Recombinant Escherichia fergusonii Glucose-6-phosphate isomerase (pgi), partial

What is Escherichia fergusonii and what is the significance of studying its glucose-6-phosphate isomerase?

Escherichia fergusonii is a Gram-negative, rod-shaped bacterium closely related to E. coli, first isolated from human blood samples. It is considered an emerging pathogen with zoonotic potential, capable of causing urinary tract infections, wound infections, bacteremia, and gastrointestinal infections3. E. fergusonii has demonstrated significant pathogenic potential and antimicrobial resistance, particularly in avian and porcine strains .

Glucose-6-phosphate isomerase (GPI or PGI) is a critical enzyme in both glycolysis and gluconeogenesis pathways, facilitating the reversible transformation of glucose-6-phosphate into fructose-6-phosphate . This enzyme serves as a key node connecting multiple metabolic pathways, including:

• Glycolysis/gluconeogenesis

• Pentose phosphate pathway

• Amino sugar metabolism

• Nucleotide sugar metabolism

Studying E. fergusonii PGI is significant because:

• It acts as a moonlighting protein with multiple cellular functions beyond sugar interconversion

• It potentially contributes to bacterial pathogenicity and virulence

• It may play a role in antimicrobial resistance mechanisms

• It could serve as a potential target for novel antimicrobial development

How does the structure and function of E. fergusonii PGI compare to homologs in other bacterial species?

The function of PGI appears to be highly conserved across bacterial species, though with species-specific adaptations. Based on comparative studies, we can infer that E. fergusonii PGI catalyzes the reversible isomerization between glucose-6-phosphate and fructose-6-phosphate, similar to its homologs in other organisms .

Functional comparison across species reveals:

While the catalytic mechanism is likely conserved, structural variations may exist in substrate binding sites and regulatory domains. These differences could potentially be exploited for the development of species-specific inhibitors targeting E. fergusonii.

What molecular techniques are recommended for cloning and expressing recombinant E. fergusonii PGI?

For successful cloning and expression of recombinant E. fergusonii PGI, the following methodological approach is recommended:

• Gene identification and isolation:

  • Extract genomic DNA from E. fergusonii clinical or environmental isolates

  • Amplify the PGI gene using PCR with primers designed based on conserved regions

  • Consider codon optimization if expression will be performed in a heterologous host

• Cloning strategy:

  • Select an appropriate expression vector with a strong, inducible promoter

  • Include a purification tag (His6, GST, or MBP) to facilitate protein purification

  • Transform into an expression strain with reduced protease activity

• Expression optimization:

  • Test multiple expression conditions (temperature, inducer concentration, expression duration)

  • Perform small-scale expression tests to optimize soluble protein yield

  • Consider using specialized strains like BL21(DE3) or Rosetta for efficient expression

• Protein purification:

  • Implement a multi-step purification strategy including:
    a. Affinity chromatography (IMAC for His-tagged proteins)
    b. Ion exchange chromatography
    c. Size exclusion chromatography

  • Verify protein purity using SDS-PAGE and Western blotting

  • Confirm protein identity with mass spectrometry

What are the standard methods for measuring recombinant E. fergusonii PGI enzyme activity?

PGI activity can be measured using several established methodological approaches:

• Spectrophotometric coupled enzyme assays:

  • Forward reaction (G6P → F6P): Couple with phosphofructokinase and aldolase

  • Reverse reaction (F6P → G6P): Couple with G6P dehydrogenase and monitor NADPH formation at 340 nm

• Direct measurement techniques:

  • HPLC analysis of substrate consumption and product formation

  • Mass spectrometry to quantify reaction products

  • NMR spectroscopy for real-time reaction monitoring

• Enzyme kinetics characterization:

  • Determine Michaelis-Menten parameters (Km, Vmax, kcat)

  • Evaluate pH optimum and buffer conditions

  • Assess temperature stability and optimal reaction temperature

  • Investigate metal ion requirements and inhibitor profiles

A standardized assay protocol might include:

• Reaction buffer: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT

• Coupled enzymes: G6P dehydrogenase (2 U/mL), NADP+ (0.5 mM)

• Substrate range: G6P or F6P (0.1-10 mM)

• Temperature: 37°C

• Monitoring: Continuous absorbance readings at 340 nm

How does E. fergusonii PGI potentially contribute to pathogenicity and virulence mechanisms?

Based on studies of PGI in related organisms, E. fergusonii PGI likely contributes to pathogenicity through multiple mechanisms :

• Metabolic adaptation:

  • PGI enables flexible carbon metabolism, allowing adaptation to nutrient-limited host environments

  • Its position at the intersection of glycolysis and the pentose phosphate pathway facilitates rapid metabolic switching in response to environmental changes

• Cell wall biosynthesis:

  • PGI likely provides precursors for cell wall components, similar to its role in Aspergillus species

  • Cell wall integrity is crucial for survival under host-induced stress conditions

  • Altered cell wall composition may affect host immune recognition

• Stress response:

  • Studies in A. flavus indicate that PGI deletion results in hypersusceptibility to osmotic, oxidative, and temperature stresses

  • E. fergusonii PGI likely plays a similar role in stress adaptation during infection

• Biofilm formation:

  • E. fergusonii strains have demonstrated biofilm formation capabilities3

  • PGI may contribute to biofilm matrix production through its role in providing precursors for exopolysaccharide synthesis

Experimental approaches to investigate these mechanisms could include:

• Generation of E. fergusonii PGI knockout mutants

• Phenotypic characterization under various stress conditions

• Virulence assessment in appropriate infection models

• Complementation studies to confirm phenotype specificity

What is known about E. fergusonii PGI's relationship to antimicrobial resistance?

E. fergusonii strains have demonstrated significant antimicrobial resistance profiles, particularly in avian and porcine isolates . While PGI is not directly associated with antibiotic resistance mechanisms, it may indirectly contribute through:

• Metabolic adaptation:

  • PGI's role in central carbon metabolism could support cellular responses to antimicrobial stress

  • Altered metabolic states may modify susceptibility to certain antibiotics

• Biofilm formation:

  • E. fergusonii can form biofilms, which significantly increase resistance to antimicrobial agents3

  • PGI likely contributes to biofilm matrix production through its metabolic functions

• Cell wall modification:

  • If E. fergusonii PGI contributes to cell wall biosynthesis (as observed in fungi ), it could influence cell envelope properties

  • The cell envelope is a primary barrier against antimicrobial agents

Research has identified multidrug-resistant E. fergusonii isolates harboring various beta-lactamase genes:

Source: Findings from carbapenem-resistant E. fergusonii clinical isolates

Experimental approaches to investigate PGI's role in antimicrobial resistance could include:

• Transcriptomic analysis of PGI expression under antimicrobial exposure

• Phenotypic characterization of PGI mutants for altered antimicrobial susceptibility

• Proteomic studies to identify PGI interaction partners during antimicrobial stress

How can genomic analysis methods be applied to study E. fergusonii PGI gene diversity across different strains?

Comprehensive genomic analysis methods for studying E. fergusonii PGI diversity include :

• Whole genome sequencing and comparative genomics:

  • Sequence multiple E. fergusonii strains from diverse sources

  • Compare PGI gene sequences to identify conserved regions and polymorphisms

  • Analyze promoter regions for potential regulatory differences

  • Examine genetic context to identify potential horizontal gene transfer events

• Phylogenetic analysis:

  • Construct phylogenetic trees based on PGI sequences to understand evolutionary relationships

  • Compare with species phylogeny to identify potential recombination events

  • Study selection pressures acting on different regions of the PGI gene

• Pangenomic analysis:

  • Determine if PGI is part of the core or accessory genome of E. fergusonii

  • Identify any strain-specific variations that might relate to niche adaptation

  • Compare with related Escherichia species to understand genus-level conservation

• Functional genomics:

  • Transcriptomic analysis of PGI expression under different environmental conditions

  • Regulatory network analysis to understand PGI gene regulation

  • Proteomics studies to confirm expression and post-translational modifications

Based on pangenomic analysis of E. fergusonii, the following insights have been reported:

Source: Comparative genomic analyses of E. fergusonii from different sources

What structural and functional differences might exist between E. fergusonii PGI and human GPI?

Understanding structural and functional differences between bacterial and human GPI is crucial for developing selective inhibitors. While specific information about E. fergusonii PGI structure is not directly available from the search results, we can infer several potential differences:

• Structural distinctions:

  • Active site architecture: Despite catalyzing the same reaction, bacterial and human enzymes often have distinct active site geometries

  • Quaternary structure: Different oligomeric states (monomeric, dimeric, or tetrameric arrangements)

  • Regulatory domains: Presence of unique regulatory sites in the bacterial enzyme

• Functional differences:

  • Moonlighting functions: Bacterial PGI may have evolved additional functions beyond its catalytic role

  • Substrate specificity: Subtle differences in substrate preference or binding affinity

  • Reaction kinetics: Different catalytic efficiencies under physiological conditions

• Post-translational modifications:

  • Human GPI undergoes glycosylation and phosphorylation

  • Bacterial PGI likely lacks these modifications or has different modification patterns

• Inhibitor sensitivity:

  • Differential sensitivity to known GPI inhibitors

  • Unique allosteric regulatory mechanisms

Research approaches to explore these differences include:

• Comparative homology modeling of E. fergusonii PGI based on related bacterial structures

• Recombinant expression and crystallization for structural determination

• Biochemical characterization of substrate specificity and inhibitor sensitivity

• Molecular dynamics simulations to identify unique structural features

What role might E. fergusonii PGI play in biofilm formation and how can this be investigated?

E. fergusonii has been reported to form biofilms, which contribute to antimicrobial resistance and environmental persistence3. The potential roles of PGI in biofilm formation include:

• Metabolic support:

  • PGI's central role in carbon metabolism provides energy and precursors needed for biofilm matrix production

  • It enables efficient utilization of available carbon sources within biofilm microenvironments

• Exopolysaccharide synthesis:

  • PGI generates precursors that feed into pathways for exopolysaccharide production

  • These exopolysaccharides form a significant component of the biofilm matrix

• Stress adaptation:

  • As demonstrated in A. flavus, PGI contributes to stress resistance

  • Enhanced stress resistance is critical for biofilm persistence

Methodological approaches to investigate PGI's role in biofilm formation:

• Genetic approaches:

  • Generate PGI knockout or knockdown mutants

  • Create conditional expression strains for controlled PGI expression

  • Complement mutants with wild-type PGI to confirm phenotype specificity

• Biofilm characterization methods:

  • Crystal violet assays for biofilm biomass quantification

  • Confocal microscopy with fluorescent stains to analyze biofilm architecture

  • Flow cell systems for dynamic biofilm formation studies

  • Scanning electron microscopy for detailed structural analysis

• Molecular and biochemical analyses:

  • Transcriptomic analysis comparing planktonic and biofilm growth

  • Proteomics to identify PGI interaction partners in biofilms

  • Metabolomic studies to track carbon flux through PGI during biofilm formation

• Inhibitor studies:

  • Test the effect of PGI inhibitors on biofilm formation and dispersal

  • Evaluate combination therapy with conventional antibiotics

What are the optimal conditions for expressing and purifying recombinant E. fergusonii PGI?

Based on general principles for bacterial enzyme expression and purification:

• Expression system optimization:

  • Host strain: E. coli BL21(DE3) or Rosetta for efficient expression

  • Vector selection: pET series vectors with T7 promoter for high-level expression

  • Fusion tags: N-terminal His6 tag for purification; MBP or SUMO tags for enhanced solubility

  • Codon optimization: Consider harmonizing codons for expression host

• Culture conditions:

  • Growth medium: Enriched media (LB, TB) for high cell density

  • Temperature: Reduced temperature (16-25°C) during induction to enhance solubility

  • Induction: Low IPTG concentration (0.1-0.5 mM) for longer periods (16-20 hours)

• Purification strategy:

  • Cell lysis: Sonication or high-pressure homogenization in buffer containing protease inhibitors

  • Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Buffer optimization: Typically 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol

• Quality control methods:

  • Purity assessment: SDS-PAGE and Western blotting

  • Activity verification: Coupled enzyme assay

  • Stability testing: Differential scanning fluorimetry

  • Identity confirmation: Mass spectrometry

How can researchers design inhibitor screening assays for E. fergusonii PGI?

Designing effective inhibitor screening assays for E. fergusonii PGI involves:

• Primary screening assay development:

  • Adapt standard PGI activity assays for high-throughput format

  • Optimize reaction conditions (buffer, pH, temperature) for maximum sensitivity

  • Develop appropriate positive and negative controls

  • Determine Z' factor to validate assay robustness

• Assay formats:

  • Spectrophotometric assays: Coupled enzyme systems monitoring NADPH production

  • Fluorescence-based assays: Higher sensitivity for detection of subtle inhibition

  • Thermal shift assays: Screen for compounds that alter protein thermal stability

• Compound library selection:

  • Natural product libraries enriched for carbohydrate analogs

  • Fragment-based libraries for initial hits

  • Structure-based virtual screening to prioritize compounds

• Hit validation and characterization:

  • Dose-response curves to determine IC50 values

  • Mechanism of inhibition studies (competitive, noncompetitive, uncompetitive)

  • Binding affinity determination using isothermal titration calorimetry or surface plasmon resonance

  • Selectivity profiling against human GPI and other related enzymes

• In vivo evaluation:

  • Antimicrobial activity against E. fergusonii

  • Effect on biofilm formation

  • Cytotoxicity testing against mammalian cells

What experimental approaches can determine if E. fergusonii PGI is essential for bacterial survival and virulence?

Multiple complementary experimental approaches can determine the essentiality of PGI for E. fergusonii survival and virulence:

• Genetic manipulation strategies:

  • CRISPR-Cas9 genome editing for targeted gene knockout

  • Conditional expression systems using inducible promoters

  • Antisense RNA approaches for gene knockdown

  • Transposon mutagenesis followed by sequencing (Tn-Seq) to assess gene essentiality

• Phenotypic characterization:

  • Growth curve analysis under different carbon sources

  • Metabolic profiling using Biolog or similar systems

  • Stress response testing (oxidative, osmotic, temperature)

  • Biofilm formation assessment

• Virulence assessment:

  • Cell culture infection models to test bacterial invasion and intracellular survival

  • Galleria mellonella infection model for preliminary in vivo testing

  • Murine models for comprehensive virulence assessment

  • Competitive index experiments comparing wild-type and PGI mutant strains

• Molecular analyses:

  • Transcriptomic profiling to identify compensatory mechanisms

  • Metabolomic analysis to identify pathway alterations

  • Proteomic studies to detect protein expression changes

• Chemical genetics:

  • Testing specific PGI inhibitors for bacteriostatic or bactericidal effects

  • Complementation with heterologous PGI enzymes to rescue phenotypes

Successful application of these methods would provide comprehensive understanding of PGI's role in E. fergusonii physiology and pathogenesis, potentially validating it as a therapeutic target.