Recombinant Bartonella quintana UPF0301 protein BQ03640 (BQ03640)

What is Bartonella quintana and why is this pathogen relevant for protein expression studies?

Bartonella quintana is a facultative intracellular bacterium that causes trench fever, endocarditis, and vasculoproliferative disorders like bacillary angiomatosis in humans. This pathogen is particularly interesting for protein studies because it alternates between two distinct environments: the human bloodstream (37°C) and the body louse gut (28°C) . This environmental adaptation requires sophisticated protein expression regulation systems that make B. quintana proteins valuable models for studying bacterial adaptation mechanisms . The pathogen reemerged in the United States and Europe in recent decades, highlighting its continued clinical significance . As a bacterium with the highest reported in vitro hemin requirement of any known bacterium, its protein systems for nutrient acquisition represent specialized adaptations worthy of study .

What expression systems are most effective for recombinant B. quintana proteins?

Multiple expression systems have been validated for B. quintana proteins, each with specific advantages:

E. coli remains the most widely used system for initial expression studies, with published protocols specifically optimized for B. quintana proteins. For proteins that are difficult to express functionally in E. coli, alternative expression systems should be considered .

What are typical yields and solubility characteristics of recombinant B. quintana proteins?

While specific yield data for BQ03640 is not available in the literature, related B. quintana proteins show varying expression profiles. Hemin-binding proteins like Pap31 express well enough in E. coli to support diagnostic applications in ELISA formats with sufficient yield for testing 137+ patient sera . Membrane proteins such as HbpA typically partition to the detergent phase of Triton X-114 extracts, indicating their hydrophobic nature . Proteins with regulatory functions like RpoE can be expressed with N-terminal tags to improve solubility . When designing expression strategies for BQ03640, researchers should anticipate potential solubility challenges if the protein contains transmembrane domains or requires specific cofactors for proper folding.

What are the optimal methods for cloning and expressing B. quintana proteins in E. coli?

A systematic approach for cloning and expressing B. quintana proteins involves:

• Gene amplification: Design PCR primers based on the B. quintana genome sequence. For example, rpoE (BQ10960), nepR (BQ10970), and phyR (BQ10980) were successfully amplified from B. quintana wild-type strain JK31 genomic DNA .

• Initial cloning vector: Use pCR2.1-TOPO-TA vector for initial cloning of PCR products .

• Expression vector selection:

  • pET-28a(+) for N-terminal 6×His-tagged proteins

  • pMAL-c2x for N-terminal maltose binding protein (MBP) fusion

  • pET24a for T7-tagged proteins

• Culture conditions: E. coli strains grown at 37°C in Luria-Bertani medium with appropriate antibiotics (kanamycin 50 μg/ml for pET vectors, ampicillin 100 μg/ml for pMAL vectors) .

• Induction parameters: IPTG induction (typically 0.5-1.0 mM) when cultures reach OD600 of 0.6-0.8, followed by 3-4 hours of expression or overnight at lower temperatures for improved solubility.

• Verification: Confirm expression by immunoblotting with antibodies against the fusion tag or the protein of interest .

This methodology has been successfully employed for multiple B. quintana proteins and can be adapted for BQ03640 expression.

How should experimental design be optimized for proteomic analysis of B. quintana proteins?

Effective proteomic analysis of B. quintana proteins requires careful experimental design with several key considerations:

• Define clear objectives: Establish a specific research question and criteria for hypothesis confirmation or rejection before beginning experimental work .

• Sample preparation optimization:

  • For bacterial cultures, harvest cells into stop solution (M199, 45% ethanol, 5% water-saturated phenol) to prevent RNA degradation if transcriptional analysis will be performed in parallel .

  • For protein extraction, use cell lysis with lysozyme (0.4 mg/ml) in 10 mM Tris and 1 mM EDTA for 5 minutes at room temperature .

  • For membrane proteins, Triton X-114 phase partitioning effectively separates integral membrane proteins .

• Statistical considerations:

  • Design experiments with sufficient biological replicates (minimum n=3)

  • Implement appropriate controls for each experimental condition

  • Plan sample size based on expected effect size and desired statistical power

• Methodological approaches:

  • 2-dimensional gel electrophoresis (2-DE) for protein separation

  • Mass spectrometry (MS) for protein identification

  • Western blotting for target verification

• Data preprocessing and normalization:

  • Address issues of missing values

  • Apply appropriate normalization methods

  • Use statistical methods designed for high-dimensional datasets

Following these guidelines ensures reliable and reproducible proteomic analysis of B. quintana proteins.

What considerations are important when designing experiments involving B. quintana membrane proteins?

Working with B. quintana membrane proteins like HbpA requires specific experimental considerations:

These considerations highlight the complexity of working with bacterial membrane proteins and the importance of multiple complementary approaches for proper characterization.

How can recombinant B. quintana proteins be utilized in developing diagnostic tests?

The development of diagnostic tests using recombinant B. quintana proteins follows this methodological approach:

• Identification of immunodominant antigens: Western blot analysis using patient sera against whole cell lysates separated on 2D gels identified Pap31 as a dominant antigen for B. quintana .

• Recombinant protein production:

  • Amplify the target gene from clinical isolates (e.g., HOSP 800-09, Peru)

  • Clone into an expression vector (pET24a) with appropriate tags

  • Express in E. coli

  • Purify using affinity chromatography

• Validation with patient samples: Test the recombinant protein with patient sera of varying antibody titers to confirm diagnostic potential. For Pap31, "Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS-PAGE" .

• ELISA development and optimization:

  • Coat plates with purified recombinant protein

  • Determine optimal antigen concentration

  • Establish cutoff values using known positive and negative sera

  • Evaluate sensitivity and specificity

• Clinical application assessment: For rPap31, "The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites" .

This methodology can be applied to BQ03640 to evaluate its potential as a diagnostic antigen for B. quintana infections.

What protein-protein interaction studies can reveal functionality of B. quintana proteins?

Several methodological approaches can be employed to study protein-protein interactions in B. quintana:

• Co-immunoprecipitation: Using tagged recombinant proteins (His-tag, MBP-tag) for pull-down assays to identify interaction partners, particularly useful for regulatory protein complexes like RpoE-NepR-PhyR .

• Bacterial two-hybrid systems: For mapping interaction networks within regulatory pathways, though specific examples are not detailed in the search results.

• In vitro binding assays: Similar to the "standard liquid binding assay" used for hemin-binding proteins, adapted to study protein-protein interactions .

• Inhibition studies: Using antibody fragments to block interactions, as demonstrated with "anti-HbpA Fab fragments" that inhibited hemin binding .

• Functional complementation: Expressing proteins in model organisms lacking specific functions to assess restoration of activity, as shown with Pap31 expression "in an E. coli K12 hemA mutant strain" .

• Comparative genomics: Using "BLAST searches" to identify homologs and predict conserved interaction networks. For example, analysis showed "that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity)" .

These complementary approaches provide insights into protein-protein interactions that are essential for understanding B. quintana protein functions in pathogenesis and environmental adaptation.

How does the structural analysis of B. quintana proteins contribute to understanding pathogenesis?

Structural analysis of B. quintana proteins reveals critical structure-function relationships that explain pathogenesis mechanisms:

• Hemin-binding proteins and nutrient acquisition:

  • HbpA/Pap31 (25-30 kDa) is "the dominant hemin-binding protein" located in the outer membrane .

  • Its "heat modifiable" structure displays "an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100°C" .

  • This protein helps B. quintana acquire hemin, essential because "B. quintana has the highest reported in vitro hemin requirement for any bacterium" .

  • The structural adaptation facilitates survival in both "hemin restricted" bloodstream and "hemin rich" body louse gut environments .

• Variably expressed outer membrane proteins (Vomps) and adhesion:

  • Vomps are "members of the trimeric autotransporter adhesin family" .

  • Each Vomp "appears to contribute a different adhesion phenotype, likely mediated by the major variable region at the adhesive tip" .

  • Deletion studies demonstrate that "the deletion of the entire vomp locus... results in a null mutant strain that is incapable of establishing bloodstream infection in vivo" .

  • Structural comparison of "VompA, VompB, and VompC protein sequences of B. quintana with the sequence of BadA of B. henselae" provides insights into adhesion mechanisms .

• RpoE extracytoplasmic function sigma factor and stress adaptation:

  • The RpoE regulatory system helps B. quintana respond to environmental stresses during infection .

  • It functions with NepR and PhyR proteins in a complex regulatory cascade .

  • This system is critical for adaptation between human host and louse vector environments .

These structural insights explain how B. quintana proteins enable the pathogen's unique lifestyle and pathogenesis strategies.

What validation methods confirm proper folding and function of recombinant B. quintana proteins?

Several complementary approaches can validate proper folding and function of recombinant B. quintana proteins:

• Functional assays: For hemin-binding proteins like rPap31 or HbpA, test hemin binding capacity in vitro. "Recombinant HbpA can bind hemin in vitro," confirming functional integrity .

• Complementation studies: Express the recombinant protein in model organisms lacking the function and assess restoration. For example, expressing Pap31 "in an E. coli K12 hemA mutant strain restored its growth when heme was added at 30 μM and above" .

• Immunological cross-reactivity: Validate proper folding by testing if antibodies against the native protein recognize the recombinant version. Verify using "immunoblots" with specific antibodies .

• Clinical sample reactivity: For proteins intended for diagnostic use, validate with patient samples. For rPap31, this was done in "an ELISA format with 137 patient sera of known IFA titers," showing clear differentiation between positive and negative samples .

• Inhibition experiments: Test if antibodies against the recombinant protein can inhibit native function. For example, "cells were preincubated for 1 h at 24°C with 40 or 80 μl (0.2 or 0.4 mg/ml, respectively) of anti-HbpA Fab fragments" to test inhibition of hemin binding .

These validation approaches ensure that recombinant proteins retain the structural and functional properties of their native counterparts, which is critical for both basic research and applications in diagnostics.

What challenges exist in crystallizing B. quintana proteins and how can they be addressed?

Crystallization of B. quintana proteins presents several challenges with corresponding mitigation strategies:

• Membrane protein crystallization difficulties:

  • Challenge: Many B. quintana proteins (HbpA, Vomps) are membrane-associated, requiring detergents that can interfere with crystal formation.

  • Solution: Consider protein engineering to create soluble domains or fusion with crystallization chaperones like T4 lysozyme.

• Protein stability issues:

  • Challenge: Some B. quintana proteins show heat modification properties, indicating potential structural instability. HbpA shows "an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100°C" .

  • Solution: Conduct thermal shift assays to identify stabilizing buffer conditions or use limited proteolysis to identify stable structural domains.

• Expression and purification optimization:

  • Challenge: Obtaining sufficient quantities of properly folded protein is essential for crystallization.

  • Solution: Screen multiple expression systems (E. coli, yeast, baculovirus, mammalian cells) as mentioned in search results and optimize purification protocols to obtain homogeneous samples.

• Crystallization condition screening:

  • Challenge: Identifying optimal crystallization conditions requires extensive screening.

  • Solution: Implement high-throughput screening with various parameters (pH, salt, precipitants) and consider addition of ligands (like hemin for hemin-binding proteins) to stabilize protein conformations.

• Alternative structural approaches:

  • Challenge: Some proteins may resist crystallization despite optimization attempts.

  • Solution: Consider complementary structural approaches such as cryo-electron microscopy (cryo-EM) for larger proteins or complexes, or NMR for smaller domains.

These strategies must be tailored to the specific properties of the target protein, considering its unique characteristics and intended applications.

How can protein expression regulation studies advance understanding of B. quintana pathogenesis?

Understanding B. quintana protein expression regulation provides critical insights into pathogenesis through several methodological approaches:

• Gene expression analysis:

  • Methodology: Perform "reverse transcriptase quantitative PCR (RT-qPCR) with an MX3000P machine" using specific primers and optimized reaction conditions .

  • Application: Quantify expression changes under different environmental conditions (temperature, hemin concentration) to understand adaptation mechanisms.

• RNA isolation techniques:

  • Methodology: Harvest bacteria "into 1 ml stop solution (M199, 45% ethanol, 5% water-saturated phenol) to prevent RNA degradation" followed by lysozyme treatment and TRIzol extraction .

  • Application: Preserve RNA integrity for accurate expression analysis across different growth conditions.

• Mutagenesis studies:

  • Methodology: Implement "A SacB Mutagenesis Strategy" allowing "in-frame, markerless deletion" of regulatory genes .

  • Application: Create knockout mutants to study regulatory pathways, as demonstrated with the vomp gene cluster deletion that "results in a null mutant strain that is incapable of establishing bloodstream infection in vivo" .

• Regulatory element identification:

  • Methodology: Conduct sequence analysis to identify potential regulatory elements, such as the "Fur box homolog with 53% identity to the Escherichia coli Fur consensus" located upstream of hbpA .

  • Application: Map regulatory networks controlling virulence factors and adaptation proteins.

• Environmental regulation studies:

  • Methodology: Analyze protein expression under varying conditions to identify environmental triggers.

  • Findings: "In B. quintana, expression of the hutA, hems, hutB, hutC, and hmuV is repressed by heme in an Irr-dependent manner" , illuminating how the pathogen adapts to different heme availability.

These approaches collectively advance understanding of how B. quintana regulates protein expression during host-pathogen interactions, environmental transitions, and disease progression.

What therapeutic potential exists in targeting B. quintana proteins?

Several B. quintana proteins represent promising therapeutic targets based on their roles in pathogenesis:

• Hemin acquisition systems:

  • Target rationale: "B. quintana has the highest reported in vitro hemin requirement for any bacterium" , making hemin acquisition systems critical for survival.

  • Potential targets: HbpA/Pap31 and the Hut system (HutA, HutB, HutC, HmuV).

  • Evidence of essentiality: "B. tribocorum and B. birtlessii hutA mutants are unable to establish bacteremia in their reservoir hosts" , suggesting these proteins are required for infection.

• Variably expressed outer membrane proteins (Vomps):

  • Target rationale: "The deletion of the entire vomp locus... results in a null mutant strain that is incapable of establishing bloodstream infection in vivo" .

  • Therapeutic approach: Blocking antibodies or small molecule inhibitors that disrupt Vomp-mediated adhesion or VEGF induction.

  • Clinical relevance: Vomps are involved in "the induction of VEGF secretion from infected host cells" , which contributes to vasculoproliferative disorders.

• Current treatment context:

  • Standard therapy: "Treatment of uncomplicated B. quintana bacteremia with a 4- to 6-week course of doxycycline (100 mg orally b.i.d.), erythromycin (500 mg orally four times a day), or azithromycin (500 mg orally once daily)" .

  • Complex infection management: For endocarditis, "4 to 6 months of therapy" is recommended, possibly with "the addition of a bactericidal agent, such as a third-generation cephalosporin or an aminoglycoside" .

  • Improvement potential: Targeted therapies against specific B. quintana proteins could enhance treatment efficacy and reduce duration.

Developing therapeutics targeting these proteins requires further validation of their essentiality, druggability, and resistance potential, but multiple promising candidates have been identified.

How might novel culture methods improve research on B. quintana proteins?

Advanced culture methodologies can significantly enhance B. quintana protein research:

• Shell vial co-culture system:

  • Methodology: "Six confluent shell vials are inoculated for each blood or tissue sample... centrifuged at 700 × g for 1 h at 22°C," washed with PBS, and incubated in appropriate medium .

  • Application: This method enables isolation of B. quintana from clinical samples with higher sensitivity than traditional methods.

  • Protein research benefit: Provides access to clinically relevant strains for comparative protein expression studies.

• Strain establishment protocol:

  • Methodology: "The supernatants of positive shell vials and the colonies obtained on agar plates were inoculated on confluent layers of ECV 304 cells in 150-cm2 culture flasks" .

  • Application: Enables establishment of stable B. quintana isolates from diverse sources.

  • Research advantage: Allows comparison of protein expression between strains with different clinical presentations.

• Growth media optimization:

  • Methodology: Use of specialized media like "M199S, consists of M199 medium supplemented with 20% fetal bovine serum, glutamine, and sodium pyruvate" .

  • Application: Supports robust growth of B. quintana under laboratory conditions.

  • Protein research impact: Improves yield and reproducibility of protein expression studies.

• Environmental condition simulation:

  • Methodology: Culture systems that can mimic the different environments B. quintana encounters (human bloodstream at 37°C vs. louse gut at 28°C).

  • Application: Study protein expression under conditions reflecting the natural lifecycle.

  • Research insight: Reveals environmental regulation of protein expression during host-vector transitions.

These advanced culture methodologies provide researchers with tools to study B. quintana proteins in contexts that more accurately reflect in vivo conditions, enhancing the relevance of findings to clinical applications.