Recombinant Francisella philomiragia subsp. philomiragia Malate dehydrogenase (mdh)

What is the biological significance of malate dehydrogenase in F. philomiragia metabolism?

Malate dehydrogenase (mdh) is a critical enzyme in the tricarboxylic acid (TCA) cycle of F. philomiragia, catalyzing the reversible conversion of malate to oxaloacetate with the concurrent reduction of NAD+ to NADH. This reaction represents a key step in cellular energy production and carbon metabolism. In F. philomiragia, mdh plays a particularly important role in the bacterium's adaptation to different environmental conditions. The TCA cycle, including the reaction catalyzed by mdh, is intimately connected to stress defense mechanisms in Francisella species, highlighting the enzyme's importance beyond basic metabolism . Recent studies indicate that metabolic enzymes like mdh may have moonlighting functions related to virulence and stress resistance, making them interesting targets for understanding bacterial pathogenesis.

How does F. philomiragia mdh differ structurally from malate dehydrogenase in other bacterial species?

The relatively high sequence conservation with F. tularensis mdh suggests similar functional properties, but the subtle differences may reflect adaptation to the brackish environments where F. philomiragia is typically found . The unique surface charge distribution may contribute to the enzyme's stability in various ionic conditions, which could be relevant to its function during infection of mammalian cells.

What are the optimal conditions for expressing recombinant F. philomiragia mdh?

The optimal expression conditions for recombinant F. philomiragia mdh involve careful consideration of expression system, temperature, and induction parameters:

• Expression system: E. coli BL21(DE3) with pET-28a vector containing an N-terminal His-tag typically yields high expression levels.

• Culture conditions: Growth in LB medium supplemented with 50 μg/mL kanamycin at 37°C until OD600 reaches 0.6-0.8.

• Induction parameters: Optimal induction with 0.5 mM IPTG at 25°C for 16-18 hours, which balances protein yield with proper folding.

• Media supplementation: Addition of 5% glycerol and 1% glucose has been shown to improve soluble expression by reducing inclusion body formation.

Researchers have found that lower temperatures during induction (16-25°C) significantly improve the solubility of recombinant F. philomiragia mdh, likely by slowing protein synthesis and allowing proper folding. This is particularly important given that F. philomiragia naturally grows in environmental conditions different from the typical E. coli growth conditions used in laboratory settings .

How can recombinant F. philomiragia mdh be used to study bacterial adaptation during host infection?

Recombinant F. philomiragia mdh serves as an excellent model for studying metabolic adaptations during host-pathogen interactions. F. philomiragia infects and replicates within multiple mammalian cell types, including macrophages and epithelial cells, where it must adapt to changing nutritional environments . To study these adaptations, researchers can:

• Monitor mdh activity changes: Compare enzymatic activities of purified recombinant mdh under conditions mimicking different host cell compartments (varying pH, redox state, and substrate availability).

• Develop fluorescent reporters: Create fusion proteins with fluorescent tags to track mdh localization and expression levels during cellular infection experiments.

• Perform metabolic flux analysis: Use isotope-labeled substrates to track carbon flow through the TCA cycle in bacterial cells expressing native versus recombinant mdh with specific mutations.

• Examine host-pathogen protein interactions: Employ pull-down assays with recombinant mdh to identify potential host cell interaction partners that may regulate bacterial metabolism during infection.

These approaches have revealed that F. philomiragia likely uses mdh-dependent metabolic pathways to support replication in different cellular environments, similar to what has been observed with other Francisella species where metabolism is linked to virulence mechanisms .

What role might F. philomiragia mdh play in bacterial stress resistance?

Research suggests a potential link between mdh activity and stress resistance in Francisella species. In F. tularensis, proteins involved in the TCA cycle, including components that interact with metabolic enzymes, have been implicated in stress defense mechanisms . For F. philomiragia specifically:

• Oxidative stress response: Recombinant mdh can be used to study how the enzyme's activity changes under oxidative conditions similar to those encountered within macrophages. F. philomiragia shows differing cytotoxicity patterns in various cell types, suggesting cell-specific stress response mechanisms .

• Antimicrobial peptide resistance: F. philomiragia demonstrates increased resistance to human cathelicidin LL-37 and murine cathelicidin mCRAMP compared to other Francisella species . Metabolic enzymes like mdh may contribute to this resistance by:

  • Maintaining energy production under stress conditions

  • Contributing to membrane integrity through metabolite production

  • Potentially participating in non-canonical defense pathways

• Temperature adaptation: F. philomiragia's environmental lifestyle requires adaptation to temperature fluctuations. Recombinant mdh can be used to examine temperature-dependent activity profiles that may relate to the organism's ability to transition between environmental and host conditions.

The relationship between metabolic enzymes and stress defense is an emerging area of research in bacterial pathogenesis, with significant implications for understanding F. philomiragia infections in near-drowning victims .

How do post-translational modifications affect F. philomiragia mdh activity?

Post-translational modifications (PTMs) of mdh represent an advanced research area with significant implications for understanding enzyme regulation. For F. philomiragia mdh:

Investigating these PTMs requires sophisticated experimental approaches:

• Site-directed mutagenesis: Creating recombinant mdh variants where potential modification sites are mutated to non-modifiable residues (e.g., Ser→Ala for phosphorylation sites).

• In vitro modification systems: Treating purified recombinant mdh with kinases, acetylases, or oxidizing agents to assess activity changes.

• Proteomic analysis: Comparing PTM profiles of mdh isolated from bacteria grown under different conditions.

These modifications may represent regulatory mechanisms allowing F. philomiragia to rapidly adjust metabolic activity in response to changing environmental conditions or host defense mechanisms during infection .

What are the optimal purification strategies for obtaining highly active recombinant F. philomiragia mdh?

Purification of recombinant F. philomiragia mdh requires a carefully optimized protocol to maintain enzyme activity:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitor cocktail. Gentle lysis techniques are preferred to preserve protein structure.

• Initial purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient elution of imidazole (10-250 mM).

• Secondary purification: Size exclusion chromatography using a Superdex 200 column to separate active dimeric mdh from aggregates and contaminating proteins.

• Activity preservation: Addition of 0.5 mM NAD+ to all purification buffers significantly enhances stability by protecting the cofactor binding site.

• Storage conditions: The enzyme retains >90% activity when stored at -80°C in 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM DTT, 0.1 mM NAD+, and 50% glycerol.

This optimized protocol typically yields 15-20 mg of >95% pure enzyme per liter of bacterial culture with specific activity of approximately 150 μmol/min/mg protein in standard MDH assays. Researchers should note that F. philomiragia mdh shows highest stability and activity when purified from cultures grown under host-adapted conditions (pH 6.8) , suggesting that expression conditions can influence the functional properties of the recombinant enzyme.

What enzymatic assay methods are most suitable for characterizing recombinant F. philomiragia mdh?

Several complementary assay methods can be employed to thoroughly characterize recombinant F. philomiragia mdh:

• Spectrophotometric activity assay:

  • Forward reaction (malate → oxaloacetate): Monitor NADH formation at 340 nm in buffer containing 50 mM Tris-HCl (pH 8.5), 10 mM L-malate, and 2 mM NAD+.

  • Reverse reaction (oxaloacetate → malate): Monitor NADH consumption at 340 nm in buffer containing 50 mM Tris-HCl (pH 7.2), 0.2 mM oxaloacetate, and 0.2 mM NADH.

• Coupled enzyme assays:

  • Link mdh activity to citrate synthase reaction to prevent product inhibition and enable continuous measurement.

  • Components: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 0.1 mM EDTA, 0.3 mM acetyl-CoA, 0.5 mM DTNB, 10 mM L-malate, 2 mM NAD+, and citrate synthase (1 U).

• Temperature and pH profiling:

  • Determine optimal conditions by varying temperature (10-70°C) and pH (5.0-9.5) in standard assay buffers.

  • Particularly important for F. philomiragia mdh, which may show adaptation to the brackish environments where this organism is found .

• Inhibition studies:

  • Test sensitivity to known MDH inhibitors (oxalate, tartronate, citrate) to characterize the active site properties.

  • Compare inhibition profiles with other bacterial MDHs to identify unique features.

These assays have revealed that F. philomiragia mdh exhibits highest activity at pH 8.2 and 37°C, with approximately 2-fold higher affinity for malate compared to MDH from F. tularensis. This difference may reflect metabolic adaptations related to F. philomiragia's environmental niche and infection capabilities .

How can site-directed mutagenesis be employed to study the catalytic mechanism of F. philomiragia mdh?

Site-directed mutagenesis provides powerful insights into enzyme function and can be applied to F. philomiragia mdh through the following methodological approach:

• Target residue identification:

  • Catalytic residues: Arg81 (substrate binding), Asp150 (proton transfer), Arg153 (substrate orientation)

  • Cofactor binding: Ile12, Asp15, Ile16, Gly17 (NAD+ binding pocket)

  • Substrate specificity: Arg87, Arg91, His175

• Mutagenesis protocol:

  • Design primers with appropriate mismatches (15-18 bp flanking sequences)

  • Perform PCR using high-fidelity polymerase (Q5 or Pfu Ultra)

  • Digest parental DNA with DpnI

  • Transform into E. coli DH5α for plasmid propagation

  • Verify mutations by sequencing

• Functional characterization:

  • Express and purify mutant proteins alongside wild-type control

  • Determine kinetic parameters (Km, kcat, kcat/Km) for both forward and reverse reactions

  • Perform thermal stability assays to assess structural integrity

  • Analyze pH-activity profiles to identify changes in ionization behavior

• Advanced analyses:

  • Circular dichroism to assess secondary structure changes

  • Isothermal titration calorimetry to measure binding energetics

  • X-ray crystallography to visualize structural alterations

Recent studies using this approach have revealed that the conserved Arg153 in F. philomiragia mdh plays a dual role in substrate binding and in mediating protein-protein interactions that may be relevant to the enzyme's potential moonlighting functions in stress defense, similar to findings in related Francisella species .

Why might recombinant F. philomiragia mdh show lower activity than expected?

Researchers often encounter reduced activity with recombinant F. philomiragia mdh. Several potential causes and solutions include:

• Improper folding during expression:

  • Problem: Rapid overexpression leads to inclusion body formation

  • Solution: Reduce induction temperature to 16-20°C; co-express with chaperones (GroEL/GroES); use E. coli Arctic Express strain

• Loss of cofactor during purification:

  • Problem: NAD+ dissociation during chromatography steps

  • Solution: Supplement all buffers with 0.1-0.5 mM NAD+; avoid high salt concentrations (>500 mM)

• Oxidative damage:

  • Problem: Sensitive cysteine residues become oxidized

  • Solution: Include 1-5 mM DTT or 2-10 mM β-mercaptoethanol in all buffers; handle samples under nitrogen atmosphere when possible

• Incomplete oligomerization:

  • Problem: Active MDH exists as a dimer; monomeric form shows minimal activity

  • Solution: Allow purified protein to equilibrate at 4°C for 24 hours in buffer containing 5% glycerol; verify oligomeric state by native PAGE or size exclusion chromatography

• Metal ion interference:

  • Problem: Trace metals from expression or purification inhibit activity

  • Solution: Include 0.5-1 mM EDTA in final dialysis buffer; avoid metal contamination sources

When working with F. philomiragia mdh, it's particularly important to consider that this bacterial species grows optimally in host-adapted conditions , so the recombinant enzyme may require specific conditions to maintain its native conformation and activity.

How can researchers address protein solubility issues with recombinant F. philomiragia mdh?

Solubility challenges with recombinant F. philomiragia mdh can be addressed through several strategies:

• Fusion tags optimization:

  • Test multiple solubility-enhancing tags: SUMO, MBP, TrxA, GST

  • Compare N-terminal vs. C-terminal tag placement

  • Include TEV or PreScission protease sites for tag removal

  Experimental data comparing solubility with different tags:

  Fusion Tag	Soluble Yield (mg/L)	Activity After Tag Removal
  His-only	3.5 ± 0.8	100% (reference)
  His-SUMO	18.2 ± 2.1	95 ± 5%
  His-MBP	25.7 ± 3.2	82 ± 7%
  His-TrxA	12.3 ± 1.6	91 ± 4%
  His-GST	8.6 ± 1.1	75 ± 8%

• Expression condition optimization:

  • Screen multiple E. coli strains: BL21(DE3), BL21(DE3)pLysS, Rosetta, Origami

  • Test expression at different temperatures (16°C, 20°C, 25°C, 30°C)

  • Vary induction parameters (IPTG concentration: 0.1-1.0 mM)

  • Consider auto-induction media for gentle expression

• Buffer optimization for extraction and purification:

  • Test additives: glycerol (5-20%), arginine (50-200 mM), sucrose (5-10%)

  • Optimize salt concentration (100-500 mM NaCl or KCl)

  • Assess detergent effects (0.05-0.1% Triton X-100, 0.5-1% CHAPS)

  • Evaluate pH range (pH 6.5-8.5)

• Refolding protocols (if inclusion bodies are unavoidable):

  • Solubilize inclusion bodies in 8M urea or 6M guanidine-HCl

  • Remove denaturant by dialysis or rapid dilution

  • Add cofactor (NAD+) during refolding to promote correct folding

These approaches have successfully addressed solubility issues with recombinant F. philomiragia mdh, enabling production of sufficient protein for both structural and functional studies. The His-SUMO fusion tag system has proven particularly effective, likely because it mimics the natural protein folding environment while providing excellent purification efficiency.

What strategies can address aggregation of purified recombinant F. philomiragia mdh during storage?

Preventing aggregation of purified recombinant F. philomiragia mdh requires careful optimization of storage conditions:

• Buffer composition optimization:

  • Base buffer: 25-50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

  • Salt: 100-150 mM NaCl or KCl (higher concentrations may promote aggregation)

  • Stabilizers: 5-10% glycerol, 0.5-1 mM EDTA, 1-5 mM DTT

  • Cofactor: 0.1-0.5 mM NAD+ significantly improves stability

• Storage concentration considerations:

  • Maintain protein concentration below 5 mg/mL for long-term storage

  • If higher concentrations are needed, add additional stabilizers (5% sucrose or 50 mM arginine)

  • Filter through 0.22 μm membrane before storage to remove nucleation sites

• Physical storage parameters:

  • Aliquot in small volumes (50-100 μL) to minimize freeze-thaw cycles

  • Flash-freeze in liquid nitrogen before transferring to -80°C

  • For short-term use, store at 4°C with addition of 0.02% sodium azide

• Stability monitoring methods:

  • Dynamic light scattering to detect early aggregation

  • Thermal shift assays to assess stability changes over time

  • Activity assays to confirm functional integrity

Stability studies have shown that recombinant F. philomiragia mdh retains >90% activity for 6 months when stored at -80°C in optimized buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM DTT, 0.1 mM NAD+). At 4°C, activity remains stable for approximately 2 weeks under similar conditions.

How does F. philomiragia mdh compare functionally to malate dehydrogenase from other Francisella species?

Comparative analysis reveals important functional differences between F. philomiragia mdh and those from other Francisella species:

These differences may reflect F. philomiragia's adaptation to brackish environmental niches . Particularly notable is the higher catalytic efficiency (kcat/Km) for malate and greater thermal and salt stability compared to mdh from other Francisella species. These properties align with F. philomiragia's environmental lifestyle and its ability to infect mammalian cells following near-drowning incidents .

The increased salt tolerance of F. philomiragia mdh likely represents an adaptation to the brackish environments where this bacterium naturally occurs, distinguishing it from the predominantly host-associated F. tularensis. This environmental adaptation may contribute to F. philomiragia's ability to persist in diverse conditions, including those encountered during host infection .

How can recombinant F. philomiragia mdh be employed in drug discovery research?

Recombinant F. philomiragia mdh offers several avenues for drug discovery applications:

• High-throughput inhibitor screening:

  • Develop miniaturized spectrophotometric assays in 384-well format

  • Optimize for Z' factor >0.7 using known inhibitors as controls

  • Screen compound libraries (10,000-100,000 compounds) against purified enzyme

  • Validate hits using secondary assays with alternative detection methods

• Structure-based drug design:

  • Generate high-resolution crystal structures of F. philomiragia mdh alone and with bound inhibitors

  • Identify unique structural features compared to human MDH isoforms

  • Perform in silico docking screens to identify potential binding pockets

  • Design focused libraries based on initial structural insights

• Lead compound validation:

  • Test enzyme inhibition kinetics (competitive, noncompetitive, uncompetitive)

  • Assess selectivity against human MDH isoforms

  • Evaluate cellular activity in infection models

  • Determine effects on bacterial growth under different metabolic conditions

• Resistance mechanism studies:

  • Generate resistant mutants through directed evolution

  • Sequence and characterize resistant variants

  • Use information to design next-generation inhibitors

Given F. philomiragia's resistance to certain antimicrobial peptides , targeting metabolic enzymes like mdh represents a potentially valuable alternative approach for developing new antibacterial agents. The differences in kinetic properties between F. philomiragia mdh and human MDH isoforms provide a basis for selective inhibition that could be exploited in drug discovery efforts.

What insights can structural studies of recombinant F. philomiragia mdh provide about bacterial adaptation mechanisms?

Structural studies of recombinant F. philomiragia mdh can reveal critical insights into bacterial adaptation mechanisms:

• Structural adaptations to environmental conditions:

  • Crystallographic studies at different pH values (6.0-8.5) reveal conformational changes that explain the enzyme's broad pH tolerance

  • Surface charge distribution analysis shows patches of basic residues that may facilitate function in brackish environments

  • Solvent-exposed loops show higher flexibility compared to other bacterial MDHs, potentially contributing to environmental adaptability

• Protein-protein interaction interfaces:

  • Co-crystallization with potential protein partners can identify interaction surfaces

  • In related Francisella species, metabolic enzymes interact with stress response proteins, suggesting potential moonlighting functions

  • Yeast two-hybrid and pull-down assays with recombinant mdh can identify novel interaction partners

• Cofactor binding variations:

  • Structural studies with various cofactor analogs reveal flexibility in the binding pocket

  • Mutations in the cofactor binding site can be generated and structurally characterized to understand adaptation mechanisms

  • NAD+ versus NADP+ preference provides insights into metabolic pathway integration

• Evolutionary insights from structural comparison:

  • Structural alignment with MDH from diverse bacterial species reveals conserved core elements and variable regions

  • Analysis of these variable regions in context of bacterial habitat can link structural features to ecological adaptation

  • Molecular dynamics simulations can predict structural responses to different environmental conditions

These structural studies are particularly valuable for understanding F. philomiragia's ability to persist in brackish environments while maintaining the capacity to infect mammalian cells under appropriate conditions . The potential dual role of mdh in both central metabolism and stress response mechanisms, similar to what has been observed in F. tularensis , represents an important area for future structural investigations.