Recombinant Coxiella burnetii Glucose-6-phosphate isomerase (pgi), partial

What is Glucose-6-phosphate isomerase in Coxiella burnetii and why is it significant for research?

Glucose-6-phosphate isomerase (pgi) in Coxiella burnetii, also known as phosphoglucose isomerase (PGI) or phosphohexose isomerase (PHI), is an enzyme (EC 5.3.1.9) that catalyzes the reversible isomerization between glucose-6-phosphate and fructose-6-phosphate in the glycolytic pathway. This enzyme is significant because:

• It represents a critical step in central carbon metabolism of C. burnetii

• It functions within the context of C. burnetii's reduced genome (~2 Mb), which has evolved specific metabolic adaptations for intracellular parasitism

• It provides insights into how this obligate intracellular pathogen manages glucose metabolism despite lacking canonical components like hexokinase

• It helps researchers understand the metabolic requirements for C. burnetii survival and pathogenesis

How does the metabolic context of C. burnetii affect the function of pgi?

C. burnetii exhibits several unique metabolic features that influence pgi function:

• Reduced genome with streamlined metabolism: Despite genome reduction to ~2 Mb due to parasitism, C. burnetii maintains a nearly complete central metabolic machinery

• Missing canonical hexokinase: C. burnetii lacks a canonical hexokinase for glucose phosphorylation, suggesting alternative mechanisms for generating glucose-6-phosphate

• Absence of oxidative pentose phosphate pathway: The bacterium lacks the oxidative branch of the pentose phosphate pathway, a major mechanism for NADPH regeneration

• Alternative glucose phosphorylation: Researchers have proposed a phosphoenolpyruvate-based phosphotransferase system or other mechanisms for glucose phosphorylation

• Adaptability to nutrient availability: C. burnetii shows metabolic plasticity in using gluconeogenic versus glycolytic carbon substrates, which affects pgi directionality

What are the recommended storage and handling conditions for recombinant C. burnetii pgi?

For optimal stability and activity of recombinant C. burnetii pgi:

How can recombinant C. burnetii pgi be used to study glucose metabolism in this pathogen?

Recombinant C. burnetii pgi serves as a valuable tool for investigating glucose metabolism:

• Enzymatic activity assays: Researchers can use purified pgi to measure kinetic parameters and compare with other bacterial species to identify unique adaptations.

• Metabolic flux analysis: By tracing the conversion between glucose-6-phosphate and fructose-6-phosphate, researchers can understand the directionality of carbon flow in C. burnetii's central metabolism.

• Host-pathogen metabolic interactions: The enzyme helps investigate how C. burnetii adapts its metabolism to the host cell environment, particularly under glucose limitation conditions.

• Comparative studies with zwf gene: As demonstrated by research, complementing C. burnetii with the zwf gene (encoding glucose-6-phosphate dehydrogenase) causes a severe metabolic fitness defect under glucose limitation, suggesting selection against certain glucose-6-phosphate utilizing enzymes. Similar studies with pgi can reveal its metabolic significance .

• Genetic complementation studies: Testing whether C. burnetii pgi can functionally complement E. coli or other bacterial mutants lacking this enzyme can reveal functional conservation or unique properties.

What methods are used to assess the purity and activity of recombinant C. burnetii pgi?

Standard protocols for quality assessment include:

Activity can be measured in a coupled assay system where the production of fructose-6-phosphate is linked to NADH oxidation through phosphofructokinase and aldolase, allowing spectrophotometric monitoring at 340 nm.

How does C. burnetii pgi compare to the enzyme in other bacterial species?

Comparative analysis reveals several distinctions:

• Sequence conservation: While the catalytic site is relatively conserved, C. burnetii pgi shows distinctive sequence features reflective of its evolutionary adaptation to intracellular lifestyle.

• Metabolic context: Unlike many other bacteria, C. burnetii pgi functions in a metabolic network lacking certain canonical components like hexokinase and the oxidative pentose phosphate pathway .

• Environmental adaptation: C. burnetii pgi likely evolved to function optimally in the acidic environment of the parasitophorous vacuole (CCV) where the bacterium replicates .

• Directionality preferences: The enzyme may show different equilibrium constants or preferences for forward/reverse reactions compared to other species, reflecting C. burnetii's need for metabolic flexibility in diverse host environments.

• Regulatory mechanisms: Regulation of pgi activity likely differs from free-living bacteria, as C. burnetii must coordinate its metabolism with available host resources.

How might pgi expression and activity be regulated in the context of C. burnetii's intracellular lifecycle?

Regulation of pgi in C. burnetii likely involves multiple sophisticated mechanisms:

• Transcriptional regulation: Expression may be coordinated with other glycolytic or gluconeogenic enzymes depending on the metabolic state of the bacterium and available carbon sources.

• Post-translational modifications: Activity modulation through phosphorylation, acetylation, or other modifications in response to environmental cues.

• Allosteric regulation: Activity control through binding of effector molecules that signal energy status or carbon availability within the host cell.

• Spatial organization: Potential inclusion in metabolic complexes or association with the bacterial membrane to enhance pathway efficiency.

• Stress response integration: Adaptation to oxidative stress, pH changes, or nutrient limitation within the phagolysosome-like parasitophorous vacuole.

• Coordination with type IV secretion system: C. burnetii uses a Dot/Icm type IV secretion system to generate its replication niche ; metabolic regulation including pgi activity may be coordinated with secretion system activity.

• Phase variation effects: C. burnetii exhibits phase variation with different LPS structures between virulent (Phase I) and avirulent (Phase II) forms , which may correlate with metabolic shifts and pgi regulation.

What experimental approaches can determine the role of pgi in C. burnetii virulence and metabolic adaptation?

Advanced experimental strategies include:

• Conditional gene expression systems:

  • Tet-regulated expression to control pgi levels during infection

  • Analysis of growth, vacuole formation, and host cell response

• Metabolomic profiling:

  • Comparison of metabolite levels between wild-type and pgi-modified C. burnetii

  • Stable isotope labeling to track carbon flux through central metabolism

• Transposon mutagenesis screens:

  • Identification of genetic interactions with pgi

  • Discovery of synthetic lethal interactions revealing metabolic dependencies

• In vivo infection models:

  • Assessment of colonization, replication, and persistence of pgi-modified strains

  • Evaluation of host immune response differences

• Structural biology approaches:

  • Crystal structure determination of C. burnetii pgi

  • Structure-guided mutagenesis to assess functional significance of unique features

• Host-pathogen interface studies:

  • Proximity labeling to identify host proteins interacting with bacterial metabolism

  • Analysis of metabolite exchange between pathogen and host

• Integration with Dot/Icm effector studies:

  • Examining potential coordination between metabolic enzymes and C. burnetii's secreted effector proteins

  • Investigation of metabolic consequences of specific effector activities

How does glucose limitation affect C. burnetii metabolism and what does this reveal about pgi's role?

Glucose limitation studies provide critical insights into C. burnetii's metabolic adaptations:

Research has shown that complementing C. burnetii with the zwf gene (encoding glucose-6-phosphate dehydrogenase) causes a severe metabolic fitness defect under glucose limitation . This suggests several important features about glucose metabolism and potentially pgi function:

• Glucose-6-phosphate allocation is critical for C. burnetii survival

  • Limited G6P must be directed to specific pathways

  • Diversion to NADPH production via G6PD becomes detrimental under glucose limitation

• Directionality of pgi reaction may shift under glucose limitation

  • May favor gluconeogenic direction (F6P → G6P) when glucose is scarce

  • Helps maintain critical G6P levels for essential pathways

• Experimental data demonstrates metabolic consequences:

These findings suggest that under glucose limitation, C. burnetii carefully regulates glucose-6-phosphate utilization, with pgi likely playing a key role in maintaining proper carbon flux distribution .

What is the relationship between pgi function and the unique intracellular niche formation of C. burnetii?

The relationship between pgi and C. burnetii's parasitophorous vacuole (CCV) formation involves complex metabolic integration:

• Acidic environment adaptation: C. burnetii uniquely thrives in an acidic phagolysosome-like CCV , and its metabolic enzymes including pgi must function optimally in this environment.

• Autophagy interaction: C. burnetii manipulates host autophagy to create its replication niche , and central metabolism must be coordinated with these host cellular manipulations.

• Metabolic support for effector function: The bacterium's type IV secretion system delivers effectors that modify host cell processes ; these effectors require energy and precursors supplied by central metabolism involving pgi.

• Nutritional immunity evasion: C. burnetii must overcome host restriction of essential nutrients, potentially requiring metabolic flexibility in which pgi's reversible reaction provides adaptability.

• Vacuole biogenesis coordination: The expansion of the CCV likely requires coordinated bacterial growth and metabolism, with pgi functioning within this highly regulated system.

• Long-term persistence mechanism: C. burnetii's ability to cause chronic infections suggests metabolic adaptations for long-term survival, potentially involving regulated activity of central metabolic enzymes like pgi.

What are the critical controls for experiments involving recombinant C. burnetii pgi?

Rigorous experimental design should include:

• Enzyme activity controls:

  • Heat-inactivated enzyme (negative control)

  • Commercial pgi from related species (positive control)

  • Substrate-only reactions (background control)

  • Enzyme concentration gradient (linearity assessment)

• Expression system considerations:

  • Empty vector controls when using recombinant expression

  • Tag-only controls if using tagged protein

  • Native versus tagged protein comparison for activity assessment

• Functional complementation controls:

  • Positive control with wild-type gene from same species

  • Negative control with inactive mutant

  • Dose-dependent expression analysis

• Storage stability assessment:

  • Fresh versus stored enzyme activity comparison

  • Multiple freeze-thaw cycle testing

  • Different storage buffer composition evaluation

• Environmental condition variables:

  • pH range testing (particularly relevant given C. burnetii's acidic niche)

  • Temperature stability assessment

  • Metal ion dependency evaluation

How can researchers effectively design mutagenesis studies to understand structure-function relationships in C. burnetii pgi?

Strategic mutagenesis approaches include:

• Catalytic site mutations:

  • Target residues involved in substrate binding and catalysis

  • Conservative substitutions to understand chemical requirements

  • Comparison with mutations in well-characterized pgi enzymes from other species

• Comparative sequence analysis-guided mutagenesis:

  • Identify residues unique to C. burnetii pgi compared to free-living bacteria

  • Create chimeric proteins swapping domains between C. burnetii and other bacterial pgi

  • Restore ancestral sequences to understand evolutionary adaptations

• Random mutagenesis with functional selection:

  • Error-prone PCR followed by complementation screening

  • Selection under different carbon source conditions

  • Deep mutational scanning with next-generation sequencing

• Structure-guided approaches:

  • Targeting surface residues potentially involved in protein-protein interactions

  • Modifications to potential allosteric sites

  • Engineering stability-enhancing mutations for biochemical studies

• Assessment methodology:

  • In vitro enzyme kinetics (Km, kcat, substrate preference)

  • In vivo complementation of pgi-deficient bacteria

  • Protein stability and folding analysis

What approaches can resolve contradictions in the experimental data regarding C. burnetii glucose metabolism?

Resolving experimental inconsistencies requires multi-faceted approaches:

• Addressing the hexokinase paradox:

  • Despite the apparent absence of a canonical hexokinase gene, "hexokinase activity" has been detected in C. burnetii extracts

  • Potential resolution strategies include:

    • Purification and identification of the non-canonical enzyme

    • Heterologous expression of candidate genes and activity testing

    • Investigation of potential host enzyme recruitment

• Resolving glucose phosphorylation mechanisms:

  • Multiple proposed mechanisms exist: phosphoenolpyruvate-based phosphotransferase system, glucose 6-phosphatase (CBU1267), or unidentified mechanisms

  • Unified experimental approach:

    • Genetic knockout/complementation of each candidate

    • Metabolic labeling with isotope-labeled glucose

    • In vitro reconstitution of the complete pathway

• Understanding the metabolic significance of the oxidative pentose phosphate pathway absence:

  • C. burnetii lacks this pathway but still requires NADPH for various cellular processes

  • Systematic investigation through:

    • Comparative transcriptomics and proteomics under oxidative stress

    • Identification of alternative NADPH-generating systems

    • Metabolic flux analysis with 13C-labeled substrates

• Methodological considerations for studying obligate intracellular pathogens:

  • Difficulty separating host and bacterial metabolism

  • Potential approaches:

    • Development of host-free cultivation systems with defined media

    • Single-cell analytical techniques

    • Selective inhibition of host pathways

How can recombinant C. burnetii pgi be utilized for developing vaccines or diagnostic tools?

Innovative applications include:

• Vaccine development strategies:

  • Recombinant pgi as a protein subunit vaccine component

  • Use in combination with other C. burnetii antigens for multi-target approach

  • Carrier protein for C. burnetii-specific polysaccharide antigens

  • Adjuvant optimization for appropriate immune response

• Diagnostic applications:

  • Development of enzyme-linked immunosorbent assays (ELISAs) for detecting anti-pgi antibodies

  • Creation of pgi-based lateral flow assays for point-of-care testing

  • Multiplex serological platforms incorporating pgi and other C. burnetii antigens

  • Development of activity-based assays for detecting active infection

• Epidemiological screening tools:

  • High-throughput serological testing for population surveillance

  • Distinguishing between acute and chronic Q fever infections

  • Differentiating vaccine-induced immunity from natural infection

• Safer research tools:

  • Use of recombinant pgi rather than whole organisms for laboratory research

  • Generation of inactive but antigenic variants for immunological studies

  • Development of in vitro activity assays to replace living organism experiments

What are the methodological considerations for using C. burnetii pgi in structural biology studies?

Successful structural characterization requires:

• Protein preparation optimization:

  • Expression system selection (bacterial, insect, mammalian)

  • Solubility enhancement through fusion partners or co-expression with chaperones

  • Purification strategy development (affinity, ion exchange, size exclusion)

  • Buffer optimization for stability and homogeneity

  • Tag design and removal considerations

• Crystallization approaches:

  • Sparse matrix screening with commercial kits

  • Optimization of initial crystallization hits

  • Co-crystallization with substrates, products, or inhibitors

  • Surface entropy reduction for challenging targets

  • Microseeding techniques for crystal improvement

• Alternative structural methods:

  • Cryo-electron microscopy for structure determination

  • Small-angle X-ray scattering for solution structure

  • Nuclear magnetic resonance for dynamics studies

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

• Structure-function correlation:

  • Enzymatic activity correlation with structural features

  • Computational docking of substrates and inhibitors

  • Molecular dynamics simulations under acidic conditions mimicking the CCV

  • Comparison with structures from related organisms

How can understanding C. burnetii pgi contribute to developing novel antimicrobial strategies?

Therapeutic targeting approaches include:

• Enzyme inhibition strategies:

  • Rational design of pgi inhibitors based on structural information

  • Virtual screening of compound libraries against the active site

  • Allosteric inhibitor development targeting C. burnetii-specific regulatory sites

  • Covalent inhibitor design for irreversible inactivation

• Metabolic vulnerability exploitation:

  • Targeting of unique metabolic dependencies revealed through pgi studies

  • Development of substrate analogues that disrupt glycolysis/gluconeogenesis balance

  • Combination approaches targeting multiple metabolic enzymes simultaneously

  • Design of pro-drugs activated by C. burnetii metabolic enzymes

• Host-directed therapeutic approaches:

  • Modulation of host cell metabolism to create unfavorable conditions for C. burnetii

  • Targeting of host-pathogen metabolic interaction points

  • Inhibition of host processes required for bacterial nutrient acquisition

• Treatment efficacy assessment:

  • Development of pgi activity-based assays to monitor treatment response

  • Creation of reporter systems based on metabolic activity

  • Biomarker identification for monitoring infection clearance

• Resistance management strategies:

  • Identification of potential resistance mechanisms through laboratory evolution

  • Design of inhibitors with high barriers to resistance

  • Combination therapy approaches targeting multiple metabolic nodes