Recombinant Salmonella agona Protein AaeX (aaeX)

What is Salmonella agona Protein AaeX and what is its function?

Protein AaeX (gene name: aaeX) is a membrane protein encoded by Salmonella agona strain SL483 with UniProt accession number B5F7M6. The protein has a full amino acid sequence of MSLFPVIVVFGLSFPPIFFELLSLSLAIFWLVRRLVPTGIYDFVWHPALFNTALYCCLFY LISRLFV, with an expression region spanning positions 1-67 . Based on its hydrophobic profile and sequence characteristics, AaeX appears to be a membrane-associated protein that may play a role in transport mechanisms or membrane integrity, though its precise function remains under investigation. The protein contains transmembrane domains, suggesting it integrates into bacterial cell membranes, potentially contributing to Salmonella's survival mechanisms during infection or persistent states .

How does AaeX relate to Salmonella agona's pathogenicity?

While direct evidence linking AaeX specifically to S. agona's pathogenicity is limited in the provided research, Salmonella agona is recognized as a significant cause of gastroenteritis with notable abilities to form biofilms, undergo genome rearrangements, and enter viable but non-culturable states while remaining metabolically active . These characteristics collectively contribute to S. agona's ability to transition from acute infection to chronic carriage in human hosts. Membrane proteins like AaeX may participate in these processes, potentially contributing to S. agona's persistence mechanisms, though specific pathogenicity relationships would require targeted investigation through knockout studies and virulence assays .

What are the structural characteristics of AaeX protein?

The AaeX protein from Salmonella agona (strain SL483) consists of 67 amino acids in its expression region . Based on its sequence (MSLFPVIVVFGLSFPPIFFELLSLSLAIFWLVRRLVPTGIYDFVWHPALFNTALYCCLFY LISRLFV), bioinformatic analysis suggests it contains hydrophobic regions consistent with transmembrane domains. The protein likely adopts an alpha-helical conformation within the membrane, with hydrophilic regions exposed to either the cytoplasm or periplasm. For detailed structural analysis, researchers would need to employ techniques such as X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy, none of which have definitively characterized the three-dimensional structure of this specific protein based on available data .

How might AaeX contribute to Salmonella agona's persistence during chronic infection?

Salmonella agona demonstrates remarkable persistence capabilities in human hosts, with research identifying convalescent carriers (3.9%), temporary carriers (1.6%), and chronic carriers (1.0%) among studied cases . This persistence may involve multiple mechanisms, including biofilm formation, genome rearrangements, and entry into viable but non-culturable states. As a membrane protein, AaeX may contribute to these processes through several possible mechanisms: (1) participation in membrane remodeling during stress adaptation, (2) involvement in transport systems that facilitate nutrient acquisition in restrictive host environments, or (3) contribution to cell surface modifications that help evade host immune responses. During the transition from acute to persistent infection, Salmonella undergoes multiple adaptations, including genomic rearrangements and phenotypic changes in biofilm formation capacity . The possible role of AaeX in these adaptations presents an intriguing avenue for future research, potentially through comparative expression studies across different infection stages.

What genomic and phenotypic changes might affect AaeX expression during persistent Salmonella agona infection?

Research on S. agona persistence has revealed intriguing patterns of genomic variation during different infection phases. During early convalescent carriage (3 weeks to 3 months), increased single nucleotide polymorphism (SNP) variation and genome structure (GS) rearrangements have been observed . These genomic changes could potentially affect AaeX expression or function. Among 207 isolates studied, 12 showed genome rearrangements from the conserved genome structure (GS1.0), primarily during the first 66 days of infection . Whether these rearrangements directly impact the aaeX gene region would require targeted analysis. Additionally, phenotypic changes occur during infection progression, with isolates from convalescent and temporary carriers showing significantly reduced biofilm formation capacity compared to acute infection isolates . This suggests potential regulatory shifts that might also influence membrane protein expression, including AaeX. Studying AaeX expression patterns specifically across these different infection stages would provide valuable insights into its potential role in persistence mechanisms.

How might AaeX interact with antibiotic resistance mechanisms in Salmonella agona?

While direct evidence connecting AaeX specifically to antibiotic resistance is not presented in the search results, multidrug-resistant S. agona isolates have been identified carrying numerous antibiotic resistance genes (ARGs) . The large plasmid pSE18-SA00377-1 (295,499 bp) from the IncHI2 family has been found to carry 16 ARGs organized in distinct clusters . As a membrane protein, AaeX could potentially interact with resistance mechanisms through several pathways: (1) influencing membrane permeability to antibiotics, (2) participating in efflux pump complexes, or (3) contributing to stress responses that enhance survival during antibiotic exposure. Research examining correlations between AaeX variants or expression levels and resistance profiles could reveal whether this protein contributes to S. agona's resistance capabilities. Additionally, investigating potential membrane-associated resistance mechanisms in AaeX-knockout strains would provide direct evidence of any functional relationship.

What are the optimal expression systems for producing recombinant Salmonella agona Protein AaeX?

Recombinant production of membrane proteins like AaeX presents significant challenges due to their hydrophobic nature and requirements for proper membrane insertion. Based on available literature, several expression systems can be considered:

The selection of an expression system should be guided by the intended application. For structural studies requiring large amounts of protein, E. coli or yeast systems may be preferable despite potential folding challenges. For functional studies where proper folding is critical, insect or mammalian cell systems might yield better results despite lower yields . Regardless of the chosen system, optimization of expression conditions (temperature, induction time, media composition) will be essential for successful AaeX production.

What purification strategies are most effective for recombinant AaeX protein?

Purifying membrane proteins like AaeX requires specialized approaches to maintain their native conformation while removing them from the lipid bilayer. A comprehensive purification strategy would typically involve:

• Membrane isolation: Differential centrifugation to separate membrane fractions from cellular debris and soluble proteins.

• Solubilization: Selection of appropriate detergents is critical. For a small membrane protein like AaeX (67 amino acids), mild detergents such as n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucoside (OG), or digitonin would be recommended to maintain native conformation.

• Affinity chromatography: Utilizing an affinity tag system. The tag type would be determined during the production process as noted in the product information . Common options include His-tag, FLAG-tag, or Strep-tag II systems.

• Size exclusion chromatography: As a polishing step to remove aggregates and ensure homogeneity.

• Storage: As recommended for the recombinant product, storage at -20°C in a Tris-based buffer with 50% glycerol . For extended storage, -80°C is preferable, with working aliquots stored at 4°C for up to one week.

The success of purification would be assessed through SDS-PAGE, Western blotting, and potentially activity assays if functional properties are established. Repeated freezing and thawing should be avoided as noted in the product recommendations .

How can researchers effectively validate the structural integrity of purified recombinant AaeX?

Validating the structural integrity of membrane proteins like AaeX presents unique challenges due to their hydrophobic nature and dependence on lipid environments. A comprehensive validation approach would include:

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure composition (alpha-helical content) and thermal stability. For AaeX, which likely contains transmembrane alpha-helices, CD should show characteristic minima at 208 and 222 nm.

• Fluorescence Spectroscopy: Intrinsic tryptophan fluorescence can provide information about the tertiary structure and folding state of the protein. The AaeX sequence contains tryptophan residues that could serve as intrinsic probes.

• Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): To determine oligomeric state and homogeneity in detergent solutions.

• Liposome Reconstitution Assays: Demonstrating successful integration into artificial lipid bilayers would strongly support proper folding of the recombinant protein.

• Limited Proteolysis: Comparing proteolytic patterns of the recombinant protein to native samples can verify structural similarity.

• Functional Assays: If specific functions of AaeX are established, activity assays would provide the most definitive validation of proper folding and function.

Each method provides complementary information, and a combination approach is recommended for comprehensive validation before proceeding to more advanced functional or structural studies.

How should researchers interpret phenotypic changes in Salmonella agona strains with modified AaeX expression?

When analyzing phenotypic changes in S. agona strains with modified AaeX expression (overexpression, knockdown, or knockout), researchers should employ a multifaceted interpretative framework:

• Growth Curve Analysis: Compare growth rates in various media conditions. Significant changes might indicate AaeX's role in nutrient acquisition or stress adaptation. Analyze not just exponential phase growth but also lag phase duration and stationary phase survival.

• Biofilm Formation: Since S. agona is known as a strong biofilm former , quantify biofilm production using crystal violet assays similar to those employed in previous studies. Research indicates that biofilm formation capacity changes during infection progression, with isolates from persistent carriers showing reduced biofilm capacity . Determine whether AaeX modification alters this pattern.

• Stress Response Profiling: Assess survival under various stressors (osmotic pressure, pH changes, nutrient limitation) that might be encountered during host infection. Compare results to wild-type responses to identify specific conditions where AaeX plays a critical role.

• Genome Stability Assessment: Given S. agona's propensity for genome rearrangements during persistence , determine whether AaeX modification influences the frequency of such rearrangements under selective pressures.

• Host Cell Interactions: Examine adhesion, invasion, and intracellular survival in relevant cell culture models to assess virulence implications.

When interpreting these results, consider that membrane proteins often participate in multiple cellular processes, so phenotypic changes may reflect both direct and indirect effects of AaeX modification. Statistical analysis should include appropriate controls and sufficient biological replicates (minimum n=3) to account for strain variability.

What comparative genomic approaches can reveal AaeX homologs and their functional significance across Salmonella strains?

To identify and analyze AaeX homologs across Salmonella strains, researchers should implement a systematic comparative genomic approach:

• Sequence-Based Homology Search: Begin with BLAST/HMMER searches using the S. agona AaeX sequence (UniProt: B5F7M6) as a query against Salmonella genome databases. This baseline approach will identify clear homologs with high sequence identity.

• Synteny Analysis: Examine the genomic context of identified homologs. Conservation of neighboring genes often indicates functional conservation. The aaeX locus in S. agona (SeAg_B3557) should serve as the reference point .

• Structural Prediction Comparison: For divergent homologs with lower sequence identity, compare predicted structural features using tools like PSIPRED, TMHMM, or AlphaFold. Membrane proteins often show structural conservation despite sequence divergence.

• Phylogenetic Analysis: Construct phylogenetic trees of identified homologs to visualize evolutionary relationships and potential functional divergence. This approach could reveal whether AaeX variants correlate with host adaptation or virulence characteristics.

• Population Genomics: Analyze single nucleotide polymorphisms (SNPs) in aaeX across clinical isolates with different infection outcomes. Previous research has shown increased SNP variation during persistent infection phases , which might extend to the aaeX locus.

For interpretation, researchers should consider that protein function conservation does not always require high sequence conservation, particularly for membrane proteins where structural motifs and physicochemical properties may be more important than exact amino acid identity. Cross-referencing genomic findings with phenotypic data from different strains can help establish functional significance of observed variations.

How can transcriptomic and proteomic data be integrated to understand AaeX regulation during Salmonella agona infection dynamics?

Integrating transcriptomic and proteomic approaches provides a comprehensive view of AaeX regulation during S. agona infection. Researchers should consider the following multi-omics integration framework:

• Time-Course Experimental Design: Sample S. agona during key infection stages identified in previous research - acute infection (≤3 weeks), early convalescent carriage (3 weeks-3 months), and chronic carriage (>3 months) . Include both in vitro stress conditions and in vivo infection models when possible.

• RNA-Seq Analysis: Quantify aaeX transcript levels alongside global transcriptome changes. Focus analysis on:

  • Correlation between aaeX expression and known virulence factors

  • Co-expression networks to identify genes with similar expression patterns

  • Differential expression between genome structure variants (GS1.0 vs. rearranged genomes)

• Proteomics Approach: Employ membrane-enriched proteomics to accurately quantify AaeX protein levels, as standard proteomics often underrepresents membrane proteins. Consider both absolute quantification and post-translational modifications.

• Integration Strategies:

  • Calculate transcript-to-protein ratios to identify post-transcriptional regulation

  • Apply pathway enrichment analysis to contextual genes with similar regulation patterns

  • Develop predictive models of AaeX regulation based on environmental cues

• Validation: Confirm key findings using targeted approaches such as RT-qPCR for transcript levels and Western blotting for protein levels under specific conditions identified as regulatory transition points.

This integrated approach would help distinguish transcriptional from post-transcriptional regulation mechanisms and identify key environmental triggers that modulate AaeX expression during the transition from acute to persistent infection states. The analysis should account for the genome rearrangements and SNP variations observed during early convalescent carriage , which might influence regulatory networks controlling AaeX expression.

What role might AaeX play in Salmonella agona biofilm formation and persistence?

Salmonella agona has been identified as a strong biofilm former, a characteristic that likely contributes to its persistence in both environmental settings and human hosts . The potential role of AaeX in this process warrants detailed investigation through several approaches:

• Expression Correlation Analysis: Research has shown that isolates from patients with convalescent and temporary carriage of S. agona exhibit significantly reduced biofilm formation capacity compared to isolates from acute infections . Examining whether AaeX expression levels correlate with these differences could provide initial evidence of involvement.

• Genetic Manipulation Studies: Developing AaeX knockout, knockdown, and overexpression strains would allow direct assessment of its contribution to biofilm formation. Quantitative crystal violet assays similar to those used in previous studies would provide comparable data on biofilm capacity.

• Localization Studies: Using fluorescently tagged AaeX to visualize its distribution within biofilm structures could reveal whether it localizes to specific regions or interfaces that might indicate functional roles in biofilm architecture.

• Interaction Analysis: Investigating whether AaeX interacts with known biofilm-related proteins such as those encoded by csgD, csgB, fimH, and other attachment-related genes previously identified as critical for Salmonella biofilm formation .

If AaeX indeed participates in biofilm formation, this would have significant implications for understanding S. agona persistence mechanisms and potentially identify new targets for disrupting chronic carriage states. The connection between membrane proteins and biofilm formation remains an underexplored area that could yield valuable insights into bacterial adaptation during long-term host association.

How might recombinant AaeX be utilized in the development of diagnostic tools for Salmonella agona detection?

Recombinant AaeX protein has potential applications in developing sensitive and specific diagnostic tools for Salmonella agona detection, addressing current diagnostic challenges:

• Antibody Development: Purified recombinant AaeX can serve as an antigen for generating specific polyclonal or monoclonal antibodies. These antibodies could then be incorporated into:

  • ELISA-based detection systems for clinical or food samples

  • Lateral flow immunoassays for rapid point-of-care testing

  • Immunofluorescence applications for direct visualization in complex samples

• Aptamer Selection: Recombinant AaeX could be used as a target for SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to develop DNA or RNA aptamers with high specificity. Aptamer-based biosensors offer advantages in stability and production costs compared to antibody-based systems.

• MS-Based Proteotyping: Using recombinant AaeX as a standard, develop mass spectrometry methods targeting AaeX-specific peptides for identification of S. agona in complex samples. This approach could be particularly valuable for distinguishing S. agona from other Salmonella serovars.

• Validation Considerations: Any diagnostic application would require:

  • Assessment of cross-reactivity with closely related proteins from other Salmonella serovars

  • Determination of sensitivity limits in realistic sample matrices

  • Comparison with gold-standard culture-based detection methods

The development of such diagnostic tools would be particularly valuable for surveillance of S. agona in high-risk settings and could potentially aid in distinguishing between acute infections and cases of persistent carriage based on quantitative detection or identification of specific AaeX variants associated with different infection stages .

What emerging technologies could advance our understanding of AaeX function in Salmonella agona pathogenesis?

Several cutting-edge technologies show promise for elucidating AaeX function in S. agona pathogenesis:

• Cryo-Electron Microscopy (Cryo-EM): Recent advances in cryo-EM have revolutionized membrane protein structural biology. Applied to AaeX, this could reveal atomic-level details of its structure, particularly in native-like lipid environments through techniques like lipid nanodisc reconstitution.

• CRISPR Interference (CRISPRi) and Activation (CRISPRa): These systems allow precise modulation of aaeX expression without permanently altering the genome. Tunable expression systems would enable the determination of dose-dependent effects on virulence, persistence, and biofilm formation.

• Single-Cell RNA-Seq and Spatial Transcriptomics: These technologies could reveal population heterogeneity in aaeX expression during infection, potentially identifying subpopulations primed for persistence. This is particularly relevant given the observed genomic and phenotypic heterogeneity during Salmonella persistence .

• Host-Pathogen Protein-Protein Interaction Mapping: Technologies like proximity labeling (BioID, TurboID) could identify host proteins that interact with AaeX during infection, potentially revealing mechanistic insights into its role in pathogenesis.

• In Situ Structural Biology: Techniques like cellular cryo-electron tomography could visualize AaeX in its native context within the bacterial membrane, providing insights into its organization and potential complex formation.

• Microfluidic Infection Models: These systems allow real-time visualization of host-pathogen interactions and could be particularly valuable for studying the transition from acute to persistent infection states, with simultaneous monitoring of AaeX expression using reporter constructs.

Each of these approaches offers unique advantages and, when integrated, could provide comprehensive insights into AaeX function that go beyond traditional genetic and biochemical approaches.

How might understanding AaeX contribute to novel antimicrobial strategies against persistent Salmonella agona infections?

The potential of AaeX as a target for novel antimicrobial strategies against persistent S. agona infections merits exploration through several research avenues:

• Structure-Based Drug Design: If structural studies confirm AaeX has a unique fold or functional site, structure-based approaches could develop small molecule inhibitors specifically targeting this protein. This would be particularly valuable if AaeX proves essential for persistence mechanisms.

• Anti-Virulence Approach: Rather than directly killing bacteria, targeting AaeX might disrupt persistence mechanisms, rendering S. agona more susceptible to conventional antibiotics or host immune clearance. This strategy could be particularly relevant given the observation that S. agona undergoes specific adaptations during the transition to persistent infection .

• Immunomodulatory Strategies: If AaeX interacts with host immune components, blocking these interactions could enhance immune recognition and clearance of persistent bacteria. Developing antibodies or peptide mimetics that interfere with such interactions represents a potential therapeutic approach.

• Biofilm Disruption: The established role of S. agona as a strong biofilm former suggests that targeting components involved in this process could help eliminate persistent infections. If AaeX contributes to biofilm formation or maintenance, it would represent a logical target.

• Combination Therapy Potential: Research into antibiotic resistance in S. agona has identified numerous resistance genes carried on plasmids like pSE18-SA00377-1 . Targeting AaeX in combination with conventional antibiotics might overcome resistance mechanisms and enhance treatment efficacy against multidrug-resistant strains.

These approaches would require thorough validation of AaeX's role in persistence and careful assessment of potential off-target effects, particularly if homologous proteins exist in commensal bacteria. The development of model systems that accurately recapitulate persistent S. agona infections would be essential for evaluating the efficacy of AaeX-targeted interventions.