial systems?

Effective analysis and interpretation of proteomics data for understanding AaeX protein interactions within bacterial systems requires a comprehensive workflow:

• Sample preparation optimization:

  • Employ differential extraction techniques to isolate membrane fractions

  • Use gentle detergents for solubilization while preserving protein-protein interactions

  • Consider crosslinking approaches to capture transient interactions

  • Include appropriate controls (wild-type, knockout, overexpression)

• Mass spectrometry approach selection:

  • For global interactome: Affinity purification-mass spectrometry (AP-MS)

  • For specific interactions: Targeted approaches like selected reaction monitoring (SRM)

  • For structural information: Hydrogen-deuterium exchange MS or crosslinking MS

  • For post-translational modifications: Enrichment strategies followed by MS/MS

• Data analysis pipeline components:

  • Protein identification: Database searching using MASCOT, SEQUEST, or X!Tandem

  • Quantification: Label-free, SILAC, TMT, or other quantitative approaches

  • Interaction scoring: Use tools like SAINT, CompPASS, or MiST to filter true interactions

  • Network analysis: Cytoscape or STRING for visualization and functional enrichment

• Statistical framework for interaction validation:

  Analysis Step	Recommended Approach	Output Interpretation
  Identification FDR	Target-decoy approach	<1% FDR at protein level
  Enrichment calculation	Fold change vs. controls	>2-fold enrichment considered significant
  Significance testing	Moderated t-tests with multiple testing correction	Adjusted p-value <0.05
  Reproducibility assessment	Correlation between biological replicates	Pearson r >0.7 indicates good reproducibility
  Network significance	Permutation testing of network properties	Empirical p-value <0.05

• Biological context integration:

  • Map interactions to known pathways using KEGG, Reactome, or similar databases

  • Correlate with transcriptomic data when available

  • Consider subcellular localization information

  • Integrate with phenotypic data from mutant studies

• Validation strategy:

  • Confirm key interactions with orthogonal methods (co-IP, FRET, BiFC)

  • Perform targeted gene knockouts of interaction partners

  • Use site-directed mutagenesis to identify critical interaction interfaces

  • Apply complementation assays to verify functional significance

For Y. pseudotuberculosis specifically, researchers should focus on interactions between AaeX and known virulence factors, including components of the virulence plasmid pYV, the high-pathogenicity island (HPI), and the Y. pseudotuberculosis-derived mitogen (YPM) . Understanding these interactions will provide insight into how AaeX contributes to pathogenicity and potential vaccine development applications.

What are the future research directions for Y. pseudotuberculosis serotype O:3 AaeX protein in vaccine development?

Future research directions for Y. pseudotuberculosis serotype O:3 AaeX protein in vaccine development should focus on several promising avenues:

• Structure-function relationship elucidation:

  • Determine high-resolution structures of AaeX using advanced structural biology approaches

  • Map immunogenic epitopes that could serve as subunit vaccine candidates

  • Identify structural motifs that contribute to immunomodulatory properties

• Genetic optimization strategies:

  • Engineer AaeX fusion constructs with enhanced immunogenicity

  • Develop regulated expression systems for optimal in vivo delivery

  • Create attenuated strains with targeted modifications in AaeX and related pathways

• Delivery system innovations:

  • Optimize oral and intranasal delivery formulations for mucosal immunity

  • Develop novel adjuvant combinations specifically synergistic with AaeX-based antigens

  • Explore prime-boost strategies combining different delivery platforms

• Broader protection assessment:

  • Evaluate cross-protection against diverse Yersinia species and serotypes

  • Test protection in multiple animal models beyond mice

  • Assess long-term immunity and memory response durability

• Translational research priorities:

  • Scale-up production under GMP conditions

  • Develop stability-indicating assays and formulation optimization

  • Design early-phase clinical trial protocols with appropriate endpoints

Building upon the success of recombinant attenuated Y. pseudotuberculosis strains like χ10069 with Δ yopK Δ yopJ Δ asd triple mutations , researchers should particularly focus on how AaeX can be integrated into existing vaccine platforms to enhance immunogenicity while maintaining safety profiles. The demonstrated protection against both subcutaneous and intranasal challenges with virulent Y. pestis and oral challenge with Y. enterocolitica WA and Y. pseudotuberculosis PB1+ provides a promising foundation for further vaccine development.