Recombinant Pasteurella haemolytica Biopolymer transport protein exbB (exbB)

What is the ExbB protein and its significance in Pasteurella haemolytica?

ExbB is a biopolymer transport protein that functions as part of a complex with ExbD and TonB proteins in Pasteurella haemolytica. These proteins collectively form a membrane-associated energy transduction system that facilitates the transport of essential nutrients, particularly iron, across the bacterial outer membrane. In P. haemolytica, the exbB-exbD-tonB genes are organized in a single locus, which differs from the genetic organization observed in Enterobacteriaceae where tonB is unlinked from the accessory genes exbB and exbD . This system is crucial for bacterial survival in iron-limited environments, such as within a host organism, making it important for virulence and pathogenicity.

What is the relationship between Pasteurella haemolytica and Mannheimia haemolytica?

Mannheimia haemolytica is the updated taxonomic classification for organisms previously identified as Pasteurella haemolytica biotype A. This reclassification occurred based on genetic analysis, though some literature still refers to the organism by its former name. The pathogen is commonly isolated from the respiratory tract of cattle and sheep, and is more frequently isolated than other Pasteurella species in animals with pneumonic conditions . The frequency of isolation is higher in young animals (OR = 1.56; 95% CI: 1.02, 2.38), in pneumonic animals (OR = 4.67; 95% CI: 3.03, 7.19), and in animals under intensive management (OR = 2.46; 95% CI: 1.12, 5.39) . Understanding this taxonomic relationship is essential when reviewing literature about ExbB, as research may use either nomenclature.

What is the genetic organization of the ExbB-ExbD-TonB locus in Pasteurella haemolytica?

The exbB-exbD-tonB genes in P. haemolytica A1 are organized in a single locus, with the genes arranged in the order: exbB, followed by exbD, followed by tonB. This organization has been confirmed through the analysis of a recombinant plasmid (pMG1) carrying P. haemolytica A1 DNA that complements a tonB mutation in Escherichia coli . The functional expression of these genes in E. coli demonstrates their conserved roles across species, as evidenced by the restoration of vitamin B12 uptake, susceptibility to bacteriophage phi 80, and sensitivity to colicin B in the complemented E. coli strain . This genetic arrangement differs from Enterobacteriaceae where tonB is not linked to exbB and exbD, suggesting a separate evolutionary lineage for this transport system in P. haemolytica.

How does ExbB interact with other components of the iron transport system?

ExbB works in concert with ExbD and TonB to form an energy transduction complex that powers the transport of iron across the outer membrane. In P. haemolytica, this complex likely interacts with iron-binding proteins such as the 35 kDa iron-regulated FbpA protein, which shows homology to the cluster 1 group of extracellular solute-binding proteins . The complete iron transport system in P. haemolytica includes additional components like FbpB and FbpC, which show homology to transmembrane and ATPase components of ATP-binding cassette (ABC)-type uptake systems, respectively . These components appear to be organized in an operonic structure with fbpA, suggesting coordinated expression and function. The ExbB-ExbD-TonB complex provides the energy for the transport process, while the Fbp system components function in binding and translocating the iron across the membrane.

What methods are used to analyze ExbB protein expression under different growth conditions?

Researchers typically employ a combination of molecular and biochemical approaches to analyze ExbB expression under various conditions:

• Iron-regulated expression analysis: Growth of P. haemolytica under iron-depleted conditions followed by protein extraction and analysis by SDS-PAGE and Western blotting using specific antibodies against ExbB .

• Quantitative PCR (qPCR): Measurement of exbB gene expression levels under different conditions using specific primers targeting the exbB gene sequence.

• Promoter fusion studies: Construction of reporter gene fusions (such as lacZ) to the exbB promoter to monitor expression levels under different environmental conditions.

• Comparative proteomics: Two-dimensional gel electrophoresis combined with mass spectrometry to identify changes in ExbB protein levels in response to environmental stimuli.

These approaches allow researchers to determine how ExbB expression is regulated in response to iron availability and other environmental factors that might be encountered during infection.

How can recombinant ExbB protein be produced and purified for research?

Production and purification of recombinant ExbB protein typically follows these methodological steps:

• Cloning: The exbB gene is amplified from P. haemolytica genomic DNA using PCR with specific primers designed based on the published sequence. The amplified gene is then cloned into an expression vector, similar to the approach used for creating the pMG1 plasmid .

• Expression system selection: Common expression systems include E. coli BL21(DE3) for high-yield protein production. The choice of vector typically includes a fusion tag (His-tag, GST, etc.) to facilitate purification.

• Expression optimization: Parameters including temperature, induction time, and inducer concentration are optimized to maximize soluble protein yield.

• Purification protocol:

  • Cell lysis using methods such as sonication or chemical lysis

  • Clarification of lysate by centrifugation

  • Affinity chromatography using the fusion tag (e.g., Ni-NTA for His-tagged proteins)

  • Size exclusion chromatography for further purification

  • Ion exchange chromatography if needed for additional purity

• Protein characterization:

  • SDS-PAGE to confirm size and purity

  • Western blotting using anti-ExbB antibodies

  • Mass spectrometry for precise molecular weight determination

  • Circular dichroism for secondary structure analysis

This methodological approach can be adapted based on specific research requirements and the intended applications of the purified protein.

What experimental designs are appropriate for studying ExbB function in vitro?

To effectively study ExbB function in vitro, researchers can employ several experimental designs:

• Membrane reconstitution assays: Purified ExbB can be reconstituted into liposomes along with ExbD and TonB to study the energy transduction properties of the complex in a controlled membrane environment.

• Protein-protein interaction studies:

  • Pull-down assays using tagged ExbB to identify binding partners

  • Surface plasmon resonance (SPR) to measure binding kinetics with ExbD and TonB

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of interactions

• Structure-function analysis:

  • Site-directed mutagenesis of conserved residues followed by functional assays

  • Limited proteolysis to identify stable domains

  • Structural characterization using X-ray crystallography or cryo-EM

• Complementation assays: Using the approach demonstrated with pMG1 in E. coli tonB mutants, researchers can conduct complementation studies to assess the functionality of ExbB variants .

• Iron transport assays: Measuring the transport of radiolabeled iron compounds in systems expressing wild-type or mutant ExbB to assess functional impacts.

The design of experiments should follow standard principles of including appropriate controls, randomization, and replication to ensure reliability of results, as emphasized in modern experimental design practices .

What are the key considerations for designing PCR primers for ExbB gene amplification?

When designing PCR primers for ExbB gene amplification, researchers should consider:

• Sequence specificity: Primers should be designed based on the published P. haemolytica exbB sequence to ensure specificity. Similar to the approach used for M. haemolytica characterization, primers should target unique regions that distinguish ExbB from related proteins .

• Primer properties:

  • Length: Typically 18-30 nucleotides

  • GC content: Aim for 40-60%

  • Melting temperature (Tm): Primers should have similar Tm values, typically between 55-65°C

  • Avoid secondary structures: Check for self-complementarity and hairpin formation

• Addition of restriction sites: Include appropriate restriction enzyme sites at the 5' ends for directional cloning into expression vectors, with additional bases (3-6) at the extreme 5' end to facilitate efficient enzyme cutting.

• Codon optimization: Consider codon usage if expressing in heterologous systems like E. coli.

• Fusion tag considerations: Design primers to maintain the reading frame with fusion tags for protein purification.

• Specificity validation: Test primers against genomic DNA from related species to ensure specificity for P. haemolytica exbB.

Following these considerations will improve the success rate of exbB amplification and subsequent cloning procedures.

How do mutations in the ExbB protein affect iron acquisition and bacterial virulence?

Mutations in ExbB can have significant impacts on iron acquisition and virulence of P. haemolytica:

• Functional domains: Mutations in transmembrane domains may disrupt the membrane topology essential for energy transduction, while mutations in cytoplasmic domains might affect interactions with ExbD or the proton motive force coupling.

• Iron acquisition impairment: Since ExbB is part of the energy transduction system for TonB-dependent transport, mutations can compromise iron uptake in iron-limited environments such as within the host. This has been demonstrated in related systems where complement experiments using recombinant plasmids like pMG1 have restored iron acquisition capabilities in mutant strains .

• Virulence attenuation: The relationship between iron acquisition and virulence is well-established in many pathogens. In P. haemolytica, which causes severe fibrinonecrotic pneumonia, impaired iron acquisition due to ExbB mutations would likely reduce bacterial survival in the host and attenuate virulence .

• Stress response: ExbB mutations may affect the bacterium's ability to respond to various stress conditions encountered during infection, further impacting virulence.

• Host-pathogen interactions: Altered ExbB function may change the expression of other virulence factors regulated by iron availability, affecting the bacterium's interactions with host immune cells.

Research methodologies to investigate these effects typically include constructing defined exbB mutants, assessing their growth under iron limitation, measuring virulence factor expression, and evaluating pathogenicity in appropriate animal models.

What is the structural relationship between ExbB and TonB in energy transduction?

The structural relationship between ExbB and TonB in energy transduction is complex:

• Membrane topology: ExbB is primarily embedded in the cytoplasmic membrane with multiple transmembrane domains, while TonB has a single transmembrane domain at its N-terminus and extends into the periplasm.

• Energy coupling mechanism: ExbB, along with ExbD, is thought to harness the proton motive force across the cytoplasmic membrane and transfer this energy to TonB. The exact mechanism of this energy transduction remains an active area of research.

• Conformational changes: The current model suggests that proton translocation through the ExbB-ExbD complex induces conformational changes in TonB, which then interacts with and energizes TonB-dependent transporters in the outer membrane.

• Stoichiometry: The functional complex is believed to consist of multiple ExbB proteins (potentially a pentamer) associated with fewer ExbD proteins and a single TonB protein, though the exact stoichiometry may vary.

• Interaction domains: Specific regions of ExbB interact with both ExbD and the transmembrane domain of TonB. These interactions are essential for the assembly and function of the energy transduction complex.

Advanced structural biology techniques such as cryo-electron microscopy, X-ray crystallography, and NMR spectroscopy are being employed to better understand these relationships, complemented by site-directed mutagenesis and cross-linking studies to identify critical interaction residues.

How can high-throughput screening be applied to identify inhibitors of ExbB function?

High-throughput screening (HTS) for ExbB inhibitors can be approached using the following methodologies:

• Target-based screening assays:

  • ATP hydrolysis assays to measure disruption of energy coupling

  • Fluorescence-based protein interaction assays to identify compounds that disrupt ExbB-ExbD or ExbB-TonB interactions

  • Conformational change assays using labeled proteins to detect inhibitors that prevent structural transitions

• Cell-based screening approaches:

  • Growth inhibition assays under iron-limited conditions where ExbB function is crucial

  • Reporter gene assays where ExbB-dependent iron acquisition is linked to expression of a detectable reporter

  • Complementation systems using the pMG1 approach, where compounds that inhibit the complementation phenotype are identified

• In silico screening strategies:

  • Structure-based virtual screening once ExbB structural data is available

  • Pharmacophore modeling based on known interaction patterns

  • Molecular docking studies to predict binding of potential inhibitors

• Screening libraries selection:

  • Natural product libraries, particularly siderophore analogs

  • Synthetic compound libraries targeting membrane proteins

  • Peptide libraries that may mimic interaction interfaces

• Validation and optimization pipeline:

  • Secondary assays to confirm hits and eliminate false positives

  • Structure-activity relationship studies to optimize lead compounds

  • Whole-cell activity confirmation in P. haemolytica and related pathogens

This systematic approach to HTS can accelerate the discovery of novel inhibitors that may have potential as antimicrobial agents against P. haemolytica and related pathogens.

How does ExbB contribute to the pathogenesis of bovine respiratory disease?

ExbB contributes to the pathogenesis of bovine respiratory disease (BRD) through several mechanisms:

• Iron acquisition: As part of the ExbB-ExbD-TonB energy transduction system, ExbB enables the uptake of iron in the iron-limited environment of the host respiratory tract. This is crucial for bacterial survival and proliferation during infection .

• Support of virulence factor expression: Many virulence factors in P. haemolytica are iron-regulated, and the function of ExbB in iron acquisition indirectly supports their expression. The 35 kDa iron-regulated protein (FbpA) identified in P. haemolytica under iron-depleted conditions is an example of such a factor .

• Bacterial persistence: Efficient iron acquisition facilitated by ExbB allows P. haemolytica to persist in the lower respiratory tract, leading to the severe fibrinonecrotic pneumonia characteristic of pneumonic pasteurellosis .

• Adaptation to stress conditions: The ExbB-ExbD-TonB system may play roles in bacterial adaptation to various stress conditions encountered during infection, beyond iron limitation.

• Potential interaction with host factors: While not directly demonstrated for ExbB, components of bacterial iron acquisition systems often interact with host proteins, potentially contributing to pathogenesis.

Research has shown that P. haemolytica, particularly the A1 serotype, is a significant etiological agent in BRD, with its ability to colonize the lower respiratory tract following stress factors or viral infection that alter the upper respiratory tract epithelium . The iron acquisition systems, including ExbB, are essential for this process.

What is the comparative expression of ExbB in different serotypes of Pasteurella haemolytica?

While specific data on ExbB expression across different serotypes is limited in the search results, we can infer some patterns based on related information:

• Serotype A1 significance: P. haemolytica A1 (now classified as Mannheimia haemolytica) is particularly associated with severe fibrinonecrotic pneumonia in cattle . The exbB-exbD-tonB locus has been specifically cloned and characterized from this serotype , suggesting its importance in the virulence of this particular strain.

• Expression patterns: Iron-regulated systems tend to be conserved across serotypes but may show variations in expression levels or regulation. The 35 kDa iron-regulated protein (FbpA) has been identified specifically in P. haemolytica grown under iron-depleted conditions , and similar regulation might be expected for the ExbB-ExbD-TonB system.

• Serotype distribution in clinical cases: Studies have shown that M. haemolytica (formerly P. haemolytica) is more frequently isolated than other Pasteurella species in pneumonic animals . From pneumonic lung samples collected from cattle and sheep, M. haemolytica was isolated at a rate of 13.07%, compared to 7.39% for B. trehalosi and 6.83% for P. multocida .

• Molecular characterization approaches: To properly compare ExbB expression across serotypes, techniques such as quantitative PCR targeting the exbB gene or proteomic analysis would be required, similar to the molecular characterization methods used for P. multocida where multiplex PCR targeting specific genes was employed to identify serovars A1 and A3 .

A comprehensive comparative study of ExbB expression would require isolating different serotypes, culturing them under standardized conditions (both iron-replete and iron-depleted), and measuring exbB transcription and ExbB protein levels using qPCR and Western blotting, respectively.

How can ExbB be targeted for vaccine development against Pasteurella haemolytica infections?

ExbB presents several attributes that make it a potential target for vaccine development against P. haemolytica infections:

• Conservation and essentiality: As part of the iron acquisition system, ExbB is likely well-conserved across strains and essential for in vivo survival, making it a potentially broad-spectrum target.

• Surface accessibility: While ExbB is primarily a cytoplasmic membrane protein, certain epitopes may be accessible to the immune system, particularly if the protein has periplasmic loops or domains.

• Vaccine strategies targeting ExbB:

  a. Subunit vaccines: Purified recombinant ExbB or immunogenic fragments could be used as subunit vaccines, potentially combining them with other virulence factors.

  b. DNA vaccines: Plasmids encoding ExbB could induce both humoral and cell-mediated immunity.

  c. Live attenuated vaccines: P. haemolytica strains with modified but not deleted ExbB (to maintain immunogenicity while reducing virulence) could serve as live attenuated vaccines.

  d. Epitope-based approaches: Identification of immunodominant B-cell and T-cell epitopes from ExbB for inclusion in multi-epitope vaccines.

• Combination approaches: ExbB could be targeted alongside other virulence factors such as leukotoxin, which plays a significant role in the pathogenesis of bovine pneumonic pasteurellosis .

• Adjuvant selection: Appropriate adjuvants would be critical to enhance immune responses against ExbB, particularly if using protein subunits.

• Evaluation parameters: Vaccine efficacy would need to be assessed through:

  • Antibody titers specific to ExbB

  • Functional assays measuring inhibition of iron acquisition

  • Challenge studies in appropriate animal models

  • Cross-protection against different serotypes

Targeting essential components of iron acquisition systems has shown promise in vaccine development against other pathogens, suggesting potential for an ExbB-based approach against P. haemolytica.

How does the ExbB-ExbD-TonB system in Pasteurella haemolytica compare to homologous systems in other pathogens?

The ExbB-ExbD-TonB system in P. haemolytica shares similarities and differences with homologous systems in other bacterial pathogens:

• Genetic organization:

  • P. haemolytica: The genes are arranged in the order exbB-exbD-tonB in a single locus

  • This organization is shared with Haemophilus influenzae, H. ducreyi, Pseudomonas putida, and Vibrio cholerae

  • In contrast, Enterobacteriaceae (including E. coli) have tonB unlinked from exbB and exbD

• Functional conservation:

  • Despite organizational differences, functional conservation is evident as demonstrated by the ability of P. haemolytica exbB-exbD-tonB to complement an E. coli tonB mutation

  • The complemented E. coli exhibits restored growth kinetics in the presence of vitamin B12, susceptibility to bacteriophage phi 80, and sensitivity to colicin B

• Sequence homology:

  • While specific percentage identities aren't provided in the search results, the functional complementation suggests significant sequence conservation in critical domains

• Evolutionary implications:

  • The linked exbB-exbD-tonB arrangement may represent a separate lineage of evolution from that of the Enterobacteriaceae

  • The flanking regions of this locus are completely different between P. haemolytica and H. influenzae, suggesting independent acquisition of this arrangement

• System redundancy:

  • Some bacteria possess multiple paralogs of ExbB, ExbD, or TonB, while others have single copies

  • The search results don't specify whether P. haemolytica has multiple copies of these genes

This comparative analysis provides insights into the evolutionary relationships between different bacterial iron acquisition systems and highlights the adaptability of these essential systems across diverse pathogens.

What methodological approaches can be used to study the interaction between ExbB and iron-regulated proteins in Pasteurella haemolytica?

Several methodological approaches can be employed to study the interactions between ExbB and iron-regulated proteins in P. haemolytica:

• Co-immunoprecipitation (Co-IP):

  • Using antibodies against ExbB to pull down the protein along with interacting partners

  • Mass spectrometry analysis of co-precipitated proteins to identify interactions

  • Western blotting to confirm specific interactions with iron-regulated proteins

• Bacterial two-hybrid systems:

  • Adaptation of yeast two-hybrid for bacterial proteins

  • Particularly useful for membrane proteins like ExbB

  • Can screen for interactions with multiple iron-regulated proteins simultaneously

• Crosslinking studies:

  • Chemical crosslinking of proteins in intact cells or membrane preparations

  • Identification of crosslinked partners by mass spectrometry

  • Site-specific crosslinking to map interaction domains

• Förster resonance energy transfer (FRET):

  • Tagging ExbB and potential interaction partners with fluorescent proteins

  • Measuring energy transfer as an indication of protein proximity

  • Live-cell imaging to study dynamics of interactions

• Surface plasmon resonance (SPR):

  • Immobilizing purified ExbB on a sensor chip

  • Measuring binding kinetics with putative interaction partners

  • Determining affinity constants for various interactions

• Genetic approaches:

  • Suppressor mutation analysis to identify functional interactions

  • Construction of chimeric proteins to map interaction domains

  • Complementation assays similar to those performed with the pMG1 plasmid

• Structural biology techniques:

  • Cryo-electron microscopy of ExbB complexes

  • X-ray crystallography of ExbB alone or in complex with interaction partners

  • NMR studies of specific domains and their interactions

These approaches can be applied to study interactions between ExbB and the iron-regulated 35 kDa FbpA protein as well as other components of the iron acquisition system in P. haemolytica.

How can experimental design principles be applied to optimize research on ExbB function in different bacterial species?

Applying experimental design principles can significantly enhance research on ExbB function across bacterial species:

• Factorial experimental designs:

  • Systematically vary multiple factors affecting ExbB function (e.g., iron concentration, pH, temperature)

  • Assess interaction effects between factors

  • Identify optimal conditions for ExbB expression and function

  • Example: A 2³ factorial design could examine effects of iron concentration (high/low), pH (acidic/neutral), and growth phase (log/stationary) on ExbB expression

• Response surface methodology (RSM):

  • Optimize quantitative responses (e.g., protein expression levels, iron transport efficiency)

  • Create mathematical models to predict optimal conditions

  • Reduce the number of experiments needed to identify optimal conditions

• Randomization and blocking:

  • Implement proper randomization to minimize bias

  • Use blocking to control for known sources of variation (e.g., different batches of media)

  • These approaches are fundamental to proper experimental design as emphasized in modern statistical approaches to experimentation

• Sample size determination:

  • Calculate appropriate sample sizes based on expected effect sizes

  • Ensure sufficient replicates to achieve statistical power

  • Account for biological and technical variability in bacterial systems

• Comparative approaches:

  • Design experiments to simultaneously compare ExbB function across multiple bacterial species

  • Use standardized protocols to minimize method-induced variations

  • Implement phylogenetically aware statistical analyses for cross-species comparisons

• Controls and validation:

  • Include appropriate positive and negative controls

  • Validate findings using complementary techniques

  • Implement recombinant complementation approaches similar to those using the pMG1 plasmid

• Data analysis and interpretation:

  • Apply appropriate statistical methods for data analysis

  • Use R or similar statistical software for complex experimental designs

  • Present results in ways that facilitate cross-species comparisons

By applying these principles, researchers can design more efficient experiments that yield more reliable and comparable data on ExbB function across different bacterial species, advancing our understanding of this important component of bacterial iron acquisition systems.

What are the current knowledge gaps in understanding ExbB function in Pasteurella haemolytica?

Despite considerable progress in understanding the ExbB protein in P. haemolytica, several significant knowledge gaps remain:

• Structural characterization: Detailed three-dimensional structures of P. haemolytica ExbB, either alone or in complex with ExbD and TonB, are currently lacking. Such structural information would provide insights into the mechanism of energy transduction and species-specific characteristics.

• Regulatory networks: The complete regulatory network controlling exbB expression in response to environmental stimuli beyond iron limitation remains to be fully elucidated. Understanding these networks would provide insights into the adaptation of P. haemolytica to various host environments.

• Host-pathogen interactions: The specific role of ExbB-mediated iron acquisition in host-pathogen interactions during P. haemolytica infection is not comprehensively characterized. How this system influences bacterial survival in different host microenvironments requires further investigation.

• Species-specific functions: While the exbB-exbD-tonB locus of P. haemolytica can complement E. coli tonB mutations , potential species-specific functions or adaptations of ExbB in P. haemolytica remain unexplored.

• Interaction with other transport systems: The interplay between the ExbB-ExbD-TonB system and other nutrient acquisition systems in P. haemolytica, such as the FbpABC system , has not been fully characterized at a molecular level.

• Therapeutic targeting: The potential of ExbB as a target for novel therapeutic interventions against P. haemolytica infections remains largely theoretical without specific inhibitors or vaccine approaches being tested.

Addressing these knowledge gaps will require integrated approaches combining structural biology, molecular genetics, biochemistry, and infection models appropriate for studying bovine respiratory disease.

What future research directions should be prioritized for ExbB studies in bacterial pathogens?

Future research on ExbB in bacterial pathogens, including P. haemolytica, should prioritize:

• Structural biology approaches: Determination of high-resolution structures of ExbB and its complexes with ExbD and TonB to understand the molecular mechanism of energy transduction. This would facilitate structure-based drug design targeting this essential system.

• Systems biology integration: Comprehensive analysis of how ExbB functions within the broader context of bacterial physiology, including interaction networks with other cellular processes beyond iron acquisition.

• In vivo expression and function: Studies examining the expression and function of ExbB during actual infection processes, using techniques such as in vivo expression technology (IVET) or RNA-seq from infected tissues.

• Comparative genomics and evolution: Expanded analysis of ExbB sequence and functional conservation across a broader range of bacterial species to better understand evolutionary adaptations and potential vulnerabilities.

• Inhibitor development and screening: High-throughput screening approaches to identify specific inhibitors of ExbB function, with subsequent optimization for potential therapeutic applications.

• Vaccine development: Assessment of ExbB and related proteins as components of subunit vaccines against P. haemolytica, particularly for prevention of bovine respiratory disease.

• Cross-talk with host systems: Investigation of potential interactions between bacterial ExbB-mediated iron acquisition and host iron sequestration mechanisms during infection.

• Application of advanced experimental designs: Implementation of sophisticated experimental designs and statistical analyses to optimize research efficiency and reproducibility in ExbB studies .

These research directions would significantly advance our understanding of ExbB function in P. haemolytica and other bacterial pathogens, potentially leading to new approaches for preventing and treating infections caused by these organisms.

How might advances in understanding ExbB function contribute to controlling Pasteurella haemolytica infections in livestock?

Advances in understanding ExbB function could significantly impact control strategies for P. haemolytica infections in livestock through several avenues:

• Novel antimicrobial development: Detailed knowledge of ExbB structure and function could enable the design of specific inhibitors that disrupt iron acquisition, potentially creating new classes of antimicrobials effective against P. haemolytica without contributing to broader antimicrobial resistance issues.

• Vaccine improvement: Understanding the immunogenicity and conservation of ExbB across P. haemolytica strains could lead to its inclusion in next-generation vaccines. Since P. haemolytica (M. haemolytica) is more frequently isolated than other Pasteurella species in pneumonic animals , targeting conserved proteins like ExbB could provide broader protection.

• Diagnostic advancements: Knowledge of ExbB expression patterns could lead to improved diagnostic tools for detecting P. haemolytica infections, potentially allowing for earlier intervention in outbreaks of bovine respiratory disease.

• Epidemiological applications: Understanding strain variations in ExbB structure and function could provide markers for tracking specific P. haemolytica lineages in epidemiological studies, improving our understanding of transmission dynamics in livestock populations.

• Preventive strategies: Insights into environmental factors that regulate ExbB expression might suggest management practices that reduce P. haemolytica virulence in livestock settings, potentially through dietary interventions affecting iron availability.

• Combination therapies: Understanding how the ExbB-ExbD-TonB system interacts with other virulence mechanisms could inform the development of combination therapies targeting multiple systems simultaneously for enhanced efficacy.

• One Health applications: Knowledge of ExbB function could have broader implications for understanding similar systems in other pathogens affecting both animals and humans, contributing to the One Health approach to disease control.