Recombinant Salmonella paratyphi A Protein AaeX (aaeX)

What is the amino acid sequence of Salmonella paratyphi A Protein AaeX?

The amino acid sequence of Salmonella paratyphi A Protein AaeX is closely related to that of Salmonella paratyphi B AaeX protein, which consists of 67 amino acids with the sequence: MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRMLVPTGIYDFVWHPALFNTALYCCLFY LISRLFV . While specific sequence variations may exist between the A and B serovars, they maintain high sequence conservation. Research indicates that comparable proteins in Salmonella species typically demonstrate high nucleotide or amino acid sequence identity, ranging from 99.2%-100.0% at the nucleotide level or 98.4%-100.0% at the amino acid level, as observed with similar outer membrane proteins .

What is the predicted structural characterization of AaeX protein in Salmonella paratyphi A?

Based on research of related proteins in Salmonella species, AaeX is predicted to be a membrane-associated protein. The amino acid composition suggests a hydrophobic profile typical of transmembrane proteins, including regions of hydrophobic residues that likely span the bacterial membrane . Structural analysis indicates the presence of alpha-helical transmembrane domains that anchor the protein within the bacterial cell envelope. This structural arrangement is consistent with its putative role in membrane integrity and potentially in virulence factor expression systems.

How does AaeX protein conservation compare across different Salmonella paratyphi A isolates?

Studies of Salmonella paratyphi proteins demonstrate remarkable conservation across clinical isolates. Similar to the outer membrane protein X (ompX), AaeX likely shows high sequence conservation across different geographic isolates, with sequence identity exceeding 98% . This conservation suggests strong evolutionary pressure to maintain the protein's structure and function, indicating its potential importance in bacterial physiology or pathogenesis. Complete genome sequence analysis of Salmonella paratyphi A isolates has revealed that virulence-associated proteins typically maintain high conservation levels within specific genotypic clades .

What are the recommended expression systems for recombinant production of Salmonella paratyphi A AaeX protein?

For recombinant expression of Salmonella paratyphi A AaeX protein, E. coli-based expression systems are most commonly employed. The protein can be expressed with an N-terminal His-tag to facilitate purification . Research protocols typically involve PCR amplification of the target gene, T-A cloning processes, and subsequent expression in an appropriate E. coli strain . For optimal expression, induction conditions should be carefully controlled, and protein extraction performed under conditions that maintain protein folding integrity. SDS-PAGE and Western blot analyses are recommended for verification of expression and purity assessment.

What experimental design approaches are most suitable for investigating AaeX protein function?

Investigation of AaeX protein function benefits from a comprehensive experimental design combining both in vitro and in vivo approaches. Independent group design with random allocation of subjects minimizes bias and increases reliability of results . Multiple experimental approaches include:

• Protein-protein interaction studies to identify binding partners

• Gene knockout studies to determine phenotypic changes

• Immunological assays to assess antigenic properties

• Structural biology techniques to determine three-dimensional conformation

• Animal model studies to evaluate pathogenicity impact

Each approach requires appropriate controls, randomization protocols, and statistical validation to ensure scientific rigor .

What are the optimal conditions for storage and stability of purified recombinant AaeX protein?

Purified recombinant AaeX protein demonstrates optimal stability when stored according to specific protocols. Based on related protein research, it is recommended to store the protein at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use scenarios to avoid repeated freeze-thaw cycles . The protein can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage. Lyophilized powder formulations offer extended shelf-life, while working aliquots can be maintained at 4°C for up to one week without significant degradation .

What is the current understanding of AaeX protein's role in Salmonella paratyphi A virulence?

Current research suggests AaeX protein may contribute to Salmonella paratyphi A virulence through membrane-associated functions. While direct experimental evidence specifically for AaeX is limited, studies of Salmonella pathogenicity islands (SPIs) indicate that membrane proteins often play crucial roles in bacterial adhesion, invasion, and host immune evasion . Genetic analysis of virulence determinants has identified several membrane-associated proteins that differ between pathogenic clades, potentially including AaeX, which may contribute to differences in virulence profiles between strains . The protein's high conservation across isolates further suggests functional importance in bacterial survival or pathogenicity.

How does AaeX interact with other virulence factors in Salmonella pathogenicity islands?

While specific interactions between AaeX and other virulence factors have not been comprehensively documented, research on Salmonella pathogenicity islands (SPIs) provides insight into potential functional relationships. Genetic analysis has revealed that virulence factors associated with SPI-1 and SPI-2, including effector proteins such as SifA, SopA, SopD2, and SopE, often demonstrate clade-specific genetic variations . Membrane proteins like AaeX potentially function within these pathogenicity networks, either as structural components or as functional elements in secretion systems. Genetic diversity in these virulence factors ranges from small mismatches to larger scale changes with multiple mutations or indels .

What immunological properties does AaeX protein demonstrate in experimental models?

Research on Salmonella paratyphi membrane proteins indicates that recombinant proteins like AaeX may possess significant immunogenic properties. Studies with similar membrane proteins have demonstrated capacity to induce high-level antibody production when administered to experimental animals . For instance, research with outer membrane proteins showed positive Western hybridization signals when recombinant proteins were combined with antisera against whole cell preparations . Similar membrane-associated proteins exhibited high sensitivity (>95%) in ELISA assays using serum samples from paratyphoid patients, suggesting potential diagnostic applications .

How can AaeX protein be utilized in developing novel vaccination strategies against Salmonella paratyphi A?

AaeX protein represents a potential candidate antigen for developing genetic engineering vaccines against Salmonella paratyphi A. Research on similar membrane proteins has demonstrated significant immunoprotective effects in mouse models, with protection rates of 93.3-100% following immunization . The potential vaccine application is supported by the protein's high conservation across clinical isolates, reducing the risk of strain-specific escape variants. Development strategies would likely involve recombinant expression of AaeX, purification, and formulation with appropriate adjuvants to enhance immunogenicity. Immunization studies should assess both humoral and cell-mediated immune responses, as well as protection against challenge with virulent S. paratyphi A strains .

What methodological approaches are most effective for investigating AaeX protein interactions with host immune systems?

Investigation of AaeX protein interactions with host immune systems requires a multifaceted methodological approach. Effective research strategies include:

• Immunodiffusion tests to assess antigenicity

• ELISA assays to quantify antibody responses in infected or immunized subjects

• Western blot analysis to confirm specific immune recognition

• Animal immunization studies to evaluate protective capacity

• Agglutination assays (such as micro-Widal's test) to measure antibody titers

These methodologies have proven effective in researching similar membrane proteins, demonstrating their utility in characterizing immune responses . For comprehensive analysis, both in vitro studies with human immune cells and in vivo studies in appropriate animal models are necessary to fully elucidate the immunological properties of AaeX protein.

How does AaeX protein compare structurally and functionally to outer membrane protein X (ompX) in Salmonella paratyphi A?

Comparative analysis between AaeX and outer membrane protein X (ompX) reveals both similarities and distinctions in these Salmonella paratyphi A proteins. While both are membrane-associated proteins with potential roles in pathogenesis, ompX has been more extensively characterized as an outer membrane protein with documented immunogenicity . OmpX shows high sequence conservation (99.2-100% nucleotide identity) across isolates, suggesting essential functionality . The structural differences between AaeX and ompX likely reflect their distinct functional roles within the bacterial membrane architecture, with ompX potentially having more direct interactions with host immune systems due to its outer membrane localization. Both proteins exhibit potential as candidate antigens for vaccine development, though ompX has demonstrated specific immunoprotective effects in experimental models .

What are the key differences in expression and regulation between AaeX in Salmonella paratyphi A versus B serovars?

The expression and regulation of AaeX likely differ between Salmonella paratyphi A and B serovars, reflecting their distinct genetic backgrounds and pathogenic mechanisms. While both serovars express AaeX proteins with similar structural characteristics, research on other virulence factors has demonstrated serovar-specific regulatory patterns . Genetic analysis of Salmonella isolates has identified various clade-specific mutations affecting virulence factor expression, suggesting potential differences in AaeX regulation as well . The specific regulatory differences may include variations in promoter regions, transcription factor binding sites, or post-transcriptional control mechanisms that influence protein expression levels or timing during infection.

What research gaps currently exist in understanding the complete functional characterization of AaeX protein?

Significant research gaps persist in the comprehensive functional characterization of AaeX protein:

• Limited direct experimental evidence for AaeX's specific role in virulence

• Incomplete understanding of its protein interaction network

• Insufficient data on expression patterns during different stages of infection

• Limited characterization of potential post-translational modifications

• Unclear relationship to antimicrobial resistance mechanisms

These knowledge gaps present opportunities for future research employing advanced methodologies such as comparative genomics, structural biology, and in vivo infection models . Additionally, systematic mutagenesis studies combined with phenotypic characterization would provide valuable insights into the protein's functional domains and essential residues. Integration of these approaches would contribute significantly to understanding AaeX's role in Salmonella paratyphi A biology and pathogenesis.