Recombinant Salmonella paratyphi C Protein AaeX (aaeX)

What is the structure and function of Recombinant Salmonella paratyphi C Protein AaeX?

Recombinant Salmonella paratyphi C Protein AaeX is a 67-amino acid protein derived from Salmonella paratyphi C strain RKS4594 with UniProt identifier C0PZR1 . The amino acid sequence (MSLFPVIVVFGLSFPPIIFFELLSLAIFWLVRRMVPTGIYDFVWHPALFNTALYCCLFYLISRLFV) contains multiple hydrophobic regions, suggesting it likely functions as a membrane-associated protein .

Functional analysis typically requires:

• Expression of the full-length protein (residues 1-67)

• Structural characterization through circular dichroism or crystallography

• Localization studies using fluorescently-tagged constructs

• Protein-protein interaction assays to identify binding partners

• Comparative analysis with homologous proteins from other Salmonella serovars

What are the optimal storage and stability conditions for this recombinant protein?

For maximum stability and activity retention, Recombinant Salmonella paratyphi C Protein AaeX should be stored according to the following protocol:

• Store stock solution at -20°C for routine use

• For extended storage periods, maintain at -20°C or preferably -80°C to prevent degradation

• Working aliquots can be stored at 4°C for up to one week only

• Avoid repeated freeze-thaw cycles which significantly compromise protein integrity

• Use a stabilizing buffer containing Tris-base and 50% glycerol optimized specifically for this protein

To monitor stability over time, researchers should periodically verify protein activity and integrity through functional assays and SDS-PAGE analysis.

How does the expression system affect recombinant AaeX production quality?

Expression system selection significantly impacts recombinant AaeX production. While specific expression data for AaeX is limited, research with similar Salmonella recombinant proteins suggests several considerations:

• Prokaryotic systems (E. coli) offer high yield but may require optimization for membrane-associated proteins

• Tag selection should be determined during the production process based on protein-specific characteristics

• Expression temperature, induction conditions, and media composition require optimization

• For functional studies, proper folding verification is essential through activity assays

• Purification strategy should account for the protein's hydrophobic nature, potentially requiring detergents

How can Recombinant Salmonella paratyphi C Protein AaeX be evaluated as a potential vaccine candidate?

Evaluation of AaeX as a vaccine candidate would follow similar protocols to those used for other Salmonella recombinant proteins like SpaO and H1a:

• Immunogenicity assessment:

  • Measure antibody responses in animal models

  • Evaluate T-cell activation and cytokine profiles

  • Determine optimal dosing and adjuvant formulations

• Protection studies:

  • Single-antigen immunization (comparable to the 41.7-66.7% protection observed with rSpaO or rH1a alone)

  • Multi-antigen formulations (comparable to the 75.0-91.7% protection with combined immunization)

  • Challenge with virulent strains to assess protective efficacy

• Distribution and conservation analysis:

  • Evaluate gene presence across clinical isolates (similar to the 97.5-100% distribution observed for spaO and h1a)

  • Sequence conservation assessment (comparable to the 99.31-99.88% similarity found in other Salmonella antigens)

  • Expression frequency verification in clinical samples

What host-pathogen interaction models are suitable for studying AaeX function?

Caenorhabditis elegans represents a valuable model organism for studying Salmonella pathogenesis and could be adapted for AaeX-specific studies . The following experimental approaches would be appropriate:

• Comparative infection studies:

  • Wild-type vs. AaeX-deficient Salmonella strains

  • Lifespan and thermotolerance assays (as S. paratyphi A reduced C. elegans lifespan and impaired thermotolerance)

  • Behavioral assessments (movement, pharyngeal pumping, food preference)

  • Reproductive capacity measurements

• Pathway analysis:

  • Genetic analysis using C. elegans mutants in MAPK and insulin signaling pathways (tir-1, nsy-1, sek-1, pmk-1, mpk-1, skn-1, daf-2, daf-16)

  • Oxidative stress evaluation through hydrogen peroxide assays

  • Expression analysis of stress-response genes via quantitative RT-PCR

• Verification in mammalian systems:

  • Cellular invasion assays with epithelial cell lines

  • Macrophage survival studies

  • Cytokine response profiles

How can researchers distinguish between the roles of AaeX and other Salmonella virulence factors?

To isolate AaeX-specific effects from other Salmonella virulence factors:

• Generate targeted deletion mutants:

  • Create AaeX knockout strains while preserving other virulence factors

  • Complementation with wild-type aaeX to confirm phenotype specificity

  • Double knockout studies to identify synergistic relationships

• Purified protein studies:

  • Expose host cells or organisms to purified recombinant AaeX

  • Compare with heat-killed bacteria and secretion fractions (as S. paratyphi A secretions and heat-killed treatments were avirulent)

  • Dose-response analyses to establish physiological relevance

• Transcriptomic/proteomic profiling:

  • Compare host responses to wild-type vs. AaeX-deficient strains

  • Identify uniquely regulated pathways

  • Validate findings through targeted gene/protein studies

What ELISA protocols are most effective for detecting AaeX-specific antibodies in research samples?

For detecting AaeX-specific antibodies, researchers should consider the following ELISA protocol elements:

• Antigen preparation:

  • Use highly purified recombinant AaeX (standard quantity: 50 μg)

  • Optimize coating concentration (typically 1-10 μg/ml)

  • Validate antigen integrity through SDS-PAGE prior to coating

• Assay optimization:

  • Determine appropriate blocking agents to minimize background

  • Establish optimal sample dilutions through titration experiments

  • Select appropriate secondary antibodies and detection systems

• Validation approaches:

  • Include known positive and negative control sera

  • Perform cross-reactivity testing with related Salmonella proteins

  • Consider paratyphoid patient sera for clinical relevance (comparable to the 94.8-98.8% detection rates observed for anti-SpaO and anti-H1a IgGs)

How can oxidative stress responses be measured in host models infected with Salmonella expressing AaeX?

Based on studies with S. paratyphi A, oxidative stress is a significant component of host-pathogen interactions . For AaeX-specific studies:

• Direct measurement approaches:

  • Hydrogen peroxide assays at multiple time points (12, 24, and 48 hours post-infection)

  • Reactive oxygen species detection using fluorescent probes

  • Lipid peroxidation product quantification

• Gene expression analysis:

  • Quantitative RT-PCR targeting oxidative stress genes:

    • Catalase genes (ctl-1, ctl-3) shown to be upregulated during Salmonella infection

    • Stress response genes (asp-12, abf-2, abf-3, clec-186, dbl-1, clec-86, C32H11.4, ugt-63, spp-1, hsp-70, cyp-35A2)

    • Downregulated genes to monitor (lys-7, clec-174, clec-218, clec-258, clec-85)

• Pathway activation assessment:

  • Western blot analysis of phosphorylated p38/PMK-1 levels (elevated during Salmonella infection)

  • Nuclear localization of transcription factors like SKN-1 and DAF-16

  • Complementary functional assays to correlate gene expression with phenotypic outcomes

What experimental design considerations are crucial when comparing AaeX with other Salmonella immunogens?

When designing comparative studies between AaeX and other Salmonella immunogens:

• Standardization requirements:

  • Protein purity (>95% by SDS-PAGE)

  • Endotoxin levels (<0.1 EU/μg protein)

  • Equivalent molar concentrations rather than weight-based dosing

  • Consistent adjuvant formulations and delivery routes

• Comprehensive immunological assessment:

  • Humoral responses (antibody titers, isotype profiles, neutralization capacity)

  • Cellular immunity (T-cell proliferation, cytokine production)

  • Memory response durability

  • Cross-protection against heterologous strains

• Statistical considerations:

  • Appropriate sample sizes based on power calculations

  • Blinded analysis where possible

  • Multiple experimental replicates

  • Appropriate statistical tests for non-parametric data often encountered in immunological studies

How can researchers troubleshoot expression difficulties with hydrophobic proteins like AaeX?

The hydrophobic nature of AaeX presents several expression challenges. Recommended troubleshooting approaches include:

• Solubility enhancement strategies:

  • Fusion with highly soluble partner proteins (MBP, SUMO, thioredoxin)

  • Codon optimization for expression host

  • Lower induction temperatures (16-25°C)

  • Specialized E. coli strains designed for membrane proteins

• Extraction optimization:

  • Detergent screening panel (ionic, non-ionic, and zwitterionic)

  • Membrane fraction isolation protocols

  • Inclusion body solubilization and refolding if necessary

• Purification refinement:

  • Tag position optimization (N vs. C-terminal)

  • On-column refolding procedures

  • Size exclusion chromatography to remove aggregates

  • Activity assays at each purification step to track functional protein yield

What strategies can resolve contradictory results in immune pathway studies involving AaeX?

When facing contradictory results in immune pathway studies:

• Model-specific considerations:

  • Different model organisms may emphasize different pathways (MAPK vs. insulin signaling)

  • Genetic background effects in C. elegans mutants can influence outcomes

  • Temperature-dependent effects (S. paratyphi showed maximal virulence at 35°C)

• Methodological reconciliation:

  • Standardize protein preparation protocols

  • Employ multiple complementary assays for each pathway

  • Consider temporal dynamics of immune responses

  • Validate antibodies and reagents across experimental systems

• Integrated analytical approaches:

  • Systems biology approaches to map pathway interactions

  • Genetic epistasis experiments to establish pathway hierarchies

  • In vivo validation of in vitro findings

  • Meta-analysis of published data with similar experimental conditions

What are the most promising future research directions for AaeX characterization and application?

Based on current knowledge of Salmonella pathogenesis and immunology, the following represent high-priority research directions:

• Structural biology approaches:

  • Crystallography or cryo-EM to determine three-dimensional structure

  • Identification of immunodominant epitopes

  • Structure-guided design of optimized vaccine constructs

• Multi-antigen vaccine formulations:

  • Combination with established immunogens (like SpaO and H1a that showed enhanced protection when combined)

  • Evaluation of novel adjuvant technologies

  • Development of delivery systems for mucosal immunity

• Fundamental biology:

  • Precise role in Salmonella pathogenesis

  • Evolution and conservation across Salmonella serovars

  • Host receptor identification and interaction mechanisms

• Diagnostic applications:

  • Development of AaeX-based serological assays

  • Multiplex detection systems combining multiple Salmonella antigens

  • Point-of-care diagnostic platforms