Recombinant Actinobacillus pleuropneumoniae serotype 3 Translation initiation factor IF-2 (infB), partial

What is translation initiation factor IF-2 (infB) and why is it significant in Actinobacillus research?

Translation initiation factor IF-2, encoded by the infB gene, is a crucial housekeeping protein involved in protein synthesis initiation, facilitating the assembly of the translation initiation complex by promoting the binding of formylmethionyl-tRNA to the ribosomal P-site. In Actinobacillus research, infB has gained significance as a phylogenetic marker due to its highly conserved nature across bacterial species while still exhibiting sufficient sequence variation to distinguish between closely related taxa . The gene shows greater intraspecies variation than 16S rRNA sequences, making it particularly valuable for taxonomic delineation within the Actinobacillus genus . Additionally, as a housekeeping gene, infB is under less selective pressure than virulence genes, providing a more reliable evolutionary signal for long-term phylogenetic relationships.

How does Actinobacillus pleuropneumoniae serotype 3 differ from other serotypes in terms of virulence and genetic characteristics?

While the search results don't explicitly detail serotype 3 characteristics, we can extrapolate from information about other serotypes. Actinobacillus pleuropneumoniae exhibits serotype-specific virulence patterns based primarily on the combination of Apx toxins produced . Different serotypes express various combinations of four defined Apx toxins, which directly influence their virulence profiles . Regional variations are significant, with European strains showing different dominant serotypes (2, 9, and 11) compared to North American ones (1 and 5) . Serotype 3 would have its own specific combination of virulence factors including potentially unique patterns of Apx toxin expression, capsula polysaccharides, biofilm production capacity, fimbriae characteristics, immunoglobulin proteases, and iron-binding mechanisms . Taxonomically, serotype 3, like other A. pleuropneumoniae serotypes, would show high genetic similarity to A. lignieresii based on infB sequence analysis, with similarity values likely exceeding 85% .

What are the current methods for isolation and purification of recombinant infB from Actinobacillus pleuropneumoniae?

The isolation and purification of recombinant infB from A. pleuropneumoniae typically follows a multi-step process combining molecular cloning and protein purification techniques:

• Gene Amplification: The partial or complete infB gene (typically a 426 bp fragment) is amplified using PCR with specific primers designed based on conserved regions of the gene .

• Cloning Strategy: The amplified fragment is inserted into an appropriate expression vector containing a suitable promoter and affinity tag sequence.

• Expression System Selection: Expression is typically performed in E. coli systems such as BL21(DE3) that are optimized for recombinant protein production.

• Induction Conditions: Protein expression is induced under controlled conditions using IPTG or similar inducers when using a T7 or lac-based expression system.

• Cell Lysis: Bacterial cells are lysed using methods such as sonication, freeze-thaw cycles, or chemical lysis buffers containing appropriate protease inhibitors.

• Purification Methodology: Affinity chromatography (typically using His-tag, GST-tag, or MBP-tag systems) is employed for initial purification, followed by size exclusion chromatography for higher purity.

• Purity Verification: SDS-PAGE and Western blotting are used to confirm the presence and purity of the recombinant protein.

This methodology draws from standard recombinant protein techniques, adapted to the specific characteristics of Actinobacillus proteins based on approaches used in similar genetic manipulation studies of this genus .

How should researchers design primers for amplifying infB genes from Actinobacillus pleuropneumoniae serotype 3?

When designing primers for infB amplification from A. pleuropneumoniae serotype 3, researchers should consider several critical factors:

• Sequence Conservation Analysis: Analyze existing infB sequences from multiple Actinobacillus strains to identify conserved regions that flank variable domains. Particularly focus on the 426 bp fragment that has proven valuable for taxonomic studies .

• Primer Specifications:

  • Design primers with optimal length (18-25 nucleotides)

  • Maintain GC content between 40-60%

  • Ensure balanced melting temperatures between forward and reverse primers (within 2-3°C of each other)

  • Avoid regions with potential secondary structures

  • Check for self-complementarity and primer-dimer formation potential

• Serotype-Specific Considerations: If targeting serotype 3 specifically, incorporate regions that distinguish this serotype from others, particularly focusing on variable regions of the infB gene that may differ between serotypes.

• Experimental Validation Strategy: Test primer specificity using:

  • In silico PCR against database sequences

  • Gradient PCR to optimize annealing temperature

  • Trial amplification from related Actinobacillus species as controls

• Cloning Compatibility: If the goal is cloning, incorporate appropriate restriction sites or adapters for directional cloning, ensuring these don't interfere with the reading frame if expression is planned.

A typical PCR protocol would include initial denaturation (94°C, 30s), followed by approximately 32 amplification cycles consisting of denaturation (94°C, 30s), annealing (around 53°C, 40s), and extension (72°C, 2min), concluding with a final extension (72°C, 10min) .

What factors should be considered when expressing recombinant infB in heterologous systems?

Several critical factors must be considered when expressing recombinant infB from Actinobacillus pleuropneumoniae in heterologous systems:

• Codon Optimization: Analyze the codon usage bias between A. pleuropneumoniae and the expression host (typically E. coli). Codon optimization may be necessary as differences in codon preference can significantly impact expression efficiency.

• Expression System Selection:

  Expression System	Advantages	Limitations	Best Use Case
  E. coli (BL21)	High yield, rapid growth	Limited post-translational modifications	Initial expression trials
  E. coli (Origami)	Enhanced disulfide bond formation	Lower yield	If protein structure requires disulfide bonds
  Yeast systems	Better for complex eukaryotic proteins	Slower, more complex	If bacterial expression fails
  Cell-free systems	Avoids toxicity issues	Lower yield, higher cost	For toxic proteins

• Protein Solubility Considerations: IF-2 is a large protein that may form inclusion bodies. Consider:

  • Using solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Lower induction temperatures (16-20°C)

  • Reduced IPTG concentrations (0.1-0.5 mM)

  • Co-expression with chaperones if necessary

• Protein Toxicity Management: If full-length infB expression is toxic to the host, consider:

  • Using tight expression control systems (pET with T7 lysozyme)

  • Expressing only the partial fragment (426 bp) used in taxonomic studies

  • Using cell-free expression systems

• Purification Strategy Design: Incorporate appropriate affinity tags (His6, GST) that don't interfere with protein function, with TEV or PreScission protease cleavage sites if tag removal is necessary.

• Functional Validation Methodology: Develop appropriate assays to confirm that the recombinant protein maintains its native functional characteristics, potentially comparing activity between different serotypes.

What quality control measures should be implemented when working with recombinant infB proteins?

Comprehensive quality control is essential when working with recombinant infB proteins to ensure experimental reliability:

• Sequence Verification:

  • Confirm the cloned sequence via bidirectional DNA sequencing

  • Verify the absence of unintended mutations, particularly in functional domains

  • Compare sequence to reference databases for confirmation of serotype-specific characteristics

• Protein Identity Confirmation:

  • Mass spectrometry analysis (MALDI-TOF or LC-MS/MS)

  • Western blot using antibodies specific to infB or to affinity tags

  • N-terminal sequencing for absolute confirmation

• Purity Assessment:

  • SDS-PAGE with densitometry analysis (aim for >90% purity)

  • Size exclusion chromatography

  • Dynamic light scattering to assess homogeneity

• Structural Integrity Evaluation:

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to determine stability

  • Limited proteolysis to test for correct folding

• Functional Validation:

  • RNA binding assays

  • GTP hydrolysis activity

  • Translation initiation assays in reconstituted systems

• Storage Stability Testing:

  • Accelerated stability studies at different temperatures

  • Freeze-thaw cycle resistance

  • Long-term activity retention analysis

• Endotoxin Testing:

  • LAL (Limulus Amebocyte Lysate) assay

  • Endotoxin removal validation if needed for functional studies

These quality control measures should be systematically documented and performed on each preparation to ensure consistency between experimental batches.

How reliable is infB sequence analysis for delineating Actinobacillus species compared to 16S rRNA analysis?

The comparative reliability of infB versus 16S rRNA for Actinobacillus species delineation reveals several important distinctions:

For optimal results, researchers should employ a polyphasic approach combining both infB and 16S rRNA sequence analyses, particularly when studying species with ambiguous taxonomic positions or strains at the periphery of the genus.

What are the challenges in distinguishing between different serotypes of Actinobacillus pleuropneumoniae using molecular methods?

Distinguishing between Actinobacillus pleuropneumoniae serotypes using molecular methods presents several significant challenges:

To overcome these challenges, researchers increasingly employ combinatorial approaches using multiple genetic markers, whole-genome sequencing, and artificial intelligence-assisted analysis for more accurate serotype determination.

How does the infB gene of Actinobacillus pleuropneumoniae serotype 3 compare to other bacterial translation initiation factors?

The infB gene of Actinobacillus pleuropneumoniae serotype 3 exhibits both conserved and unique features when compared to translation initiation factors from other bacterial species:

• Structural Conservation Patterns:

  • As a translation initiation factor, infB in A. pleuropneumoniae maintains the core functional domains found across bacterial species

  • These include the GTP-binding domain, the formylmethionyl-tRNA binding domain, and ribosome interaction domains

  • The conservation level varies across the protein, with higher conservation in functional domains and greater variability in connecting regions

• Taxonomic Positioning:

  • Within the Pasteurellaceae family, A. pleuropneumoniae infB shows closest homology to A. lignieresii, with the two species being nearly indistinguishable by infB sequence analysis alone

  • This suggests a very recent evolutionary divergence between these species

  • The 426 bp fragment of infB used in taxonomic studies provides sufficient information for phylogenetic placement while being manageable for routine sequencing

• Comparative Analysis Across Bacterial Phyla:

  Bacterial Group	Similarity to A. pleuropneumoniae infB	Key Differences
  Other Pasteurellaceae	>85% for core Actinobacillus species	Minor sequence variations, primarily in variable regions
  Enterobacteriaceae	Moderate to high similarity	Variations in GTP binding domain architecture
  Gram-positive bacteria	Lower sequence similarity	Significant structural differences in ribosome binding domains
  Distant bacterial phyla	Conserved functional motifs only	Major structural and sequence divergence

• Functional Implications of Sequence Variations:

  • Despite sequence variations, the core function of translation initiation is maintained across bacteria

  • Species-specific and serotype-specific variations may reflect adaptations to different tRNA populations or ribosomal structures

  • These variations could potentially influence translation efficiency under different environmental conditions relevant to the pathogen's lifecycle

• Evolutionary Rate Analysis:

  • The infB gene generally evolves at a moderate rate compared to other bacterial genes

  • It shows greater intraspecies variation than 16S rRNA genes, making it valuable for finer taxonomic discrimination

  • The evolutionary rate appears consistent with its role as a housekeeping gene under purifying selection

This comparative analysis demonstrates that while infB maintains its essential functional conservation across bacteria, it contains sufficient variation to serve as an effective phylogenetic marker, particularly for studying relationships within the Actinobacillus genus.

How can recombinant infB be utilized in developing diagnostic tools for Actinobacillus pleuropneumoniae infections?

Recombinant infB offers several promising applications for developing next-generation diagnostic tools for Actinobacillus pleuropneumoniae infections:

• Serological Diagnostics Enhancement:

  • Recombinant infB can serve as a target antigen in ELISA-based assays, offering a complementary approach to the existing ApxIIA ELISA that's currently used for discriminating between infected and immunized herds

  • The constitutive expression of infB across all growth conditions makes it a reliable diagnostic target regardless of the infection stage

  • By combining infB detection with other serological markers, researchers can develop multiplex assays with improved sensitivity and specificity

• Molecular Beacon Development:

  • Sequence variations in the infB gene can be exploited to design molecular beacons or TaqMan probes for real-time PCR assays

  • These assays can potentially differentiate between A. pleuropneumoniae serotypes with greater accuracy than current methods

  • The partial 426 bp fragment of infB that has been well-characterized can serve as the foundation for designing these molecular diagnostics

• Isothermal Amplification Platforms:

  • LAMP (Loop-mediated isothermal amplification) assays targeting infB could enable rapid field testing without sophisticated laboratory equipment

  • This approach could be particularly valuable for monitoring outbreaks in resource-limited settings

  • The design would focus on regions of the infB gene that show serotype-specific variations

• Biosensor Integration:

  • Recombinant infB antibodies can be incorporated into electrochemical or optical biosensors

  • Such devices could enable rapid, point-of-care detection of A. pleuropneumoniae

  • This application would build upon the specificity of antibody-antigen interactions while providing quantitative results

• Genetic Marker for Virulence Prediction:

  • While infB itself is not a virulence factor, specific variations in its sequence might correlate with strains of different virulence potential

  • Machine learning algorithms could potentially identify patterns in infB sequences that predict virulence characteristics

  • This could enable risk stratification of infections based on molecular typing

These diagnostic applications would significantly advance our ability to rapidly detect and characterize A. pleuropneumoniae infections, potentially reducing the economic impact of this pathogen on the swine industry while improving animal welfare.

What are the challenges in developing attenuated vaccine strains of A. pleuropneumoniae serotype 3 using recombinant infB technology?

Developing attenuated vaccine strains of A. pleuropneumoniae serotype 3 using recombinant infB technology presents several significant challenges:

• Essential Gene Manipulation Constraints:

  • infB is an essential housekeeping gene, making complete deletion lethal

  • Modification strategies must preserve sufficient functionality for bacterial viability while ensuring attenuation

  • Potential approaches include:

    • Partial gene deletions of non-essential domains

    • Point mutations in specific functional regions

    • Conditional expression systems

• Genetic Stability Concerns:

  • Attenuated strains must maintain stable genetic characteristics through multiple generations

  • Selective pressure may favor reversion to virulence through compensatory mutations

  • Long-term stability testing is essential before clinical application

• Balancing Attenuation and Immunogenicity:

  Attenuation Level	Immunogenicity	Safety Profile	Development Complexity
  Minimal modification	High	Potential residual virulence	Lower
  Moderate modification	Moderate	Good	Moderate
  Extensive modification	Potentially lower	Excellent	Higher
  Multiple gene targets	Variable based on targets	Dependent on specific modifications	Highest

• Technical Manipulation Hurdles:

  • Not all A. pleuropneumoniae isolates are equally amenable to genetic manipulation

  • Successful genetic modification systems developed for one serotype (e.g., serotype 2) may require significant optimization for serotype 3

  • Countermeasures against host restriction systems may be necessary

• Vaccine Efficacy Assessment:

  • Modifying infB might alter bacterial fitness and presentation of other immunogens

  • Cross-protection against heterologous serotypes must be evaluated

  • Correlation between immunogenicity in laboratory models and protection in target species is not always predictable

• Serological Differentiation Requirements:

  • Vaccines must allow for differentiation between vaccinated and infected animals (DIVA capability)

  • If infB is modified rather than a virulence gene like apxIIA, alternative markers for serological differentiation would be needed

  • This might necessitate additional genetic modifications beyond infB

• Regulatory and Safety Considerations:

  • Recombinant live vaccines face stringent regulatory scrutiny

  • The ideal attenuated strain would contain no foreign DNA, as exemplified by the urec/apxIIA double mutant approach

  • Environmental safety assessments would be required to ensure the attenuated strain cannot revert to virulence or transfer genetic material to other organisms

These challenges highlight the complexity of developing effective attenuated vaccines and emphasize the need for comprehensive testing protocols that address genetic stability, immunogenicity, and safety concerns.

How can comparative genomics of infB sequences contribute to understanding the evolution of virulence in Actinobacillus species?

Comparative genomics of infB sequences provides valuable insights into the evolutionary trajectory of virulence in Actinobacillus species, offering a unique lens through which researchers can understand pathogen adaptation:

• Evolutionary Context for Virulence Acquisition:

  • While infB itself is not a virulence factor, its evolutionary history creates a reliable phylogenetic framework against which virulence gene acquisition can be mapped

  • As a housekeeping gene under purifying selection, infB evolves at a relatively constant rate, providing a molecular clock for timing virulence gene acquisition events

  • The greater intraspecies variation observed in infB compared to 16S rRNA allows for finer resolution of evolutionary relationships

• Horizontal Gene Transfer Detection:

  • Discrepancies between infB-based phylogenies and virulence gene distributions can identify instances of horizontal gene transfer

  • The observation that A. lignieresii and A. pleuropneumoniae cannot be clearly separated by infB analysis despite differences in pathogenicity suggests that virulence factors were acquired after minimal divergence of the core genome

  • This highlights the importance of mobile genetic elements in virulence evolution

• Serotype Emergence Patterns:

  • Comparative analysis of infB sequences across the 16 known serotypes can reveal:

    • The order in which serotypes emerged

    • Whether certain serotypes evolved multiple times independently

    • If particular genetic backgrounds are more amenable to specific virulence traits

• Correlation with Virulence Factor Distribution:

  Virulence Factor	Relationship to infB Phylogeny	Evolutionary Implication
  Apx toxins	Variable correlation	Likely acquired via horizontal transfer
  Capsular genes	Higher correlation	Potentially evolved via modification of existing genes
  Iron acquisition systems	Mixed correlation	Multiple evolutionary mechanisms
  Adhesins	Often correlates with core phylogeny	May represent adaptations of ancestral structures

• Geographic Distribution Analysis:

  • Combining infB sequence data with geographical metadata reveals patterns of pathogen spread

  • The observation that different serotypes predominate in different regions (serotypes 2, 9, 11 in Europe vs. 1, 5 in North America) can be contextualized within the infB phylogeny

  • This approach can identify whether regional differences represent separate evolutionary lineages or recent dispersal events

• Selection Pressure Mapping:

  • While the infB gene itself is under strong purifying selection, comparing synonymous vs. non-synonymous substitution rates across the Actinobacillus genus can identify regions experiencing different selection pressures

  • These patterns may correlate with functional adaptations to different host environments or transmission dynamics

  • Such analysis can identify whether certain lineages are evolving more rapidly, potentially indicating adaptation to new niches

• Future Virulence Prediction:

  • By establishing patterns between infB genetic backgrounds and virulence acquisition, researchers may eventually develop predictive models for emergent virulence

  • This could allow for proactive surveillance and vaccine development strategies

  • Machine learning approaches integrating infB sequence data with virulence phenotypes may identify subtle genetic signatures associated with virulence potential

This multifaceted approach to comparative genomics provides a comprehensive framework for understanding the complex evolutionary history of virulence in Actinobacillus species.

What are common technical difficulties encountered when working with recombinant Actinobacillus proteins and how can they be addressed?

Researchers working with recombinant Actinobacillus proteins frequently encounter several technical challenges that require systematic troubleshooting approaches:

• Low Expression Levels:

  • Problem: A. pleuropneumoniae proteins often express poorly in heterologous systems due to codon bias and toxicity issues.

  • Solutions:

    • Optimize codon usage for the expression host

    • Use stronger promoters (T7, tac) or inducible systems with tight regulation

    • Decrease induction temperature (16-20°C) to slow protein synthesis and improve folding

    • Express as fusion proteins with solubility enhancers (MBP, SUMO, TrxA)

    • Consider cell-free expression systems for toxic proteins

• Protein Insolubility:

  • Problem: Formation of inclusion bodies, particularly common with larger proteins like IF-2.

  • Solutions:

    • Reduce induction temperature and IPTG concentration

    • Add solubility enhancers to culture media (sorbitol, arginine)

    • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

    • Express soluble domains separately if full-length protein proves recalcitrant

    • Develop refolding protocols using gradual dialysis against decreasing concentrations of denaturants

• Proteolytic Degradation:

  • Problem: Recombinant proteins from A. pleuropneumoniae may be susceptible to proteolysis.

  • Solutions:

    • Use protease-deficient expression strains (BL21, Rosetta)

    • Include protease inhibitors in all purification buffers

    • Optimize purification workflow to minimize handling time

    • Consider adding stabilizing agents (glycerol, specific metal ions)

    • Identify and potentially modify proteolytically sensitive regions

• Protein Toxicity to Host:

  • Problem: Some A. pleuropneumoniae proteins may be toxic to expression hosts.

  • Solutions:

    • Use tightly controlled expression systems with minimal leaky expression

    • Consider specialized strains designed for toxic protein expression

    • Express partial protein domains rather than full-length proteins

    • Utilize in vitro translation systems for highly toxic proteins

• Purification Challenges:

  Challenge	Technical Solution	Methodological Approach
  Low binding to affinity resins	Optimize tag position and linker length	Test both N and C-terminal tags with various linkers
  Co-purification of contaminants	Include additional purification steps	Combine affinity chromatography with ion exchange and size exclusion
  Protein aggregation during purification	Modify buffer conditions	Screen various pH, salt, and additive combinations
  Tag cleavage inefficiency	Optimize protease accessibility	Ensure adequate linker length and proper folding
  Batch-to-batch variability	Standardize expression and purification	Develop detailed SOPs with quality control checkpoints

• Functional Characterization Difficulties:

  • Problem: Recombinant proteins may lack proper post-translational modifications or correct folding.

  • Solutions:

    • Validate protein structure using circular dichroism, thermal shift assays

    • Develop activity assays specific to the protein's function

    • Compare to native protein where possible

    • Consider eukaryotic expression systems if bacterial systems prove inadequate

These troubleshooting approaches should be implemented systematically, documenting outcomes at each stage to develop optimal protocols for specific Actinobacillus proteins.

How can researchers resolve discrepancies between infB sequence data and other molecular markers in Actinobacillus taxonomy?

Resolving discrepancies between infB sequence data and other molecular markers in Actinobacillus taxonomy requires a systematic approach:

• Source Validation and Sequencing Quality Assessment:

  • Validation Process:

    • Confirm strain identity through phenotypic characterization

    • Verify culture purity through repeated isolation

    • Sequence bidirectionally with high coverage

    • Compare technical replicates to eliminate sequencing artifacts

  • Quality Control Measures:

    • Implement rigorous sequence quality filtering

    • Manually inspect chromatograms at positions of discrepancy

    • Re-sequence problematic regions with alternative primers

• Comprehensive Multi-Locus Analysis:

  • Analyze multiple independent genetic markers simultaneously to establish a consensus phylogeny

  • The specific observation that infB-based phylogeny is "essentially congruent with relationships inferred from 16S rRNA sequence comparisons and DNA hybridization studies" with "discrepancies encountered with single strains or taxa at the periphery of the genus" suggests that most inconsistencies involve outlier taxa

  • For taxa showing discrepant positions, expand analysis to include additional housekeeping genes (e.g., recA, rpoB, gyrB)

• Horizontal Gene Transfer Investigation:

  • Assess potential horizontal gene transfer events through:

    • Anomalous GC content analysis

    • Codon usage pattern comparison

    • Phylogenetic incongruence testing

    • Analysis of flanking mobile genetic elements

  • Horizontal gene transfer may explain discrepancies between markers with different evolutionary histories

• Addressing RNA Operon Heterogeneity:

  • The observation that "apparent subdivision of some species by 16S rRNA analysis was most likely caused by RNA operon heterogeneity" highlights a specific source of discrepancy

  • Solutions include:

    • Sequencing multiple 16S rRNA operons from the same strain

    • Using techniques that target specific operons consistently

    • Developing composite analyses that account for operon heterogeneity

• Statistical Approaches to Reconcile Discrepancies:

  Method	Application	Advantage	Limitation
  Bayesian inference	Integrates multiple data sources	Accounts for uncertainty	Computationally intensive
  Supertree construction	Combines trees from different markers	Synthesizes conflicting data	May obscure genuine conflicts
  Split decomposition	Visualizes conflicting signals	Shows reticulate relationships	Complex interpretation
  Consensus networks	Depicts competing phylogenetic signals	Highlights true incongruence	Requires extensive data

• Phenotypic Correlation Analysis:

  • When molecular markers conflict, correlate phylogenetic assignments with phenotypic characteristics

  • For Actinobacillus, relevant phenotypes include:

    • Host specificity

    • Virulence factor production

    • Biochemical profiles

    • Serological characteristics

• Whole Genome Sequencing Resolution:

  • For particularly problematic taxa, whole genome sequencing provides the most comprehensive resolution

  • Approaches include:

    • Core genome phylogeny construction

    • Average nucleotide identity (ANI) calculation

    • Genome-wide SNP analysis

    • Pan-genome comparison

By systematically implementing these approaches, researchers can resolve most discrepancies and develop a more robust taxonomic framework for Actinobacillus species that accounts for the complex evolutionary history of this genus.

What analytical methods should be employed to compare data from recombinant infB experiments with native protein studies?

• Structural Equivalence Assessment:

  • Biophysical Characterization:

    • Circular dichroism (CD) spectroscopy to compare secondary structure elements

    • Differential scanning calorimetry (DSC) to analyze thermal stability profiles

    • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to assess oligomeric state

    • Small-angle X-ray scattering (SAXS) for low-resolution structural comparison

  • Structural Visualization:

    • Limited proteolysis patterns to identify correctly folded domains

    • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to compare solution dynamics

    • NMR spectroscopy for detailed structural comparison when feasible

• Functional Equivalence Evaluation:

  • Biochemical Activity Analysis:

    • GTP binding affinity comparison using isothermal titration calorimetry (ITC)

    • GTPase activity kinetics (Km, Vmax, kcat) through colorimetric or fluorescent assays

    • tRNA binding capability using electrophoretic mobility shift assays (EMSA)

    • Ribosome interaction studies through co-sedimentation assays

  • Translation Initiation Capacity:

    • In vitro translation systems to measure functional competence

    • Reconstitution assays with purified translation components

    • Complementation studies in appropriate genetic backgrounds

• Post-Translational Modification Mapping:

  Modification Type	Detection Method	Significance Assessment
  Phosphorylation	Phos-tag SDS-PAGE, LC-MS/MS	Compare modification patterns between native and recombinant
  Methylation	Immunoblotting, MS analysis	Evaluate impact on function if present in native only
  Acetylation	MS analysis, specific antibodies	Determine functional consequences of differences
  Other modifications	Comprehensive MS profiling	Assess whether modifications affect critical domains

• Statistical Comparison Frameworks:

  • Establish equivalence testing rather than difference testing where appropriate

  • Implement Bland-Altman plots for method comparison

  • Use appropriate statistical tests with correction for multiple comparisons

  • Consider Bayesian approaches for integrating multiple data types

• Interactome Analysis:

  • Pull-down assays with cellular extracts to compare binding partners

  • Surface plasmon resonance (SPR) to compare interaction kinetics with known partners

  • Protein microarrays to assess broader interaction profiles

  • Crosslinking mass spectrometry to map interaction interfaces

• Computational Validation:

  • Molecular dynamics simulations to compare conformational flexibility

  • Docking studies to assess interaction potential with binding partners

  • Sequence-structure-function relationship modeling

  • Energy landscape comparison

• In Vivo Functional Correlation:

  • Complementation studies in infB mutant backgrounds

  • Assessment of phenotypic rescue in appropriate cellular contexts

  • Comparison of immunological properties if relevant to research goals

  • Evaluation of cross-species functionality if studying evolutionary aspects

These analytical methods should be applied in a hierarchical fashion, beginning with basic structural and functional comparisons and proceeding to more sophisticated analyses as needed. The goal is to establish a quantitative understanding of the similarities and differences between recombinant and native infB, enabling researchers to determine which aspects of the native protein are faithfully recapitulated in the recombinant system.

What are promising new approaches for studying the role of infB in translation regulation within Actinobacillus species?

Several innovative approaches are emerging for investigating infB's role in translation regulation within Actinobacillus species:

• Cryo-Electron Microscopy Applications:

  • High-resolution structural studies of Actinobacillus IF-2 bound to ribosomes in various states

  • Visualization of conformational changes during translation initiation

  • Comparative structural analysis between different Actinobacillus species and serotypes

  • This approach would provide unprecedented insights into species-specific translation mechanisms

• Ribosome Profiling Technology:

  • Global analysis of translation efficiencies and initiation site selection

  • Comparison between wild-type and IF-2 variant strains

  • Identification of genes particularly dependent on optimal IF-2 function

  • Correlation between translation patterns and virulence gene expression

  • This technique could reveal how infB influences the Actinobacillus translatome under different conditions

• CRISPR-Based Approaches:

  • Development of conditional IF-2 mutants using CRISPRi technology

  • Creation of domain-specific mutations to dissect functional regions

  • Analysis of growth and virulence phenotypes associated with specific infB variants

  • Assessment of compensatory mechanisms activated upon IF-2 depletion

  • This strategy would overcome challenges associated with manipulating essential genes

• Single-Cell Translation Dynamics:

  • Real-time monitoring of translation initiation using fluorescent reporters

  • Analysis of cell-to-cell variability in translation rates

  • Correlation between translation efficiency and bacterial phenotypes

  • Investigation of translation dynamics during host infection

  • This approach would reveal heterogeneity in translation regulation within bacterial populations

• Integrative Multi-Omics Frameworks:

  Approach	Application to infB Research	Expected Insights
  Transcriptomics + Proteomics	Correlate mRNA and protein levels	Identify post-transcriptional regulation by IF-2
  Proteomics + Metabolomics	Link protein synthesis to metabolic state	Reveal how IF-2 connects translation to metabolism
  Genomics + Translatome analysis	Compare genomic variations with translation patterns	Determine how infB sequence affects global translation
  Network analysis	Integrate multiple data types	Map IF-2's position in regulatory networks

• Host-Pathogen Interaction Studies:

  • Examination of infB regulation during different stages of infection

  • Analysis of host factors that interact with bacterial IF-2

  • Investigation of whether IF-2 is a target of host immune responses

  • Assessment of how translation regulation contributes to bacterial adaptation within the host

  • This would provide context for infB function during pathogenesis

• Comparative Evolutionary Approaches:

  • Analysis of selective pressures on different infB domains across Actinobacillus species

  • Investigation of co-evolution between infB and interacting translation components

  • Correlation between infB variants and ecological niches of different Actinobacillus species

  • This evolutionary perspective would contextualize functional studies within the broader adaptive history of the genus

• Microfluidics and High-Throughput Phenotyping:

  • Rapid screening of large libraries of infB variants

  • Assessment of growth phenotypes under diverse environmental conditions

  • Correlation between specific infB mutations and stress responses

  • This approach would efficiently map the sequence-function landscape of infB

These emerging approaches promise to transform our understanding of infB's role beyond its canonical function in translation initiation, potentially revealing species-specific regulatory mechanisms that could be targeted for therapeutic intervention or diagnostic applications.

How might tools from synthetic biology be applied to engineer novel functions in Actinobacillus pleuropneumoniae using infB as a target?

Synthetic biology offers innovative approaches to engineer novel functions in Actinobacillus pleuropneumoniae using infB as a target:

• Translational Control Switches:

  • Engineer modified infB variants with altered regulation for controlled protein expression

  • Design riboswitches that modulate infB activity in response to specific small molecules

  • Create conditional expression systems where infB function depends on environmental triggers

  • This could enable precise control of bacterial growth and protein production for vaccine development

• Attenuated Vaccine Design:

  • Create temperature-sensitive infB mutants that restrict growth at host body temperature

  • Design conditionally functional infB variants that become attenuated in specific host tissues

  • Develop strains with modified infB that maintain immunogenicity while reducing virulence

  • These approaches build upon the successful double-mutant strategy demonstrated for serotype 2 but with more sophisticated regulation

• Orthogonal Translation Systems:

  • Engineer modified infB variants that recognize alternative initiation codons

  • Create specialized ribosomes that work exclusively with engineered infB

  • Develop systems for selective translation of specific mRNA subsets

  • This would allow for expression of synthetic genes without interfering with normal bacterial physiology

• Biosensor Development:

  • Design infB fusion proteins that respond to environmental signals

  • Create reporter systems where translation initiation efficiency correlates with target molecule presence

  • Develop whole-cell biosensors using infB-regulated expression systems

  • These systems could be used for environmental monitoring or diagnostic applications

• Heterologous Protein Production Optimization:

  Approach	Design Principle	Application
  Codon-optimized infB	Match tRNA availability	Enhance protein production efficiency
  Chimeric infB variants	Combine domains from different species	Optimize translation of specific mRNA classes
  Multiplexed infB systems	Express multiple infB variants	Allow parallel translation of different protein sets
  Site-directed engineering	Modify specific functional residues	Fine-tune translation initiation rates

• Multi-Functional Fusion Proteins:

  • Create infB fusions with additional enzymatic or binding domains

  • Design bifunctional proteins that combine translation initiation with RNA modification capabilities

  • Develop infB variants with expanded substrate recognition

  • These fusion proteins could connect translation to other cellular processes in novel ways

• Biocontainment Strategies:

  • Engineer strains dependent on synthetic infB variants

  • Design genetic circuits where survival requires specific non-natural inputs

  • Create conditional lethality systems based on modified infB function

  • These approaches would enhance the safety of engineered Actinobacillus strains for research or biotechnological applications

• Protein Evolution Platforms:

  • Develop directed evolution systems targeting infB

  • Create selection schemes for infB variants with novel properties

  • Implement continuous evolution systems that adapt translation to changing conditions

  • This would generate novel infB variants with potentially valuable properties for biotechnology

These synthetic biology applications represent a paradigm shift from studying infB as a phylogenetic marker to utilizing it as a versatile platform for engineering novel functions in Actinobacillus pleuropneumoniae, potentially leading to new vaccines, diagnostics, and biotechnological tools.

What potential exists for targeting infB in the development of novel antimicrobial strategies against Actinobacillus infections?

The essential nature of infB in bacterial translation makes it a compelling target for novel antimicrobial strategies against Actinobacillus infections:

• Structure-Based Drug Design:

  • Leverage structural differences between bacterial IF-2 and eukaryotic eIF2

  • Target GTP-binding pocket with high-affinity small molecules

  • Design compounds that interfere with tRNA binding without affecting host translation

  • Focus on regions of IF-2 that are highly conserved among Actinobacillus species but distinct from mammalian counterparts

  • This approach could yield narrow-spectrum antibiotics with reduced selective pressure for resistance development

• Translation Initiation Inhibitors:

  • Develop peptide mimetics that compete with natural binding partners

  • Create small molecules that lock IF-2 in non-functional conformations

  • Design compounds that accelerate GTP hydrolysis, preventing productive initiation

  • These strategies would disrupt bacterial protein synthesis while potentially minimizing effects on host cells

• Nucleic Acid-Based Therapeutics:

  • Antisense oligonucleotides targeting infB mRNA

  • CRISPR-Cas systems programmed to target the infB gene

  • Peptide nucleic acids (PNAs) designed to interfere with infB transcription or translation

  • RNA-targeting approaches could offer high specificity based on sequence differences between bacterial species

• Combination Therapy Approaches:

  Strategy	Mechanism	Advantage
  infB inhibitor + traditional antibiotic	Simultaneous targeting of translation and other processes	Reduced resistance development
  Sub-inhibitory infB targeting + host immunity enhancement	Weakening bacteria without selecting for resistance	Leverages host defense mechanisms
  Dual-targeting of different translation factors	Simultaneous inhibition of multiple steps in translation	Higher barrier to resistance
  infB-targeting phage therapy	Delivery of infB inhibitors via bacteriophage	Precise targeting of pathogenic species

• Immunotherapeutic Strategies:

  • Antibodies targeting surface-exposed regions of IF-2 in gram-negative bacteria

  • Vaccine development using conserved epitopes from IF-2

  • T-cell based therapies targeting IF-2 peptides presented on infected cells

  • While IF-2 is primarily intracellular, these approaches could target populations where the protein becomes accessible

• Allosteric Modulators:

  • Design compounds that bind to allosteric sites on IF-2

  • Develop molecules that trap IF-2 in inactive conformations

  • Create inhibitors that prevent necessary protein-protein interactions

  • Allosteric approaches might offer higher specificity than active site inhibitors

• Resistance Mitigation Strategies:

  • Target multiple domains of IF-2 simultaneously

  • Develop cycling protocols that alternate between different targeting mechanisms

  • Create inhibitor libraries that can be rapidly adapted to emerging resistance

  • These approaches acknowledge the likelihood of resistance development and proactively address it

• Therapeutic Delivery Innovations:

  • Nanoparticle encapsulation for targeted delivery to infection sites

  • Siderophore-conjugated inhibitors for bacterial uptake

  • Cell-penetrating peptides to enhance intracellular delivery

  • These delivery systems would improve the pharmacokinetics and target specificity of IF-2 inhibitors

The development of infB-targeting antimicrobials represents a promising approach for addressing Actinobacillus infections, particularly as conventional antibiotic resistance becomes increasingly problematic. The essential nature of IF-2, combined with structural differences from eukaryotic counterparts, creates opportunities for selective inhibition with potentially reduced side effects compared to broader-spectrum antibiotics.

What are the key insights from current research on Actinobacillus pleuropneumoniae infB and what questions remain unresolved?

Current research on Actinobacillus pleuropneumoniae infB has yielded several crucial insights while highlighting important unresolved questions. This field has evolved from basic taxonomic studies to advanced molecular characterization with implications for diagnosis, vaccine development, and antimicrobial strategies.

Key Research Insights:

• Taxonomic Utility: The infB gene has proven valuable for delineating Actinobacillus species, showing greater intraspecies variation than 16S rRNA sequences while maintaining sufficient conservation for reliable phylogenetic analysis . This has helped clarify relationships within the Pasteurellaceae family.

• Genus Structure: Comparative analysis of infB sequences has revealed that core Actinobacillus species (including A. pleuropneumoniae, A. equuli, A. suis, A. ureae, A. arthritidis, and A. hominis) share >85% similarity with the type species A. lignieresii . This establishes a well-defined genus boundary.

• Serotype Differentiation Challenges: The inability to clearly separate even distinct species like A. lignieresii and A. pleuropneumoniae by infB sequence analysis alone highlights limitations in using single genetic markers for fine taxonomic discrimination . This observation informs multi-locus approaches.

• Phylogenetic Congruence: The phylogeny based on infB analysis is generally consistent with 16S rRNA sequences and DNA hybridization studies, providing cross-validation of taxonomic frameworks . Discrepancies are primarily limited to peripheral taxa.

• Genetic Manipulation Potential: While not directly studied for serotype 3, research on serotype 2 has demonstrated the feasibility of genetic manipulation in A. pleuropneumoniae for creating attenuated vaccine strains . These techniques could potentially be applied to infB.

• Virulence Factor Diversity: Different serotypes express various combinations of four defined Apx toxins, which directly influence their virulence profiles . This diversity must be considered when developing broadly effective interventions.

Unresolved Questions:

• Serotype 3 Specificities:

  • What are the unique genetic characteristics of infB in serotype 3 compared to other serotypes?

  • How do sequence variations in infB correlate with serotype-specific virulence profiles?

  • Do these variations affect translation efficiency of specific virulence factors?

• Functional Implications:

  • How do naturally occurring variations in infB affect translation initiation efficiency across different Actinobacillus species?

  • Does infB play a regulatory role in stress response or virulence expression?

  • Are there condition-specific changes in infB expression during infection?

• Recombination and Horizontal Transfer:

  • To what extent has recombination shaped infB evolution within Actinobacillus?

  • Has horizontal gene transfer contributed to infB diversity or is its evolution primarily vertical?

  • How stable is the infB gene compared to other housekeeping genes?

• Taxonomic Outliers:

  • What accounts for the unresolved taxonomic position of A. capsulatus despite infB analysis?

  • Why do some taxa at the periphery of the genus show discrepancies between different molecular markers?

  • Do these outliers represent transitional forms or misclassified species?

• Translation Regulation:

  • How does translation initiation factor IF-2 in Actinobacillus interact with host cellular environments?

  • Are there host factors that specifically target or modify bacterial IF-2 during infection?

  • Does IF-2 contribute to environmental adaptation through selective translation?

• Applied Biotechnology:

  • Can infB be effectively targeted for antimicrobial development without affecting host translation?

  • How can infB be utilized in diagnostic applications for specific serotype identification?

  • What is the potential for infB-based vaccine development strategies?

These unresolved questions represent fertile ground for future research, particularly as technologies for genetic manipulation, high-throughput sequencing, and structural biology continue to advance. Addressing these questions would not only enhance our fundamental understanding of Actinobacillus biology but also inform the development of novel diagnostic, preventive, and therapeutic approaches.

How can researchers best integrate findings about infB with broader understanding of bacterial translation and pathogenesis?

Integrating infB research with broader understanding of bacterial translation and pathogenesis requires a multidisciplinary approach that connects molecular mechanisms to clinical outcomes:

• Systems Biology Integration:

  • Incorporate infB into comprehensive models of translation regulation

  • Map interactions between translation initiation and other cellular processes

  • Develop computational frameworks that predict how infB variations affect global protein synthesis

  • This holistic approach would place infB within its broader functional context rather than studying it in isolation

• Comparative Pathogenomics:

  • Analyze infB across diverse pathogens to identify convergent or divergent evolution

  • Compare translation initiation mechanisms between pathogens and commensals

  • Identify whether specific infB variants correlate with pathogenic potential

  • This comparative approach would reveal whether patterns in infB evolution contribute to pathogenesis across bacterial species

• Host-Pathogen Interface Analysis:

  • Investigate how host environments influence bacterial translation dynamics

  • Examine whether host defenses target translation initiation as an antibacterial strategy

  • Study how translation efficiency affects expression of virulence factors during infection

  • This perspective recognizes that infB function occurs within the complex environment of host-pathogen interactions

• Translational Medicine Applications:

  Research Area	Integration Approach	Potential Impact
  Diagnostics	Correlate infB variations with clinical presentations	More precise identification of infection types
  Therapeutics	Target translation as part of multimodal treatment strategies	Overcome antibiotic resistance mechanisms
  Vaccines	Design live attenuated strains with modified translation efficiency	Balanced attenuation and immunogenicity
  Epidemiology	Use infB as part of molecular typing schemes	Better tracking of outbreak strains

• One Health Framework Adoption:

  • Consider infB evolution across animal hosts, humans, and environments

  • Track how translation factors adapt as pathogens cross species barriers

  • Analyze whether agricultural practices influence translation factor evolution

  • This approach acknowledges the interconnectedness of human, animal, and environmental health

• Interdisciplinary Collaboration Enhancement:

  • Foster partnerships between molecular biologists, structural biologists, clinicians, and epidemiologists

  • Develop shared resources like strain collections with well-characterized infB sequences

  • Create databases that link infB variations to phenotypic and clinical data

  • This collaborative infrastructure would accelerate translation from basic infB research to clinical applications

• Technological Convergence:

  • Combine cryo-EM, single-molecule studies, and in vivo imaging to study translation in action

  • Integrate genomics, transcriptomics, and proteomics to track from gene to function

  • Apply artificial intelligence to identify subtle patterns in infB sequence-function relationships

  • This technological synthesis would provide unprecedented insights into translation dynamics

• Evolutionary Medicine Perspective:

  • Frame infB variations within the context of pathogen adaptation

  • Consider how translation optimization contributes to bacterial fitness in changing environments

  • Analyze the coevolution of translation factors and the genes they help express

  • This evolutionary lens would enhance our understanding of pathogen emergence and adaptation