Recombinant Pasteurella haemolytica Integration host factor subunit beta (ihfB)

What is Integration Host Factor (IHF) and what role does the beta subunit play?

Integration Host Factor is a heterodimeric nucleoid-associated protein composed of alpha (IhfA) and beta (IhfB) subunits that plays crucial roles in bacterial nucleoid architecture and genome-wide gene regulation. The beta subunit (IhfB) works in conjunction with IhfA to form the functional heterodimer. IHF acts as an architectural component in many bacterial systems by bending DNA up to 180°, which promotes contact between DNA sequences located upstream and downstream of the IHF-bound site and between proteins attached to those flanking sequences . These sharp bends facilitate the formation of nucleoprotein complexes that are essential for processes like transcription regulation and site-specific recombination.

What is the molecular structure of recombinant IhfB?

Recombinant IhfB typically consists of 94 amino acids (for example, in the Yersinia pestis version) with a sequence that includes: "MTKSELIERL AGQQSHVPAK VVEDAVKEML EHMAGTLAEG ERIEIRGFGS FSLHYRAPRV GRNPKTGDKV ELEGKYVPHF KPGKELRDRA NIYG" . The protein has a molecular weight of approximately 60.8 kDa when expressed as part of a larger recombinant construct . The structure facilitates DNA binding and protein-protein interactions that are critical for its architectural function within bacterial cells.

Why is recombinant P. haemolytica IhfB of interest in vaccine development?

Recombinant P. haemolytica antigens, potentially including IhfB, are of interest in vaccine development because they can elicit protective immune responses against bovine respiratory disease (BRD). Studies have demonstrated that vaccines containing recombinant P. haemolytica components can significantly reduce BRD morbidity (by 20-24%) and mortality (by up to 88-100% for fibrinous pneumonia) . Specifically, P. haemolytica vaccines consisting of outer membrane proteins and genetically attenuated leukotoxin produced by recombinant DNA technology have shown efficacy in protecting cattle from respiratory disease . While the search results don't explicitly connect IhfB to these vaccines, research on recombinant P. haemolytica antigens generally aims to identify components that can provide robust protection.

How does recombinant IhfB influence DNA binding and transcriptional regulation?

Recombinant IhfB, as part of the IHF heterodimer, influences DNA binding and transcriptional regulation by introducing significant structural changes to DNA. The heterodimer bends DNA by up to 180°, creating architecture that promotes contacts between DNA-bound transcription factors and RNA polymerase. This architectural role can have either positive or negative effects on transcription initiation, depending on the distribution of the participating proteins along the bent DNA . The DNA-bending property of IHF allows it to serve as a central component in many nucleoprotein complexes that regulate gene expression across the bacterial genome.

What roles does IhfB play in Pasteurella haemolytica pathogenicity?

While the search results don't directly address the role of IhfB in P. haemolytica pathogenicity, we can infer based on IHF functions in other bacteria that it likely plays important roles in regulating virulence gene expression. In Salmonella, for example, IHF influences the expression of Salmonella Pathogenicity Islands 1 and 2 (SPI-1 and SPI-2), which are critical for virulence . By extension, IhfB in P. haemolytica may similarly regulate expression of virulence factors involved in causing bovine respiratory disease. As a nucleoid-associated protein that influences genome-wide gene expression, IhfB likely contributes to the regulation of genes involved in host adaptation, immune evasion, and other pathogenicity-related functions.

What expression systems are most effective for producing recombinant IhfB?

Based on the available information, effective expression systems for recombinant IhfB production include:

• Baculovirus expression systems: The recombinant IhfB product described in search result was produced using baculovirus, which is advantageous for expressing proteins that require post-translational modifications or are challenging to express in bacterial systems.

• E. coli expression systems: As indicated in search result , E. coli clones carrying recombinant plasmids have been successfully used to express P. haemolytica antigens. For IhfB specifically, coordinated expression with IhfA may improve stability and solubility of the recombinant protein .

When designing expression systems, researchers should consider that IhfB expressed without IhfA may form insoluble aggregates, necessitating co-expression strategies or specialized solubilization methods .

What purification strategies yield the highest purity and activity for recombinant IhfB?

While specific purification strategies for P. haemolytica IhfB aren't detailed in the search results, general approaches for similar recombinant proteins typically include:

• Affinity chromatography: Using tag systems (His-tag, GST, etc.) for initial capture

• Ion exchange chromatography: For further purification based on charge properties

• Size exclusion chromatography: For final polishing and removal of aggregates

For optimal results, purification should aim for >85% purity as assessed by SDS-PAGE . Considering IhfB's tendency to form insoluble aggregates when expressed without IhfA, purification protocols may need to include solubilization steps or co-purification with IhfA to maintain proper folding and activity.

What are the optimal storage conditions for maintaining recombinant IhfB stability?

According to the product information for recombinant IhfB, the following storage recommendations apply :

• The shelf life of liquid form is generally 6 months at -20°C/-80°C

• The shelf life of lyophilized form is 12 months at -20°C/-80°C

• Repeated freezing and thawing is not recommended

• Working aliquots can be stored at 4°C for up to one week

• For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage at -20°C/-80°C

These conditions help maintain protein stability and prevent degradation during storage.

How should researchers design experiments to study IhfB-DNA interactions?

When designing experiments to study IhfB-DNA interactions, researchers should consider:

• Protein preparation: Ensure that functional IHF heterodimers are formed by co-expressing IhfA and IhfB, as IhfB alone forms insoluble aggregates .

• DNA binding assays: While homodimers of IhfB can bind in vitro to the same DNA sequences as heterodimeric IHF, these complexes are much less stable than those formed by the heterodimer . Therefore, experimental designs should account for this reduced stability when using IhfB homodimers.

• Single-Subject Experimental Design (SSED): For studies evaluating the effects of IhfB on specific cellular processes, SSED approaches may be valuable. These designs feature controlled introduction and withdrawal of the experimental variable (IhfB) to establish causality .

• Controls: Include proper controls such as:

  • DNA without protein

  • Non-specific proteins with DNA

  • Known IHF binding sequences versus non-binding sequences

• Binding conditions: Optimize salt concentration, pH, and temperature to reflect physiological conditions in the host organism.

What controls should be included when evaluating recombinant IhfB function in gene regulation studies?

When evaluating recombinant IhfB function in gene regulation studies, the following controls should be included:

• Positive controls: Known IHF-regulated promoters with well-characterized responses.

• Negative controls: Promoters not known to be regulated by IHF or with mutated IHF binding sites.

• Complementation controls: If using knockout strains (ΔihfB), include experiments with wild-type ihfB complementation to confirm phenotype rescue.

• IhfA controls: Since proper function depends on heterodimer formation, include conditions with IhfA alone to distinguish effects specific to IhfB.

• Rewired expression controls: As demonstrated in the study with Salmonella, comparing wild-type expression patterns with strains where ihfA and ihfB regulatory regions have been swapped can reveal the importance of coordinated expression .

• Growth phase controls: IHF effects may vary with growth phase, as seen in the Salmonella study where rewired strains showed growth-phase-specific reductions in SPI-1 and SPI-2 gene expression .

How can researchers effectively design vaccination studies using recombinant P. haemolytica antigens including IhfB?

Based on the successful P. haemolytica vaccine study described in the search results, effective vaccination study design should include :

• Randomized controlled trial approach: Use a double-blind study design with randomized assignment of subjects to test groups.

• Adequate sample size: The referenced study used 2,324 high-risk calves, providing sufficient statistical power.

• Multiple vaccine test groups: Design should include various combinations of antigens to identify synergistic or antagonistic effects (e.g., the study had four vaccine test groups with different combinations of P. haemolytica vaccine and BHV-1 vaccines).

• Appropriate timing: Consider whether vaccination should occur upon arrival at high-risk locations or prior to expected risk periods.

• Extended monitoring period: The referenced study monitored calves for 90 days after vaccination to assess long-term protection.

• Clear outcome measures: Define specific endpoints such as:

  • Morbidity from all bovine respiratory disease

  • Mortality from all BRD

  • Mortality specifically from fibrinous pneumonia

  • Antibody response to specific antigens

• Statistical analysis: Include appropriate statistical methods (p-value < 0.05 was used in the referenced study).

What analytical techniques are most appropriate for characterizing recombinant IhfB?

For comprehensive characterization of recombinant IhfB, researchers should consider the following analytical techniques:

• SDS-PAGE: For assessing purity (>85% is considered acceptable) and approximate molecular weight confirmation.

• Western immunoblot analysis: As used in the P. haemolytica antigen studies, this technique can confirm the expression of specific antigens using sera from vaccinated animals .

• Mass spectrometry (MS): Valuable for confirming the exact molecular weight, sequence coverage, and potential post-translational modifications. MS was used in the Salmonella study to show that IhfA is produced in excess over IhfB in the rewired strain .

• Circular dichroism (CD) spectroscopy: For assessing secondary structure and proper folding.

• DNA binding assays: To evaluate the functional activity of the recombinant protein, including electrophoretic mobility shift assays (EMSA) or surface plasmon resonance (SPR).

• Stability testing: Monitoring degradation over time under different storage conditions as mentioned in the product information .

These techniques together provide a comprehensive profile of the recombinant protein's physical, chemical, and functional properties.

How should researchers interpret conflicting data on IhfB function in different bacterial species?

When faced with conflicting data on IhfB function across bacterial species, researchers should:

• Consider evolutionary conservation and divergence: While IHF is conserved across many bacterial species, specific functions may have evolved differently. For example, the effects of IHF on pathogenicity islands in Salmonella may differ from its roles in P. haemolytica.

• Examine experimental contexts: Differences in growth conditions, genetic backgrounds, or experimental methodologies can lead to apparently conflicting results. The study on rewired ihf genes in Salmonella showed that strains grew at similar rates to wild-type but had reduced motility and growth-phase-specific changes in gene expression .

• Apply appropriate statistical methods: For experimental designs like SSED, visual analysis should examine changes in level, trend, and variability between baseline and intervention phases . When effects are ambiguous, as shown in Figure 2 of result , careful analysis is needed to rule out threats to internal validity.

• Conduct replication studies: Within-study replication is a primary characteristic of well-designed SSEDs and is essential for making internally valid inferences, especially when there are delays in observed effects .

• Use the WWCH panel criteria: These criteria assess the adequacy of experimental design and include visual analysis of results to determine experimental effects .

What statistical approaches are recommended for analyzing vaccine efficacy data involving recombinant P. haemolytica antigens?

Based on the vaccine efficacy study described in the search results, recommended statistical approaches include :

• Significance testing: Analysis using p-value thresholds (p < 0.05 was used in the referenced study) to determine statistically significant differences between vaccine groups.

• Comparative percentage reduction: Calculating percentage reductions in morbidity and mortality between different vaccine formulations (e.g., 20% reduction in BRD morbidity, 88% reduction in BRD mortality, 100% reduction in fibrinous pneumonia mortality).

• Antibody response measurement: Quantitative analysis of antibody responses to specific antigens, such as P. haemolytica leukotoxin, can provide mechanistic insights into vaccine efficacy.

• Multivariate analysis: To account for potential confounding factors such as animal age, weight, prior health status, or environmental conditions.

• Survival analysis: Methods like Kaplan-Meier curves and Cox proportional hazards models may be appropriate for time-to-event data (e.g., time to disease onset or death).

• Sample size determination: Proper power analysis should be conducted to ensure adequate sample size for detecting clinically meaningful differences.

How does P. haemolytica IhfB compare structurally and functionally to IhfB from other bacterial pathogens?

While the search results don't provide direct comparative data on P. haemolytica IhfB versus other bacterial IhfB proteins, we can infer some comparisons based on information about IhfB from Yersinia pestis and the Salmonella studies :

• Sequence conservation: The IhfB protein is generally conserved across different bacterial species, with the Yersinia pestis version consisting of 94 amino acids . Sequence comparison would likely reveal conserved domains involved in DNA binding and dimerization with IhfA.

• Functional similarities: Across different bacterial species, IHF functions as a nucleoid-associated protein that bends DNA and influences genome architecture. In Salmonella, IHF affects motility and expression of pathogenicity islands , suggesting similar roles in regulating virulence in various pathogens.

• Regulatory context: The regulatory relationship between ihfA and ihfB appears important across species. In Salmonella, rewiring the expression system of these genes affected the ratio of IhfA to IhfB and had consequences for pathogenicity island expression and macrophage engulfment . Similar regulatory interconnections likely exist in P. haemolytica.

• Heterodimer formation: The mutual dependency of IhfA and IhfB for stability observed in Salmonella is likely a conserved feature across bacterial species, suggesting similar structural requirements for functional IHF formation in P. haemolytica.

How can IhfB be integrated into systems biology approaches for understanding P. haemolytica pathogenesis?

Integration of IhfB into systems biology approaches could include:

• Genome-wide binding profiles: ChIP-seq or similar techniques to map IhfB binding sites across the P. haemolytica genome, identifying genes directly regulated by IHF.

• Transcriptomics integration: RNA-seq comparing wild-type and ihfB mutant strains to identify genes differentially expressed in response to IhfB activity, as was done for the rewired Salmonella strains .

• Protein interaction networks: Identifying proteins that interact with IhfB beyond IhfA, potentially including other transcription factors or regulatory proteins.

• Regulatory circuit modeling: Mathematical modeling of the regulatory relationships between IhfA and IhfB and their target genes, similar to the analysis of the rewired ihf gene circuit in Salmonella .

• Host-pathogen interaction studies: Systematic analysis of how IhfB-regulated genes influence interactions with host cells, such as the increased macrophage engulfment observed with rewired Salmonella strains .

• Vaccine component integration: Evaluation of IhfB as part of a systems vaccinology approach, considering its potential contributions to protective immunity alongside other P. haemolytica antigens.

What are the implications of altering IhfB expression on bacterial fitness and virulence?

Based primarily on the Salmonella rewiring study , altering IhfB expression can have several implications:

• Growth and fitness effects: When ihfA and ihfB genes were repositioned and rewired in Salmonella, the strain grew at a similar rate to wild-type and showed similar competitive fitness, suggesting some robustness to altered IhfB expression ratios.

• Virulence factor expression: The rewired Salmonella strain had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression, indicating that altered IhfB expression patterns can affect virulence factor regulation.

• Host interaction changes: The rewired Salmonella strain was engulfed at a higher rate by RAW macrophages, suggesting changes in surface properties or other factors affecting host-pathogen interactions.

• Motility impacts: Reduced motility was observed in the rewired Salmonella strain, indicating that altered IhfB expression can affect complex cellular functions beyond simple growth.

• IhfA:IhfB ratio effects: In the rewired Salmonella strain, IhfA was produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB–ihfA mRNA. This suggests that the ratio between the two subunits, not just their absolute levels, is important for proper cellular function.

By extension, similar effects might be expected when altering IhfB expression in P. haemolytica, potentially impacting virulence and host interactions relevant to bovine respiratory disease.

What are the most promising applications for recombinant P. haemolytica IhfB in vaccine development?

Future research directions for recombinant P. haemolytica IhfB in vaccine development could include:

• Combination vaccine strategies: Building on the successful P. haemolytica vaccine study , investigate combinations of IhfB with other antigens to create more comprehensive protection against bovine respiratory disease.

• Antigen delivery systems: Explore novel delivery platforms for recombinant IhfB that maximize immune response while minimizing the number of doses required.

• Cross-protection potential: Evaluate whether IhfB-based vaccines can provide protection against multiple serotypes of P. haemolytica and possibly related pathogens.

• Adjuvant optimization: Investigate different adjuvant formulations to enhance the immunogenicity of recombinant IhfB antigens.

• Single-dose efficacy: The P. haemolytica vaccine study noted that "vaccination prior to expected risk would be more appropriate" than vaccination upon arrival , suggesting research into timing and durability of protection from recombinant antigens.

• Interference mitigation: The study found that MLV vaccines interfered with the protective capacity of the P. haemolytica vaccine , indicating a need to understand and mitigate vaccine component interactions.

What genetic engineering approaches might enhance the utility of recombinant IhfB for research purposes?

Promising genetic engineering approaches include:

• Site-directed mutagenesis: Creating specific mutations in IhfB to study structure-function relationships and identify critical domains for DNA binding and IhfA interaction.

• Fusion protein construction: Developing tagged versions (fluorescent, affinity, etc.) of IhfB for easier tracking, purification, and functional studies without compromising activity.

• Inducible expression systems: Creating constructs with tightly regulated expression to study the effects of IhfB level modulation on bacterial physiology and gene regulation.

• Domain swapping: Exchanging domains between IhfB proteins from different bacterial species to understand species-specific functions.

• Rewiring experiments: Building on the Salmonella study , creating P. haemolytica strains with rewired ihfA and ihfB expression to understand the importance of their coordinated regulation.

• CRISPR-Cas9 approaches: Using precise genome editing to create chromosomal mutations or regulatory modifications without introducing resistance markers that might interfere with IHF-mediated site-specific recombination.

How might advanced structural biology techniques enhance our understanding of IhfB function in P. haemolytica?

Advanced structural biology techniques could advance understanding of P. haemolytica IhfB through:

• Cryo-electron microscopy: Determining high-resolution structures of the IHF heterodimer bound to DNA from P. haemolytica virulence gene promoters.

• X-ray crystallography: Resolving atomic-level structures of IhfB alone, in complex with IhfA, and in nucleoprotein complexes with other regulatory proteins.

• NMR spectroscopy: Investigating the dynamics of IhfB-DNA interactions and conformational changes upon binding.

• Single-molecule techniques: Directly observing IHF-induced DNA bending and its effects on the assembly of transcriptional complexes.

• Hydrogen-deuterium exchange mass spectrometry: Mapping protein-protein and protein-DNA interaction surfaces in solution.

• Molecular dynamics simulations: Predicting the effects of mutations or environmental changes on IhfB structure and function.

• AlphaFold and related AI approaches: Generating structural predictions to guide experimentation, especially for variant forms of IhfB not readily amenable to experimental structure determination.

These advanced approaches would provide deeper insights into how IhfB contributes to P. haemolytica pathogenicity and potentially identify novel targets for intervention.

Comparative Table of IhfB Properties Across Studies