Recombinant Salmonella schwarzengrund Protein AaeX (aaeX)

What is the biological function of Protein AaeX in Salmonella schwarzengrund?

Protein AaeX in Salmonella schwarzengrund is a membrane-associated protein comprised of 67 amino acids with a transmembrane domain. Based on sequence analysis, AaeX functions as a putative membrane protein involved in cellular transport mechanisms and potentially in bacterial adaptation to environmental stresses. The amino acid sequence (MSLFPVIVVFGLSFPPIFFFELLSLAILFWLVRRLVPTGIYDFVWHPALFNTALYCCLFY LISRLFV) suggests a hydrophobic profile consistent with membrane integration . Research indicates that AaeX may contribute to the bacterium's ability to adapt to different environmental conditions, though full functional characterization requires further investigation through gene knockout studies and complementation assays.

What are the optimal storage and handling conditions for recombinant AaeX protein preparations?

Recombinant AaeX protein requires careful storage to maintain its structural integrity and functional properties. The optimal storage conditions include:

For handling, it is recommended to centrifuge the vial briefly before opening to collect the contents at the bottom. Reconstitution should be performed in deionized sterile water to achieve concentrations between 0.1-1.0 mg/mL . Adding glycerol to a final concentration of 50% helps maintain protein stability during storage. Researchers should always confirm protein activity after extended storage periods using appropriate functional assays.

How can researchers verify the identity and purity of recombinant AaeX protein?

Verification of recombinant AaeX protein identity and purity involves multiple complementary analytical approaches:

• SDS-PAGE Analysis: Confirm the expected molecular weight (~7.5 kDa based on the 67 amino acid sequence) and assess purity (should exceed 90%) .

• Western Blot Analysis: Use antibodies specific to either AaeX or the fusion tag (if present) to confirm identity.

• Mass Spectrometry: Perform peptide mass fingerprinting to verify the amino acid sequence and identify any post-translational modifications.

• Circular Dichroism: Assess the secondary structure profile to confirm proper protein folding.

• Size Exclusion Chromatography: Evaluate the homogeneity of the protein preparation and detect potential aggregation.

Researchers should maintain detailed records of each validation step, including gel images, chromatograms, and spectral data, to ensure reproducibility across experiments and proper quality control.

What expression systems yield the highest functional activity for recombinant AaeX protein?

While the search results don't explicitly specify the optimal expression system for AaeX, comparable recombinant Salmonella proteins have been successfully produced in E. coli expression systems with high yield and functionality . For membrane proteins like AaeX, the following expression strategies have proven effective:

To optimize AaeX expression, researchers should consider:

• Using low induction temperatures (16-25°C)

• Employing specialized vectors containing mild promoters

• Incorporating fusion partners that enhance solubility

• Testing detergent panels for extraction efficiency

The expression region for AaeX has been identified as amino acids 1-67, representing the full-length protein , which should be considered when designing expression constructs.

How does AaeX contribute to Salmonella schwarzengrund virulence and pathogenicity?

The role of AaeX in Salmonella schwarzengrund virulence remains under investigation, but several research approaches can help elucidate its function:

• Infection Models: Comparing wild-type and AaeX-deficient strains in cellular and animal infection models can reveal its contribution to virulence. Recent case reports of S. schwarzengrund causing severe infections, including sacroiliac joint infection with septic shock in immunocompetent individuals, highlight the importance of understanding virulence factors .

• Transcriptomic Analysis: Examining expression changes during different infection stages can identify when AaeX is upregulated, suggesting critical infection phases where it may play a role.

• Protein-Protein Interaction Studies: Identifying binding partners using techniques like pull-down assays or bacterial two-hybrid systems can reveal functional associations with known virulence pathways.

S. schwarzengrund has been associated with severe extraintestinal infections even in young, healthy individuals without immunocompromise , suggesting robust virulence mechanisms that may involve membrane proteins like AaeX. Research into specific virulence determinants is essential given the increasing prevalence of S. schwarzengrund infections globally .

What structural characteristics of AaeX influence its membrane localization and function?

AaeX's primary sequence (MSLFPVIVVFGLSFPPIFFFELLSLAILFWLVRRLVPTGIYDFVWHPALFNTALYCCLFY LISRLFV) contains several hydrophobic regions consistent with membrane integration . Advanced structural analysis reveals:

• Transmembrane Domain Prediction:

  • N-terminal region (approximately residues 5-25) contains a predicted transmembrane helix

  • Central hydrophobic region (approximately residues 30-50) may form a second membrane-associated domain

• Structural Motifs:

  • The VPTGIYDF sequence (residues 32-39) contains a potential functional motif

  • C-terminal region contains charged residues that may interact with cytoplasmic factors

• Topology Models:

  • Type I membrane protein (N-terminus outside, C-terminus inside)

  • Potential for forming homo-oligomeric structures

Researchers investigating AaeX structure-function relationships should consider employing:

• Site-directed mutagenesis of key residues

• Fluorescent protein fusions to confirm membrane localization

• Cross-linking studies to identify potential oligomerization

• Molecular dynamics simulations to model membrane interactions

Understanding these structural characteristics is essential for developing strategies to modulate AaeX function in antimicrobial research.

What methodological approaches are most effective for studying AaeX interactions with host immune system components?

Investigating interactions between AaeX and host immune components requires specialized techniques due to the membrane-associated nature of this protein:

• Recombinant Protein Production Strategies:

  • Expression with removable solubility tags

  • Reconstitution into liposomes or nanodiscs to maintain native conformation

  • Use of detergent-solubilized preparations for binding studies

• Interaction Analysis Methods:

  • Surface Plasmon Resonance (SPR) using immobilized immune components

  • Enzyme-Linked Immunosorbent Assays (ELISA) with recombinant AaeX

  • Flow cytometry for cell-based interaction studies

  • Immunoprecipitation followed by mass spectrometry

• Functional Immunological Assays:

  • Cytokine release quantification using ELISA or multiplex assays

  • Neutrophil activation assays

  • Inflammasome activation assessment

  • Antigen presentation studies with dendritic cells

The study of host-pathogen interactions is particularly relevant given the recent reports of severe S. schwarzengrund infections including pyogenic sacroiliitis with septic shock, even in immunocompetent individuals . Understanding how membrane proteins like AaeX interact with host defenses may help explain the pathogen's ability to cause extraintestinal infections.

What are the challenges and solutions in purifying functional recombinant AaeX for structural studies?

Obtaining pure, functional AaeX for structural studies presents several challenges due to its membrane-associated nature:

For crystallography or cryo-EM studies, researchers should consider:

• Using fusion partners that facilitate crystallization

• Implementing limited proteolysis to identify stable domains

• Testing lipid cubic phase crystallization for membrane proteins

• Employing nanobodies to stabilize specific conformations

Successful structural studies will require careful optimization of expression and purification protocols, with validation of structural integrity at each step. The recommended storage in Tris-based buffer with 50% glycerol provides a starting point for stability optimization .

How can researchers effectively design experiments to understand AaeX's role in antibiotic resistance mechanisms?

Investigating AaeX's potential role in antibiotic resistance requires a multi-faceted experimental approach:

• Gene Expression Analysis:

  • Quantify aaeX expression changes in response to antibiotic exposure

  • Compare expression levels between antibiotic-sensitive and resistant strains

  • Use RT-qPCR to measure transcriptional responses to different classes of antibiotics

• Genetic Manipulation Strategies:

  • Generate aaeX knockout strains and assess changes in antibiotic susceptibility

  • Create point mutations in key functional domains

  • Implement CRISPR-Cas9 for precise genome editing

  • Develop complementation systems to confirm phenotypic changes

• Phenotypic Assays:

  • Minimum Inhibitory Concentration (MIC) determination for various antibiotics

  • Time-kill kinetics to assess the dynamics of antibiotic action

  • Biofilm formation assays to evaluate community resistance mechanisms

  • Membrane permeability studies using fluorescent dyes

• Molecular Interaction Studies:

  • Assess direct binding between AaeX and antibiotics using fluorescence-based assays

  • Evaluate impacts on membrane integrity using artificial membrane systems

  • Investigate effects on efflux pump activity

The increasing prevalence of multidrug-resistant Salmonella strains globally underscores the importance of understanding all potential resistance mechanisms, including the roles of membrane proteins like AaeX that may influence bacterial membrane permeability or contribute to efflux systems.

What are the most reliable approaches for generating antibodies against AaeX for research applications?

Developing specific antibodies against AaeX requires strategic approaches due to its small size (67 amino acids) and membrane-associated nature:

• Antigen Design Strategies:

  • Use the full-length recombinant protein with appropriate tags

  • Identify immunogenic epitopes through in silico prediction

  • Synthesize peptides corresponding to exposed regions

  • Consider KLH or BSA conjugation to enhance immunogenicity

• Antibody Production Methods:

  • Polyclonal antibody generation in rabbits or goats

  • Monoclonal antibody development focusing on specific epitopes

  • Recombinant antibody technologies (phage display, yeast display)

  • Single-domain antibodies (nanobodies) for accessing cryptic epitopes

• Validation Protocols:

  • Western blot against recombinant protein and native extracts

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence to confirm subcellular localization

  • Neutralization assays to assess functional blocking

• Optimization Approaches:

  • Affinity purification against immobilized antigen

  • Cross-adsorption to eliminate cross-reactivity

  • Isotype selection for specific applications

For researchers working with AaeX, generating reliable antibodies is crucial for studying its expression, localization, and interactions in different experimental contexts.

How can researchers effectively utilize recombinant AaeX protein in vaccine development studies?

Utilizing recombinant AaeX in vaccine research requires systematic evaluation of its immunogenic potential and protective efficacy:

• Antigen Formulation Strategies:

  • Purified recombinant protein with adjuvants

  • DNA vaccines encoding the aaeX gene

  • Viral vector-based delivery systems

  • Outer membrane vesicle incorporation

• Immunogenicity Assessment:

  • Antibody titer measurement (IgG, IgA) in serum and mucosal surfaces

  • T-cell response evaluation (proliferation, cytokine production)

  • Memory B-cell quantification

  • Dendritic cell activation and antigen presentation analysis

• Protection Studies:

  • Challenge models with virulent S. schwarzengrund strains

  • Bacterial burden quantification in tissues

  • Survival rate assessment

  • Immunopathology evaluation

• Cross-protection Analysis:

  • Evaluation against heterologous Salmonella serovars

  • Assessment of cross-reactive antibodies

  • T-cell epitope conservation analysis

The relevance of S. schwarzengrund as a pathogen capable of causing severe infections highlights the potential value of developing effective vaccines against this organism. Membrane-associated proteins like AaeX represent potential vaccine candidates, particularly if they are surface-exposed and conserved across strains.

What analytical techniques provide the most accurate structural information about recombinant AaeX?

Obtaining high-resolution structural information about a small membrane protein like AaeX requires specialized techniques:

For AaeX specifically, researchers should consider:

• Using NMR as the primary approach due to the protein's small size (67 amino acids)

• Implementing membrane mimetics (nanodiscs, bicelles) to maintain native-like environment

• Combining multiple techniques for comprehensive structural characterization

• Validating structural models through functional studies of key residues

Understanding the structure of AaeX could provide insights into its role in S. schwarzengrund pathogenicity and potential as a therapeutic target.

What are the common pitfalls in functional studies of AaeX and how can they be addressed?

Functional characterization of AaeX presents several challenges that researchers should anticipate and address:

• Expression and Purification Challenges:

  • Pitfall: Low yield or aggregation during expression

  • Solution: Optimize expression conditions (temperature, induction time); use specialized strains; implement fusion partners

• Protein Stability Issues:

  • Pitfall: Loss of activity during storage

  • Solution: Use stabilizing agents; store at -20°C/-80°C with 50% glycerol; avoid freeze-thaw cycles

• Functional Assay Limitations:

  • Pitfall: Lack of established functional readouts

  • Solution: Develop binding assays with predicted interaction partners; use complementation studies in knockout strains

• Membrane Environment Reconstitution:

  • Pitfall: Non-native behavior in solution

  • Solution: Utilize lipid nanodiscs or liposomes; optimize detergent selection; validate function in membrane mimetics

• Structural Constraints:

  • Pitfall: Interference from tags or fusion partners

  • Solution: Compare activity with and without tags; use cleavable tags; verify structure is not perturbed

• Heterologous Expression Artifacts:

  • Pitfall: Post-translational modification differences

  • Solution: Compare protein from different expression systems; verify modification status by mass spectrometry

How can researchers differentiate between direct and indirect effects when studying AaeX's role in bacterial physiology?

Distinguishing direct from indirect effects is critical for accurately characterizing AaeX function:

• Genetic Approaches:

  • Create clean deletion mutants using scarless techniques

  • Implement conditional expression systems (inducible promoters)

  • Use point mutations targeting specific functional domains

  • Develop complementation systems with wild-type and mutant alleles

• Biochemical Validation:

  • Perform direct binding assays with purified components

  • Implement proximity labeling techniques (BioID, APEX)

  • Use crosslinking followed by mass spectrometry to identify interaction partners

  • Reconstitute minimal systems in vitro to test direct effects

• Temporal Analysis:

  • Monitor rapid responses to perturbation using real-time assays

  • Implement time-course experiments to establish causality

  • Use pulse-chase approaches to track dynamic processes

• Control Experiments:

  • Include structurally similar but functionally distinct proteins as controls

  • Test effects across multiple genetic backgrounds

  • Validate key findings using orthogonal methodologies

• Systems Biology Approaches:

  • Perform transcriptomic analysis of knockout strains

  • Use metabolomic profiling to identify pathway disruptions

  • Implement network analysis to separate direct from downstream effects

Since AaeX is a membrane protein potentially involved in adaptation to environmental stresses or transport processes, distinguishing its direct functional role from secondary effects is particularly important for understanding its contribution to S. schwarzengrund physiology and pathogenicity.

How can AaeX be utilized as a potential biomarker for Salmonella schwarzengrund detection in clinical and environmental samples?

The application of AaeX as a biomarker for S. schwarzengrund detection offers promising research opportunities:

• Antibody-Based Detection Methods:

  • Develop ELISA systems using anti-AaeX antibodies for quantitative detection

  • Implement lateral flow assays for point-of-care diagnostics

  • Create biosensor platforms with immobilized antibodies

  • Design multiplex detection systems incorporating multiple Salmonella biomarkers

• Nucleic Acid-Based Detection:

  • Design PCR primers targeting the aaeX gene with species-specific regions

  • Develop LAMP (Loop-mediated isothermal amplification) for field detection

  • Implement NGS approaches for metagenomic detection

  • Create RNA-based detection systems for viable cell identification

• Performance Optimization:

  • Determine detection limits in various matrices (clinical samples, food, environmental)

  • Assess specificity against other Salmonella serovars and related bacteria

  • Evaluate robustness in complex sample types

  • Compare sensitivity to gold standard methods

• Validation Studies:

  • Test performance with clinical isolates from different geographic regions

  • Evaluate detection in artificially contaminated samples

  • Perform field testing in relevant environmental contexts

The increasing prevalence of S. schwarzengrund in various regions and its association with serious infections highlight the need for specific and sensitive detection methods. Utilizing species-specific proteins like AaeX could improve diagnostic specificity compared to genus-level detection systems.

What emerging technologies show promise for studying the function and interactions of AaeX in living systems?

Several cutting-edge technologies offer new approaches to investigate AaeX in biological contexts:

• Advanced Imaging Techniques:

  • Super-resolution microscopy for precise localization

  • Single-molecule tracking to monitor dynamics

  • Correlative light and electron microscopy for structural context

  • Expansion microscopy for enhanced resolution in bacterial cells

• Genetic Manipulation Technologies:

  • CRISPR interference for precise transcriptional control

  • CRISPRa for controlled overexpression

  • Base editing for specific amino acid substitutions

  • Optogenetic control of expression or activity

• Protein Engineering Approaches:

  • Split protein complementation for interaction studies

  • Optogenetic protein control systems

  • Chemogenetic regulation of protein function

  • Nanobody-based detection in live cells

• Systems Biology Integration:

  • Multi-omics approaches combining proteomics, transcriptomics, and metabolomics

  • Machine learning for pattern recognition in complex datasets

  • Network analysis to position AaeX within cellular pathways

  • Predictive modeling of protein function based on integrated data

• Structural Biology Innovations:

  • Cryo-electron tomography for in situ structural determination

  • Integrative structural biology combining multiple data types

  • AlphaFold and related AI prediction tools for structure modeling

  • Time-resolved structural studies to capture conformational changes

These technologies can help elucidate the function of AaeX in the context of S. schwarzengrund pathogenicity, particularly given the organism's ability to cause severe infections even in immunocompetent hosts .