Recombinant Coxiella burnetii Single-stranded DNA-binding protein (ssb)

Basic Research Questions

• What is the structure and function of Coxiella burnetii single-stranded DNA-binding protein (ssb)?

Coxiella burnetii ssb is a critical component of the bacterium's DNA metabolism machinery. It plays an important role in DNA replication, recombination, and repair processes by binding to ssDNA and recruiting partner proteins to their sites of action . The protein likely forms homooligomeric structures similar to other bacterial SSBs that coat and protect ssDNA intermediates during genome maintenance reactions .

C. burnetii ssb binds to ssDNA during replication, recombination, and repair processes, protecting it from nuclease degradation and preventing the formation of secondary structures. The cooperative binding mechanism involves a "bridge interface" that links adjacent SSB tetramers on the ssDNA, which is critical for its function in maintaining genomic integrity, especially in the hostile environment of the parasitophorous vacuole within host cells .

• How does C. burnetii DNA repair differ from other bacterial systems?

C. burnetii possesses unique DNA repair adaptations that distinguish it from other bacterial systems:

C. burnetii has evolved unique repair adaptations, including constitutive SOS gene expression due to the lack of LexA and induction of AddAB under oxidative stress, unlike the non-inducible E. coli recBCD . These adaptations likely reflect the bacterium's need to maintain genomic integrity in the harsh environment of the macrophage parasitophorous vacuole with lysosomal characteristics .

• What methods are recommended for expressing and purifying recombinant C. burnetii ssb?

Based on published protocols for C. burnetii proteins, recombinant ssb can be efficiently expressed and purified using the following optimized methodology:

• Cloning strategy:

  • Amplify the ssb gene (CBU_1779/CBUD_1779) from C. burnetii genomic DNA

  • Clone into an expression vector with an affinity tag (His-tag or GST-tag)

• Expression conditions:

  • Transform into E. coli BL21(DE3) or similar expression strain

  • Induce with 0.5-1.0 mM IPTG at OD₆₀₀ of 0.6-0.8

  • Express at lower temperatures (16-25°C) to enhance solubility

• Purification protocol:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF

  • Purify using affinity chromatography (Ni-NTA for His-tagged proteins)

  • Further purify by ion-exchange and/or size-exclusion chromatography

  • Concentrate protein and store in buffer with 50% glycerol at -80°C

This approach has been successful for other C. burnetii proteins and should be adaptable for ssb .

• What is the role of C. burnetii ssb in bacterial adaptation to environmental stresses?

C. burnetii is exposed to various stresses throughout its lifecycle, including the oxidative environment within macrophages and temperature fluctuations during transmission. The ssb protein plays a critical role in maintaining genomic integrity under these conditions by:

• Protecting ssDNA during stress responses: During temperature stress, C. burnetii exhibits minor changes in gene regulation , but DNA repair mechanisms remain essential.

• Supporting lifecycle transitions: C. burnetii transitions between a metabolically active large-cell variant (LCV) and a spore-like, quiescent small-cell variant (SCV) . These transitions involve DNA replication and repair processes where ssb is likely crucial.

• Oxidative stress response: C. burnetii's DNA repair systems, including ssb-mediated processes, help maintain genomic integrity against oxidative damage in the parasitophorous vacuole .

The constitutive expression of SOS genes in C. burnetii (due to lexA absence) suggests that ssb and other DNA repair proteins are constantly available to respond to ongoing environmental stresses, representing a unique adaptation to its intracellular lifestyle .

Advanced Research Questions

• How does cooperative DNA binding by C. burnetii ssb differ from other bacterial SSBs?

Cooperative DNA binding is a critical property of bacterial SSB proteins. While specific data for C. burnetii ssb is limited, insights can be drawn from structural studies of other bacterial SSBs:

To determine C. burnetii-specific properties, researchers should:

• Express and purify recombinant C. burnetii ssb

• Perform DNA binding assays using fluorescently labeled ssDNA oligonucleotides

• Compare binding parameters with well-characterized SSBs from E. coli or B. subtilis

• Attempt crystallization of C. burnetii ssb with ssDNA to resolve the structure of the bridge interface

Mutations in the bridge interface residues of C. burnetii ssb would likely reduce binding cooperativity and alter DNA binding modes, similar to observations with E. coli SSB variants .

• What experimental approaches can determine how the absence of LexA affects ssb expression in C. burnetii?

The absence of LexA in C. burnetii creates a unique regulatory environment for DNA repair genes. To investigate its impact on ssb expression:

• Transcriptional analysis:

  • Quantitative RT-PCR to compare ssb expression levels between C. burnetii and other bacteria with LexA

  • RNA-seq to analyze the global transcriptional profile under normal conditions and after DNA damage

• Reporter gene assays:

  • Clone the ssb promoter region upstream of a reporter gene (e.g., GFP or luciferase)

  • Introduce this construct into C. burnetii and a model organism with LexA (e.g., E. coli)

  • Compare baseline expression and response to DNA-damaging agents

• Complementation studies:

  • Express E. coli LexA in C. burnetii using a CRISPR interference system similar to that described for other C. burnetii genes

  • Determine if introduced LexA can bind to potential SOS boxes in the ssb promoter region

  • Assess changes in ssb expression and response to DNA damage

• ChIP-seq analysis:

  • Perform chromatin immunoprecipitation followed by sequencing to identify potential regulatory proteins that might bind to the ssb promoter in the absence of LexA

These approaches would help elucidate how ssb expression is regulated in C. burnetii despite lacking the canonical LexA-mediated SOS response system .

• How does C. burnetii ssb interact with other DNA repair proteins like RecA and AddAB?

Understanding the interaction network of C. burnetii ssb is crucial for deciphering its role in DNA metabolism. Based on knowledge from other bacterial systems and the unique DNA repair mechanisms in C. burnetii:

To characterize these interactions:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged ssb in C. burnetii or a surrogate host

  • Purify ssb complexes under near-native conditions

  • Identify interacting partners by mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize purified ssb on a sensor chip

  • Flow potential partners (RecA, AddAB) over the surface

  • Measure binding kinetics and affinity constants

• Bacterial Two-Hybrid:

  • Test binary interactions between ssb and other DNA repair proteins

  • Map interaction domains through truncation analysis

The AddAB system in C. burnetii, unlike E. coli's RecBCD, is upregulated under oxidative stress , suggesting potential coordinated regulation with ssb during DNA repair responses.

• What are the potential applications of recombinant C. burnetii ssb in developing diagnostic tools for Q fever?

Recombinant C. burnetii proteins have shown promise as antigens for Q fever diagnostics. While ssb has not been specifically identified in immunoproteomic studies as a major immunodominant antigen, its potential utility can be evaluated:

• Serological assay development:

  • Express and purify C. burnetii ssb with high purity

  • Develop ELISA, immunoblot, or protein microarray assays using the recombinant protein

  • Test against sera from acute and chronic Q fever patients

• Advantages over whole-cell antigens:

  • Reduced cross-reactivity with other bacterial pathogens

  • No need to culture hazardous C. burnetii organisms

  • Potential differentiation between infection stages based on antibody profiles

• Multiplex approach:

  • Combine ssb with other identified immunoreactive proteins (e.g., CBU0891, CBU1398, CBU0664)

  • Create a panel of recombinant antigens for improved sensitivity and specificity

• Potential for DIVA (Differentiating Infected from Vaccinated Animals) diagnostics:

  • If ssb generates a different antibody response in infected versus vaccinated individuals

  • Particularly valuable for livestock testing in endemic areas

Immunoproteomic studies have identified approximately 50 C. burnetii proteins that strongly react with immune sera . Testing recombinant ssb alongside these known immunodominant antigens would determine its diagnostic value.

• What structural modifications could enhance the stability and functionality of recombinant C. burnetii ssb?

Enhancing recombinant C. burnetii ssb stability and functionality requires rational protein engineering approaches:

Experimental approach:

• Structure prediction and modeling:

  • Generate homology models based on known bacterial SSB structures

  • Identify regions for potential modification using computational tools

• Stability screening:

  • Create a library of variants with different modifications

  • Screen for improved thermal stability using differential scanning fluorimetry

  • Assess functional stability through DNA binding assays

• Activity optimization:

  • Test DNA binding properties using electrophoretic mobility shift assays

  • Verify cooperative binding behavior is maintained or enhanced

  • Confirm ability to interact with partner proteins remains intact

These modifications could prove valuable for both research applications and potential diagnostic or therapeutic development using C. burnetii ssb.

• How can C. burnetii ssb be utilized as a target for antimicrobial development?

The essential role of ssb in DNA metabolism makes it a potential target for novel antimicrobials against C. burnetii:

• Target validation:

  • Demonstrate essentiality using CRISPR interference or antisense RNA approaches

  • Identify whether ssb is accessible to inhibitors within the parasitophorous vacuole

  • Determine if inhibition of ssb function can clear persistent infection

• Inhibitor screening strategies:

  • Develop high-throughput assays measuring ssb-ssDNA binding

  • Screen small molecule libraries for compounds that disrupt this interaction

  • Test peptide mimetics that interfere with ssb-protein interactions

• Structure-based drug design:

  • Use structural information from crystallography or homology modeling

  • Identify potential binding pockets for small molecule inhibitors

  • Design compounds that specifically target C. burnetii ssb over human SSB

• Evaluation in infection models:

  • Test candidate inhibitors in cell culture models of C. burnetii infection

  • Assess efficacy in animal models of acute and chronic Q fever

  • Determine pharmacokinetic properties and tissue distribution

The considerable differences between bacterial and eukaryotic single-stranded DNA-binding proteins provide an opportunity for selective targeting of C. burnetii ssb. Additionally, the unique properties of C. burnetii DNA repair systems, including constitutive SOS expression , may make ssb inhibition particularly effective against this pathogen.

Methodology Questions

• What techniques can be used to study the protein-protein interactions of C. burnetii ssb?

Multiple complementary techniques can be employed to comprehensively map the protein interaction network of C. burnetii ssb:

• Affinity Purification coupled with Mass Spectrometry (AP-MS):

  • Express His-tagged or FLAG-tagged ssb in heterologous systems

  • Perform gentle cell lysis to preserve protein-protein interactions

  • Capture ssb complexes using affinity resins

  • Identify binding partners by LC-MS/MS

  • Quantify enrichment relative to control pulldowns

• Crosslinking Mass Spectrometry:

  • Treat samples with chemical crosslinkers to stabilize transient interactions

  • Digest crosslinked complexes and identify crosslinked peptides by MS

  • Map interaction interfaces at amino acid resolution

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize purified ssb on sensor surfaces

  • Measure real-time binding kinetics with potential partners

  • Determine association and dissociation rate constants

  • Calculate binding affinities (KD values)

• Fluorescence-based techniques:

  • Förster Resonance Energy Transfer (FRET) between labeled proteins

  • Microscale Thermophoresis (MST) for binding affinity measurements

  • Fluorescence Polarization (FP) for measuring interactions with small molecules

• Computational prediction:

  • Utilize protein docking simulations

  • Predict interaction surfaces based on conserved domains

  • Guide experimental validation of predicted interactions

The integration of these techniques would provide a comprehensive understanding of how C. burnetii ssb interfaces with the DNA repair machinery and other cellular processes in this unique intracellular pathogen.

• How should experiments be designed to analyze C. burnetii ssb expression under different stress conditions?

To systematically analyze C. burnetii ssb expression under different stress conditions relevant to its lifecycle:

Experimental Design Framework:

• Stress Conditions to Test:

  • Oxidative stress (H₂O₂, paraquat)

  • Acid stress (pH 4.5-5.5, mimicking the parasitophorous vacuole)

  • Nutrient limitation

  • Temperature stress (heat shock at 42°C, cold shock at 4°C)

  • DNA-damaging agents (UV, mitomycin C, methyl methanesulfonate)

• Cell Culture Models:

  • Axenic ACCM-2 medium culture of C. burnetii

  • Infected cell lines (THP-1 macrophages, Vero cells)

  • Primary macrophages from different sources

• Expression Analysis Methods:

  • qRT-PCR for ssb mRNA quantification

  • Western blotting with anti-ssb antibodies

  • Promoter-reporter fusions (if genetic manipulation is available)

  • RNA-seq for global transcriptional response

• Experimental Protocol:

  • Grow C. burnetii to mid-log phase

  • Apply stress treatments for varying durations

  • Extract RNA for transcript analysis

  • Harvest protein for Western blot analysis

  • Compare results to unstressed controls and other DNA metabolism genes

• Data Analysis:

  • Normalize expression to reference genes stable under stress conditions

  • Perform statistical analysis (ANOVA with post-hoc tests)

  • Correlate ssb expression with other DNA repair genes

  • Compare responses to those observed in other bacteria with different SOS regulation

Since C. burnetii exhibits minor changes in gene regulation under short exposure to temperature stress , extended stress periods and multiple stress types should be evaluated to capture the full regulatory response of ssb.

• What are the recommended protocols for assessing C. burnetii ssb-DNA binding properties?

To comprehensively characterize the DNA binding properties of C. burnetii ssb:

Protocol Suite for DNA Binding Characterization:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Prepare fluorescently labeled ssDNA oligonucleotides of varying lengths

  • Incubate with increasing concentrations of purified ssb protein

  • Resolve complexes on native polyacrylamide gels

  • Quantify bound vs. unbound DNA to determine binding affinities

• Fluorescence Anisotropy:

  • Label ssDNA with fluorescent dyes

  • Measure changes in anisotropy as ssb binds

  • Determine binding constants and cooperativity parameters

  • Test various salt concentrations to assess binding mode transitions

• Single-Molecule Techniques:

  • Fluorescence Resonance Energy Transfer (FRET) to monitor protein-induced DNA conformational changes

  • Total Internal Reflection Fluorescence (TIRF) microscopy to visualize individual ssb-DNA complexes

  • Magnetic or optical tweezers to measure ssb binding dynamics in real-time

• Atomic Force Microscopy (AFM):

  • Visualize ssb-DNA complexes at nanometer resolution

  • Assess protein binding patterns and cooperativity

  • Monitor structural changes in DNA upon ssb binding

• Isothermal Titration Calorimetry (ITC):

  • Determine thermodynamic parameters of binding

  • Measure stoichiometry, binding affinity, enthalpy, and entropy

  • Compare data with other bacterial SSBs

• Analytical Ultracentrifugation:

  • Characterize ssb oligomerization state

  • Analyze ssb-DNA complex formation

  • Determine stoichiometry of binding

These complementary approaches would provide a comprehensive understanding of how C. burnetii ssb interacts with ssDNA, its binding modes, cooperativity, and potential unique properties compared to other bacterial SSBs .

• How can researchers effectively study C. burnetii ssb function in the context of the bacterial SOS response?

Studying C. burnetii ssb within the context of its unique SOS response system requires specialized approaches:

• Genetic complementation studies:

  • Express C. burnetii ssb in E. coli ssb mutants

  • Assess functional complementation under DNA damage conditions

  • Introduce C. burnetii AddAB system to evaluate coordinated function with ssb

• DNA damage response analysis:

  • Expose C. burnetii to DNA-damaging agents (UV, mitomycin C, MMS)

  • Monitor ssb expression relative to known SOS genes

  • Compare with E. coli response to identify LexA-independent regulation

• Protein interaction mapping:

  • Identify binding partners of ssb after DNA damage

  • Determine if interaction networks change during stress response

  • Map regulatory proteins that might control ssb in absence of LexA

• Axenic culture system for direct manipulation:

  • Utilize ACCM-2 medium for growing C. burnetii outside host cells

  • Apply CRISPR interference to modulate ssb expression

  • Monitor effects on growth and survival after DNA damage

• Host cell infection models:

  • Infect macrophages with wild-type C. burnetii and ssb-modulated strains

  • Assess intracellular survival and replication

  • Measure oxidative stress response and DNA damage repair efficiency

This integrated approach would help elucidate how C. burnetii ssb functions within the bacterium's unique constitutive SOS expression system, particularly in relation to the AddAB recombinational repair system that is upregulated under oxidative stress .

• What approaches should be used to develop C. burnetii ssb as a vaccine antigen candidate?

Although not identified among the top immunodominant C. burnetii antigens in previous studies , ssb could be systematically evaluated as a vaccine antigen using the following approach:

• Immunogenicity assessment:

  • Express and purify recombinant C. burnetii ssb

  • Immunize mice using different adjuvants

  • Measure antibody titers using ELISA

  • Assess T-cell responses through cytokine profiling and proliferation assays

• Protective efficacy evaluation:

  • Challenge immunized animals with virulent C. burnetii

  • Monitor bacterial load in tissues

  • Assess pathological changes and disease symptoms

  • Compare protection with existing vaccines (e.g., Q-Vax, Coxevac)

• Antigen optimization:

  • Identify immunodominant epitopes within ssb

  • Create epitope-focused constructs for enhanced immunogenicity

  • Test multi-epitope vaccines combining ssb with other immunodominant antigens

  • Evaluate different delivery platforms (protein, DNA, viral vectors)

• Safety profile characterization:

  • Assess reactogenicity in previously sensitized animals

  • Evaluate autoimmune potential through cross-reactivity studies

  • Determine stability and optimal formulation

• DIVA capability testing:

  • Develop serological assays to differentiate vaccinated from infected animals

  • Evaluate in field studies in endemic regions

Previous attempts to develop Q fever vaccines using recombinant C. burnetii proteins (including Omp, HspB, and others) have shown limited success , suggesting that ssb would likely need to be part of a multi-antigen approach rather than used as a single antigen.

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Reference Table: Key C. burnetii DNA Metabolism Proteins