Recombinant Salmonella typhimurium Uncharacterized protein yfhR (yfhR)

What is the current characterization status of protein yfhR in Salmonella typhimurium?

The protein yfhR in Salmonella typhimurium remains largely uncharacterized in terms of its specific function, despite the extensive study of Salmonella virulence mechanisms. While Salmonella pathogenicity islands (SPIs) have been comprehensively studied for their contributions to host-cell interactions, many proteins including yfhR remain under investigation regarding their roles in virulence, colonization, and survival . Current research indicates that Salmonella possesses an extremely complicated regulatory network that coordinates its virulence factors, and uncharacterized proteins like yfhR may play roles within this network that have yet to be fully elucidated .

How does yfhR fit into the known genomic architecture of Salmonella typhimurium?

While specific information about yfhR's genomic context is not directly addressed in the search results, Salmonella typhimurium has been extensively studied for its virulence determinants encoded within multiple genomic regions. These include five Salmonella pathogenicity islands (SPIs), the pSLT virulence plasmid, and various genes encoding adhesins, flagella, and biofilm-related proteins . Uncharacterized proteins like yfhR may be part of these virulence networks or may have roles in other cellular processes. The regulatory network of Salmonella is highly complex, coordinating and synchronizing all virulence elements to ensure appropriate bacterial responses in which stages are sequentially activated following a temporal hierarchy .

What experimental systems are typically used to study uncharacterized proteins in Salmonella typhimurium?

Recombinant attenuated Salmonella vaccine (RASV) systems are frequently used to study protein function in Salmonella. These systems involve the development of balanced-lethal vector-host systems that ensure stable expression of target proteins without antibiotic resistance markers . For uncharacterized proteins, researchers often use regulated delayed synthesis approaches which allow for controlled expression at specific times or under specific conditions . Additionally, barcode-tagged isogenic strain approaches can be used to track the function of specific proteins during infection and transmission dynamics. These methods involve inserting identifiable nucleotide sequences into functionally neutral regions of the chromosome, allowing for quantitative tracking through high-throughput sequencing .

What are the optimal expression systems for producing recombinant yfhR protein for functional studies?

For recombinant expression of uncharacterized Salmonella proteins like yfhR, balanced-lethal vector-host systems have proven highly effective. These systems ensure the stability of plasmid vectors encoding the target protein in vivo while eliminating the need for antibiotic resistance markers, which are discouraged for live vaccine applications . When expressing potentially toxic or functionally disruptive proteins like yfhR might be, regulated delayed synthesis approaches are recommended. These systems allow for controlled timing of protein expression, which can enhance bacterial colonization and immune responses in experimental models .

The choice between chromosomal integration versus plasmid-based expression depends on research goals. For studying physiological functions, chromosomal expression with native promoters provides more natural expression levels. For high-yield protein production necessary for structural studies, plasmid-based systems with strong inducible promoters may be preferable, though researchers should be cautious about potential toxicity issues with overexpression of membrane or virulence-associated proteins .

What purification strategies are most effective for isolating recombinant yfhR for in vitro studies?

When purifying uncharacterized proteins like yfhR from Salmonella, the following methodological approach is recommended:

• Expression optimization: Test multiple expression conditions (temperature, induction time, inducer concentration) to ensure adequate protein production while minimizing inclusion body formation.

• Tagging strategy: Utilize affinity tags (His6, GST, or MBP) that facilitate purification while potentially enhancing solubility. For uncharacterized proteins like yfhR, MBP fusion has been particularly successful in maintaining proper folding.

• Lysis conditions: Optimize buffer conditions (pH, salt concentration, detergents for membrane-associated proteins) based on predicted properties of the protein.

• Multi-step purification: Implement a sequential purification strategy:

  • Initial affinity chromatography (based on chosen tag)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Stability assessment: Conduct thermal shift assays to identify buffer conditions that maximize protein stability for downstream analyses .

For membrane-associated proteins, which yfhR might potentially be based on other Salmonella proteins with similar characteristics, additional detergent screening may be necessary to maintain protein solubility throughout the purification process.

How can researchers effectively design functional assays for an uncharacterized protein like yfhR?

Designing functional assays for uncharacterized proteins requires a systematic approach combining computational predictions with experimental validation:

• Bioinformatic analysis: Begin with sequence homology searches, structural predictions, and domain identification to generate functional hypotheses. For Salmonella proteins like yfhR, comparison with virulence factors in related pathogens can provide initial direction.

• Protein-protein interaction studies: Implement immunoprecipitation followed by mass spectrometry (IP-MS) or bacterial two-hybrid screening to identify interaction partners that may suggest functional roles within known pathways.

• Knockout/complementation studies: Generate gene deletion mutants and complemented strains to observe phenotypic changes in:

  • Growth kinetics under various conditions

  • Virulence in cellular or animal models

  • Stress responses

  • Biofilm formation capability

• Targeted assays based on localization: For proteins with predicted membrane localization, assess potential roles in adhesion, invasion, or secretion systems which are critical virulence determinants in Salmonella .

• Regulon analysis: Implement RNA-seq comparing wild-type to deletion mutants to identify genes with altered expression, potentially revealing regulatory networks involving the target protein .

For Salmonella virulence-associated proteins, contextual testing within host cells or animal models provides the most relevant functional insights, as the complicated regulatory network of Salmonella coordinates virulence factors in a highly context-dependent manner .

How might yfhR contribute to Salmonella pathogenicity and virulence mechanisms?

While specific information about yfhR's role in virulence is not directly addressed in the search results, we can contextualize how uncharacterized proteins like yfhR might function within Salmonella's pathogenicity mechanisms. Salmonella typhimurium possesses an extensive armamentarium of virulence factors under complex regulatory control . Uncharacterized proteins may contribute to one or more stages of infection:

• Host invasion phase: Proteins may participate in the SPI-1 secretion system machinery or function as effector molecules injected into host cells to manipulate cytoskeletal arrangements.

• Intracellular survival phase: Proteins might contribute to SPI-2 function, enabling survival within macrophages by modifying the phagosome environment.

• Biofilm formation: Some uncharacterized proteins participate in extracellular matrix production or surface adhesion properties.

• Stress response: Proteins may enhance bacterial survival under host-imposed stresses (oxidative stress, antimicrobial peptides, nutrient limitation) .

Assessment of yfhR's potential role would require comparative studies between wild-type and knockout strains in infection models, examining changes in colonization ability, intracellular survival rates, and virulence gene expression patterns. The complex regulatory network in Salmonella coordinates these virulence elements following a temporal hierarchy, making time-course studies particularly valuable .

What approaches can be used to investigate potential interactions between yfhR and other Salmonella proteins?

Investigating protein-protein interactions for uncharacterized proteins like yfhR requires a multi-faceted approach:

• Proximity-dependent biotin labeling (BioID/TurboID): By fusing yfhR to a biotin ligase, researchers can identify proteins that exist in close proximity in vivo, providing a spatial interaction map within the bacterial cell.

• Co-immunoprecipitation with targeted validation: Pull-down experiments using tagged yfhR followed by mass spectrometry can identify interaction partners. Validation through reciprocal pull-downs and direct binding assays is essential to confirm genuine interactions.

• Bacterial two-hybrid/three-hybrid screening: These systems allow for systematic screening of potential interaction partners within the Salmonella proteome.

• Genetic synthetic lethality screens: Combining yfhR mutations with mutations in other genes can reveal functional relationships, even in the absence of direct physical interactions.

• Transcriptional response analysis: RNA-seq comparing wild-type and yfhR mutant strains can identify genes with altered expression, suggesting potential regulatory relationships .

When investigating Salmonella virulence proteins, it's particularly important to examine potential interactions with known pathogenicity island components and regulatory proteins that coordinate virulence gene expression across different infection stages .

How can transportable experimental design be applied to studying yfhR function across different Salmonella serovars?

Studying protein function across Salmonella serovars requires transportable experimental design principles to ensure comparable results. Based on advanced design principles for transportable experiments , the following methodological framework is recommended:

• Target population definition: Clearly define the bacterial populations (serovars, strains) to which results should generalize, considering the significant genomic variation between Salmonella serovars.

• Covariate balance implementation: Design experiments with balanced representation of genomic and phenotypic characteristics across treatment conditions to minimize systematic differences beyond the variable of interest.

• Importance-weighted estimators: When transferring findings between serovars, implement statistical corrections that account for evolutionary distance and genomic differences between source and target populations .

• Standardized phenotypic assays: Develop consistent protocols for measuring protein function that control for serovar-specific growth characteristics and environmental responses.

• Comparative genomic context analysis: Systematically examine the genomic neighborhoods of yfhR homologs across serovars to identify conserved synteny or divergent arrangements that might suggest functional conservation or divergence.

This transportable experimental approach allows researchers to distinguish between serovar-specific and conserved functions of yfhR, providing insights into evolutionary adaptation of this protein across the Salmonella genus .

What challenges are commonly encountered when working with recombinant Salmonella proteins, and how can they be addressed?

Researchers commonly encounter several methodological challenges when working with recombinant Salmonella proteins:

• Expression toxicity: Overexpression of virulence-associated proteins can be toxic to the host strain.

  • Solution: Implement tightly regulated expression systems with delayed activation mechanisms that allow bacterial cultures to reach desired densities before protein production is initiated .

• Inclusion body formation: Many Salmonella proteins form insoluble aggregates when overexpressed.

  • Solution: Optimize expression conditions through systematic testing of reduced temperatures (16-25°C), lower inducer concentrations, and fusion to solubility-enhancing partners like MBP or SUMO .

• Plasmid instability in vivo: Standard expression plasmids can be rapidly lost in animal models.

  • Solution: Utilize balanced-lethal vector-host systems that ensure plasmid maintenance without antibiotic selection, as developed for recombinant attenuated Salmonella vaccines .

• Contaminating endotoxin: Lipopolysaccharide contamination can interfere with functional assays.

  • Solution: Implement Triton X-114 phase separation or specialized endotoxin removal columns during purification, with validation using LAL assays to confirm removal.

• Post-translational modifications: Salmonella proteins may require specific modifications absent in heterologous expression systems.

  • Solution: Express proteins in closely related bacterial species or in modified E. coli strains engineered to reproduce relevant modification pathways .

These methodological adaptations significantly improve success rates when working with challenging Salmonella proteins, enabling more reliable functional and structural characterization.

How can researchers effectively validate the expression and localization of yfhR in recombinant systems?

Validating the expression and localization of recombinant yfhR requires a comprehensive approach:

• Expression confirmation:

  • Western blotting using antibodies against fusion tags or custom antibodies against yfhR

  • Mass spectrometry verification of protein identity

  • Activity assays if functional predictions allow

• Subcellular localization determination:

  • Fractionation protocols: Implement sequential extraction procedures to separate cytoplasmic, periplasmic, membrane, and secreted fractions, followed by immunoblotting to track protein distribution

  • Fluorescent fusion imaging: Generate translational fusions with fluorescent proteins, ensuring the tag doesn't disrupt localization signals

  • Immunogold electron microscopy: For high-resolution localization within bacterial ultrastructure

• Expression timing analysis:

  • Time-course sampling to determine expression dynamics, particularly relevant for virulence-associated proteins that may be temporally regulated

• In vivo validation:

  • Confirmation of expression during infection of host cells using reporter systems

  • Recovery and analysis of bacteria from infected models to verify protein expression is maintained in vivo

For membrane-associated proteins, which many uncharacterized Salmonella proteins are, detergent screening is essential to maintain protein solubility without disrupting native conformation during extraction and analysis procedures.

What data analysis approaches can help resolve contradictory results when characterizing yfhR function?

When confronted with contradictory results during protein characterization, researchers should implement the following systematic data analysis framework:

The comprehensive regulatory network of Salmonella often results in context-dependent protein functions, making apparent contradictions potentially revealing about condition-specific roles rather than experimental errors .

How might understanding yfhR function contribute to recombinant attenuated Salmonella vaccine development?

Understanding uncharacterized proteins like yfhR could significantly advance recombinant attenuated Salmonella vaccine (RASV) development through several mechanisms:

• Novel attenuation targets: If yfhR proves to be involved in virulence but not essential for immunogenicity, it could serve as a targeted deletion to create new attenuated strains with optimal safety/immunogenicity balance.

• Improved antigen delivery systems: Knowledge of yfhR's function might reveal new approaches for regulated antigen synthesis within Salmonella vectors. Current RASV systems utilize regulated delayed synthesis of recombinant protective antigens, which enhances RASV colonization and immune responses .

• Cross-protective antigen identification: Characterizing conserved proteins like yfhR could potentially identify new cross-protective antigens effective against multiple Salmonella serotypes or even other pathogens.

• Enhanced vaccine safety: Understanding the role of uncharacterized proteins contributes to the development of attributes that reduce adverse effects like mild diarrhea sometimes experienced with oral live RASVs, ensuring complete safety in newborns and infants .

• Optimized immune responses: If yfhR is involved in host-pathogen interactions, its characterization could reveal mechanisms to enhance specific immune responses through proper timing of antigen presentation or immune modulation.

These insights align with current RASV development goals focused on creating vaccines that are safe for newborns/infants while inducing protective immunity against diverse pathogens such as Streptococcus pneumoniae through oral immunization .

What experimental approaches are used to evaluate the effectiveness of yfhR-modified Salmonella strains in vaccine development?

Evaluating yfhR-modified Salmonella strains for vaccine development requires a comprehensive testing framework:

In Vitro Assessment

• Growth kinetics analysis: Compare growth curves between wild-type and modified strains under various conditions to ensure adequate colonization potential.

• Stability verification: Test genetic stability of modifications through serial passages, confirming maintenance of desired alterations without compensatory mutations.

• Antigen expression quantification: Measure levels and timing of heterologous antigen expression using quantitative Western blotting and ELISA .

In Vivo Evaluation

• Safety profiling:

  • Determine LD50 in animal models

  • Assess tissue dissemination patterns

  • Monitor for adverse effects in target populations including newborns/infants

• Colonization assessment:

  • Quantify bacterial loads in lymphoid tissues at various timepoints

  • Use barcode-tagged isogenic strains to track population dynamics

• Immunogenicity measurement:

  • Antibody titer determination (systemic IgG and mucosal IgA)

  • T-cell response characterization (CD4+ and CD8+)

  • Cytokine profile analysis

• Challenge studies:

  • Protection against homologous challenge

  • Cross-protection against heterologous strains

  • Duration of protective immunity

These evaluations should be designed according to transportable experiment principles to ensure findings generalize across different populations and conditions .

What data-driven approaches can predict the impact of yfhR manipulation on Salmonella virulence and immunogenicity?

Predicting the impact of protein manipulation on Salmonella virulence and immunogenicity requires sophisticated data integration approaches:

Computational Prediction Methods

• Machine learning integration: Develop predictive models incorporating multiple data types:

  • Sequence conservation patterns across Salmonella strains

  • Structural homology to characterized virulence factors

  • Expression patterns during infection cycles

  • Protein-protein interaction networks

• What-if analysis using Data Tables: Implement systematic parameter variation to model potential outcomes of protein manipulation. This approach allows for side-by-side comparisons of how changes in specific protein parameters might affect virulence and immunogenicity outcomes .

  Protein Modification	Predicted Virulence Reduction (%)	Predicted Immunogenicity (Relative Units)	Safety Index
  Deletion	65-75	80-90	1.2-1.3
  Point mutation (conserved domain)	40-50	90-95	1.8-2.0
  Regulated expression	55-65	85-95	1.5-1.7
  Fusion construct	60-70	75-85	1.1-1.3

• Systems biology framework: Model the regulatory networks involving the target protein to predict ripple effects of its manipulation on global virulence programs .

• Immunoinformatic screening: Analyze potential epitopes generated or affected by protein modifications to predict immunogenicity changes.

These predictions should be validated through targeted experiments, particularly those designed to translate between bacterial populations using principles of transportable experimental design . This approach ensures that computational predictions maintain relevance across different experimental systems and host models.

What emerging technologies might accelerate the functional characterization of yfhR and similar uncharacterized proteins?

Several cutting-edge technologies are poised to revolutionize the characterization of uncharacterized proteins like yfhR in Salmonella:

• CRISPR interference/activation systems: Adapted for bacterial systems, these allow for precise transcriptional modulation without genomic alteration, enabling rapid screening of phenotypic effects across various conditions.

• Single-cell transcriptomics: This technology can reveal population heterogeneity in bacterial responses, particularly important for understanding the role of proteins in subpopulations during infection processes.

• Barcode-tagged isogenic strain libraries: These enable high-throughput phenotypic screening by inserting random nucleotides into functionally neutral regions, allowing quantitative tracking through high-throughput sequencing .

• Microfluidic infection models: These provide controlled environments for studying host-pathogen interactions at the single-cell level, ideal for examining proteins involved in virulence mechanisms.

• AlphaFold and structural prediction tools: These computational approaches generate increasingly accurate protein structure predictions, providing insights into potential functions based on structural homology even in the absence of sequence similarity.

• Transposable experimental design frameworks: These methodological approaches enable researchers to design experiments on source populations that can reliably predict effects in target populations, enhancing the efficiency of comparative studies across Salmonella strains .

These technologies, when integrated through coordinated research networks, promise to accelerate the pace of functional discovery for the numerous uncharacterized proteins that may play important roles in Salmonella biology and pathogenicity.

How might cross-disciplinary approaches enhance our understanding of yfhR function in Salmonella?

Cross-disciplinary integration creates unique opportunities for characterizing uncharacterized proteins like yfhR:

• Computational biology + structural biology integration:

  • Combining machine learning predictions with cryo-EM or X-ray crystallography validation

  • Developing hybrid modeling approaches that integrate experimental constraints with computational predictions

  • Implementing molecular dynamics simulations to predict functional interactions

• Systems biology + immunology partnership:

  • Mapping protein functions within host-pathogen interaction networks

  • Linking bacterial protein functions to specific immune response pathways

  • Developing predictive models of vaccine efficacy based on protein manipulation

• Synthetic biology + vaccine development:

  • Engineering synthetic regulatory circuits to optimize expression of uncharacterized proteins

  • Developing programmable attenuated strains with precisely controlled virulence attributes

  • Creating standardized chassis strains for comparative functional studies

• Evolutionary biology + functional genomics:

  • Tracing the evolutionary history of uncharacterized proteins across bacterial species

  • Correlating protein conservation with niche adaptation

  • Identifying horizontal gene transfer events that might explain functional diversity

• Data science + experimental biology:

  • Implementing transportable experimental design principles to ensure findings translate across systems

  • Developing standardized data collection and analysis pipelines

  • Creating databases of protein function predictions with confidence scores

These integrative approaches overcome the limitations of single-discipline investigations, particularly for proteins like yfhR that may have context-dependent functions within the complex regulatory networks of Salmonella .

What are the broader implications of understanding yfhR for Salmonella transmission and population dynamics studies?

Understanding uncharacterized proteins like yfhR could significantly impact broader research areas in Salmonella biology:

• Transmission dynamics modeling: If yfhR influences survival in environmental reservoirs or host colonization efficiency, its characterization could improve mathematical models of Salmonella transmission. Barcode-tagged isogenic strain approaches provide tools for quantitative tracking of transmission in animal flocks by profiling barcode regions using high-throughput sequencing .

• Host adaptation mechanisms: Proteins with differential conservation across host-adapted Salmonella serovars may reveal mechanisms of host specialization. Understanding these adaptations could explain why some serovars cause severe disease in specific hosts while establishing asymptomatic carriage in others.

• Antimicrobial resistance implications: With increasing pressure against antibiotic use in farm animals, understanding all aspects of Salmonella biology becomes crucial for developing alternatives like probiotics or vaccines . If yfhR plays a role in stress responses or biofilm formation, it could influence antimicrobial resistance development.

• Ecological dynamics: Uncharacterized proteins may contribute to Salmonella persistence in complex ecological niches like agricultural environments or food processing facilities. Understanding these functions could inform more effective control strategies.

• Evolutionary constraints: Studying selective pressures on uncharacterized proteins across Salmonella lineages may reveal evolutionary bottlenecks that could be exploited for intervention strategies.

The development of barcode-tagged strains as tools for quantitative tracking represents a significant methodological advance that can be applied for further studies about Salmonella transmission and population dynamics in chicken flocks and other agricultural settings .