Recombinant Treponema denticola Glucose-6-phosphate isomerase (pgi), partial

What are the basic molecular characteristics of T. denticola PGI?

T. denticola PGI is a key metabolic enzyme that catalyzes the reversible isomerization of glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P). While specific molecular weight data for T. denticola PGI isn't extensively documented in the provided literature, PGIs from other organisms typically exist as dimeric enzymes with subunit molecular weights around 60 kDa . The enzyme belongs to the PGI superfamily, though interestingly, archaeal PGIs have been found to represent a novel type with no significant similarity to the conserved PGI superfamily of eubacteria and eucarya .

For context, the recombinant PGI from Mycobacterium tuberculosis was found to have a molecular mass of 61.45 kDa when analyzed by mass spectroscopy . This provides a reference point for what might be expected for T. denticola PGI.

What expression systems are suitable for producing recombinant T. denticola PGI?

Based on research with similar enzymes and T. denticola proteins, several expression systems can be considered:

• E. coli expression systems: Common vectors like pET-22b(+) have been successfully used for expressing recombinant PGI from M. tuberculosis . For T. denticola proteins, E. coli has been used to express other proteins such as dentilisin complex components .

• T. denticola shuttle vectors: Several T. denticola shuttle plasmids have been developed that allow gene expression in both E. coli and T. denticola. These include vectors with various promoters that provide different expression levels:

  • P-tap1: A flagellar operon promoter that drives relatively high expression levels

  • P-ermB: A weaker promoter suitable when overexpression might be problematic

  • P-msp: A strong promoter from the major surface protein gene

Methodological approach:
When choosing an expression system, consider:

• The need for native folding and post-translational modifications

• Required expression levels

• Cytoplasmic vs. periplasmic expression

• The presence of transmembrane domains or signal peptides

• Codon optimization based on the host organism

How can you purify recombinant T. denticola PGI effectively?

While specific purification protocols for T. denticola PGI are not detailed in the provided literature, a general methodological approach based on similar enzymes would include:

• Cell lysis: Use methods appropriate for the expression host (sonication, French press, or enzymatic lysis)

• Initial separation: Ammonium sulfate precipitation or heat treatment (if the protein is thermostable)

• Chromatographic techniques:

  • Ion-exchange chromatography (as used for M. tuberculosis PGI )

  • Affinity chromatography using His-tags or other fusion tags

  • Size exclusion chromatography for final polishing

For recombinant PGI from M. tuberculosis expressed in E. coli, purification to near homogeneity was achieved using ion-exchange chromatography, resulting in enzymatically active protein with specific activity of 600 U/mg .

What are the kinetic properties of bacterial PGI enzymes, and how might T. denticola PGI compare?

Bacterial PGI enzymes show various kinetic properties depending on the species. While specific data for T. denticola PGI is not provided in the search results, comparative data from other bacterial PGIs is informative:

Kinetic parameters from different PGIs:

For T. denticola PGI characterization, researchers would likely employ similar analytical methods:

• For the forward reaction (F6P to G6P): Couple the reaction with G6P dehydrogenase and monitor NADPH production spectrophotometrically

• For the reverse reaction (G6P to F6P): Either use high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) or couple with additional enzymes like phosphofructokinase, aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase to monitor NADH oxidation

How does T. denticola PGI potentially contribute to its pathogenicity and survival in periodontal pockets?

While direct evidence of T. denticola PGI's role in pathogenicity is not explicitly described in the provided literature, several connections can be inferred based on related research:

• Metabolic adaptation to the periodontal environment: PGI is essential for both glycolysis and gluconeogenesis, allowing T. denticola to utilize different carbon sources available in the periodontal pocket. This metabolic flexibility likely contributes to survival in the dynamic subgingival environment .

• Energy production for virulence factor expression: PGI's role in central carbon metabolism provides the energy required for expression of virulence factors like dentilisin, a proteolytic complex involved in host tissue destruction, penetration of epithelial barriers, and immunomodulation .

• Connection with the "Red Complex": T. denticola forms a bacterial consortium with Porphyromonas gingivalis and Tannerella forsythia, known as the "Red Complex," strongly associated with periodontitis progression. Metabolic coordination between these species may be facilitated by central carbon metabolism enzymes like PGI .

• Biofilm formation: The motility of T. denticola, which requires energy generated through metabolic pathways involving PGI, is implicated in synergistic biofilm development with other periodontal pathogens .

A research approach to investigating this connection would involve gene knockout or knockdown studies, coupled with assessment of virulence, biofilm formation, and metabolic profiles.

What structural features distinguish bacterial PGI enzymes, and what might be expected for T. denticola PGI?

Bacterial PGIs exhibit several structural characteristics, although specific information on T. denticola PGI structure is not provided in the search results. Based on studies of PGIs from other organisms:

• Domain organization: Most bacterial PGIs consist of two domains with a catalytic site located at the domain interface. Some bacterial PGIs may have additional domains or extensions.

• Active site residues: The catalytic mechanism typically involves conserved residues. For instance, in subtilisin-like proteases (which are different enzymes but illustrate the principle), mutation of a single active site residue (Ser447→Ala) in T. denticola dentilisin significantly reduced but did not completely eliminate enzymatic activity .

• Oligomeric state: Most PGIs function as dimers, although the interfaces and specific interactions between subunits can vary between species.

• Unique features: Some bacterial PGIs have unique structural features not found in eukaryotic counterparts. For example, the archaeal PGI from P. furiosus represents a novel type with no significant similarity to the conserved PGI superfamily .

For structural studies of T. denticola PGI, approaches would include:

• X-ray crystallography

• Cryo-electron microscopy

• Homology modeling based on structurally characterized PGIs

• Site-directed mutagenesis to identify functionally important residues

How do genetic variations in T. denticola strains affect PGI properties and function?

Genetic variability between T. denticola strains is well-documented, suggesting potential variation in PGI properties among different isolates:

• Strain diversity: Studies of T. denticola have revealed significant genetic diversity among clinical isolates. For example, research using the pyrH gene as a marker identified multiple distinct genotypes in both periodontitis and gingivitis subjects . This suggests potential variations in metabolic enzymes like PGI.

• Functional implications: Genome sequencing of a mutant T. denticola strain derived from ATCC 35405 revealed over 200 mutations compared to the original strain, resulting in altered phenotypic characteristics including growth density and colony formation . Such mutations, if affecting PGI, could alter metabolic capabilities.

• Impact on enzymatic properties: While not specifically addressing PGI, studies of T. denticola's dentilisin protease complex showed that proteolytic activity varied considerably between strains, possibly due to differences in expression levels or sequence variations .

A methodological approach to studying PGI variation would include:

• Comparative sequence analysis of pgi genes from multiple T. denticola strains

• Expression and characterization of PGI variants

• Correlation of enzymatic properties with strain virulence or metabolic capabilities

What are the most effective methods for measuring T. denticola PGI activity in vitro?

Based on established methods for measuring PGI activity in other organisms, the following approaches would be effective for T. denticola PGI:

Discontinuous assays:

• F6P to G6P direction:

  • Incubate PGI with F6P at optimal temperature (likely 37°C for T. denticola enzymes)

  • Stop reaction at defined time points

  • Measure G6P formation by coupling to glucose-6-phosphate dehydrogenase (G6PDH) and NADP+ reduction

  • Monitor NADPH formation spectrophotometrically at 340 or 365 nm

• G6P to F6P direction:

  • Incubate PGI with G6P

  • Couple product formation to a series of enzymes:

    • Phosphofructokinase (PFK)

    • Fructose-1,6-bisphosphate (FBP) aldolase

    • Triosephosphate isomerase (TIM)

    • Glycerol-3-phosphate dehydrogenase

  • Monitor NADH oxidation at 340 or 365 nm

Continuous assays:

• For F6P formation:

  • Assay mixture containing G6P, NADP+, and G6PDH

  • Monitor NADPH formation continuously at 340 nm

• For G6P formation:

  • Assay mixture containing F6P, ATP, MgCl₂, NADH, PFK, FBP aldolase, TIM, and glycerol-3-phosphate dehydrogenase

  • Monitor NADH oxidation continuously at 340 nm

These methods have been successfully applied to PGI from various organisms, including P. furiosus and A. fumigatus .

What genetic tools are available for manipulating the pgi gene in T. denticola?

Several genetic tools have been developed for gene manipulation in T. denticola that could be applied to pgi:

• Shuttle vectors: A variety of T. denticola shuttle plasmids have been characterized, including:

  • pBFC and derivatives with the P-tap1 promoter

  • Vectors with P-ermB for lower expression levels

  • Vectors with P-msp for enhanced expression

• Gene knockout systems:

  • Allelic replacement mutagenesis using linear DNA fragments with selectable markers (ermB or aphA2) flanked by homologous sequences

  • Electroporation protocols optimized for T. denticola transformation

• Inducible expression systems:

  • Tet-inducible calibrated promoter system has been applied to T. denticola, allowing regulated gene expression through the addition of anhydrotetracycline (ATc)

• Site-directed mutagenesis:

  • Methods like QuickChange XL kit have been used for introducing specific mutations in T. denticola genes

  • Overlap extension PCR and FastCloning techniques have been employed for more complex modifications

An optimal approach for pgi manipulation would include:

• Constructing a knockout mutant to assess the essentiality of the gene

• Complementation with wild-type or modified pgi variants

• Expression analysis using RT-qPCR to confirm transcription levels

• Phenotypic characterization of mutants, including growth curves, carbon source utilization, and virulence assays

How can metabolic flux analysis be applied to understand the role of PGI in T. denticola carbon metabolism?

Metabolic flux analysis would provide valuable insights into the role of PGI in T. denticola carbon metabolism. While not explicitly described for T. denticola in the search results, a comprehensive approach would include:

• 13C-labeled substrate experiments:

  • Cultivate T. denticola with 13C-labeled glucose or other carbon sources

  • Extract and analyze metabolites using mass spectrometry to determine labeling patterns

  • Calculate flux distributions through central carbon metabolism pathways

• Comparative metabolomics:

  • Compare metabolite profiles between wild-type and pgi mutant strains

  • Identify accumulated or depleted metabolites to infer pathway alterations

• Integration with genomic data:

  • Construct a genome-scale metabolic model of T. denticola

  • Use flux balance analysis to predict the impact of PGI perturbation

Example data from A. fumigatus pgi mutant shows significant metabolic changes compared to wild-type:

Similar analysis in T. denticola would reveal the metabolic consequences of PGI disruption and its impact on energy generation and precursor synthesis .

What approaches can be used to investigate the potential role of T. denticola PGI in periodontal disease progression?

Investigating the role of T. denticola PGI in periodontal disease progression would require multifaceted approaches:

• In vitro virulence assays with pgi mutants:

  • Generate T. denticola pgi knockout or knockdown strains

  • Assess changes in:

    • Growth characteristics in different media

    • Biofilm formation capacity

    • Motility using semisolid agar swimming assays

    • Co-aggregation with other periodontal pathogens like P. gingivalis

    • Proteolytic activity of virulence factors like dentilisin

    • Epithelial cell invasion and cytotoxicity

• Co-infection models:

  • Evaluate the role of PGI in polymicrobial interactions using:

    • In vitro biofilm models with multiple periodontal pathogens

    • Animal models of periodontal disease using wild-type vs. pgi mutant T. denticola

    • Analysis of alveolar bone loss and inflammatory markers

• Clinical correlations:

  • Compare pgi expression levels in T. denticola isolated from:

    • Healthy sites vs. periodontitis sites

    • Different stages of periodontal disease progression

  • Correlate with clinical parameters like pocket depth, attachment loss, and bleeding on probing

• Immunological studies:

  • Assess the impact of PGI on host immune responses:

    • Cytokine production by host cells

    • Neutrophil recruitment and function

    • Pattern recognition receptor activation

These approaches would provide comprehensive insights into whether PGI contributes to T. denticola pathogenicity beyond its basic metabolic role.

How does T. denticola PGI compare with PGI enzymes from other oral pathogens?

While the search results don't provide direct comparisons of PGI enzymes between T. denticola and other oral pathogens, a methodological approach to this question would include:

• Sequence and structural comparisons:

  • Conduct phylogenetic analysis of PGI sequences from T. denticola, P. gingivalis, T. forsythia, and other oral bacteria

  • Identify conserved domains and catalytic residues

  • Analyze potential structural differences that might affect substrate specificity or catalytic efficiency

• Biochemical characterization:

  • Express and purify PGI enzymes from multiple oral pathogens

  • Compare kinetic parameters (Km, kcat, substrate specificity)

  • Analyze pH and temperature optima, which might reflect adaptation to specific microenvironments in periodontal pockets

• Expression analysis:

  • Determine if PGI expression varies between species under different environmental conditions

  • Assess if PGI is differentially regulated during biofilm formation or polymicrobial growth

• Functional complementation:

  • Test if PGI from one species can functionally replace PGI from another species in genetic complementation experiments

This comparative analysis would provide insights into potential metabolic specializations among oral pathogens and how these might contribute to their ecological roles in periodontal disease.

What research challenges are specific to studying T. denticola metabolic enzymes compared to other bacteria?

Studying T. denticola metabolic enzymes presents several unique challenges compared to research on other bacteria:

• Growth and cultivation difficulties:

  • T. denticola is an obligate anaerobe with complex nutritional requirements

  • It has a relatively slow growth rate, taking days rather than hours to reach optimal density

  • Requires specialized media like OBGM (oral bacterial growth medium) with specific supplements

  • Anaerobic culture conditions must be strictly maintained throughout experiments

• Genetic manipulation challenges:

  • Limited genetic tools compared to model organisms like E. coli

  • Lower transformation efficiency, though improvements have been made with modified protocols

  • Challenges in selecting transformants due to spontaneous antibiotic resistance

  • Limited number of selectable markers (primarily ermB for erythromycin resistance and aphA2 for kanamycin resistance)

• Protein expression considerations:

  • Specialized promoters needed for expression in T. denticola

  • Potential toxicity of overexpressed proteins

  • Need for appropriate controls due to different codon usage patterns

  • Challenges in purifying sufficient amounts of protein due to slow growth

• Enzyme assay complications:

  • Need to maintain anaerobic conditions during enzyme preparation and assays

  • Potential interference from other T. denticola enzymes or compounds

  • Limited specific antibodies available for detection and immunoprecipitation

Researchers addressing these challenges typically employ:

• Optimized transformation protocols with reduced washing steps

• Shuttle vectors with regulated expression systems to control protein levels

• Recombinant expression in heterologous hosts like E. coli followed by detailed characterization

• Careful purification strategies to maintain enzyme activity

How might inhibitors of T. denticola PGI be designed and tested for potential therapeutic applications?

Designing and testing inhibitors of T. denticola PGI would involve several methodological steps:

• Inhibitor design strategies:

  • Structure-based approaches using homology models or crystal structures (if available)

  • Fragment-based screening to identify initial binding scaffolds

  • Modification of known PGI inhibitors (e.g., 6-phosphogluconate, which inhibits M. tuberculosis PGI with a Ki of 0.8 mM )

  • Computer-aided drug design to identify compounds that might selectively target bacterial PGI over human counterparts

• In vitro inhibition assays:

  • Enzyme kinetic studies to determine inhibition constants and mechanisms (competitive, non-competitive, uncompetitive)

  • Structure-activity relationship (SAR) analysis to optimize lead compounds

  • Selectivity assays comparing inhibition of T. denticola PGI versus human PGI

• Antimicrobial activity testing:

  • Antimicrobial gradient strip tests similar to those used for other antibiotics against T. denticola

  • Growth inhibition assays in liquid culture

  • Assessment of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

  • Biofilm inhibition assays to determine efficacy against T. denticola in biofilm state

• Specificity and combination studies:

  • Testing effects on other oral bacteria to determine spectrum of activity

  • Combination studies with established antibiotics like tetracycline, metronidazole, or clindamycin, which have shown efficacy against T. denticola

  • Assessment of potential synergistic effects when combined with inhibitors targeting other metabolic pathways

• Preliminary safety assessment:

  • In vitro cytotoxicity testing using gingival epithelial cells and fibroblasts

  • Selectivity index calculation (ratio of cytotoxic concentration to antimicrobial concentration)

  • Hemolysis assays to assess potential effects on red blood cells