Recombinant Burkholderia cenocepacia Glucose-6-phosphate isomerase (pgi), partial

What is Glucose-6-phosphate isomerase in Burkholderia cenocepacia and what is its metabolic significance?

Glucose-6-phosphate isomerase (GPI, also known as PGI, EC 5.3.1.9) in Burkholderia cenocepacia is a key metabolic enzyme that catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. This reaction represents a critical step in both glycolysis and gluconeogenesis pathways, making it essential for bacterial energy metabolism and carbon utilization.

In B. cenocepacia, a member of the Burkholderia cepacia complex (Bcc), this enzyme likely contributes to the bacterium's remarkable metabolic versatility and adaptability in diverse environments. B. cenocepacia is known to colonize various ecological niches including soil, water, plant rhizosphere, and the human respiratory tract, particularly in cystic fibrosis (CF) patients . The metabolic flexibility enabled by enzymes like PGI may contribute to the bacterium's ability to thrive in these varied environments.

From a research perspective, understanding PGI function is valuable as it represents a metabolic node that potentially influences bacterial virulence, survival during infection, and interaction with other pathogens such as Pseudomonas aeruginosa, which is often co-isolated with B. cenocepacia from CF patient lungs .

What are the properties and handling recommendations for recombinant B. cenocepacia PGI?

The recombinant form of B. cenocepacia PGI (Uniprot No. Q1BH79) has several specific properties that researchers should consider when designing experiments:

Physical and biochemical properties:

• Protein classification: Glucose-6-phosphate isomerase (EC 5.3.1.9)

• Alternative names: GPI, Phosphoglucose isomerase (PGI), Phosphohexose isomerase (PHI)

• Expression source: Mammalian cell system

• Purity: >85% as determined by SDS-PAGE

• Structure: Partial protein rather than full-length enzyme

• Tag information: Variable, determined during manufacturing process

Handling and storage recommendations:

• Reconstitution protocol: Briefly centrifuge the vial before opening; reconstitute in deionized sterile water to 0.1-1.0 mg/mL; add 5-50% glycerol (with 50% being standard) for long-term storage

• Storage conditions:

  • Short-term (up to one week): 4°C for working aliquots

  • Long-term (liquid form): Up to 6 months at -20°C/-80°C

  • Long-term (lyophilized form): Up to 12 months at -20°C/-80°C

Critical experimental considerations:

• Avoid repeated freeze-thaw cycles as they significantly reduce enzyme activity

• The partial nature of the recombinant protein may affect certain structural or functional studies

• The shelf life depends on multiple factors including storage state, buffer ingredients, temperature, and the intrinsic stability of the protein itself

• When designing activity assays, consider the optimal buffer conditions for bacterial PGIs (typically pH 7.0-8.0 with divalent cations such as Mg²⁺)

These specifications will help researchers maintain optimal enzyme quality and activity for experiments involving this recombinant protein.

Why is B. cenocepacia significant in cystic fibrosis research?

Burkholderia cenocepacia holds significant importance in cystic fibrosis (CF) research for several critical reasons:

Clinical severity and prognosis:
B. cenocepacia is an opportunistic pathogen particularly dangerous for CF patients. Infections can cause severe decline in lung function and potentially develop into a life-threatening systemic infection known as "cepacia syndrome" . Understanding the molecular basis of its pathogenicity is essential for developing effective interventions.

Extraordinary antibiotic resistance:
This bacterium demonstrates extreme resistance to multiple antibiotics and antimicrobial peptides, making infections particularly difficult to treat . For example, B. cenocepacia shows high resistance to antimicrobial peptides like polymyxin B through mechanisms involving L-Ara4N modifications of lipopolysaccharide (LPS) and the alternative sigma factor RpoE . This resistance profile severely limits therapeutic options.

Complex polymicrobial interactions:
B. cenocepacia engages in intricate interactions with other pathogens commonly found in CF lungs, particularly Pseudomonas aeruginosa. These interactions involve:

• Stimulation of P. aeruginosa biofilm production with increased biomass

• Utilization of P. aeruginosa-produced rhamnolipids to enable B. cenocepacia swarming motility

• Complex competitive and cooperative relationships that influence colonization patterns

Genetic diversity and epidemiology:
Research has revealed high genetic diversity among B. cenocepacia clinical isolates. A multilocus sequence typing study of 57 clinical isolates identified 21 sequence types, including 12 novel sequence types, demonstrating significant genetic variation in the population . This diversity has implications for transmission, virulence, and treatment strategies.

Research on specific B. cenocepacia proteins like PGI contributes to the broader goal of understanding this pathogen's metabolism, which may reveal new therapeutic targets to improve management of CF-related infections and ultimately enhance patient outcomes.

How might PGI activity influence B. cenocepacia antibiotic resistance mechanisms?

The potential relationship between PGI activity and B. cenocepacia's exceptional antibiotic resistance presents a fascinating research area. While direct evidence linking this specific enzyme to resistance mechanisms is limited, several hypothetical pathways warrant investigation:

Energy-dependent resistance mechanisms:
Many antibiotic resistance mechanisms require energy, including efflux pumps that actively export antibiotics from bacterial cells. As a key glycolytic enzyme, PGI influences ATP production, potentially affecting energy-dependent resistance mechanisms. Researchers could investigate this by:

• Creating PGI knockdown strains and assessing changes in efflux pump activity

• Measuring intracellular ATP levels in relation to antibiotic resistance profiles

• Combining PGI inhibitors with efflux pump inhibitors to test for synergistic effects

Metabolic adaptation and persistence:
B. cenocepacia demonstrates extremely high resistance to antimicrobial peptides like polymyxin B through mechanisms involving LPS modifications and the RpoE sigma factor . The synthesis of modified LPS components requires metabolic precursors from central carbon metabolism. PGI, as a central metabolic enzyme, could influence:

• Production of precursors for LPS modifications

• Metabolic shifts that support persistence during antibiotic stress

• Adaptive responses to antimicrobial challenges

Experimental approaches to investigate these connections:

This research direction could reveal previously unrecognized connections between central metabolism and antibiotic resistance in B. cenocepacia, potentially identifying novel therapeutic targets for this difficult-to-treat pathogen.

What role might PGI play in B. cenocepacia interactions with other pathogens in polymicrobial infections?

The role of PGI in B. cenocepacia interactions with other pathogens, particularly in the context of cystic fibrosis polymicrobial infections, represents an intriguing research direction. Based on documented interactions between B. cenocepacia and Pseudomonas aeruginosa , several research hypotheses emerge:

Metabolic cross-feeding and competition:
In polymicrobial environments like CF lungs, bacteria can exchange metabolites, potentially enhancing collective survival. PGI, as a central metabolic enzyme, may influence:

• Production of secreted metabolites that benefit or inhibit co-infecting species

• Utilization of resources in competitive or cooperative scenarios

• Metabolic adaptation to the presence of other species

Surface motility and colonization:
B. cenocepacia and P. aeruginosa engage in fascinating co-dependent surface motility behaviors. Research has shown that P. aeruginosa provides rhamnolipids that enable B. cenocepacia swarming, while non-motile P. aeruginosa can "hitchhike" along with B. cenocepacia cells . PGI activity may influence:

• Energy availability for flagellar movement

• Metabolic responses to surfactants produced by other species

• Adaptation to shared niches during polymicrobial colonization

Experimental design for investigating PGI's role in polymicrobial interactions:

Molecular mechanisms to investigate:

• How PGI activity influences production of secreted factors that mediate inter-species interactions

• Whether metabolic adaptations regulated by PGI affect expression of genes involved in species interaction

• If energy status controlled by central metabolism affects cooperative behaviors

This research direction could reveal how fundamental metabolic enzymes like PGI contribute to the complex social behaviors observed between B. cenocepacia and other pathogens in clinically relevant polymicrobial infections.

How can structural studies of recombinant B. cenocepacia PGI contribute to drug discovery efforts?

Structural studies of recombinant B. cenocepacia PGI can significantly advance drug discovery efforts against this highly resistant pathogen through several methodological approaches:

X-ray crystallography protocol optimization:

A systematic approach to obtaining the crystal structure would include:

• Protein preparation optimization:

  • Express with a cleavable purification tag in a mammalian cell system

  • Implement multi-step purification (affinity chromatography followed by size exclusion)

  • Achieve >95% purity (higher than the standard 85% reported for commercial preparations )

  • Screen multiple buffer conditions for optimal stability

• Crystallization strategy:

  • Perform initial screens using commercial sparse matrix kits

  • Include substrate/product molecules to capture different conformational states

  • Optimize promising conditions by varying precipitant concentration, pH, and additives

  • Consider surface entropy reduction mutations if crystallization proves challenging

Structure-based drug design applications:

Active site analysis:
Detailed mapping of the B. cenocepacia PGI active site could reveal unique features distinguishing it from human PGI, enabling the design of selective inhibitors. Key areas to analyze include:

• Substrate binding pocket architecture

• Catalytic residue orientation and microenvironment

• Species-specific adjacent pockets that could be exploited for selectivity

Virtual screening and fragment-based approach:

Addressing B. cenocepacia-specific challenges:
The extreme antibiotic resistance of B. cenocepacia creates unique drug discovery challenges. Structural studies can help address these by:

• Identifying potential allosteric sites unique to bacterial PGIs

• Revealing structural features that could be exploited to overcome efflux mechanisms

• Providing templates for designing compounds with appropriate properties to penetrate the B. cenocepacia cell envelope

This methodological framework demonstrates how structural studies of recombinant B. cenocepacia PGI can systematically contribute to drug discovery against this challenging pathogen.

What are the optimal enzymatic assay conditions for measuring recombinant B. cenocepacia PGI activity?

Establishing optimal enzymatic assay conditions is crucial for accurate characterization of recombinant B. cenocepacia PGI activity. The following comprehensive protocol outlines the methodological approach:

Standard coupled spectrophotometric assay:

This assay measures PGI activity by coupling the formation of fructose-6-phosphate to NADPH production through glucose-6-phosphate dehydrogenase, which can be monitored at 340 nm.

Reagents and reaction conditions:

• Assay buffer: 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT

• Substrate (glucose-6-phosphate): Test range 0.1-10 mM for Km determination

• NADP+: 0.5 mM

• Glucose-6-phosphate dehydrogenase (coupling enzyme): 0.5-1.0 U/mL

• Recombinant B. cenocepacia PGI: Start with 0.1-1 μg per reaction

• Temperature: 30°C (standard) with comparison at 37°C (physiologically relevant)

Parameter optimization table:

Data analysis and kinetic characterization:

• Generate Michaelis-Menten plots using varied substrate concentrations

• Calculate Km and Vmax using non-linear regression

• Determine kcat by dividing Vmax by enzyme concentration

• Calculate catalytic efficiency (kcat/Km)

• Compare kinetic parameters with PGI from other bacterial species and human PGI

Critical validation steps:

• Include commercial PGI from other species as positive controls

• Run no-enzyme controls to account for background reactions

• Confirm activity in both forward and reverse directions (glucose-6-phosphate ↔ fructose-6-phosphate)

• Verify product formation using an orthogonal method (e.g., HPLC)

This comprehensive methodological approach provides the foundation for accurate characterization of recombinant B. cenocepacia PGI activity, essential for subsequent inhibitor development and structure-function studies.

How can researchers effectively use recombinant B. cenocepacia PGI to study bacterial metabolism during host-pathogen interactions?

Leveraging recombinant B. cenocepacia PGI to study bacterial metabolism during host-pathogen interactions requires sophisticated experimental designs that bridge biochemistry, cell biology, and infection biology. The following methodological framework provides a comprehensive approach:

In vitro infection models with metabolic analysis:

• Cell culture infection system:

  • Culture relevant host cells (human bronchial epithelial cells, macrophages)

  • Infect with B. cenocepacia wild-type and PGI-modulated strains

  • Isolate bacteria from infected cells at various timepoints

  • Measure PGI activity in recovered bacteria

  • Correlate activity changes with intracellular survival and replication

• Isotope tracing during infection:

  • Supply 13C-labeled glucose to culture medium during infection

  • Extract metabolites from bacteria isolated from host cells

  • Analyze isotopologue distribution using mass spectrometry

  • Determine flux changes through glycolysis vs. pentose phosphate pathway

  • Compare metabolic profiles between extracellular and intracellular bacteria

Specialized experimental models:

Functional approaches using recombinant PGI:

• Enzymatic rescue experiments:

  • Create PGI-deficient B. cenocepacia strains

  • Supplement with purified recombinant PGI during infection

  • Determine whether exogenous enzyme can restore virulence traits

  • Identify potential extracellular roles of PGI during infection

• Antibody inhibition studies:

  • Generate antibodies against recombinant B. cenocepacia PGI

  • Test effects of antibody treatment on bacterial metabolism and virulence

  • Evaluate potential as therapeutic approach

• Integration with multi-omics data:

  • Correlate PGI activity with transcriptomic changes during infection

  • Identify co-regulated metabolic and virulence genes

  • Map protein-protein interactions of PGI in different infection stages

  • Conduct targeted metabolomics focusing on glycolysis and related pathways

This comprehensive methodological framework enables researchers to effectively use recombinant B. cenocepacia PGI as a tool to understand the metabolic basis of host-pathogen interactions, potentially revealing new therapeutic targets for this challenging pathogen.

What are the key considerations for developing inhibitors targeting B. cenocepacia PGI as potential therapeutic agents?

Developing inhibitors targeting B. cenocepacia PGI as potential therapeutic agents requires a systematic approach addressing several critical considerations. The following methodological framework outlines the key steps and special considerations for this drug discovery process:

Target validation and druggability assessment:

• Essentiality evaluation:

  • Determine whether PGI is essential for B. cenocepacia growth and virulence

  • Create conditional knockdown strains to evaluate impact on survival

  • Assess growth in different carbon sources to identify bypass mechanisms

  • Validate in both laboratory and infection-relevant conditions

• Selectivity analysis:

  • Compare bacterial PGI with human homolog to identify structural differences

  • Assess sequence conservation among bacterial species

  • Identify unique features of B. cenocepacia PGI that can be exploited

Inhibitor discovery pipeline:

Special considerations for B. cenocepacia:

• Inherent resistance mechanisms:
  B. cenocepacia is known for its extreme antibiotic resistance . Inhibitor development should address:

  • Efflux pump-mediated extrusion

  • Permeability barriers (including the LPS structure that contributes to antimicrobial peptide resistance )

  • Potential metabolic adaptations and bypass pathways

• Delivery challenges in CF context:

  • Penetration through CF mucus

  • Activity in anaerobic/microaerobic conditions found in CF lungs

  • Efficacy in biofilm-embedded bacteria (B. cenocepacia forms biofilms in CF lungs)

• Combination approaches:

  • Evaluate PGI inhibitors in combination with existing antibiotics

  • Assess synergy with compounds targeting other metabolic pathways

  • Consider adjuvants that enhance bacterial membrane permeability

• Resistance development risk:

  • Monitor potential resistance mechanisms

  • Design inhibitors with high barrier to resistance

  • Target conserved residues essential for catalysis

This comprehensive methodological framework addresses the key considerations for developing B. cenocepacia PGI inhibitors as potential therapeutic agents against this challenging pathogen, particularly in the context of cystic fibrosis infections where new treatment options are urgently needed .