Recombinant Bartonella quintana Deoxyguanosinetriphosphate triphosphohydrolase-like protein (BQ07690)

What is Bartonella quintana and what diseases does it cause?

Bartonella quintana is a gram-negative bacterium that causes trench fever and chronic bacteremia. This pathogen is primarily transmitted through body lice and has been reported in populations experiencing homelessness and in Indigenous communities with limited access to water . The bacterium has been documented in multiple Canadian provinces and territories, with increasing detection rates in recent years .

B. quintana infection can manifest in several clinical forms:

• Trench fever, characterized by relapsing febrile episodes

• Chronic bacteremia

• Endocarditis, which can be fatal (19% mortality rate in documented cases)

• Vascular proliferative lesions

Unlike many gram-negative bacteria, B. quintana lipopolysaccharide (LPS) demonstrates unique immunomodulatory properties, acting as a potent antagonist of Toll-like receptor 4 (TLR4) and inhibiting the production of proinflammatory cytokines in human monocytes .

What is the structure and function of Deoxyguanosinetriphosphate triphosphohydrolase-like protein (BQ07690)?

BQ07690 is classified as a deoxyguanosinetriphosphate triphosphohydrolase-like protein in Bartonella quintana . While the search results don't provide comprehensive structural data specifically for BQ07690, this enzyme likely belongs to the family of nucleotide triphosphatases that hydrolyze dGTP to deoxyguanosine and triphosphate.

The protein's functional role in B. quintana may involve:

• Regulation of dGTP pools within the bacterium

• Maintenance of nucleotide homeostasis

• Potential involvement in DNA replication processes

• Possible role in pathogenesis or stress response mechanisms

Based on similar proteins in other bacterial species, BQ07690 may play a role in bacterial survival under various environmental conditions, though specific research on this particular protein is needed to confirm its precise functions.

How is recombinant BQ07690 typically expressed and purified?

Recombinant BQ07690 can be expressed in various host systems, with E. coli being the most commonly used for bacterial proteins. Based on similar recombinant Bartonella proteins, the following expression and purification approach can be employed:

Expression Systems:

• E. coli expression systems (most common)

• Yeast expression systems

• Baculovirus expression systems

• Mammalian cell expression systems

Purification Process:

• Cell lysis using mechanical disruption or chemical methods

• Clarification of lysate by centrifugation

• Initial capture using affinity chromatography (if tagged)

• Polishing steps using ion exchange or size exclusion chromatography

• Purity assessment by SDS-PAGE (target purity: ≥85%)

Storage Recommendations:

• Store liquid form at -20°C/-80°C for up to 6 months

• Store lyophilized form at -20°C/-80°C for up to 12 months

• Avoid repeated freezing and thawing

• Store working aliquots at 4°C for up to one week

What optimization strategies can be employed for the expression of recombinant BQ07690?

Response Surface Methodology (RSM) with Box-Behnken design (BBD) represents an effective approach for optimizing recombinant protein expression, including proteins like BQ07690. This methodology allows researchers to identify optimal cultivation conditions by systematically varying multiple parameters simultaneously.

Key Parameters for Optimization:

• Induction temperature

• Post-induction time

• Pre-induction optical density (OD600)

• Inducer concentration (e.g., IPTG)

RSM-BBD Optimization Process:

• Establish experimental design (typically a four-factor-three-level BBD)

• Conduct experimental runs with varying parameter combinations

• Analyze results using statistical software (e.g., Design-Expert)

• Develop predictive models for protein expression

• Validate optimal conditions experimentally

Previous studies have demonstrated significant increases in recombinant protein production using RSM-BBD optimization. For example, anti-keratin scFv TS1-218 expression showed a 21-fold increase after optimization of pH, methanol concentration, and temperature parameters . Similarly, DsbA-IGF1 expression and purification were effectively enhanced using this approach .

What analytical techniques are most appropriate for characterizing BQ07690?

Comprehensive characterization of BQ07690 requires multiple analytical techniques to assess its structural and functional properties:

Structural Characterization:

• SDS-PAGE for purity assessment and molecular weight determination

• Circular dichroism (CD) spectroscopy for secondary structure analysis

• X-ray crystallography for high-resolution 3D structure determination

• Nuclear magnetic resonance (NMR) for solution structure analysis

• Mass spectrometry for accurate mass determination and post-translational modifications

Functional Characterization:

• Enzymatic activity assays (dGTPase activity)

• Substrate specificity analysis

• Kinetic parameter determination (Km, Vmax, kcat)

• Inhibition studies

• Temperature and pH stability profiles

Interaction Analysis:

• Surface plasmon resonance (SPR) for binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Pull-down assays for identifying interaction partners

• Analytical ultracentrifugation for oligomerization state determination

How can researchers effectively design experiments to investigate the interaction between BQ07690 and host cells?

Investigating the interaction between BQ07690 and host cells requires a multifaceted experimental approach:

In Vitro Interaction Studies:

• Express and purify recombinant BQ07690 (≥85% purity)

• Prepare relevant human cell lines (e.g., monocytes, macrophages)

• Expose cells to varying concentrations of BQ07690

• Analyze cellular responses:

  • Cytokine production (TNF-α, IL-1β, IL-6)

  • Gene expression changes via microarray or RNA-seq

  • Signaling pathway activation (e.g., TLR pathways)

  • Cell viability and morphological changes

Mechanistic Studies:

• Identify potential host cell receptors or binding partners

• Perform co-immunoprecipitation experiments

• Conduct cellular localization studies using fluorescently tagged BQ07690

• Generate BQ07690 variants with specific domain mutations to map interaction regions

Host Response Analysis:
Based on the findings with B. quintana LPS, which acts as a TLR4 antagonist and inhibits proinflammatory cytokine production , researchers should investigate whether BQ07690 exhibits similar immunomodulatory properties. This could involve comparative studies between BQ07690 and other Bartonella proteins to determine their relative contributions to immune evasion.

What data presentation approaches are most effective for BQ07690 research?

Effective data presentation is crucial for communicating research findings. The following guidelines can help researchers determine the most appropriate format for presenting BQ07690-related data:

Table 1. Guidelines for Choosing Between Tables, Figures, and Text for Data Presentation

Regardless of the presentation method chosen, researchers should ensure that:

• Tables have clear titles that describe their content in the past tense

• Column headings clearly indicate the nature of the data presented

• Each table is understandable without reference to the text

• Only relevant results are included

• Large tables are broken into smaller, more focused ones if necessary

What are the optimal storage and handling procedures for recombinant BQ07690?

Proper storage and handling of recombinant BQ07690 are essential for maintaining its stability and activity:

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot for long-term storage

Storage Recommendations:

• Liquid form: Store at -20°C/-80°C for up to 6 months

• Lyophilized form: Store at -20°C/-80°C for up to 12 months

• Working aliquots: Store at 4°C for up to one week

• Avoid repeated freezing and thawing

Stability Factors:
The shelf life of recombinant BQ07690 depends on several factors:

• Storage temperature

• Buffer composition

• Presence of stabilizing agents (e.g., glycerol)

• Initial protein concentration

• Inherent stability of the protein

How can RSM-BBD be specifically applied to optimize BQ07690 expression?

To apply RSM-BBD for optimizing BQ07690 expression, researchers should follow this systematic approach:

Experimental Design Setup:

• Identify critical parameters affecting BQ07690 expression:

  • Induction temperature (e.g., 18°C, 25°C, 30°C)

  • Post-induction time (e.g., 4h, 8h, 16h)

  • Pre-induction OD600 (e.g., 0.6, 0.8, 1.0)

  • IPTG concentration (e.g., 0.1mM, 0.5mM, 1.0mM)

• Design experiments using Box-Behnken methodology:

  • For four factors at three levels, 29 experimental runs are typically needed

  • Include 5 center point replicates

  • Use statistical software like Design-Expert (Version 7.0.0) for design and analysis

Table 2. Example RSM-BBD Experimental Design for BQ07690 Expression Optimization

• Perform expression experiments and measure protein yield

• Analyze data to identify significant factors and optimal conditions

• Validate the model with confirmation experiments

Analysis and Validation:

• Assess model significance using F-value and P-value (e.g., F= 9.8, P < 0.0001 indicates a significant model)

• Evaluate lack of fit values (non-significant values indicate good model fit)

• Generate response surface plots to visualize parameter interactions

• Conduct validation experiments under the predicted optimal conditions

What are potential applications of BQ07690 in understanding Bartonella pathogenesis?

Understanding the role of BQ07690 in Bartonella quintana pathogenesis could provide valuable insights into:

• Nucleotide metabolism during infection

• Bacterial adaptation to host environments

• Mechanisms of persistent infection

• Potential role in immune evasion

Research approaches could include:

• Creating knockout mutants of BQ07690 and assessing virulence

• Investigating BQ07690 expression during different growth phases and infection stages

• Examining host cellular responses to purified BQ07690

• Analyzing structural similarities with dGTPases from other pathogens

Given the increasing prevalence of B. quintana infections, as documented in Canadian surveillance data showing cases across seven provinces and one territory with rising incidence , understanding the role of specific proteins like BQ07690 in pathogenesis is increasingly important.

How does BQ07690 compare to similar proteins in other bacterial pathogens?

Comparative analysis of BQ07690 with similar proteins in other bacterial species could reveal evolutionary relationships and functional conservation:

Comparative Analysis Approaches:

• Sequence alignment and phylogenetic analysis

• Structural comparison through homology modeling

• Functional characterization of enzymatic activities

• Expression pattern comparison under similar conditions

• Host response comparison studies

Such comparative studies could help identify:

• Conserved functional domains

• Species-specific adaptations

• Potential targets for broad-spectrum antimicrobial development

• Evolutionary patterns in bacterial nucleotide metabolism enzymes

What are the current technical challenges in studying BQ07690 and how might they be overcome?

Researchers face several technical challenges when studying BQ07690:

Expression and Purification Challenges:

• Potential toxicity to expression hosts

• Inclusion body formation

• Limited solubility

• Activity loss during purification

Solutions:

• Use specialized expression systems (e.g., SHuffleTM T7 strain)

• Optimize expression conditions using RSM-BBD methodology

• Employ fusion tags to enhance solubility (e.g., Thioredoxin tag)

• Develop refolding protocols if inclusion bodies form

• Utilize high-throughput screening to identify optimal buffer conditions

Functional Analysis Challenges:

• Limited knowledge of natural substrates

• Lack of established activity assays

• Difficulty in reproducing in vivo conditions

Solutions:

• Develop sensitive enzyme assays for dGTPase activity

• Employ substrate screening approaches

• Establish cell-based functional assays

• Use structural predictions to guide functional studies