Recombinant Bartonella henselae Protein grpE (grpE)

What is the biological role of grpE in Bartonella henselae?

The grpE protein in Bartonella henselae functions as a critical co-chaperone in the heat shock response system. As part of the molecular chaperone machinery, grpE works cooperatively with DnaK (Hsp70) and DnaJ (Hsp40) to facilitate proper protein folding under stress conditions . During temperature shifts that B. henselae experiences when transitioning from vector environments (cooler) to mammalian hosts (warmer), the grpE protein plays an essential role in helping the bacteria adapt to thermal stress . This adaptation mechanism is particularly relevant given that B. henselae must transition between arthropod vectors and mammalian hosts during its lifecycle, facing temperature changes from approximately 28°C to 37°C . The protein contributes to bacterial survival and pathogenicity by preventing protein aggregation and assisting in the refolding of denatured proteins during stress conditions.

How does B. henselae grpE differ from other bacterial heat shock proteins?

B. henselae grpE shares fundamental structural and functional characteristics with other bacterial heat shock proteins but exhibits sequence variations that may reflect adaptation to the unique intracellular lifestyle of this pathogen . While the core nucleotide exchange factor functionality remains conserved across bacterial species, B. henselae grpE likely contains specialized domains that facilitate its particular role in the organism's adaptation between vector and host environments . Unlike better-characterized heat shock responses in model organisms like E. coli, the B. henselae heat shock system appears to be specifically tailored to support its intracellular invasion and persistence capabilities . Transcriptomic analyses have shown that B. henselae exhibits distinct gene expression patterns between extracellular and intracellular states, suggesting that heat shock proteins including grpE may be regulated differently depending on the bacterial microenvironment .

What expression systems are most effective for producing recombinant B. henselae grpE?

Based on successful approaches with other Bartonella recombinant proteins, the pET expression system in E. coli BL21(DE3) offers an efficient platform for recombinant B. henselae grpE production . When designing the expression construct, researchers should consider:

• Codon optimization for E. coli expression

• Inclusion of an appropriate tag (His-tag or T7-tag) for purification

• Selection of the correct reading frame and orientation

The expression protocol should involve:

• PCR amplification of the grpE gene from B. henselae genomic DNA

• Cloning into a vector such as pET200D/TOPO or pET24a

• Transformation into competent E. coli BL21(DE3) cells

• Confirmation of successful cloning by Sanger sequencing

• Protein expression induction with IPTG at optimal concentrations

The success of this approach is supported by similar methodologies applied to the expression of B. henselae Pap31 and B. bacilliformis Pap31 proteins, which have been successfully produced as recombinant proteins for diagnostic applications .

What are the optimal conditions for purifying recombinant B. henselae grpE protein?

Purification of recombinant B. henselae grpE protein can be achieved through affinity chromatography, with specific conditions tailored to maximize yield and purity. Based on successful purification strategies for other Bartonella recombinant proteins, the following protocol is recommended:

• Cell lysis: Sonication in buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and protease inhibitors

• Clarification: Centrifugation at 12,000× g for 30 minutes at 4°C

• Affinity purification: Using nickel-nitrilotriacetic acid (Ni-NTA) resin for His-tagged proteins

• Washing: Gradient washing with increasing imidazole concentrations (20-50 mM) to remove nonspecific binding

• Elution: With buffer containing 250-300 mM imidazole

• Buffer exchange: Dialysis against PBS or similar buffer to remove imidazole

• Quality control: SDS-PAGE and Western blot analysis to confirm purity and identity

Protein folding should be verified through circular dichroism spectroscopy to ensure the recombinant protein maintains its native conformation. If purified protein shows evidence of aggregation, optimization of buffer conditions or the addition of chaperone co-expression systems may be necessary.

What methods are most effective for analyzing the structure-function relationship of B. henselae grpE?

Comprehensive analysis of B. henselae grpE structure-function relationships requires a multi-method approach:

• Biophysical characterization:

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

  • Differential scanning calorimetry (DSC) to determine thermal stability

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

• Functional assays:

  • ATPase activity assays to measure nucleotide exchange function

  • Protein refolding assays with denatured substrate proteins

  • Thermal aggregation prevention assays

• Structural analysis:

  • X-ray crystallography or cryo-electron microscopy for high-resolution structure

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify flexible regions

  • Site-directed mutagenesis combined with functional assays to identify critical residues

• Interaction studies:

  • Co-immunoprecipitation with DnaK homologs

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

These approaches would help determine how the protein responds to temperature shifts between vector (28°C) and host (37°C) environments, which is particularly relevant given B. henselae's lifecycle .

How does the nucleotide exchange activity of B. henselae grpE compare with other bacterial homologs?

The nucleotide exchange activity of B. henselae grpE likely exhibits specialized features adapted to the organism's unique lifestyle. While detailed comparative studies are still emerging, preliminary functional characterization suggests:

The nucleotide exchange activity should be assessed through:

• Direct measurement of ATP/ADP exchange rates in reconstituted systems with purified B. henselae DnaK

• Comparative analysis with homologous systems from E. coli and other well-characterized bacteria

• Evaluation of activity under varying temperature conditions that mimic the transition between vector (28°C) and mammalian host (37°C)

Differences in nucleotide exchange activity may reflect adaptations to B. henselae's intracellular lifestyle and particular thermal stress conditions encountered during host infection.

What is the potential of recombinant B. henselae grpE as a diagnostic antigen?

The potential of recombinant B. henselae grpE as a diagnostic antigen should be evaluated based on its immunogenicity and specificity. Current evidence from studies with other Bartonella recombinant proteins suggests a structured approach to this assessment:

• Initial evaluation with Western blot:

  • Test against sera from confirmed B. henselae-infected patients

  • Compare with control sera from healthy individuals

  • Determine sensitivity and specificity at this preliminary level

• ELISA development and optimization:

  • Establish optimal coating concentration of recombinant grpE

  • Determine appropriate serum dilutions

  • Establish cutoff values using ROC curve analysis

• Comparative analysis with established antigens:

  • Side-by-side comparison with currently used diagnostic antigens

  • Calculation of concordance rates with gold standard tests

  • Determination of positive and negative predictive values

While studies with recombinant Pap31 from B. henselae showed 72% sensitivity and 61% specificity for human bartonellosis diagnosis , the potential of grpE remains to be fully evaluated. As a highly conserved heat shock protein, grpE may present cross-reactivity challenges that would need to be addressed through careful epitope selection or combination with other antigens in a multiplex approach.

How can cross-reactivity be minimized when using B. henselae grpE for serodiagnosis?

Minimizing cross-reactivity when using B. henselae grpE for serodiagnosis requires strategic approaches to enhance specificity:

• Epitope mapping and selection:

  • Identify B. henselae-specific epitopes within the grpE protein

  • Use bioinformatic analysis to predict unique regions

  • Experimentally validate these regions with epitope mapping techniques

  • Consider using only specific fragments rather than the whole protein

• Recombinant protein modification:

  • Create chimeric constructs containing only species-specific regions

  • Introduce mutations in conserved regions to enhance specificity

  • Remove highly conserved domains that contribute to cross-reactivity

• Assay optimization:

  • Implement pre-adsorption steps with heterologous proteins

  • Use high stringency washing conditions

  • Employ competitive inhibition methods with soluble cross-reactive proteins

• Validation against diverse serum panels:

  • Test against sera from patients with other bacterial infections

  • Include sera from patients with other Bartonella species infections

  • Calculate specificity across different clinical scenarios

These strategies should be systematically evaluated, as studies with other Bartonella recombinant proteins have shown that whole proteins may not achieve ideal diagnostic performance. For example, recombinant Pap31 fragments showed varied specificity values, with certain cutoff adjustments improving specificity to 94% at the cost of reduced sensitivity (39%) .

How does B. henselae grpE contribute to intracellular survival in host cells?

B. henselae grpE likely plays a crucial role in facilitating bacterial adaptation to the intracellular environment of host cells. The protein's contribution to intracellular survival can be investigated through several experimental approaches:

• Transcriptomic profiling:

  • Compare grpE expression levels between extracellular and intracellular bacteria

  • Analyze temporal expression patterns during different stages of infection

  • Correlate expression with stress response gene networks

• Gene knockout/knockdown studies:

  • Create conditional mutants with reduced grpE expression

  • Assess intracellular survival rates compared to wild-type bacteria

  • Measure bacterial replication in endothelial cells and monocytes

• Protein interaction studies:

  • Identify host proteins that interact with bacterial grpE

  • Characterize how these interactions affect host cell signaling

  • Determine if grpE is secreted or remains intracellular

• Stress response analysis:

  • Evaluate thermal tolerance of bacteria with altered grpE levels

  • Assess resistance to oxidative stress encountered in phagocytes

  • Measure proteomic changes dependent on grpE function

Recent transcriptomic analyses have shown that B. henselae undergoes significant gene expression changes upon intracellular invasion, with virulence factors and adhesion molecules being differentially regulated . While specific data on grpE regulation is emerging, its role as a heat shock protein suggests it could be critical for bacterial adaptation to the intracellular niche, particularly in helping the bacterium cope with host-induced stress conditions.

What role does B. henselae grpE play in bacterial adaptation between vector and mammalian host environments?

B. henselae grpE functions as a key adaptive protein during the transition between arthropod vector (28°C) and mammalian host (37°C) environments. This adaptation process can be characterized through:

• Temperature-dependent expression studies:

  • Quantify grpE expression at different temperatures using qPCR

  • Compare protein levels using Western blot analysis

  • Conduct promoter activity assays at different temperatures

• Functional characterization at different temperatures:

  • Measure nucleotide exchange activity at 28°C versus 37°C

  • Assess protein stability and oligomeric state at different temperatures

  • Evaluate interaction with DnaK under varying temperature conditions

• In vivo transition models:

  • Develop experimental systems to model vector-to-host transition

  • Monitor grpE expression during this transition

  • Correlate grpE activity with bacterial survival rates

• Comparative analysis with vector-borne pathogens:

  • Compare with adaptation mechanisms in related alpha-proteobacteria

  • Identify conserved and species-specific features of temperature adaptation

  • Develop models of evolutionary adaptation to the vector-host lifecycle

Research with B. quintana has shown that the expression of heat shock genes, including the rpoE gene, is upregulated when bacteria transition from body louse temperature (28°C) to human host temperature (37°C) . Similar temperature-specific transcriptional profiles have been documented in Bartonella species, suggesting that grpE likely participates in a coordinated stress response system that enables successful host infection following transmission from arthropod vectors.

How can recombinant B. henselae grpE be used to develop novel therapeutic approaches?

Recombinant B. henselae grpE offers several potential avenues for therapeutic development:

• Inhibitor discovery and development:

  • High-throughput screening for small molecule inhibitors of grpE function

  • Structure-based drug design targeting the nucleotide exchange activity

  • Identification of peptide inhibitors that disrupt grpE-DnaK interactions

• Vaccine development:

  • Evaluation of recombinant grpE as a subunit vaccine candidate

  • Assessment of protective immunity in animal models

  • Design of epitope-based vaccines targeting immunogenic regions

• Immunomodulatory approaches:

  • Characterization of host immune responses to grpE

  • Development of immunotherapeutic strategies targeting these responses

  • Investigation of grpE-derived peptides with immunomodulatory properties

• Combination therapeutic strategies:

  • Identification of synergistic effects with conventional antibiotics

  • Development of dual-targeting approaches affecting both grpE and other bacterial systems

  • Creation of nanoparticle-based delivery systems for anti-grpE molecules

This approach is particularly relevant given the reduced antibiotic susceptibility of intracellular Bartonella reported in previous studies . As essential components of bacterial stress response systems, heat shock proteins like grpE represent promising targets for new antimicrobial strategies, especially against intracellular pathogens where conventional antibiotics often show limited efficacy.

What techniques are most effective for studying the interaction between B. henselae grpE and the host immune system?

Investigating the interaction between B. henselae grpE and the host immune system requires sophisticated immunological techniques:

• T-cell response characterization:

  • ELISPOT assays to measure T-cell activation by grpE epitopes

  • Intracellular cytokine staining to identify T-cell subsets responding to grpE

  • T-cell proliferation assays to quantify antigen-specific responses

• B-cell and antibody studies:

  • Epitope mapping to identify immunodominant regions using:

    • Peptide arrays covering the complete grpE sequence

    • Phage display libraries expressing grpE fragments

    • Hydrogen-deuterium exchange mass spectrometry with anti-grpE antibodies

  • Antibody affinity maturation analysis using surface plasmon resonance

  • Memory B-cell ELISpot assays to quantify B-cell responses

• Innate immune recognition:

  • Reporter cell assays for TLR activation by recombinant grpE

  • Inflammasome activation studies in macrophage models

  • Neutrophil activation and NETosis assessment

• In vivo imaging of immune responses:

  • Tracking labeled grpE in animal models using PET or fluorescence imaging

  • Monitoring cellular immune responses using transgenic reporter animals

  • Two-photon microscopy of immune cell interactions with grpE-expressing bacteria

Preliminary studies with other Bartonella proteins have identified linear B-cell epitope regions that contribute to serological responses . For grpE, similar epitope mapping approaches using tools like IEDB Analysis Resource software (BepiPred 2.0) would help identify immunodominant regions that could be exploited for diagnostic or therapeutic applications.

What are the common challenges in producing soluble recombinant B. henselae grpE protein?

Producing soluble recombinant B. henselae grpE protein presents several technical challenges that require systematic troubleshooting:

• Expression-related challenges:

  • Inclusion body formation: Common with overexpressed proteins

  • Toxicity to host cells: May occur if protein disrupts E. coli metabolism

  • Premature termination: Could result from rare codon usage

• Purification difficulties:

  • Aggregation during purification steps

  • Co-purification of contaminating E. coli proteins

  • Protein instability in standard buffer conditions

• Methodological solutions:

  • Expression optimization:

    • Reduce induction temperature to 16-20°C

    • Lower IPTG concentration (0.1-0.5 mM)

    • Use specialized E. coli strains (e.g., Rosetta for rare codons, Arctic Express for cold-adaptation)

    • Co-express with chaperone systems

  • Solubility enhancement:

    • Test multiple solubility tags (MBP, SUMO, thioredoxin)

    • Optimize lysis buffer conditions (test various detergents, salt concentrations)

    • Consider on-column refolding protocols

  • Stability improvement:

    • Screen buffer conditions using differential scanning fluorimetry

    • Add stabilizing agents (glycerol, trehalose, specific ions)

    • Optimize protein concentration to prevent concentration-dependent aggregation

Experience with other Bartonella recombinant proteins has shown that careful optimization of expression and purification conditions is essential. For example, while recombinant Pap31 and some fragments yielded single bands on SDS-PAGE, the middle domain fragment showed purification challenges requiring additional optimization .

How can the antigenic integrity of recombinant B. henselae grpE be verified for research applications?

Verifying the antigenic integrity of recombinant B. henselae grpE requires a comprehensive validation approach:

• Structural integrity assessment:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Thermal denaturation profiles to assess stability

  • Tryptophan fluorescence spectroscopy to evaluate tertiary structure

  • Dynamic light scattering to detect aggregation

• Functional validation:

  • Nucleotide exchange activity assays with B. henselae DnaK

  • Thermal protection assays with model substrate proteins

  • ATPase stimulation of the DnaK chaperone system

• Immunological characterization:

  • Western blot with patient sera from confirmed B. henselae infections

  • Cross-reactivity testing with sera from patients with other infections

  • Epitope accessibility analysis using monoclonal antibodies

  • Comparison with native protein using B. henselae lysates

• Quantitative metrics:

  Validation Parameter	Acceptable Range	Method
  Secondary structure similarity	>80% compared to predicted	Far-UV CD spectroscopy
  Nucleotide exchange activity	>70% of theoretical	Fluorescence-based exchange assay
  Antibody recognition	Recognition by >80% of confirmed positive sera	Western blot/ELISA
  Thermal stability	Tm within 5°C of native protein	Differential scanning calorimetry
  Aggregation state	>90% monomeric/native oligomeric state	Size exclusion chromatography

These validation steps are essential for ensuring that research findings with recombinant grpE accurately reflect the biological properties of the native protein. Studies with other recombinant Bartonella antigens have demonstrated the importance of thorough validation for both research and diagnostic applications .