Recombinant Mycobacterium marinum Triosephosphate isomerase (tpiA)

What is the biochemical function of TPI in Mycobacterium marinum metabolism?

Triosephosphate isomerase (encoded by tpiA) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is critical for both glycolysis and gluconeogenesis pathways in mycobacteria . The enzyme plays a central role in carbon metabolism, allowing the organism to utilize various carbon sources efficiently. Based on studies with M. tuberculosis, TPI appears essential for growth when the organism is cultured with a single carbon source, as deletion mutants cannot survive in such conditions . The enzyme's activity is fundamental to energy production and carbon utilization strategies in mycobacteria.

Why is M. marinum used as a model for studying TPI instead of M. tuberculosis?

M. marinum serves as an excellent model organism for studying mycobacterial pathogenesis for several important reasons:

• It is a close genetic relative of the obligate human pathogen M. tuberculosis

• M. marinum has a significantly faster growth rate (generation time ~4 hours) compared to M. tuberculosis (generation time ~20 hours)

• It requires only biosafety level 2 containment rather than the higher levels needed for M. tuberculosis

• M. marinum causes similar intracellular infections, allowing researchers to study host-pathogen interactions in various models

• Genetic manipulation techniques have been successfully adapted for M. marinum, including transposon mutagenesis tools originally developed for M. tuberculosis

These advantages make M. marinum TPI research more accessible and tractable while still providing relevant insights applicable to tuberculosis research.

What is known about the essentiality of TPI in mycobacterial species?

Studies with M. tuberculosis have revealed interesting nuances regarding TPI essentiality. While tpi was initially predicted to be essential for growth, conditional knockdown experiments demonstrated that:

• TPI depletion reduces growth in media containing a single carbon source

• Surprisingly, TPI depletion does not affect growth in media containing both glycolytic and gluconeogenic carbon sources

• A complete tpi deletion (Δtpi) mutant cannot survive with single carbon substrates but grows like wild-type in the presence of both glycolytic and gluconeogenic carbon sources

Metabolic tracing experiments confirmed the absence of alternative triosephosphate isomerases or bypass reactions in M. tuberculosis . Importantly, despite growth in dual-carbon media in vitro, the Δtpi strain was severely attenuated in mouse models of tuberculosis, suggesting that mycobacteria cannot simultaneously access sufficient quantities of both types of carbon substrates during infection . This pattern is likely conserved in M. marinum given their close phylogenetic relationship.

What expression systems are most effective for producing recombinant M. marinum TPI?

Based on mycobacterial expression system developments, the following approaches are recommended for recombinant M. marinum TPI production:

Expression System Considerations:

• E. coli-based expression: Commonly using pET vector systems with T7 promoters for high-yield expression

• Mycobacterial expression: Using adapted TM4-derived mycobacteriophage systems which have been shown to work effectively with M. marinum

• Temperature optimization: Expression should be conducted at 30-33°C rather than 37°C to match M. marinum's natural growth temperature requirements

When designing expression constructs, it's important to consider that transformation efficiencies for M. marinum have historically been low . The conditionally replicating mycobacteriophage phAE94, originally developed for M. tuberculosis, has been adapted specifically for M. marinum to overcome this limitation and can be useful for both gene expression and mutagenesis approaches .

How can researchers effectively purify active recombinant M. marinum TPI?

A methodological approach to purification should include:

• Initial clarification: Cell lysis using mechanical disruption (sonication or bead-beating) in buffer optimized for mycobacterial proteins (typically containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and protease inhibitors)

• Affinity chromatography: Using histidine or other affinity tags for initial capture

• Activity preservation: Including reducing agents (1-5 mM DTT or β-mercaptoethanol) to protect active site cysteines

• Secondary purification: Size exclusion chromatography to ensure proper oligomeric state (TPI typically functions as a dimer)

• Activity verification: Using coupled enzyme assays that monitor the conversion between DHAP and G3P spectrophotometrically

For highest enzymatic activity, purification should be performed at lower temperatures (4°C) and the final preparation should be stored with glycerol (10-20%) at -80°C to maintain long-term stability and activity.

What host-pathogen models are suitable for studying M. marinum TPI function in infection contexts?

Several host-pathogen models have been validated for studying M. marinum infections and can be applied to TPI research:

• Mast cell model: Both human mast cell line (HMC-1) and primary murine mast cells have been demonstrated to support M. marinum infection, where the bacteria survive, replicate, and cause dose-dependent cell damage

• Dictyostelium discoideum model: A high-throughput infection model using D. discoideum amoebae expressing mCherry and bioluminescent M. marinum allows simultaneous monitoring of bacterial growth and host cell viability

• Zebrafish model: The natural host for M. marinum, providing an excellent in vivo system to study mycobacterial pathogenesis

• Mouse model: Systemic infections can be established to evaluate virulence attenuation, as demonstrated with TPI-deficient mycobacteria

The D. discoideum-M. marinum infection model is particularly valuable for high-throughput screening, enabling researchers to differentiate between antibiotics and anti-infective compounds through quantitative measurements such as IC50 and MIC calculations .

How can metabolic tracing be implemented to study carbon flux through the TPI reaction in M. marinum?

Metabolic flux analysis using isotope labeling provides critical insights into TPI function and can be implemented as follows:

Methodological approach:

• Culture M. marinum in media containing 13C-labeled carbon sources (e.g., [13C]glucose or [13C]acetate)

• Extract metabolites using cold methanol quenching

• Analyze isotopomer distribution of glycolytic and gluconeogenic intermediates using LC-MS/MS

• Quantify labeled DHAP and G3P to assess TPI activity and potential accumulation of substrates

When conducting these experiments, researchers should compare wild-type M. marinum with conditional or partial TPI knockdown strains to detect metabolic bottlenecks. Based on studies with M. tuberculosis, TPI depletion should result in accumulation of its substrates when grown on single carbon sources, confirming the absence of metabolic bypass reactions .

What strategies can be employed to generate and characterize TPI-deficient M. marinum strains?

Creating TPI-deficient strains requires careful experimental design due to its potential essentiality:

• Conditional knockdown approach:

  • Use tetracycline-regulated expression systems

  • Culture bacteria in dual carbon source media (containing both glycolytic and gluconeogenic substrates)

  • Gradually deplete TPI by removing inducer

  • Monitor growth rates, metabolite profiles, and bacterial viability

• Complete deletion strategy:

  • Utilize mycobacteriophage-based transposon mutagenesis systems adapted for M. marinum

  • Perform deletion in media containing both glycolytic and gluconeogenic carbon sources

  • Verify deletion by PCR, Western blotting, and enzyme activity assays

  • Test growth on various carbon sources to confirm phenotype

• Characterization methods:

  • Growth curve analysis in different carbon sources

  • Intracellular replication within host cells (mast cells or macrophages)

  • Virulence assessment in animal models

  • Metabolomic profiling to identify accumulated intermediates

Based on M. tuberculosis studies, researchers should expect TPI-deficient M. marinum to grow normally in dual-carbon media but fail to survive with single carbon substrates .

How do metabolic profiles differ between wild-type and TPI-deficient M. marinum strains?

Based on studies with related mycobacteria, the following metabolic differences should be observed when comparing wild-type and TPI-deficient strains:

When analyzing metabolomic data, researchers should focus on:

What are the key considerations for assessing TPI enzyme kinetics in recombinant versus native forms?

When analyzing enzyme kinetics, researchers should consider:

• Kinetic parameters comparison:

  • Km values for both DHAP and G3P substrates

  • Catalytic efficiency (kcat/Km)

  • pH and temperature optima specific to M. marinum

• Potential differences affecting interpretation:

  • Effects of purification tags on enzyme activity

  • Buffer composition impact on measured kinetics

  • Allosteric regulators that may be present in native but not recombinant environments

  • Temperature sensitivity (M. marinum's natural environment is cooler than standard laboratory conditions)

• Experimental validation approaches:

  • Use both forward and reverse reaction measurements

  • Include negative controls with heat-inactivated enzyme

  • Perform sufficient technical and biological replicates

  • Compare with purified TPI from related mycobacterial species

How can researchers address difficulties in expressing soluble, active recombinant M. marinum TPI?

Common challenges and solutions include:

• Insoluble protein expression:

  • Lower expression temperature to 16-20°C

  • Use solubility-enhancing fusion partners (MBP, SUMO, etc.)

  • Optimize induction conditions (lower IPTG concentrations)

  • Try mycobacterial expression hosts instead of E. coli

• Low enzyme activity:

  • Include reducing agents throughout purification

  • Ensure proper metal cofactors if required

  • Check for correct oligomeric state using size exclusion chromatography

  • Verify protein folding using circular dichroism

• Unstable enzyme preparations:

  • Optimize buffer composition (pH, salt, additives)

  • Add stabilizing agents (glycerol, specific substrates)

  • Avoid freeze-thaw cycles

  • Consider storage as ammonium sulfate precipitate

What strategies can overcome challenges in generating TPI-deficient M. marinum for in vivo studies?

Based on experiences with mycobacterial genetics:

• Addressing essentiality barriers:

  • Always use dual carbon source media for transformation and selection of TPI-deficient strains

  • Consider complementation with temperature-sensitive TPI variants

  • Use merodiploid approaches where appropriate

• Improving transformation efficiency:

  • Utilize adapted mycobacteriophage systems specific for M. marinum

  • Optimize electroporation conditions (voltage, resistance, cell preparation)

  • Prepare highly competent cells from mid-log phase cultures

• Confirming TPI depletion:

  • Use multiple verification methods (genomic PCR, RT-qPCR, Western blot)

  • Perform enzyme activity assays on cell extracts

  • Conduct metabolomic profiling to confirm substrate accumulation

• Addressing attenuated growth in vivo:

  • Start with higher inoculum doses for animal infection studies

  • Consider using modified infection routes

  • Monitor bacterial burden using bioluminescent reporters