Recombinant Coxiella burnetii Chaperone protein ClpB (clpB), partial

How does ClpB contribute to bacterial survival during infection?

ClpB contributes to bacterial survival during infection through multiple mechanisms:

• Thermotolerance: ClpB enables bacteria to survive elevated temperatures encountered during infection. This function has been demonstrated in several pathogens including Francisella tularensis, Helicobacter pylori, and Campylobacter species .

• Protein disaggregation: ClpB resolubilizes aggregated proteins that accumulate during stress conditions within the host environment. This function is critical for maintaining essential cellular processes during infection .

• Virulence regulation: In some pathogens like F. tularensis, ClpB has been shown to regulate secretion of bacterial effector molecules through the type VI secretion system (T6SS), though this specific function has not been conclusively demonstrated in C. burnetii .

• Response to oxidative stress: Within host cells, bacteria encounter reactive oxygen species and other stressors. ClpB likely helps C. burnetii withstand these challenges by preventing irreversible protein aggregation .

Based on studies in related bacteria, ClpB deletion mutants typically show severe defects in survival at elevated temperatures and increased sensitivity to other stressors, which collectively impair their ability to establish successful infections .

What is the structural organization of C. burnetii ClpB?

While the specific structural details of C. burnetii ClpB have not been comprehensively characterized in the provided search results, we can infer its structure based on highly conserved bacterial ClpB proteins. Generally, bacterial ClpB proteins share the following structural features:

• N-terminal domain: Functions in substrate recognition and binding.

• Two AAA+ domains: Contains ATP-binding and hydrolysis sites that power the disaggregation activity.

• Middle domain (M-domain): A coiled-coil structure that extends from the first AAA+ domain and regulates the disaggregase activity.

• C-terminal domain: Involved in oligomerization and substrate interaction.

C. burnetii ClpB likely assembles into hexameric ring structures, similar to other bacterial ClpB proteins, with the central pore serving as the translocation channel for substrate proteins . This structure is distinct from mammalian homologs like Skd3 (human ClpB), which lacks the characteristic microbial ClpB coiled-coil domain and contains a unique ankyrin-repeat domain instead .

How does recombinant C. burnetii ClpB interact with nucleic acids and ribonucleoprotein complexes?

Recent research indicates that bacterial ClpB family proteins demonstrate significant nucleic acid binding properties, particularly with RNA. Meta-analyses of protein interactions suggest that ClpB preferentially associates with ribonucleoprotein complexes and translation components . In human mitochondrial systems, CLPB has been shown to interact predominantly with RNA granules and translation initiation components .

The nucleic acid interaction of ClpB appears to be functionally significant, as recent data suggests that ClpB and ClpXP are crucial for counteracting misfolded insoluble protein assemblies that contain nucleotides . For C. burnetii specifically, this RNA-binding capability may be particularly important during intracellular replication within acidified vacuoles, where the pathogen must regulate gene expression in response to hostile conditions.

Experimental data from protein interaction studies show that even when overexpressed in non-native compartments, ClpB maintains its preferential interaction with ribonucleoprotein complexes, suggesting an inherent affinity for these structures . This RNA-binding property may represent an underexplored aspect of ClpB function in C. burnetii pathogenesis and stress response.

What genetic mechanisms underlie C. burnetii virulence, and how might ClpB contribute?

C. burnetii virulence is closely associated with lipopolysaccharide (LPS) structure. Virulent phase I bacteria possess smooth, full-length LPS, while avirulent phase II bacteria have rough, truncated LPS . The transition from phase I to phase II occurs during in vitro passage and involves mutations in LPS biosynthesis genes .

While ClpB has not been directly implicated in LPS biosynthesis, as a major stress response chaperone, it may indirectly influence virulence through:

• Maintaining protein homeostasis: ClpB likely ensures proper folding and function of virulence-associated proteins, including those involved in LPS biosynthesis.

• Stress adaptation: C. burnetii must adapt to various stressors within host cells, and ClpB contributes to this adaptation, which is prerequisite for establishing infection.

• Potential secretion system regulation: Based on studies in F. tularensis, where ClpB functions as an energizer for the Type VI secretion system, C. burnetii ClpB might play roles in protein secretion mechanisms .

Genetic diversity studies have identified that approximately 7% of the C. burnetii genome is polymorphic among isolates, consisting mainly of complete ORF deletions, partial deletions, point mutations, and insertions . The relationship between this genetic diversity, ClpB function, and virulence represents an important area for future research.

What methodologies are most effective for analyzing functional activity of recombinant C. burnetii ClpB?

For effective functional analysis of recombinant C. burnetii ClpB, several complementary methodologies should be employed:

Table 1: Recommended Methodologies for C. burnetii ClpB Functional Analysis

When conducting these analyses, it's essential to include appropriate controls, such as heat-denatured ClpB and ATPase-deficient mutants. The co-chaperone DnaK system (DnaK, DnaJ, GrpE) should be included in disaggregation assays, as ClpB typically functions in collaboration with this system .

How should researchers optimize expression and purification of recombinant C. burnetii ClpB?

Optimizing expression and purification of recombinant C. burnetii ClpB requires careful consideration of multiple factors:

Expression System Selection:

• E. coli BL21(DE3) is generally preferred due to its reduced protease activity

• Consider using specialized strains for toxic proteins (e.g., C41/C43 or Rosetta for rare codon optimization)

• Alternative systems like insect cells may be necessary if E. coli yields insoluble protein

Expression Conditions:

• Lower induction temperature (16-25°C) to enhance solubility

• Reduced IPTG concentration (0.1-0.5 mM) for slower induction

• Rich media (2xYT or TB) for higher cell density

• Addition of chaperones (GroEL/ES co-expression) to aid folding

• Test multiple affinity tags (His6, GST, MBP) to identify optimal solubility

Purification Strategy:

• Initial capture using affinity chromatography (typically IMAC for His-tagged protein)

• Secondary purification via ion exchange chromatography

• Final polishing through size exclusion chromatography to ensure homogeneity

• Addition of ATP to purification buffers (1-5 mM) to stabilize oligomeric state

• Include reducing agents (DTT or TCEP) to prevent oxidation

Importantly, because ClpB forms hexamers in its active state, optimization of buffer conditions to maintain the correct oligomeric state is crucial. Consider adding non-hydrolyzable ATP analogs (ATPγS) during later purification stages to stabilize the hexameric form .

What genetic approaches are available for studying C. burnetii ClpB function in vivo?

Several genetic approaches can be employed to study C. burnetii ClpB function:

Targeted Mutagenesis:
Recent advances have made genetic manipulation of C. burnetii possible. Targeted mutagenesis can be performed using:

• Homologous recombination-based techniques

• Himar1 transposon mutagenesis

• CRISPR-Cas9 genome editing

A nutritional selection system based on lysine auxotrophy has been developed for C. burnetii genetic manipulation, which could be adapted for ClpB studies . This approach allows for both gene disruption and complementation without antibiotic selection markers.

Complementation Studies:
For validating phenotypes associated with ClpB deletion, complementation can be performed using:

• Chromosomal integration of the wild-type gene

• Plasmid-based expression systems

• Conditional expression systems (e.g., tetracycline-inducible promoters)

Domain Analysis:
To understand the contribution of specific ClpB domains:

• Create domain deletion constructs

• Generate point mutations in critical residues (particularly in ATPase domains)

• Create chimeric proteins with domains from other bacteria to assess specificity

Reporter Systems:
To monitor ClpB expression and activity:

• Fluorescent protein fusions for localization studies

• Luciferase reporters for transcriptional analysis

• Split-protein complementation for interaction studies

When designing genetic studies, researchers should consider that full virulence requires phase I bacteria with complete LPS, while genetic manipulation is often easier in phase II strains that can be handled under less stringent biosafety conditions . The genetic approaches used should carefully balance these considerations.

How can researchers differentiate between direct and indirect effects of ClpB in C. burnetii pathogenesis?

Differentiating between direct and indirect effects of ClpB in C. burnetii pathogenesis requires a multi-faceted experimental approach:

Direct Substrate Identification:

• Substrate trapping: Use ATPase-deficient ClpB mutants that bind but cannot release substrates

• Crosslinking studies: Employ chemical crosslinkers to capture transient ClpB-substrate interactions

• Co-immunoprecipitation: Pull down ClpB complexes followed by mass spectrometry

• Proximity labeling: Use BioID or APEX2 fusions to identify proteins in close proximity to ClpB

Time-resolved Studies:

• Monitor changes immediately following ClpB inactivation (before secondary effects occur)

• Use inducible systems for rapid depletion or activation of ClpB

• Compare early vs. late timepoints after infection to distinguish primary from secondary effects

Complementation Analysis:

• Test domain-specific mutants that selectively impair specific ClpB functions

• Perform cross-species complementation with ClpB from other bacteria

• Use point mutants that affect specific substrate interactions but maintain general disaggregase activity

In vitro Validation:

• Reconstitute proposed ClpB-substrate interactions with purified components

• Develop biochemical assays to directly measure ClpB activity on candidate substrates

• Compare kinetics of different reactions to identify primary pathways

Comparative Approaches:

• Analyze ClpB function across different C. burnetii strains with varying virulence

• Compare ClpB-dependent processes between C. burnetii and related pathogens

• Examine ClpB activity under different infection conditions to identify context-specific roles

By combining these approaches, researchers can build a comprehensive understanding of which phenotypes are directly attributable to ClpB activity versus those that arise as secondary consequences of its role in proteostasis.

How does ClpB interact with other chaperone systems in bacterial stress response networks?

ClpB functions within an integrated network of chaperone systems to maintain proteostasis. The interactions between these systems are highly coordinated:

• DnaK System Cooperation: ClpB works synergistically with the DnaK-DnaJ-GrpE system. DnaK and DnaJ first interact with aggregated proteins, partially remodeling them and preparing them for ClpB-mediated disaggregation . This requires direct physical interaction between ClpB and DnaK.

• Functional Overlap with ClpX: Both ClpB and ClpX are ATP-powered unfoldases/disaggregases, but they target different substrate pools. While ClpX typically works with ClpP to degrade proteins, ClpB acts independently to resolubilize aggregates without degradation .

• Complementary Heat Shock Response: During heat shock, ClpB cooperates with small heat shock proteins (sHsps) like IbpA and IbpB, which bind to aggregates and maintain them in a disaggregation-competent state .

• RNA-associated Functions: Recent research indicates that ClpB and ClpXP are enriched in association with RNA-binding proteins and may be crucial for counteracting misfolded insoluble protein assemblies containing nucleotides . This suggests a specialized role in maintaining the integrity of ribonucleoprotein complexes.

Meta-analyses of protein interactions show that bacterial CLPB is significantly enriched in association with mitochondrial RNA granules and translation components . This specialized association suggests that beyond general protein quality control, ClpB may have evolved specific functions in protecting nucleic acid-associated protein complexes during stress.

What potential exists for targeting C. burnetii ClpB as a therapeutic approach against Q fever?

ClpB represents a promising therapeutic target against C. burnetii infections for several reasons:

• Essential for Stress Survival: ClpB is crucial for bacterial survival under stress conditions, including those encountered during infection .

• Structural Divergence from Human Homologs: Unlike many bacterial proteins, ClpB has limited homology to human proteins. The human mitochondrial homolog Skd3/human ClpB differs significantly from bacterial ClpB, lacking the characteristic microbial coiled-coil domain and containing a unique ankyrin-repeat domain . This structural divergence creates opportunities for selective targeting.

• Central Role in Virulence: While not directly proven for C. burnetii, studies in related pathogens show that ClpB deletion leads to significant attenuation of virulence .

• Druggability: As an ATP-dependent enzyme with well-defined functional domains, ClpB offers multiple potential sites for small molecule inhibition.

Therapeutic Strategies:

Table 2: Potential ClpB-targeting Therapeutic Approaches

The therapeutic potential of targeting ClpB is supported by successful development of inhibitors against other bacterial chaperones and proteases. For instance, inhibition of the related ClpP protease has proven effective against multiple bacterial pathogens . Development of ClpB inhibitors could provide a novel class of antibiotics with activity against C. burnetii and potentially other intracellular pathogens.

How should researchers interpret contradictory findings in ClpB functional studies across different experimental systems?

When confronted with contradictory findings regarding ClpB function, researchers should consider several factors that may explain discrepancies:

• Strain Variability: C. burnetii exhibits genetic diversity with approximately 7% of its genome being polymorphic among isolates . Different research groups may use different strains with variations in ClpB sequence or regulation.

• Phase Variation: The transition between phase I (virulent) and phase II (avirulent) states in C. burnetii involves numerous genetic changes . ClpB function or importance may differ between these phases.

• Experimental Conditions: In vitro conditions rarely fully recapitulate the complex environment encountered during infection. Temperature, pH, nutrient availability, and oxidative stress levels all influence ClpB activity.

• Methodological Differences: Various approaches to studying ClpB (biochemical assays, genetic knockouts, overexpression systems) have inherent limitations that may lead to apparently contradictory results.

• Context-Dependent Functions: ClpB may have different roles depending on the specific stress condition or growth phase being studied.

Recommended Approach for Resolving Contradictions:

• Standardize Experimental Conditions: When comparing results across studies, ensure comparable growth conditions, strain backgrounds, and methodologies.

• Perform Comprehensive Controls: Include appropriate positive and negative controls, especially when using recombinant proteins or heterologous expression systems.

• Use Multiple Complementary Techniques: Combine genetic, biochemical, and structural approaches to build a more complete understanding.

• Consider Regulatory Networks: Examine how ClpB interacts with other stress response systems and how these interactions might differ across experimental systems.

• Validate in Relevant Models: Confirm findings in models that closely mimic natural infection conditions whenever possible.

By systematically analyzing variables and integrating multiple lines of evidence, researchers can reconcile apparent contradictions and develop a more nuanced understanding of ClpB function in C. burnetii.

What are the critical considerations when designing experiments to assess the impact of ClpB mutations on C. burnetii pathogenesis?

Designing robust experiments to assess the impact of ClpB mutations requires careful consideration of multiple factors:

Mutation Design Strategy:

• Target selection: Consider creating multiple types of mutations:

  • Complete gene deletion

  • Point mutations in ATPase domains

  • M-domain mutations affecting DnaK interaction

  • Domain deletion mutants

• Complementation controls: Always include complemented strains to confirm phenotype specificity

• Marker effects: Consider the impact of selection markers on bacterial fitness

Phenotypic Assays:

• In vitro stress resistance:

  • Heat shock survival (42-45°C)

  • Oxidative stress (H₂O₂ challenge)

  • pH stress (acidic conditions)

  • Nutrient limitation

• Intracellular growth:

  • Multiple cell types (macrophages, epithelial cells)

  • Time-course experiments (attachment, invasion, replication, persistence)

  • Vacuole formation and maintenance

• Animal models:

  • Consider both acute and chronic infection models

  • Assess bacterial burden in multiple tissues

  • Evaluate host immune response

Mechanistic Analyses:

• Protein aggregation: Assess protein aggregation levels in wild-type vs. mutant strains

• Proteome analysis: Compare protein expression profiles under normal and stress conditions

• Transcriptome analysis: Evaluate changes in gene expression patterns

• Interaction studies: Identify alterations in protein-protein interactions

Potential Confounding Factors:

• Polar effects: Deletion of clpB may affect expression of neighboring genes

• Compensatory mutations: Prolonged passage of ClpB mutants may select for secondary mutations

• Growth rate differences: Slower growth of mutants may confound infection studies

• Phase variation: Ensure consistent LPS phase status when comparing strains

Table 3: Recommended Controls for ClpB Mutation Studies

By incorporating these considerations, researchers can generate more reliable and interpretable data on the role of ClpB in C. burnetii pathogenesis.

What emerging technologies might advance our understanding of C. burnetii ClpB function?

Several cutting-edge technologies offer promising avenues for deeper insights into C. burnetii ClpB function:

• Cryo-Electron Microscopy (Cryo-EM): High-resolution structural analysis of ClpB in different conformational states and in complex with substrates could reveal mechanistic details of its disaggregase activity. Recent advances allowing visualization of dynamic protein complexes are particularly relevant for studying ClpB's ATP-driven conformational changes .

• Single-Molecule Techniques: Methods such as optical tweezers and FRET can track individual ClpB molecules as they engage with and process substrate proteins, providing unprecedented insights into the mechanics and kinetics of disaggregation.

• Proximity-Dependent Biotinylation (BioID/TurboID): These approaches can identify proteins that transiently interact with ClpB in living cells, helping to map the complete interactome under different stress conditions .

• RNA-Protein Interaction Mapping: Given the emerging evidence for ClpB association with RNA and ribonucleoprotein complexes, techniques like CLIP-seq could identify specific RNA targets of ClpB in C. burnetii .

• Microfluidics and Single-Cell Analysis: These approaches can track how individual bacteria respond to stressors and how ClpB activity varies across a population of C. burnetii cells during infection.

• CRISPR Interference (CRISPRi): For conditional depletion of ClpB at specific stages of infection to determine temporal requirements for its activity.

• Synthetic Biology Approaches: Designer ClpB variants with engineered specificity or activity could help dissect the precise mechanisms of substrate recognition and processing.

• In situ Structural Analysis: Techniques like cellular cryo-electron tomography could visualize ClpB activity directly within C. burnetii cells during infection.

These technologies, particularly when used in combination, promise to overcome current limitations in understanding the complex and dynamic functions of ClpB in C. burnetii pathogenesis.

How might comparative analysis of ClpB across different C. burnetii strains inform our understanding of pathogen evolution?

Comparative analysis of ClpB across C. burnetii strains offers valuable insights into pathogen evolution and adaptation:

• Genetic Diversity Assessment: Studies have shown that C. burnetii exhibits genetic diversity with approximately 7% of its genome being polymorphic among isolates . Analyzing ClpB conservation versus variation across strains could identify selectively constrained regions essential for core functions versus variable regions that may reflect adaptation to specific niches.

• Strain-Specific Adaptations: Different C. burnetii strains are associated with varied clinical presentations and host specificities. Comparing ClpB sequences, expression levels, and functional properties across these strains may reveal adaptations to particular host environments or transmission patterns.

• Evolutionary Pressure Analysis: Calculating the ratio of nonsynonymous to synonymous substitutions (dN/dS) in ClpB across strains can identify regions under positive or purifying selection, indicating functional importance.

• Horizontal Gene Transfer Assessment: While ClpB is highly conserved, analysis of flanking sequences and codon usage patterns could reveal evidence of horizontal gene transfer events that influenced ClpB evolution in C. burnetii.

• Host Adaptation Signatures: Comparing ClpB from strains with different host preferences might reveal adaptations to specific host pressures, such as temperature ranges or immune mechanisms.

Genomic studies have identified that C. burnetii isolates contain various genomic polymorphisms consisting of 1 to 18 ORFs each, representing deletions, mutations, and insertions . Determining whether ClpB is contained within these variable regions or remains highly conserved could provide important insights into its essentiality for C. burnetii survival across diverse environments.

What potential applications exist for recombinant C. burnetii ClpB beyond basic research?

Recombinant C. burnetii ClpB has several potential applications beyond fundamental research:

• Diagnostic Applications:

  • Development of serological assays for Q fever diagnosis based on ClpB as an antigen

  • Creation of molecular probes for detecting C. burnetii in clinical or environmental samples

  • Potential biomarker for disease progression or treatment response

• Vaccine Development:

  • Component in subunit vaccines against Q fever

  • Adjuvant properties of bacterial heat shock proteins could enhance immune responses

  • Carrier protein for C. burnetii-specific antigens

• Therapeutic Applications:

  • Target for development of new antimicrobials specific to C. burnetii

  • Structure-based drug design focusing on unique features of C. burnetii ClpB

  • Development of inhibitors that could attenuate bacterial survival during infection

• Biotechnological Applications:

  • Use as a tool for solubilizing aggregated proteins in biotechnological processes

  • Development of protein purification methods utilizing ClpB's disaggregase activity

  • Creation of biosensors for detecting specific stress conditions

• Research Tools:

  • Development of in vitro assay systems for studying protein aggregation and disaggregation

  • Creation of reporter systems for monitoring bacterial stress responses

  • Use as a selective marker for genetic manipulation of C. burnetii

The unique properties of C. burnetii ClpB, particularly its ability to function in acidic environments and its potential RNA-binding capabilities, may make it especially valuable for specialized biotechnological applications requiring activity under extreme conditions .

How does our current understanding of C. burnetii ClpB integrate with broader knowledge of bacterial stress responses and host-pathogen interactions?

Our understanding of C. burnetii ClpB contributes to a more integrated view of bacterial stress responses and host-pathogen interactions in several key ways:

• Conserved Stress Response Mechanisms: ClpB represents a highly conserved component of bacterial stress response systems. Studies across multiple pathogens, including F. tularensis, H. pylori, and Campylobacter species, consistently demonstrate ClpB's crucial role in surviving environmental stresses, particularly heat shock . This conservation highlights fundamental requirements for bacterial survival across diverse niches.

• Pathogen Adaptation to Host Environments: C. burnetii uniquely replicates within acidified vacuoles, an environment that poses specific protein folding challenges. ClpB likely plays a crucial role in maintaining protein homeostasis under these conditions, illustrating how pathogens have adapted core stress response systems to specialized intracellular niches.

• Beyond Protein Quality Control: Emerging evidence suggests that bacterial ClpB proteins have roles beyond simple protein disaggregation. The association with RNA-binding proteins and nucleic acid complexes indicates a more nuanced role in maintaining cellular functions during stress .

• Virulence Regulation Networks: In some pathogens, ClpB has been directly linked to virulence through regulation of secretion systems . While this connection has not been conclusively demonstrated for C. burnetii, it suggests that stress response chaperones may serve as regulatory hubs connecting environmental sensing to virulence expression.

• Evolution of Host-Pathogen Interactions: The structural differences between bacterial ClpB and its mammalian homolog Skd3 reflect evolutionary divergence that may be exploited therapeutically . These differences have likely evolved through the selective pressures of host-pathogen interactions.