Recombinant Bordetella pertussis HPr kinase/phosphorylase (hprK)

How do the kinase and phosphatase activities of HprK/P regulate bacterial metabolism?

HprK/P plays a crucial regulatory role in bacterial carbon metabolism through its dual catalytic activities:

• Kinase activity: Catalyzes the ATP-dependent phosphorylation of HPr (histidine-containing phosphocarrier protein), a component of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) .

• Phosphatase activity: Dephosphorylates P-Ser-HPr, effectively reversing the kinase reaction and enabling dynamic control of HPr phosphorylation status .

This bifunctional capacity allows precise regulation of carbon catabolite repression (CCR), a process where preferred carbon sources repress the utilization of secondary carbon sources. Research has demonstrated that mutations affecting the phosphatase activity of HprK/P can significantly impact CCR-sensitive gene expression . For example, studies with L. casei hprK alleles revealed that mutations lowering phosphatase activity while maintaining normal kinase function altered the expression of a CCR-sensitive reporter gene fusion .

The regulatory impact of HprK/P extends beyond simple metabolic control. In pathogenic bacteria like B. pertussis, metabolic regulation is increasingly recognized as interconnected with virulence. During infection, B. pertussis encounters variable nutrient environments in the respiratory tract, requiring metabolic adaptations that may involve HprK/P-mediated regulation . Deep longitudinal multi-omics analysis of B. pertussis cultures has revealed complex metabolic shifts during growth phases, including putative cysteine and proline starvations that triggered significant transcriptomic and proteomic changes .

For researchers investigating B. pertussis metabolism, understanding the balance between HprK/P kinase and phosphatase activities provides critical insights into how this pathogen adapts to changing environments during colonization and infection.

What expression systems are most effective for producing functional recombinant B. pertussis HprK/P?

Several expression systems have demonstrated success for producing recombinant proteins from Bordetella species, each with distinct advantages for different research applications:

• E. coli expression systems: This approach has been successfully employed for various bacterial HprK/P proteins. For example, L. casei hprK alleles have been amplified by PCR and cloned into the expression vector pQE30 for efficient production in E. coli . This system offers rapid growth, high protein yields, and established protocols for optimization.

• Yeast expression systems: For Bordetella proteins specifically, the industrial yeast Pichia pastoris has proven highly effective. Studies with recombinant B. pertussis pertactin (P69) demonstrated that multi-copy transformants of P. pastoris could achieve remarkable protein yields exceeding 3 g per liter of culture in high-density fermentations . The recombinant protein maintained proper immunogenicity, suggesting preservation of native structure . This precedent suggests P. pastoris could be an excellent choice for HprK/P expression when high yields of properly folded protein are required.

• Alternative systems: For challenging proteins, Baculovirus or mammalian cell expression systems may be considered, although these typically involve greater complexity and cost compared to microbial systems .

For researchers selecting an expression system, key considerations include:

• Codon optimization: Adapting the B. pertussis hprK gene sequence for the selected expression host can significantly improve yields

• Fusion tags: Strategic selection of affinity or solubility tags (His, GST, MBP) can facilitate both expression and subsequent purification

• Expression conditions: Optimization of induction parameters (temperature, inducer concentration, time) is essential for maximizing functional protein yield

The choice of expression system should align with specific research objectives, balancing considerations of yield, purity, functionality, and experimental timeline.

What purification strategies and analytical methods are most appropriate for characterizing recombinant HprK/P?

Effective purification and characterization of recombinant HprK/P require a strategic combination of techniques:

Purification Approach:

• Initial capture: Affinity chromatography using fusion tags (His-tag, GST) provides an efficient first step. Conditions must be optimized to maintain enzyme stability while minimizing non-specific binding.

• Intermediate purification: Ion exchange chromatography leverages the unique charge characteristics of HprK/P to separate it from remaining contaminants.

• Polishing: Size exclusion chromatography serves to both enhance purity and provide information about the oligomeric state of the purified protein, which is critical for functional studies.

Analytical Characterization:

• Enzymatic Activity Assays:

  For kinase activity:

  • ATP consumption measurement

  • Detection of phosphorylated HPr substrate using phospho-specific antibodies

  • Radioactive assays with [γ-³²P]ATP to quantify phosphate transfer

  For phosphatase activity:

  • Measurement of phosphate release from P-Ser-HPr

  • Monitoring substrate dephosphorylation by mobility shifts in gel electrophoresis

• Structural Characterization:

  Data collection and processing statistics for structural characterization should include:

  Parameter	Specifications
  Resolution (Å)	2.8-3.0
  Observations	54,000-95,000
  Unique reflections	4,800-6,000
  Completeness (%)	>99
  I/σ	5.1-10.7
  R sym (%)	4.5-9.3
  R cryst (%)	~23.5
  R free (%)	~28.6
  Ramachandran plot (most favored %)	~81

  These parameters are based on published structural characterizations of HprK/P proteins and serve as benchmarks for assessing structural data quality.

• Protein-Protein Interaction Analysis:

  • Pull-down assays to identify interaction partners

  • Surface plasmon resonance (SPR) to determine binding kinetics with HPr

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

• Stability Assessment:

  • Thermal shift assays to determine melting temperature and buffer optimization

  • Limited proteolysis to identify stable domains

  • Dynamic light scattering to assess homogeneity and aggregation propensity

Comprehensive characterization using these methodologies provides critical information about enzyme quality and functionality that directly impacts the reliability of subsequent research applications.

How should experiments be designed to study the impact of HprK/P mutations on enzymatic function and bacterial physiology?

Designing robust experiments to study HprK/P mutations requires a systematic approach that addresses both enzymatic function and physiological impacts:

1. Mutation Design Strategy:

• Structure-guided approach: Utilize crystal structures of HprK/P to identify key residues in catalytic domains. Targeting these residues can generate mutations with selective effects on either kinase or phosphatase activity.

• Functional domain targeting: Previous research has successfully generated mutations that lower phosphatase activity while maintaining kinase function . For example, L. casei hprK alleles were amplified by PCR under mutagenic conditions using reduced concentrations of specific dNTPs (200 μM dGTP, dTTP, dCTP and 20 μM dATP in one reaction, and altered ratios in a second) .

• Disease-relevant mutations: For B. pertussis specifically, consider targeting residues that may influence adaptation to host environments or interact with virulence regulatory networks.

2. In vitro Enzymatic Characterization:

• Parallel activity assays: Design assays that can independently measure kinase and phosphatase activities of the same enzyme preparation to directly compare activity ratios.

• Substrate specificity analysis: Test mutant enzymes with both natural substrates and synthetic peptides to assess changes in substrate recognition.

• Comparative kinetics: Determine key enzymatic parameters (Km, kcat, kcat/Km) for wild-type and mutant enzymes under standardized conditions.

3. Bacterial Physiology Assessment:

• Strain construction: Generate B. pertussis strains carrying hprK mutations, including complemented strains as controls. For validation, follow approaches similar to those used to replace B. subtilis hprK with L. casei hprK variants .

• Growth phenotyping: Systematically evaluate growth characteristics under varying carbon source conditions to detect metabolic impacts.

• Virulence factor expression: Monitor production of key virulence factors (pertussis toxin, filamentous hemagglutinin, pertactin) to identify any regulatory connections between metabolism and virulence .

• Multi-omics profiling: Apply transcriptomic, proteomic, and metabolomic analyses to comprehensively characterize the impact of hprK mutations on bacterial physiology, similar to approaches used in longitudinal studies of B. pertussis cultures .

4. Infection Models:

• Animal selection: Prior research on B. pertussis infection has utilized various mouse strains, including recombinant congenic strains to identify susceptibility loci . For example, studies have examined 12 different CcS/Dem strains and 21 HcB/Dem strains with approximately 10 mice per strain .

• Standardized infection protocol: Establish consistent inoculation procedures and timing of assessments. Previous work determined bacterial colonization in lungs 1 week post-inoculation .

• Control for variability: Include appropriate controls in each experiment, as demonstrated by including BALB/c control mice inoculated in the same way on several experimental days .

• Quantitative readouts: Use colony-forming unit (CFU) determinations and additional markers of infection persistence and severity to assess phenotypic impacts.

By systematically addressing both enzyme function and bacterial physiology, researchers can establish mechanistic links between HprK/P mutations and their impacts on B. pertussis metabolism and potentially virulence.

What analytical and bioinformatic approaches are most effective for interpreting complex data sets involving HprK/P function?

Interpreting complex data sets related to HprK/P function requires sophisticated analytical pipelines that integrate multiple data types and leverage computational approaches:

1. Multi-omics Data Integration:

• Quality control procedures: Implement rigorous quality assessment to ensure data reliability. For example, principal component analysis (PCA) can verify sample clustering by experimental time points and assess biological replicate consistency, as demonstrated in multi-omics studies of B. pertussis .

• Temporal trajectory analysis: Apply longitudinal clustering to identify genes and proteins with similar expression patterns over time. Previous research identified four main cluster trajectories (clusters A-D) in B. pertussis transcriptomic and proteomic data, revealing coordinated expression patterns .

• Cross-omics correlation: Assess consistency between transcriptomic and proteomic data by measuring the percentage of proteins from a given cluster profile found within matching transcripts in the same cluster profile. In B. pertussis studies, this consistency ranged from 51-70% across different clusters .

2. Pathway and Network Analysis:

• Pathway enrichment analysis: Apply over-representation analysis (ORA) to identify enriched pathways in Gene Ontology and KEGG databases. This approach can reveal biological processes associated with specific expression clusters .

• Visualization of enrichment results: Present pathway enrichment using spot visualizations where colors (blue to red) indicate significance and spot sizes represent gene ratios, facilitating interpretation of complex relationships .

• Regulatory network reconstruction: Infer potential regulatory relationships between HprK/P and other metabolic or virulence factors based on co-expression patterns and known regulatory mechanisms.

3. Structural and Functional Analysis:

• Crystallographic data interpretation: Evaluate structural data quality using established parameters such as those in Table I from HprK/P structural studies :

  Parameter	Data Quality Metrics
  Resolution range	2.8-3.0 Å
  Completeness	>99%
  R factors	Rfree <30%, Rcryst <25%
  Stereochemical quality	>80% residues in favored Ramachandran regions

• Comparative sequence analysis: Utilize genomic databases such as the Bordetella database (containing 2,582 isolates and 2,085 genomes) to identify conservation patterns and variations in hprK across Bordetella species .

• Structure-function correlation: Map experimental findings onto structural models to identify structural features responsible for observed functional differences between wild-type and mutant proteins.

4. Statistical Approaches for Genetic Studies:

• Linkage analysis: For studies involving genetic variations in host or pathogen, apply appropriate statistical methods to identify quantitative trait loci (QTLs). Previous research with F2 hybrid mice generated from recombinant congenic strains identified susceptibility loci for B. pertussis infection through linkage analysis .

• Genotype-phenotype correlations: Compare genotypes with phenotypes (e.g., bacterial lung colonization levels) to identify significant associations. In B. pertussis infection studies, bacterial counts in F2 hybrid mice ranged from detection limits (1.0 × 10²) to 1.8 × 10⁷ CFU per lung, providing quantitative phenotypes for correlation with genotypes .

5. Integrated Systems Biology Approaches:

• Metabolic flux analysis: Integrate metabolomic data with enzyme activity measurements to model changes in metabolic flux resulting from HprK/P mutations.

• Causality network inference: Apply directed graph approaches to infer causal relationships between HprK/P activity, metabolic shifts, and downstream physiological changes.

• Predictive modeling: Develop computational models that predict the impact of HprK/P mutations on bacterial metabolism and potentially virulence, generating testable hypotheses for experimental validation.

By applying these analytical approaches, researchers can extract meaningful biological insights from complex datasets and establish mechanistic understanding of HprK/P function in the context of B. pertussis metabolism and pathogenesis.

How does HprK/P potentially contribute to B. pertussis virulence and pathogenesis?

The relationship between HprK/P and B. pertussis virulence represents an emerging area of research at the intersection of metabolism and pathogenesis:

Metabolic Regulation and Virulence Connections:

While HprK/P is primarily characterized as a metabolic regulator, growing evidence suggests potential connections to virulence mechanisms in B. pertussis:

• Coordinated regulation during infection: Deep longitudinal multi-omics analysis of B. pertussis has revealed that nutrient starvation conditions trigger significant molecular changes, including alterations in metabolism and virulence factor production. Specifically, putative cysteine and proline starvations observed at different growth phases coincided with negative effects on specific total PT (pertussis toxin), PRN (pertactin), and Fim2 (fimbrial protein) antigen production .

• Beyond classical virulence regulation: Intriguingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) appears not to be the sole virulence regulator in certain growth conditions. Novel intermediate regulators have been identified as potentially involved in the expression of some virulence-activated genes (vags) . Metabolic regulators like HprK/P could function within these alternative regulatory networks.

• Adaptation to host environments: During infection, B. pertussis encounters varying nutrient conditions in different regions of the respiratory tract. HprK/P-mediated metabolic adaptation likely contributes to the bacterium's ability to persist and express virulence factors under these changing conditions.

Clinical and Epidemiological Context:

B. pertussis causes whooping cough, a serious respiratory illness that has reemerged as a public health threat despite vaccination . Studies have shown that individuals with comorbidities may experience more severe pertussis disease , suggesting that host-pathogen metabolic interactions may influence disease outcomes.

Genomic epidemiology resources for Bordetella include comprehensive databases with thousands of isolates and genomes , providing opportunities to investigate whether variations in hprK correlate with virulence or clinical outcomes across different strains.

Research Approaches for Investigating HprK/P in Pathogenesis:

• Genetic susceptibility studies: Building on approaches used to identify host susceptibility loci , researchers could investigate whether B. pertussis strains with variant hprK genes show altered virulence in different host genetic backgrounds.

• Infection model systems: Established mouse infection models can be leveraged to compare colonization, persistence, and disease severity between wild-type B. pertussis and isogenic strains with altered hprK genes.

• Virulence factor monitoring: Quantitative assessment of key virulence factors like pertussis toxin, filamentous hemagglutinin, and pertactin in relation to HprK/P activity can elucidate regulatory relationships .

Understanding the relationship between HprK/P and virulence could potentially inform novel therapeutic strategies targeting metabolic vulnerabilities of B. pertussis during infection.

How can recombinant HprK/P be utilized in vaccine development or therapeutic research?

Recombinant B. pertussis HprK/P offers several potential applications in vaccine and therapeutic research, representing an innovative direction beyond traditional approaches:

1. Adjuvant or Carrier Protein Applications:

Bacterial proteins with dual enzymatic functions like HprK/P could serve as potential adjuvants or carrier proteins in vaccine formulations. Previous research with other B. pertussis proteins provides a template for this approach:

• Recombinant B. pertussis pertactin (P69), when expressed in Pichia pastoris, yielded high levels (>3 g/L) of immunologically active protein .

• Purified recombinant pertactin was able to enhance the incomplete protection afforded by pertussis toxoid to reach the protective level of whole-cell vaccines, as demonstrated in the Kendrick test .

• This precedent suggests that other B. pertussis proteins, potentially including HprK/P, could be explored for similar immunomodulatory properties.

2. Novel Vaccine Target Exploration:

Acellular pertussis vaccines have traditionally focused on a limited set of antigens:

• Current acellular vaccines are based primarily on pertussis toxoid .

• Their effectiveness may be increased by adding other B. pertussis antigens .

• While conventional vaccines include proteins like filamentous hemagglutinin (FHA), pertactin, and fimbrial proteins , metabolic regulators like HprK/P represent unexplored territory.

Research approaches could include:

• Immunogenicity screening of recombinant HprK/P

• Animal protection studies comparing standard acellular formulations with those including HprK/P

• Analysis of human immune responses to HprK/P during natural infection

3. Small Molecule Inhibitor Development:

As a bifunctional enzyme with distinct catalytic activities, HprK/P presents an attractive target for therapeutic intervention:

• Structure-based virtual screening approaches, similar to those used for other bacterial targets , could identify potential inhibitors of either kinase or phosphatase activity.

• Homology modeling and sequence alignment can support target validation, as demonstrated for other bacterial enzymes .

• Molecular docking approaches using defined binding pockets could identify compounds with potential inhibitory activity.

For virtual screening, established parameters include:

• Coordination of active sites grid box set to encompass the entire binding pocket

• Grid box centroid and dimensions based on protein structure and SiteMap analysis

• Complete coverage of potential interaction space

4. Diagnostic Applications:

Recombinant HprK/P could be explored for diagnostic applications:

• Serological assays to detect anti-HprK/P antibodies during infection

• Development of rapid diagnostic tests based on HprK/P detection

• Monitoring vaccine responses through analysis of anti-HprK/P antibodies

Current diagnostic approaches often combine multiple B. pertussis antigens. For example, filamentous hemagglutinin (FHA) is used in combination with pertussis toxin for diagnostic screening purposes . Similar approaches could potentially incorporate HprK/P if it proves to have diagnostic value.

5. Systems Biology Platform for Vaccine Enhancement:

Multi-omics studies involving HprK/P regulation networks could inform vaccine development:

• Longitudinal multi-omics analysis has emerged as a powerful tool for characterizing and optimizing vaccine antigen production .

• Understanding the regulatory connections between metabolism and virulence factor expression could lead to improved cultivation conditions for antigen production.

• Insights from HprK/P regulation might reveal optimal timing for antigen harvesting during in vitro culture processes.

By integrating HprK/P research into vaccine and therapeutic development pipelines, researchers may discover novel approaches to address the ongoing challenges posed by B. pertussis infections despite widespread vaccination.

What are the most promising unexplored aspects of B. pertussis HprK/P that warrant further investigation?

Several high-potential research directions remain largely unexplored for B. pertussis HprK/P:

1. Structural Characterization of B. pertussis-Specific Features:

While crystal structures exist for HprK/P from other bacterial species , B. pertussis-specific structural features remain uncharacterized. Future research should:

• Determine high-resolution crystal structures of B. pertussis HprK/P in different functional states

• Investigate potential conformational changes associated with kinase versus phosphatase activities

• Compare structural features with HprK/P from both closely related Bordetella species and more distant bacterial relatives

Such structural information would provide crucial insights for both basic understanding and applied research, including rational drug design.

2. Identification of B. pertussis-Specific Substrates and Partners:

• Proteomic approaches to identify phosphorylation targets of HprK/P in B. pertussis

• Investigation of potential interactions with virulence regulatory networks

• Characterization of protein complexes involving HprK/P during different growth phases and environmental conditions

These studies could reveal unexpected regulatory connections between metabolism and virulence.

3. Environmental Regulation of HprK/P Activity:

The dual functionality of HprK/P likely responds to environmental signals encountered during infection:

• Systematic characterization of how environmental factors (pH, temperature, nutrient availability) influence the kinase-phosphatase activity balance

• Investigation of potential post-translational modifications affecting HprK/P function during host adaptation

• Analysis of hprK expression patterns during different stages of infection

Understanding these regulatory mechanisms could reveal key transition points in B. pertussis pathogenesis.

4. Evolutionary Analysis Across the Bordetella Genus:

The comprehensive Bordetella genomic database containing 2,582 isolates and 2,085 genomes provides an unprecedented opportunity for evolutionary analysis:

• Comparative genomic analysis of hprK across classical bordetellae (B. pertussis, B. parapertussis, B. bronchiseptica) and newly described species

• Investigation of selection pressures on hprK during adaptive evolution to different hosts

• Correlation of hprK variants with phenotypic characteristics and clinical outcomes

Such studies could reveal how metabolic regulation through HprK/P has contributed to the evolution of host specificity and virulence in the Bordetella genus.

5. Integration with Host-Pathogen Interaction Studies:

The role of HprK/P in host-pathogen interactions remains virtually unexplored:

• Investigation of how host metabolic conditions influence HprK/P activity

• Analysis of potential interactions between HprK/P-regulated metabolic pathways and host immune responses

• Exploration of how HprK/P-mediated adaptations influence persistence in vaccinated hosts

These studies would place HprK/P function in the broader context of B. pertussis pathogenesis.

How might emerging technologies advance our understanding of HprK/P and its applications?

Emerging technologies offer powerful new approaches for investigating HprK/P function and applications:

1. Advanced Structural Biology Techniques:

Next-generation structural biology methods provide unprecedented insights into protein function:

• Cryo-electron microscopy (cryo-EM): Enables visualization of HprK/P complexes in different conformational states without crystallization constraints

• Time-resolved X-ray crystallography: Captures structural intermediates during catalytic cycles

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps conformational dynamics and protein-protein interaction surfaces

These approaches could reveal how structural changes coordinate the dual kinase/phosphatase activities and identify potential allosteric regulation sites.

2. Genome Editing and Synthetic Biology:

Precise genetic manipulation technologies enable sophisticated functional studies:

• CRISPR-Cas9 genome editing: Creates precise mutations in hprK to dissect structure-function relationships

• Inducible expression systems: Controls HprK/P levels and activity timing during infection

• Biosensors: Reports real-time HprK/P activity in living cells during environmental transitions

These tools would enable unprecedented dissection of HprK/P function in native contexts.

3. Single-Cell and Spatial Technologies:

New single-cell approaches reveal population heterogeneity and spatial dynamics:

• Single-cell RNA-seq: Identifies subpopulations with distinct metabolic states during infection

• Spatial transcriptomics: Maps gene expression patterns in infected tissues

• Super-resolution microscopy: Visualizes HprK/P localization and protein complex formation

These technologies could reveal how HprK/P contributes to bacterial population heterogeneity during infection, potentially identifying persister subpopulations.

4. Advanced Computational Approaches:

Computational methods enable integration of diverse data types:

• Molecular dynamics simulations: Models conformational changes and substrate interactions

• Machine learning: Predicts functional outcomes of hprK mutations

• Systems biology modeling: Integrates HprK/P function into genome-scale metabolic models

For example, structure-based virtual screening approaches similar to those used for other bacterial targets could identify potential HprK/P inhibitors. This would typically involve defining a coordination grid box centered at specific coordinates (e.g., 45.4, 69.6, 0.9) with appropriate dimensions (e.g., 15 Å × 15 Å × 15 Å) to ensure complete coverage of the potential interaction space .

5. Multi-omics Integration and Longitudinal Analysis:

Comprehensive omics approaches provide system-level insights:

• Integrated multi-omics: Combines transcriptomics, proteomics, lipidomics, and metabolomics data to map regulatory networks

• Temporal trajectory analysis: Reveals dynamic responses to changing environments

• Pathway enrichment analysis: Identifies biological processes associated with specific expression patterns

Previous multi-omics studies of B. pertussis identified coordinated expression patterns across four main cluster trajectories in transcriptomic and proteomic data, with consistency between proteins and transcripts ranging from 51-70% across different clusters . Similar approaches focused specifically on HprK/P networks could reveal previously unrecognized regulatory relationships.

By leveraging these emerging technologies, researchers can address fundamental questions about HprK/P function while simultaneously exploring applications in vaccine development, diagnostics, and therapeutic interventions against pertussis.