Recombinant Salmonella enteritidis PT4 UPF0266 membrane protein yobD (yobD)

What expression systems are optimal for producing recombinant YobD?

For recombinant production of YobD, Escherichia coli expression systems have proven effective. The protein can be successfully expressed as an N-terminal His-tagged fusion protein in E. coli, which facilitates subsequent purification steps. When designing expression constructs, researchers should consider:

• Using cold-inducible expression vectors (such as pCold) that can reduce inclusion body formation

• Selecting E. coli strains optimized for membrane protein expression (e.g., C41(DE3) or C43(DE3))

• Employing low induction temperatures (16-18°C) to enhance proper folding

• Including solubilizing tags such as His-tag for purification purposes

The expression methodology should be adapted to ensure the recombinant YobD maintains its native conformation and functionality, which is crucial for downstream applications and analyses .

What are the recommended storage conditions for recombinant YobD protein?

For optimal stability and activity retention of recombinant YobD protein, the following storage conditions are recommended:

Repeated freeze-thaw cycles significantly reduce protein stability and should be avoided. For working solutions, storing at 4°C for up to one week is recommended. The addition of 6% trehalose to the storage buffer can enhance protein stability during freeze-thaw and reconstitution processes .

What methodologies are appropriate for investigating potential roles of YobD in Salmonella pathogenesis?

To investigate YobD's role in Salmonella pathogenesis, researchers should employ a multi-faceted approach:

• Gene knockout studies: Create yobD deletion mutants in S. enteritidis using homologous recombination techniques similar to those employed for yafD studies in S. enteritidis . This allows for functional assessment through phenotypic analysis.

• Infection models: Compare wild-type and ΔyobD mutant strains in cellular and animal infection models to assess virulence, invasion, and intracellular survival.

• Complementation studies: Reintroduce the yobD gene into knockout strains to confirm observed phenotypes are specifically due to yobD deletion.

• Protein-protein interaction studies: Employ pull-down assays, bacterial two-hybrid systems, or co-immunoprecipitation to identify YobD interaction partners that may provide functional insights.

• Localization studies: Use fluorescent protein fusions or immunostaining to determine YobD's subcellular localization during different stages of infection.

The approach should be systematic, with appropriate controls, including consideration of strain backgrounds and experimental conditions that mimic the host environment during Salmonella infection .

How can protein-protein interactions of YobD be effectively studied?

Investigating protein-protein interactions of membrane proteins like YobD requires specialized techniques that preserve the membrane environment. The following methodologies are recommended:

• Cross-linking mass spectrometry (XL-MS): This technique involves chemical cross-linking of proteins in their native environment followed by mass spectrometric analysis. For YobD, formaldehyde or DSP (dithiobis(succinimidyl propionate)) can be used as crosslinkers due to their membrane permeability.

• Bacterial two-hybrid systems: Modified for membrane proteins, these systems can identify potential interaction partners in vivo.

• Co-immunoprecipitation with membrane solubilization: Using mild detergents like DDM (n-dodecyl β-D-maltoside) or digitonin to solubilize membranes while preserving protein-protein interactions.

• Proximity-based labeling: BioID or APEX2 fusion proteins can identify proteins in close proximity to YobD in living cells.

• Surface plasmon resonance (SPR): For investigating interactions between purified YobD and potential binding partners in reconstituted membrane environments.

Each method has specific advantages and limitations, and researchers should employ multiple complementary approaches to build a comprehensive interaction network for YobD .

How does YobD compare structurally and functionally to homologous proteins in other bacterial species?

Comparative analysis of YobD requires both bioinformatic and experimental approaches:

• Sequence alignment and phylogenetic analysis: Multiple sequence alignment of YobD with homologs from other bacterial species reveals conserved domains and evolutionary relationships.

• Structural prediction and comparison: Using homology modeling tools like AlphaFold or Phyre2 to predict the tertiary structure of YobD and compare it with known structures of homologous proteins.

• Functional complementation: Express YobD homologs from other species in ΔyobD S. enteritidis to assess functional conservation.

• Expression pattern analysis: Compare expression patterns of yobD and its homologs under various growth conditions and stress responses across bacterial species.

While specific information about YobD homologs is limited in the provided search results, researchers commonly find that membrane proteins with similar structures may have divergent functions depending on the bacterial species and their particular ecological niches or pathogenic strategies.

What protocols are recommended for reconstitution of lyophilized YobD protein?

For optimal reconstitution of lyophilized YobD protein, follow this methodological approach:

• Pre-reconstitution preparation:

  • Briefly centrifuge the vial containing lyophilized YobD to bring contents to the bottom

  • Allow the vial to reach room temperature before opening

• Reconstitution procedure:

  • Add deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL

  • Gently mix by rotating the vial rather than vortexing to avoid protein denaturation

  • Allow the protein to rehydrate completely for 15-30 minutes at room temperature

• Post-reconstitution processing:

  • For long-term storage, add glycerol to a final concentration of 50%

  • Aliquot the reconstituted protein to minimize freeze-thaw cycles

  • Perform quality control testing (e.g., SDS-PAGE) to confirm protein integrity

• Experimental considerations:

  • For membrane protein studies, consider adding compatible detergents at concentrations above their critical micelle concentration (CMC)

  • Verify protein functionality using appropriate activity assays before experimental use

This protocol maintains protein stability while minimizing aggregation and denaturation that can occur during the reconstitution process .

What experimental approaches can verify the structural integrity of recombinant YobD after purification?

To ensure that purified recombinant YobD maintains its structural integrity, researchers should employ a combination of biophysical and biochemical techniques:

• Circular Dichroism (CD) Spectroscopy: Provides information about secondary structure content (α-helices, β-sheets) and can be compared to theoretical predictions based on the amino acid sequence.

• Size Exclusion Chromatography (SEC): Assesses the homogeneity of the protein preparation and detects aggregation or degradation.

• Dynamic Light Scattering (DLS): Measures the hydrodynamic radius of particles in solution, helping to identify protein aggregates.

• Limited Proteolysis: Properly folded proteins often show resistance to proteolytic digestion at specific sites, creating a characteristic digestion pattern.

• Thermal Shift Assays: Measures protein stability by monitoring unfolding in response to increasing temperature.

• Native PAGE: Evaluates protein homogeneity under non-denaturing conditions.

• Functional Assays: Although specific for YobD functions that may not be fully characterized, binding assays with known interaction partners can indicate proper folding.

Data from these techniques should be analyzed collectively to provide comprehensive validation of structural integrity before proceeding with functional studies .

How can researchers study the cellular localization of YobD in Salmonella enteritidis?

To investigate the cellular localization of YobD in Salmonella enteritidis, researchers should employ multiple complementary approaches:

• Immunofluorescence microscopy:

  • Generate specific antibodies against YobD or use anti-His antibodies for tagged versions

  • Fix bacteria with paraformaldehyde (2-4%) to preserve membrane structure

  • Permeabilize cells selectively using detergents like Triton X-100 (0.1-0.5%)

  • Visualize using confocal or super-resolution microscopy

• Fluorescent protein fusions:

  • Generate C- or N-terminal fusions with fluorescent proteins (GFP, mCherry)

  • Ensure the fusion doesn't disrupt membrane localization by comparing with immunolocalization

  • Use time-lapse microscopy to monitor dynamic localization during cell cycle or stress

• Subcellular fractionation:

  • Separate bacterial cellular components (cytoplasm, inner membrane, outer membrane)

  • Analyze fractions by Western blotting using anti-YobD antibodies

  • Include controls for each cellular compartment (e.g., OmpA for outer membrane)

• Electron microscopy with immunogold labeling:

  • Provides high-resolution localization at the ultrastructural level

  • Use gold-conjugated secondary antibodies against anti-YobD primary antibodies

• Protease accessibility assays:

  • Determine membrane topology by testing protease accessibility in intact cells versus permeabilized cells

This multi-method approach provides robust verification of YobD localization while minimizing artifacts associated with any single technique.

What bioinformatic approaches can predict YobD function based on its sequence?

To predict YobD function using bioinformatic approaches, researchers should implement a comprehensive analysis pipeline:

• Sequence homology analysis:

  • BLAST against non-redundant protein databases to identify homologs

  • Multiple sequence alignment to identify conserved residues

  • Phylogenetic analysis to understand evolutionary relationships

• Domain and motif prediction:

  • InterProScan to identify conserved domains

  • MOTIF Search for functional motifs

  • TPRpred for detection of tetratricopeptide repeats or other structural features

• Structural prediction:

  • Transmembrane topology prediction (TMHMM, Phobius)

  • Secondary structure prediction (PSIPRED)

  • 3D structure modeling (AlphaFold2, I-TASSER)

• Genomic context analysis:

  • Investigation of gene neighborhood conservation

  • Operon prediction to identify functionally related genes

  • Analysis of co-occurrence patterns across bacterial genomes

• Protein-protein interaction prediction:

  • Co-expression analysis

  • Interface prediction tools (PIER, ProMate)

  • Molecular docking simulations with potential partners

• Integration of multiple prediction methods:

  • Consensus function prediction using COFACTOR or similar tools

  • Machine learning approaches incorporating multiple features

This multi-layered approach provides greater confidence in functional predictions than any single method alone .

What methods are appropriate for investigating YobD in the context of bacterial stress responses?

To investigate YobD's potential role in bacterial stress responses, researchers should employ:

• Expression analysis under stress conditions:

  • qRT-PCR to measure yobD transcript levels under various stresses (oxidative, acidic, osmotic, nutritional)

  • Western blotting to monitor YobD protein levels during stress

  • Transcriptomics (RNA-seq) to place yobD in the context of global stress responses

• Phenotypic characterization of yobD mutants:

  • Growth curves of wild-type vs. ΔyobD strains under stress conditions

  • Survival assays following exposure to antimicrobial compounds

  • Competition assays between wild-type and mutant strains under stress

• Stress-specific functional assays:

  • Measurement of reactive oxygen species (ROS) accumulation

  • Membrane integrity assays using fluorescent dyes

  • Determination of intracellular pH in response to acid stress

• Protein modification analysis:

  • Post-translational modifications of YobD during stress (phosphorylation, acetylation)

  • Changes in YobD localization under stress conditions

  • Alterations in protein-protein interactions during stress

• Comparative analysis with known stress response systems:

  • Epistasis experiments with genes in established stress response pathways

  • Double mutant analyses to identify genetic interactions

This systematic approach, similar to methodologies used in studying YafD's role in DNA repair , will help elucidate YobD's specific contributions to stress adaptation in Salmonella enteritidis.

How can researchers develop and validate antibodies against YobD for experimental applications?

Developing and validating antibodies against YobD requires a methodical approach:

• Antigen design and production:

  • Identify antigenic regions using epitope prediction algorithms

  • Consider using recombinant full-length His-tagged YobD

  • Alternatively, use synthesized peptides from predicted antigenic regions

  • Ensure proper folding of antigens, particularly for conformational epitopes

• Antibody production:

  • Polyclonal antibodies: Immunize rabbits or other animals with purified antigen

  • Monoclonal antibodies: Screen hybridoma clones for specificity and sensitivity

  • Recombinant antibodies: Phage display selection against YobD

• Rigorous validation procedures:

  • Western blot analysis using:

    • Purified recombinant YobD as positive control

    • Wild-type Salmonella enteritidis lysates

    • ΔyobD knockout strain lysates as negative control

    • Cross-reactivity testing with related bacterial species

  • Immunoprecipitation efficiency testing

  • Immunofluorescence specificity validation using:

    • Wild-type vs. ΔyobD strains

    • Peptide competition assays

• Validation data documentation:

This comprehensive validation ensures antibody specificity and reliability for experimental applications in YobD research .

What methodologies are recommended for studying YobD interactions with host cells during infection?

To investigate interactions between YobD and host cells during Salmonella infection, researchers should employ a multi-faceted approach:

• Infection models:

  • In vitro: Human intestinal epithelial cell lines (Caco-2, HT-29) or macrophage cell lines (THP-1, RAW264.7)

  • Ex vivo: Primary intestinal organoids or explants

  • In vivo: Mouse models of Salmonella infection

• YobD detection during infection:

  • Immunofluorescence microscopy to visualize YobD localization

  • Bacterial transcriptomics to monitor yobD expression during infection

  • Reporter fusions (yobD promoter-GFP) to track expression in real-time

• Functional analysis techniques:

  • Compare wild-type and ΔyobD mutant strains for:

    • Invasion efficiency

    • Intracellular survival

    • Host cell response modifications

  • Complementation with wild-type yobD to confirm phenotypes

• Host interaction partners identification:

  • Bacterial adenylate cyclase two-hybrid system

  • Crosslinking followed by immunoprecipitation and mass spectrometry

  • Yeast two-hybrid screening against host protein libraries

• Host response analysis:

  • Transcriptomics/proteomics of infected host cells

  • Cytokine/chemokine profiling

  • Signaling pathway activation assessment

This approach is similar to methodologies used to study other Salmonella virulence factors, such as SseB, which has been investigated as a vaccine candidate .

How can structure-function relationships of YobD be experimentally determined?

Determining structure-function relationships for YobD requires a systematic approach combining structural analysis with functional assays:

• High-resolution structural determination:

  • X-ray crystallography of purified YobD

  • Cryo-electron microscopy for membrane-embedded YobD

  • NMR spectroscopy for dynamic structural elements

  • If challenging, use computational structure prediction (AlphaFold2) as a starting point

• Site-directed mutagenesis strategy:

  • Identify conserved residues through multiple sequence alignment

  • Target predicted functional sites based on structural analysis

  • Create alanine scanning libraries or targeted mutations

  • Express mutant proteins in ΔyobD backgrounds

• Functional characterization of mutants:

  • Growth phenotypes under various conditions

  • Bacterial fitness assays in competition experiments

  • Membrane localization and topology verification

  • Protein-protein interaction profiling

• Structure-guided domain analysis:

  • Generate truncation variants targeting specific domains

  • Create domain-swapped chimeras with homologous proteins

  • Assess domain-specific contributions to function

• Correlation analysis:

  • Map functional defects to structural features

  • Identify critical residues and structural motifs

  • Develop predictive models for structure-function relationships

This methodological framework allows researchers to systematically identify crucial structural elements of YobD that contribute to its biological function, similar to approaches used in studying other Salmonella membrane proteins involved in pathogenesis .