Recombinant Salmonella dublin UPF0266 membrane protein yobD (yobD)

What is the UPF0266 membrane protein YobD and what is its significance in Salmonella dublin?

The UPF0266 membrane protein YobD belongs to a family of uncharacterized proteins found in various bacterial species. While the computed structure model available in the RCSB PDB (AF_AFB7L6U8F1) provides insights into the protein's structure in Escherichia coli strain 55989, its specific role in Salmonella dublin requires further investigation .

The significance of YobD in S. dublin may be related to bacterial persistence and adaptation, particularly given that S. dublin is a cattle-adapted serovar that causes both intestinal and systemic infections in bovine hosts while also posing a serious threat to human health . Research into membrane proteins like YobD is critical for understanding how S. dublin persists in cattle populations for extended periods, as demonstrated by phylogenetic analyses showing persistence of closely related isolates within the same herd for over 20 years .

How does S. dublin differ from other Salmonella serovars in terms of genetic composition and host adaptation?

S. dublin is a cattle-adapted serovar with a relatively conserved genome compared to other Salmonella serovars. Phylogenetic analyses of 197 Danish cattle isolates from 1996 to 2016 revealed three major clades corresponding to distinct geographical regions, suggesting limited genetic diversity within the S. dublin population .

Despite being highly clonal, S. dublin can acquire variation through plasmids carrying additional virulence and resistance genes, which may enhance its adaptability to changing environments . This adaptation ability is evidenced by multiple reports of resistance plasmids in S. dublin from cattle and humans across different countries, including Germany, the United States, Canada, and Peru .

The host adaptation of S. dublin to cattle is a key characteristic that distinguishes it from many other Salmonella serovars, enabling it to cause both intestinal and systemic infections in bovine hosts while maintaining the ability to infect humans .

What are the critical factors in designing recombinant expression systems for membrane proteins like YobD from S. dublin?

Designing recombinant expression systems for membrane proteins like YobD requires careful consideration of several critical factors:

• Expression host selection: The choice between prokaryotic (E. coli) or eukaryotic expression systems depends on protein complexity and post-translational modification requirements. For bacterial membrane proteins like YobD, E. coli often serves as an appropriate host, though codon optimization may be necessary .

• Vector design: Vectors should include appropriate promoters, selection markers, and fusion tags (like His-tags) to facilitate purification. For membrane proteins, fusion partners that enhance solubility or membrane targeting may be beneficial .

• Expression conditions: Membrane proteins often require specialized conditions to prevent misfolding or aggregation. Lower expression temperatures (16-25°C), specific media compositions, and induction parameters must be optimized .

• Solubilization strategy: Effective extraction from membranes requires careful selection of detergents that maintain protein structure and function. Different detergents should be screened for optimal results .

• Purification approach: Multi-step purification strategies typically involve affinity chromatography followed by size exclusion chromatography. The entire process must maintain the native conformation of the membrane protein .

When designing these systems, researchers should employ a systematic experimental design approach with clearly defined dependent and independent variables to optimize expression and purification conditions .

How can recombination analysis be applied to study the evolution of proteins like YobD in S. dublin populations?

Recombination analysis for studying the evolution of proteins like YobD in S. dublin populations involves several methodological approaches:

• Whole genome sequencing: As demonstrated in the study of 197 isolates of S. Dublin from Danish cattle, WGS provides the foundation for identifying potential regions of recombination occurring throughout the genome .

• Phylogenetic analysis: Construction of phylogenetic trees helps identify distinct clades and evolutionary relationships between strains. For S. dublin, this revealed three major clades corresponding to distinct geographical regions .

• Single Nucleotide Polymorphism (SNP) detection: Identifying SNPs across multiple isolates helps track evolutionary changes. The presence of >106 SNPs separating different S. dublin populations suggests distinct evolutionary paths rather than continuous evolution .

• Plasmid analysis: Investigation of plasmid acquisition, which can carry resistance genes and virulence factors. In S. dublin, certain clades were found to harbor specific plasmids (49-kb and 87-kb) carrying antibiotic resistance genes .

• Recombination detection algorithms: Software tools specifically designed to detect genetic recombination events can identify regions of horizontal gene transfer that may affect protein evolution.

The application of these approaches revealed that S. dublin evolves primarily through point mutations in the whole genome and by single gene gain and loss, with plasmid acquisition playing a role in adaptation to changing environments .

What experimental design would be most appropriate for studying the function of recombinant YobD protein in S. dublin?

An appropriate experimental design for studying the function of recombinant YobD protein in S. dublin would include the following components:

• Research question definition: Clearly define variables - independent variable (YobD expression/modification) and dependent variables (bacterial phenotype, virulence, persistence) .

• Hypothesis formulation: Develop a specific, testable hypothesis about YobD function based on structural predictions and homology to characterized proteins .

• Gene knockout and complementation:

  • Generate a YobD knockout strain in S. dublin

  • Create complementation strains with wild-type and modified YobD variants

  • Include appropriate controls (wild-type and empty vector)

• Phenotypic characterization:

  Experimental group	Treatment	Measurements
  Wild-type S. dublin	No modification	Growth rate, stress response, virulence
  ΔyobD S. dublin	YobD knockout	Growth rate, stress response, virulence
  ΔyobD + pYobD	Complementation with wild-type YobD	Growth rate, stress response, virulence
  ΔyobD + pYobD-mutant	Complementation with modified YobD	Growth rate, stress response, virulence

• Stress response assays: Test resistance to various stressors (pH, temperature, antimicrobials) to elucidate protein function .

• Localization studies: Use fluorescent tags or antibodies to confirm membrane localization and potential interaction partners.

• In vitro and in vivo virulence assays: Assess the impact of YobD modification on bacterial invasion, persistence, and pathogenicity in cell culture and animal models.

This between-subjects experimental design with multiple controls allows for systematic investigation of YobD function while controlling for extraneous variables that could influence the results .

What are the key considerations for optimizing purification of recombinant membrane proteins like YobD?

Optimizing purification of recombinant membrane proteins like YobD requires addressing several key considerations:

• Membrane extraction optimization:

  • Screening of detergent types (ionic, non-ionic, zwitterionic)

  • Detergent concentration optimization

  • Buffer composition adjustment (pH, salt concentration, glycerol content)

  • Extraction time and temperature determination

• Affinity chromatography parameters:

  Parameter	Options to test	Considerations
  Tag type	His-tag, GST, MBP	Tag size, position (N/C-terminal), cleavability
  Column matrix	Ni-NTA, Cobalt, Glutathione	Binding capacity, specificity, background
  Elution conditions	Imidazole gradient, pH shift	Protein stability, yield, purity
  Flow rate	0.2-2 ml/min	Binding efficiency vs. processing time

• Secondary purification steps:

  • Size exclusion chromatography for oligomeric state determination

  • Ion exchange chromatography for charge variant separation

  • Optimization of buffer conditions to maintain protein stability

• Detergent exchange or reconstitution:

  • Gradual detergent exchange during purification

  • Reconstitution into lipid nanodiscs or liposomes for functional studies

  • Assessment of protein stability in different membrane-mimetic environments

• Quality control criteria:

  • SDS-PAGE and Western blot for purity assessment

  • Mass spectrometry for identity confirmation

  • Circular dichroism for secondary structure analysis

  • Dynamic light scattering for aggregation evaluation

• Scale-up considerations:

  • Optimization of culture volume and protein expression conditions

  • Adjustment of purification protocols for larger column capacities

  • Implementation of automation where possible

A systematic experimental design approach should be employed, where each variable is tested independently while keeping others constant, followed by optimization of the combined parameters .

How should researchers interpret structural data of membrane proteins like YobD in the context of bacterial adaptation?

Researchers should apply the following analytical framework when interpreting structural data of membrane proteins like YobD in the context of bacterial adaptation:

• Structure-function relationship analysis:

  • Identify conserved structural motifs across related proteins

  • Map potential functional domains based on structural features

  • Compare confidence scores (like pLDDT scores in AlphaFold models) across different regions to assess reliability of structural predictions

• Evolutionary conservation mapping:

  • Analyze sequence conservation patterns in the context of the 3D structure

  • Identify highly conserved regions that may be functionally important

  • Compare conservation across different bacterial species and strains

• Membrane topology analysis:

  • Determine transmembrane regions and orientation

  • Identify potential interaction interfaces with other membrane components

  • Analyze exposure of functional domains to different cellular compartments

• Structural comparison in different environments:

  • Evaluate structural differences in various membrane compositions

  • Assess how environmental stressors might affect protein conformation

  • Consider how structural features contribute to bacterial persistence in host environments

• Integration with population genomic data:

  • Correlate structural features with phylogenetic clades identified in population studies

  • Assess whether structural variations align with geographic or host-specific adaptations

  • Examine how point mutations or recombination events might impact protein structure and function

For YobD specifically, researchers should note that the available AlphaFold model has a global pLDDT score of 72.34, placing it in the "Confident" category (70 < pLDDT ≤ 90), suggesting reliable structural prediction for most regions but potentially with some uncertainty in specific areas .

What statistical approaches are most appropriate for analyzing the impact of YobD mutations on S. dublin virulence and persistence?

When analyzing the impact of YobD mutations on S. dublin virulence and persistence, researchers should consider these statistical approaches:

• Experimental design considerations:

  • Use a factorial design to test multiple YobD variants simultaneously

  • Include appropriate controls (wild-type, knockout, complemented strains)

  • Determine adequate sample sizes through power analysis

  • Plan for both between-subjects and within-subjects comparisons where appropriate

• Primary statistical methods:

  Data type	Statistical test	Application
  Continuous (normally distributed)	ANOVA, t-tests	Growth rates, biofilm formation
  Non-parametric	Mann-Whitney U, Kruskal-Wallis	When normality assumptions are violated
  Categorical	Chi-square, Fisher's exact	Survival/mortality data
  Time-to-event	Kaplan-Meier, Cox regression	Persistence over time, survival analysis

• Multifactorial analysis approaches:

  • MANOVA for analyzing multiple dependent variables simultaneously

  • Principal Component Analysis (PCA) for dimension reduction in complex datasets

  • Hierarchical clustering to identify patterns across multiple phenotypic measures

• Longitudinal data analysis:

  • Repeated measures ANOVA for time-series data

  • Mixed-effects models to account for individual variation and time effects

  • Growth curve modeling for bacterial persistence studies

• Specialized approaches for genomic-phenotypic correlations:

  • Genome-wide association studies (GWAS) to correlate genetic variations with phenotypic outcomes

  • Bayesian networks to model complex relationships between genetic factors and phenotypes

  • Machine learning approaches for predicting virulence based on genetic signatures

When applying these methods, researchers should carefully control for confounding variables, such as growth conditions, host factors, and experimental batch effects. Multiple test correction (e.g., Bonferroni, Benjamini-Hochberg FDR) should be applied when performing numerous statistical tests to minimize type I errors while maintaining statistical power .

How can recombinant YobD protein be utilized to improve biosecurity measures against S. dublin in cattle herds?

Recombinant YobD protein could potentially enhance biosecurity measures against S. dublin in cattle herds through several research-driven applications:

• Diagnostic tool development:

  • Development of YobD-based ELISA or lateral flow assays for rapid detection

  • Creation of antibody panels targeting YobD epitopes for improved sensitivity

  • Implementation as a biomarker to distinguish between S. dublin strains or clades

• Vaccination strategies:

  • Use as a subunit vaccine component if YobD proves immunogenic

  • Design of attenuated vaccine strains with modified YobD expression

  • Development of DNA vaccines encoding immunogenic YobD epitopes

• Monitoring herd transmission dynamics:

  • Tracking strain-specific YobD variants to monitor transmission between herds

  • Identification of persistent strains based on YobD sequence variations

  • Investigation of geographical distribution patterns of different YobD variants

• Internal biosecurity improvement:

  • Development of targeted interventions based on YobD function in bacterial persistence

  • Implementation of monitoring systems for early detection of persistent strains

  • Design of environment-specific control measures based on YobD structure-function relationships

The research on S. dublin epidemiology has demonstrated that long-term persistence within herds for periods exceeding 20 years is a significant challenge, indicating that improved internal biosecurity measures are essential . Molecular tools based on YobD characterization could provide targeted approaches to address this persistence issue, particularly as phylogenetic analysis has shown herd-specific clustering of S. dublin isolates over extended periods .

What are the potential applications of studying YobD in understanding antimicrobial resistance in S. dublin?

Studying YobD in the context of antimicrobial resistance (AMR) in S. dublin offers several potential applications:

• Membrane permeability and drug efflux:

  • Investigation of YobD's role in membrane structure and permeability

  • Assessment of potential interactions with efflux pump systems

  • Development of membrane-targeting compounds that may overcome resistance mechanisms

• Plasmid-associated resistance connections:

  • Analysis of relationships between YobD expression and plasmid-mediated resistance

  • Investigation of potential regulatory interactions between YobD and plasmid-encoded resistance factors

  • Study of co-evolution patterns between membrane proteins and resistance plasmids

• Biofilm formation and persistence:

  Research focus	Methodological approach	Potential outcomes
  YobD role in biofilm formation	Comparative biofilm assays with wild-type and YobD mutants	Identification of biofilm-associated resistance mechanisms
  Stress response modulation	Gene expression analysis under antibiotic pressure	Understanding adaptive responses involving YobD
  Membrane integrity regulation	Membrane permeability assays with fluorescent dyes	Novel targets for combination therapies

• Clonal distribution of resistance:

  • Correlation of YobD variants with resistance patterns across S. dublin populations

  • Mapping of resistance profiles to phylogenetic clades identified in population studies

  • Tracking the spread of resistance in relation to YobD evolutionary patterns

• Resistance mechanism elucidation:

  • Structure-based investigations of YobD interactions with antimicrobials

  • Identification of potential binding sites or conformational changes affecting drug access

  • Development of structure-based drug design strategies targeting YobD or related proteins

The research on S. dublin has shown that resistance genes are not commonly found in Danish bovine isolates, but specific clades often harbor plasmids carrying resistance genes like bla TEM-1, tetA, strA, and strB . Understanding the relationship between membrane proteins like YobD and these resistance mechanisms could provide valuable insights for addressing the increasing concern of antibiotic resistance in S. dublin infections .