Recombinant Staphylococcus aureus Bone sialoprotein-binding protein (bbp), partial

What is Bone sialoprotein-binding protein (Bbp) and what is its role in Staphylococcus aureus?

Bone sialoprotein-binding protein (Bbp) is a cell-surface protein expressed by Staphylococcus aureus with a molecular weight of approximately 97 kDa. This protein specifically interacts with bone sialoprotein (BSP), a glycoprotein found in bone and dentine extracellular matrix. Bbp is encoded by a gene that produces a protein consisting of 1171 amino acids . As a member of the MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecule) family, Bbp facilitates bacterial adherence to host tissues, particularly bone tissue . This adherence mechanism is critical in the pathogenesis of osteomyelitis and other bone and joint infections caused by S. aureus .

How does Bbp relate to other S. aureus surface proteins?

Bbp displays significant structural and functional similarity to members of the Sdr (serine-aspartate repeat) family of proteins in S. aureus, including SdrC, SdrD, and SdrE. These proteins share similar organizational patterns and are characterized by a serine-aspartic acid repeat sequence located near their C-terminal ends, close to the cell-wall-anchoring Leu-Pro-Xaa-Thr-Gly sequence . Bbp also shows similarity to Fbe, a fibrinogen-binding protein expressed by Staphylococcus epidermidis . While these proteins share structural features, they may have distinct binding specificities and roles in bacterial pathogenesis. The Sdr family members are part of the larger MSCRAMM group that mediates bacterial adherence to host matrix components .

What are the binding properties of Bbp?

Bbp primarily binds to bone sialoprotein (BSP), a component of bone and dentine extracellular matrix . Additionally, Bbp has been shown to interact with fibrinogen-α (Fg α) . The binding region of Bbp is located in the N-terminal portion of the protein, specifically between residues 273 and 598. This region is subdivided into domains N2 and N3, which form a structure composed of two layers of β-sheets with an open groove at the C-terminus where primary ligand binding occurs . The binding mechanism follows a "dock, lock, and latch" model similar to other MSCRAMM proteins . The binding property of Bbp to bone matrix components likely explains the tropism of S. aureus for bone tissue in osteomyelitis and related infections .

How can recombinant Bbp be produced for research purposes?

For producing recombinant Bbp (rBbp), researchers typically employ an Escherichia coli expression system. The bbp gene or a portion of it (for partial rBbp) can be cloned into an expression vector, such as one containing a glutathione S-transferase (GST) fusion tag . The process involves:

• PCR amplification of the bbp gene from S. aureus genomic DNA

• Cloning into an appropriate expression vector with a fusion tag for purification

• Transformation of E. coli expression strain

• Induction of protein expression (e.g., with IPTG)

• Cell lysis and extraction of the recombinant protein

• Affinity purification using the fusion tag

• Verification of the purified protein by SDS-PAGE, immunoblotting, and functional assays

The functional activity of rBbp can be verified by testing its ability to bind radiolabelled native BSP and to inhibit the binding of BSP to staphylococcal cells . Similar approaches have been used for other S. aureus proteins like PBP2B, where recombinant proteins are produced with histidine tags for purification purposes .

What methodologies are optimal for studying Bbp-host protein interactions in vitro?

For investigating Bbp-host protein interactions, several complementary methodologies yield comprehensive insights:

• Surface Plasmon Resonance (SPR): This technique allows real-time measurement of binding kinetics between Bbp and potential ligands. Purified recombinant Bbp can be immobilized on a sensor chip, and potential binding partners (e.g., BSP, fibrinogen-α) flowed over the surface to quantify association and dissociation rates.

• Isothermal Titration Calorimetry (ITC): This provides thermodynamic parameters of binding interactions, including binding affinity, enthalpy changes, and stoichiometry. ITC measurements can reveal the energetic basis of Bbp-ligand interactions.

• Fluorescence-based binding assays: Fluorescently labeled Bbp or host proteins can be used to measure binding interactions through changes in fluorescence anisotropy or FRET (Fluorescence Resonance Energy Transfer).

• Differential Scanning Fluorimetry (DSF): This method can assess protein stability and binding effects. As demonstrated with related proteins, DSF reveals how factors like calcium affect protein folding and stability .

• Co-crystallization and structural analysis: X-ray crystallography of Bbp-ligand complexes provides atomic-level insights into binding mechanisms. The structures of N2 and N3 domains complexed with their ligands can reveal specific interaction residues .

• Immunoprecipitation assays: Similar to techniques used for other S. aureus proteins, immunoprecipitation with anti-Bbp antibodies followed by testing for co-precipitated host proteins can confirm interactions in complex mixtures .

These methodologies should be employed in combination for a thorough characterization of Bbp-host interactions, as each provides distinct but complementary information about binding mechanisms.

How can the mechanostability of Bbp-ligand complexes be experimentally assessed?

The mechanostability of Bbp-ligand complexes can be assessed using several sophisticated biophysical techniques:

• Single-Molecule Force Spectroscopy (SMFS): Using atomic force microscopy (AFM), researchers can directly measure the forces required to rupture individual Bbp-ligand bonds. This approach involves:

  • Immobilizing Bbp on an AFM cantilever tip

  • Attaching the binding partner (e.g., fibrinogen-α) to a substrate

  • Measuring the force-distance curves during approach and retraction cycles

  • Analyzing rupture forces at various loading rates to determine bond characteristics

• Biomembrane Force Probe (BFP): This technique offers high temporal resolution for measuring short-lived interactions and can complement AFM-based SMFS.

• Optical Tweezers: For measuring lower force ranges with higher precision than AFM, optical tweezers can be employed to study Bbp-ligand interactions.

• Magnetic Tweezers: These provide excellent stability for long-duration measurements of bond lifetimes under constant force.

• Computational approaches: As described in the research, combining all-atom and coarse-grained steered molecular dynamics (SMD) simulations can provide detailed insights into the mechanostability of Bbp-fibrinogen complexes . These computational methods can predict:

  • Force thresholds for bond rupture

  • Structural changes under mechanical stress

  • Key residues involved in mechanical resistance

Studies have shown that Bbp exhibits extraordinary mechanostability, with rupture forces beyond 2 nN at typical experimental SMFS pulling rates . This high mechanostability appears to be physiologically relevant, as high force loads during bacterial infection may actually stabilize the protein structure, making it more rigid and enhancing host-pathogen interactions .

What are the challenges in developing anti-adhesion strategies targeting Bbp, and how can they be addressed?

Developing effective anti-adhesion strategies targeting Bbp faces several significant challenges:

• Structural adaptation under mechanical force: Research indicates that Bbp undergoes conformational changes under force loads, becoming more rigid and potentially enhancing binding . This mechanoadaptation complicates the design of inhibitors that must be effective under various mechanical conditions.

• Functional redundancy: S. aureus expresses multiple MSCRAMMs with overlapping functions, including SdrC, SdrD, and SdrE . Targeting Bbp alone may not sufficiently prevent bacterial adhesion due to this redundancy.

• Strain-to-strain variability: While the bbp gene is present across multiple S. aureus strains, sequence variations may affect inhibitor efficacy across different clinical isolates.

These challenges can be addressed through:

• Structure-based drug design: Using crystal structures of Bbp N2-N3 domains in complex with ligands to design inhibitors that specifically block the binding interface.

• Force-responsive inhibitors: Developing inhibitors that maintain or increase efficacy under mechanical stress, targeting the "locked" conformation of Bbp.

• Multi-target approaches: Creating cocktails of inhibitors targeting multiple MSCRAMMs simultaneously to overcome functional redundancy.

• Allosteric inhibitors: Designing molecules that bind to regions outside the primary binding site but induce conformational changes that prevent ligand binding.

• Antibody-based approaches: Developing antibodies that recognize conserved epitopes across different S. aureus strains, as evidenced by the detection of anti-Bbp antibodies in patients with bone and joint infections .

• Peptide mimetics: Creating peptides that mimic the binding region of bone sialoprotein but cannot be displaced by mechanical force, effectively competing with natural ligands.

Experimental validation would involve in vitro adhesion assays under flow conditions to simulate physiological shear forces, followed by in vivo testing in animal models of osteomyelitis to assess efficacy in preventing bone colonization.

What is the relationship between Bbp expression and biofilm formation in S. aureus strains?

The relationship between Bbp expression and biofilm formation in S. aureus involves complex interactions:

• Adhesion initiation: Bbp facilitates the initial attachment of S. aureus to host components, particularly fibrinogen-α, which serves as a conditioning film on surfaces . This initial attachment is a critical first step in biofilm formation.

• Mechanosensing and signaling: The mechanical forces experienced by bacteria during attachment may trigger Bbp-mediated signaling that regulates biofilm-associated genes. The extraordinary mechanostability of Bbp (>2 nN rupture forces) suggests it may function as a mechanosensor during early infection stages .

• Matrix incorporation: Bbp-bound host proteins may become incorporated into the biofilm matrix, contributing to its structural integrity and providing additional binding sites for bacterial attachment.

• Strain-dependent effects: The contribution of Bbp to biofilm formation likely varies among S. aureus strains, depending on the expression levels of Bbp and other adhesins.

To investigate this relationship, researchers should:

• Compare biofilm formation between wild-type and bbp-deletion mutants under static and flow conditions

• Examine the effect of anti-Bbp antibodies or peptide inhibitors on biofilm development

• Analyze the spatial distribution of Bbp within established biofilms using immunofluorescence microscopy

• Correlate Bbp expression levels with biofilm biomass and architecture across clinical isolates

• Investigate the composition of biofilm extracellular matrix in strains with varying Bbp expression levels

Understanding the role of Bbp in biofilm formation has significant implications for developing strategies to prevent implant-associated and bone infections, where biofilms contribute substantially to antibiotic resistance and persistent infection .

How can Bbp be utilized as a vaccine candidate against S. aureus bone and joint infections?

Developing Bbp as a vaccine candidate against S. aureus bone and joint infections requires a methodical approach:

• Antigen design strategies:

  • Full-length recombinant Bbp may contain all relevant epitopes but could present purification challenges due to its size (1171 amino acids)

  • The N2-N3 binding domains (residues 273-598) represent a focused target that contains the functional binding regions

  • Multiple epitope vaccines combining conserved regions from Bbp and other MSCRAMMs might provide broader protection

• Adjuvant selection:

  • Aluminum-based adjuvants typically induce strong antibody responses

  • TLR agonists may enhance cell-mediated immunity, which is crucial for S. aureus protection

  • Combination adjuvants might provide optimal immune stimulation

• Immune response assessment:

  • Measure anti-Bbp antibody titers and assess their functional capacity to block BSP and fibrinogen binding

  • Evaluate T-cell responses, particularly Th1/Th17 responses associated with protection against S. aureus

  • Test opsonophagocytic activity of induced antibodies against Bbp-expressing S. aureus

• Protection correlates:

  • The presence of anti-Bbp antibodies in patients with bone and joint infections suggests natural immunogenicity

  • Protection could be correlated with:

    • Antibody titers against conformational epitopes in the N2-N3 domains

    • Capacity of antibodies to block Bbp-ligand interactions under mechanical force

    • Ability to reduce bacterial burden in animal models

• Validation approaches:

  • Mouse and rabbit models of osteomyelitis and implant-associated infection

  • Assessment of bacterial burden, inflammatory markers, and tissue damage

  • Comparative studies with other S. aureus vaccine candidates

  • Combination approaches with antibiotics to assess synergistic protection

The development pathway should include careful assessment of cross-protection against diverse clinical isolates, as the conservation of Bbp across S. aureus strains suggests it could provide broad protection. Similar approaches have shown promise with other S. aureus proteins like EpiP, where both full-length and mutant forms provided comparable protection in mouse models .

What are the optimal conditions for producing high-yield, soluble recombinant Bbp in E. coli?

Optimizing the production of high-yield, soluble recombinant Bbp in E. coli requires careful consideration of several factors:

• Expression vector selection:

  • T7-based expression systems (pET series) often provide high yields

  • Fusion tags that enhance solubility: GST, MBP, SUMO, or thioredoxin

  • Previous successful approaches used GST fusion for Bbp expression

• E. coli strain optimization:

  • BL21(DE3) derivatives are common for T7-based expression

  • Strains with enhanced disulfide bond formation (Origami, SHuffle)

  • Strains containing additional tRNAs for rare codons (Rosetta, CodonPlus)

• Expression conditions table:

• Domain-based approach:

  • Express functional domains (e.g., N2-N3 region, residues 273-598) rather than full-length protein

  • Remove signal peptides and membrane-anchoring regions that may interfere with solubility

  • Similar approaches were successful with other S. aureus proteins like PBP2B

• Purification strategy:

  • Two-step purification: affinity chromatography followed by size exclusion

  • For GST fusion proteins, on-column cleavage with PreScission protease

  • Include protease inhibitors during lysis (PMSF, EDTA-free protease inhibitor cocktail)

  • Consider including low concentrations of non-ionic detergents (0.1% Triton X-100) during lysis

• Quality control:

  • SDS-PAGE and western blotting to confirm identity and purity

  • Dynamic light scattering to assess aggregation state

  • Circular dichroism to confirm proper folding

  • Functional assays: testing binding to native BSP or fibrinogen

If inclusion bodies form despite optimization attempts, protocols for refolding from inclusion bodies can be employed, though these typically result in lower yields of active protein.

How can the binding kinetics and affinity of Bbp to bone sialoprotein be accurately measured?

Accurate measurement of binding kinetics and affinity between Bbp and bone sialoprotein requires sophisticated biophysical techniques and careful experimental design:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified recombinant Bbp (or its N2-N3 domains) on a CM5 sensor chip using amine coupling

  • Flow varying concentrations of bone sialoprotein (5-500 nM) over the surface

  • Measure association and dissociation phases at different flow rates (30-60 μL/min)

  • Fit the data to appropriate binding models (1:1 Langmuir, heterogeneous ligand, etc.)

  • Extract kinetic parameters (ka, kd) and equilibrium dissociation constant (KD)

• Bio-Layer Interferometry (BLI):

  • Alternative to SPR with similar principles but different detection method

  • Immobilize Bbp on biosensors and measure wavelength shifts during binding

  • Offers advantage of higher throughput than traditional SPR

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of heat changes during binding

  • Provides complete thermodynamic profile (ΔH, ΔS, ΔG)

  • Typically requires larger amounts of protein than SPR/BLI

  • No immobilization required, measuring binding in solution

• Microscale Thermophoresis (MST):

  • Measures changes in movement of fluorescently labeled molecules in microscopic temperature gradients

  • Requires minimal sample amounts

  • Works well for studying interactions in complex biological fluids

• Fluorescence Anisotropy:

  • Label BSP with fluorescent dye

  • Measure changes in rotational diffusion upon binding to Bbp

  • Effective for studying smaller ligands binding to larger proteins

For all methods, consider these critical controls and parameters:

• Include positive controls (known binding partners) and negative controls (non-binding proteins)

• Account for non-specific binding by including reference surfaces or control proteins

• Ensure buffer matching between analyte and ligand solutions to minimize bulk refractive index changes

• Validate results using multiple techniques when possible

• Test binding under different calcium concentrations, as calcium affects folding of related proteins

• Consider the effect of glycosylation on BSP, using either native BSP or properly glycosylated recombinant BSP

The binding mechanism may involve multivalent interactions or conformational changes. Therefore, global fitting of data from multiple techniques provides the most comprehensive characterization of the Bbp-BSP interaction.

What approaches can be used to identify and validate the key residues in Bbp responsible for bone sialoprotein binding?

Identifying and validating key residues in Bbp responsible for bone sialoprotein binding requires a systematic approach combining computational prediction and experimental validation:

• Computational approaches:

  • Homology modeling of the N2-N3 domains based on structures of related proteins

  • Molecular docking simulations with bone sialoprotein

  • Molecular dynamics simulations to identify stable interaction interfaces

  • Computational alanine scanning to predict energetically important residues

  • Conservation analysis across Bbp sequences from different S. aureus strains

• Site-directed mutagenesis strategy:

  • Based on computational predictions, create single and multiple point mutants

  • Focus on residues in the N2-N3 domains (residues 273-598)

  • Include mutations in predicted binding grooves and at the interface between domains

  • Create conservative (similar amino acid) and non-conservative mutations

  • Include controls by mutating residues predicted not to affect binding

• Expression and purification of mutants:

  • Produce wild-type and mutant proteins under identical conditions

  • Verify proper folding using circular dichroism or thermal shift assays

  • Differential Scanning Fluorimetry can reveal if mutations affect protein stability

• Binding assays for mutant proteins:

  • SPR or BLI to quantitatively compare binding kinetics and affinity

  • ELISA-based binding assays for higher throughput screening

  • Pull-down assays with immobilized BSP or Bbp

  • Inhibition assays measuring the ability of mutants to block wild-type Bbp-BSP interaction

• Structural validation:

  • X-ray crystallography of key mutants in complex with BSP peptides

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • NMR analysis of chemical shift perturbations upon ligand binding

• Functional validation:

  • Cell-based adhesion assays comparing wild-type and mutant Bbp

  • S. aureus strains expressing mutant Bbp versions to test for altered bone tissue binding

  • Biofilm formation assays to correlate binding mutations with functional outcomes

• Validation across mechanical conditions:

  • Given Bbp's mechanostability properties, test binding of critical mutants under mechanical force

  • Single-molecule force spectroscopy to compare rupture forces of wild-type and mutant complexes

  • Steered molecular dynamics simulations to visualize the effect of mutations on force resistance

This comprehensive approach would identify both the specific residues critical for BSP binding and provide insight into the structural basis of the interaction, potentially revealing targets for therapeutic intervention.

How can researchers detect and quantify Bbp expression in clinical S. aureus isolates?

Detection and quantification of Bbp expression in clinical S. aureus isolates can be performed using several complementary techniques:

• Genetic screening:

  • PCR amplification of the bbp gene using specific primers

  • Real-time quantitative PCR to measure bbp mRNA levels

  • Whole-genome sequencing to identify bbp gene variants

  • Similar approaches have been used successfully for screening other S. aureus genes across multiple strains

• Protein detection:

  • Western blotting of bacterial cell lysates or membrane fractions

    • Prepare membrane fractions from clinical isolates

    • Separate proteins by SDS-PAGE

    • Transfer to membrane and probe with anti-Bbp antibodies

    • Include recombinant Bbp as a positive control

  • Flow cytometry of intact bacteria labeled with fluorescent anti-Bbp antibodies

  • Immunofluorescence microscopy to visualize Bbp on the bacterial surface

• Quantitative analysis protocol:

  a. Sample preparation:

  • Grow clinical isolates to standardized OD₆₀₀ (mid-log phase)

  • Harvest cells by centrifugation

  • For membrane extraction:

    • Resuspend in buffer with lysostaphin (20 μg/ml)

    • Incubate at 37°C for 30 minutes

    • Sonicate and ultracentrifuge to isolate membrane fraction

  b. Western blot quantification:

  • Run samples alongside a dilution series of recombinant Bbp

  • Detect with specific anti-Bbp antibodies

  • Use chemiluminescence and densitometry software for quantification

  • Express as ng Bbp per μg total membrane protein or per 10⁸ CFU

  c. Flow cytometry quantification:

  • Fix bacteria with 2% paraformaldehyde

  • Label with anti-Bbp antibodies and fluorescent secondary antibodies

  • Analyze mean fluorescence intensity

  • Compare to calibrated beads with known antibody binding capacity

• Functional assays:

  • Measure binding of clinical isolates to immobilized bone sialoprotein

  • Correlate binding capacity with Bbp expression levels

  • Inhibition with anti-Bbp antibodies to confirm specificity

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) for targeted Bbp quantification

  • Identify unique peptide signatures for Bbp

  • Use isotopically labeled peptide standards for absolute quantification

This multi-faceted approach allows researchers to determine both the presence of the bbp gene and its level of expression across clinical isolates, providing insights into the relationship between Bbp expression and clinical manifestations such as osteomyelitis or implant-associated infections.

What are the key considerations for designing in vivo experiments to study the role of Bbp in S. aureus bone infections?

Designing effective in vivo experiments to study the role of Bbp in S. aureus bone infections requires careful planning:

• Animal model selection:

  • Mouse models:

    • Advantages: well-characterized immune system, available genetic knockouts, cost-effective

    • Limitations: anatomical differences from humans, rapid disease progression

  • Rabbit models:

    • Advantages: larger size for surgical procedures, closer bone physiology to humans

    • Limitations: fewer immunological tools available, higher costs

  • Rat models:

    • Advantages: intermediate size, established osteomyelitis protocols

    • Limitations: fewer genetic tools than mice

• Infection model types:

  Model type	Advantages	Limitations	Best for studying
  Post-traumatic osteomyelitis	Mimics surgical site infections	Variability in wound healing	Acute infection following trauma
  Implant-associated infection	Clinically relevant	Technical complexity	Biofilm formation on prosthetic materials
  Hematogenous osteomyelitis	Models blood-borne spread	Less controlled infection site	Systemic dissemination to bone
  Diabetic osteomyelitis	Models comorbidity effects	Requires diabetic animals	Host factors in infection

• Bacterial strain preparation:

  • Use isogenic mutant pairs: wild-type S. aureus vs. bbp-deletion mutant

  • Complement mutant with plasmid-expressed Bbp to confirm phenotype specificity

  • Consider bioluminescent or fluorescent strains for in vivo imaging

  • Standardize inoculum preparation and verify bacterial counts

• Infection procedure:

  • Surgical exposure of target bone followed by direct inoculation

  • Implantation of contaminated foreign body (e.g., pin, screw)

  • For implant studies, pre-coat materials with bone sialoprotein or fibrinogen

  • Intravenous injection for hematogenous models

• Assessment parameters:

  • Bacterial burden: CFU counts from homogenized bone tissue

  • Imaging: X-ray, micro-CT for bone destruction, PET-CT for metabolic activity

  • Histopathology: H&E, Gram staining, immunohistochemistry for Bbp in situ

  • Immunological parameters: Cytokine profiles, immune cell infiltration

  • Biomechanical testing: Bone strength assessment for chronic infections

• Experimental design considerations:

  • Include appropriate sample sizes based on power analysis

  • Blind investigators to experimental groups during analysis

  • Include sham-operated controls

  • Monitor animals for pain and distress with validated scoring systems

  • Consider time course experiments to capture different infection phases

• Therapeutic interventions:

  • Test anti-Bbp antibodies or peptide inhibitors as preventive or therapeutic agents

  • Combine with conventional antibiotics to assess synergistic effects

  • Use passive immunization to assess protective capacity of anti-Bbp antibodies

• Translational relevance:

  • Compare findings with analyses of clinical samples from osteomyelitis patients

  • Correlate experimental outcomes with antibody responses against Bbp in patients

  • Consider host factors (age, sex, comorbidities) that might influence outcomes

These considerations will help researchers design robust in vivo experiments that can elucidate the specific contribution of Bbp to bone infections and evaluate potential therapeutic strategies targeting this virulence factor.

What are promising new approaches for targeting Bbp in the prevention and treatment of S. aureus bone infections?

Several promising approaches are emerging for targeting Bbp to prevent and treat S. aureus bone infections:

• Structure-guided inhibitor development:

  • Rational design of small molecule inhibitors targeting the binding groove in the N2-N3 domains

  • Peptide mimetics based on the binding motifs of bone sialoprotein or fibrinogen-α

  • Fragment-based drug discovery to identify chemical scaffolds that bind to key regions of Bbp

  • Computer-aided drug design leveraging the unique mechanical properties of Bbp

• Antibody-based therapeutics:

  • Monoclonal antibodies targeting conformational epitopes in the binding regions

  • Antibody-antibiotic conjugates for targeted delivery of antimicrobials

  • Bispecific antibodies targeting Bbp and other S. aureus surface proteins simultaneously

  • Nanobodies or single-chain variable fragments with enhanced tissue penetration

• Innovative vaccine strategies:

  • Multi-antigen vaccines combining Bbp with other MSCRAMM proteins

  • RNA-based vaccines encoding Bbp epitopes

  • Virus-like particles displaying Bbp binding domains

  • Adjuvant optimization to enhance mucosal immunity at potential infection sites

• Material science approaches:

  • Surface modification of implants to prevent Bbp binding

  • Bioactive coatings that release Bbp inhibitors

  • Smart biomaterials that respond to bacterial attachment by releasing antimicrobials

  • Bone cement formulations containing anti-Bbp antibodies or peptides

• Combination therapies:

  • Anti-adhesion compounds combined with conventional antibiotics

  • Biofilm-disrupting agents plus Bbp inhibitors

  • Immunomodulatory approaches to enhance host defense against S. aureus

• Precision medicine strategies:

  • Screening patients for Bbp antibody levels to guide treatment decisions

  • Genotyping clinical isolates for bbp variants to predict virulence

  • Developing therapies targeting specific Bbp variants or expression patterns

• Delivery systems:

  • Bone-targeting nanoparticles carrying Bbp inhibitors

  • Controlled-release systems for sustained local delivery

  • Cell-penetrating peptides to enhance intracellular delivery in osteoblasts and osteocytes

Each of these approaches presents unique advantages and challenges, but the most successful strategies will likely combine multiple modalities to overcome the redundancy in S. aureus virulence factors and the challenges of treating established bone infections.

How might the mechanobiology of Bbp interactions inform new therapeutic strategies?

The unique mechanobiological properties of Bbp offer novel insights for therapeutic development:

• Force-sensitive therapeutic targeting:

  • Design inhibitors specifically targeting the force-stabilized conformation of Bbp

  • Develop compounds that interfere with mechanosensing properties of Bbp

  • Create molecules that increase rather than decrease Bbp rigidity, potentially locking it in non-functional conformations

• Mechanically triggered release systems:

  • Design drug delivery systems that release inhibitors in response to mechanical forces similar to those that activate Bbp

  • Create materials that sense bacterial attachment forces and respond by releasing antimicrobials

• Disruption of force transmission pathways:

  • Target the interface between N2 and N3 domains that transmits mechanical force

  • Develop compounds that interfere with force-induced conformational changes

  • Research indicates that high force loads make Bbp more rigid, suggesting that compounds stabilizing flexible conformations might reduce adhesion

• Mechanoresponsive biomaterials:

  • Design implant surfaces with controlled nanoscale topography that minimizes force-enhanced binding

  • Develop materials that change surface properties under mechanical stress to prevent bacterial adhesion

• Exploitation of mechanical thresholds:

  • Identify the force threshold required for Bbp activation and design therapies that operate below this threshold

  • Create flow systems that generate shear forces unfavorable for Bbp-mediated adhesion but favorable for host cell attachment

• Combining mechanical and biochemical approaches:

  • Develop dual-action therapies that both block the binding site and prevent mechanically induced conformational changes

  • Design therapeutic antibodies that bind preferentially to mechanically stretched conformations of Bbp

• Diagnostic applications:

  • Develop biosensors that detect the mechanical activation state of Bbp in clinical samples

  • Create assays measuring bond strength between Bbp and host ligands as prognostic tools

The extraordinary mechanostability of Bbp, with rupture forces beyond 2 nN in typical experimental SMFS pulling rates, suggests that mechanical forces play a crucial role in infection . Understanding how these forces influence bacterial adhesion could lead to entirely new classes of anti-infective therapies that target the mechanical aspects of host-pathogen interactions rather than conventional biochemical pathways.

What fundamental questions remain about the structure and function of Bbp in S. aureus pathogenesis?

Despite significant advances in understanding Bbp, several fundamental questions remain unresolved:

• Structural determinants of binding specificity:

  • What are the atomic-level interactions that determine Bbp's preference for bone sialoprotein over other matrix proteins?

  • How does the three-dimensional structure of Bbp accommodate binding to both bone sialoprotein and fibrinogen-α?

  • Are there additional, undiscovered ligands for Bbp in bone and other tissues?

• Mechanistic questions:

  • What is the precise mechanism by which Bbp mediates bacterial attachment to bone tissue?

  • How does force-induced conformational change in Bbp affect downstream signaling in S. aureus?

  • Does Bbp serve functions beyond adhesion, such as immune evasion or biofilm formation?

• Regulation of Bbp expression:

  • What environmental signals trigger Bbp expression in vivo?

  • How is Bbp expression coordinated with other virulence factors during infection?

  • Are there tissue-specific cues that modulate Bbp expression in bone environments?

• Evolution and diversity:

  • How diverse are Bbp variants across clinical S. aureus isolates?

  • What is the evolutionary history of Bbp in relation to other MSCRAMM proteins?

  • Do different Bbp variants confer tissue-specific advantages during infection?

• Host-pathogen interactions:

  • How does the host immune system recognize and respond to Bbp?

  • Are there host genetic factors that influence susceptibility to Bbp-mediated infection?

  • Does Bbp interact with osteoblasts, osteoclasts, or other bone cells beyond simple adhesion?

• Biofilm contributions:

  • What is the precise role of Bbp in biofilm initiation, maturation, and dispersal?

  • How does Bbp contribute to antibiotic tolerance in biofilms?

  • Does Bbp-mediated attachment influence the spatial organization of cells within biofilms?

• Therapeutic targeting:

  • What is the minimum inhibitory concentration of anti-Bbp agents needed to prevent infection?

  • How can the redundancy of adhesins in S. aureus be overcome when targeting Bbp?

  • What combination of targets would synergize with Bbp inhibition?

Addressing these questions will require interdisciplinary approaches combining structural biology, bacterial genetics, advanced imaging techniques, and in vivo models of infection. The answers will not only enhance our understanding of S. aureus pathogenesis but also inform the development of more effective strategies to prevent and treat bone infections.

How might advanced molecular techniques be applied to better understand the role of Bbp in bacterial adaptation to bone environments?

Advanced molecular techniques offer powerful approaches to elucidate Bbp's role in S. aureus adaptation to bone environments:

• Single-cell technologies:

  • Single-cell RNA sequencing to identify heterogeneity in bbp expression within bacterial populations during bone infection

  • Time-lapse microscopy with fluorescent bbp reporters to track expression dynamics in real-time

  • Correlative light-electron microscopy to visualize Bbp distribution on the bacterial surface during attachment

• CRISPR-based approaches:

  • CRISPR interference (CRISPRi) for tunable repression of bbp expression

  • CRISPR activation (CRISPRa) to artificially upregulate bbp in controlled environments

  • CRISPR-Cas9 genome editing to introduce specific mutations in bbp binding domains

  • CRISPR screening to identify genes that interact functionally with bbp

• Advanced proteomics:

  • Proximity-dependent biotin identification (BioID) to map Bbp protein interactions on the bacterial surface

  • Phosphoproteomics to identify signaling pathways activated by Bbp-ligand interactions

  • Crosslinking mass spectrometry to capture transient interactions between Bbp and host proteins

  • Targeted proteomics to quantify Bbp expression relative to other adhesins in bone tissue

• Structural biology techniques:

  • Cryo-electron microscopy to visualize Bbp structure on intact bacterial surfaces

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon ligand binding

  • NMR spectroscopy to study dynamic aspects of Bbp-ligand interactions

  • Native mass spectrometry to analyze intact Bbp complexes with host proteins

• In situ approaches:

  • RNA-FISH to visualize bbp transcripts during infection

  • Intravital microscopy to observe S. aureus interactions with bone in real-time

  • Spatial transcriptomics to correlate bbp expression with microenvironmental factors

  • 3D bioprinting of bone tissue models with controlled matrix composition

• Systems biology integration:

  • Multi-omics integration combining transcriptomics, proteomics, and metabolomics data

  • Network analysis to identify regulatory hubs controlling bbp expression

  • Mathematical modeling of force-dependent binding kinetics

  • Artificial intelligence approaches to predict Bbp binding sites in novel host proteins

• Innovative animal models:

  • Humanized mouse models expressing human bone matrix proteins

  • Ex vivo bone infection models maintaining living bone tissue architecture

  • Organ-on-a-chip microfluidic devices mimicking bone microenvironments

These advanced techniques, used in combination, would provide unprecedented insights into how Bbp facilitates S. aureus adaptation to bone environments, from initial attachment to persistent infection. The molecular mechanisms revealed could identify new intervention points for preventing and treating osteomyelitis and implant-associated infections.

How does Bbp compare structurally and functionally with other members of the Sdr protein family?

A comprehensive comparison of Bbp with other Sdr family proteins reveals important structural and functional similarities and differences:

Structural Comparison

Functional Comparison

Key Differences

• Bbp shows extraordinary mechanostability compared to other family members, with rupture forces exceeding 2 nN .

• Each Sdr protein has distinct ligand specificity, with Bbp uniquely targeting bone sialoprotein .

• The number of B repeats varies significantly between family members.

• Expression patterns differ, with Bbp expression particularly associated with bone and joint infections .

• Bbp binding to fibrinogen-α occurs at a different site than ClfB binding to the same protein.