Recombinant Yersinia pestis bv. Antiqua L-threonine 3-dehydrogenase (tdh)

What is L-threonine 3-dehydrogenase (tdh) and what is its functional role in Yersinia pestis?

L-threonine 3-dehydrogenase (tdh) is an enzyme with EC number 1.1.1.103 that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate using NAD+ as a cofactor. In Yersinia pestis, tdh is part of the threonine degradation pathway, which plays a significant role in amino acid metabolism and energy production. The enzyme is particularly important for bacterial survival under nutrient-limited conditions, as it allows the bacterium to utilize threonine as an alternative carbon and nitrogen source. This metabolic versatility may contribute to Y. pestis's ability to adapt to different host environments during infection.

The Yersinia pestis bv. Antiqua strain (Angola) expresses this enzyme as a 341-amino acid protein with a molecular weight of approximately 37 kDa. The recombinant form is frequently used in research to understand bacterial metabolism, potential drug targets, and pathogenicity mechanisms .

How is recombinant Yersinia pestis bv. Antiqua L-threonine 3-dehydrogenase typically expressed and purified?

Recombinant Yersinia pestis bv. Antiqua tdh is commonly expressed using baculovirus expression systems in insect cells. This expression system is preferred for producing complex bacterial proteins as it provides eukaryotic post-translational modifications while maintaining high expression levels. The commercially available recombinant protein (CSB-BP023350YAM) is produced using this system, which allows for proper folding and enhanced solubility .

For purification, a multi-step chromatography approach is typically employed. First, affinity chromatography using a tag (the specific tag type varies and is determined during the manufacturing process) enables initial capture of the protein. This is followed by either ion-exchange chromatography or size exclusion chromatography to remove contaminants and improve purity. The final product typically achieves >85% purity as verified by SDS-PAGE analysis .

For laboratory-scale expression, researchers may also use E. coli expression systems with optimization of induction conditions, temperature, and media composition to improve solubility and yield. When using E. coli, the addition of zinc to the culture medium is often necessary to ensure proper folding of the enzyme, as zinc is a critical cofactor for tdh function.

What are the optimal storage and reconstitution conditions for recombinant Yersinia pestis tdh?

The optimal storage conditions for recombinant Yersinia pestis tdh depend on both the formulation and intended use duration. For long-term storage, the protein should be kept at -20°C or preferably at -80°C for extended preservation. The lyophilized form offers greater stability with a shelf life of approximately 12 months at these temperatures, while the liquid form has a reduced shelf life of about 6 months .

For reconstitution of lyophilized protein, the recommended protocol includes:

• Brief centrifugation of the vial before opening to collect contents at the bottom

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (optimally 50%) for cryoprotection

• Aliquoting into smaller volumes to prevent repeated freeze-thaw cycles

What assays are recommended for measuring the enzymatic activity of recombinant Yersinia pestis tdh?

Several assay methods can be employed to measure the enzymatic activity of recombinant Yersinia pestis tdh, with spectrophotometric NAD+ reduction assays being the most common. The standard assay involves:

• Monitoring the reduction of NAD+ to NADH at 340 nm, which exhibits an increase in absorbance

• Conducting reactions in a buffer containing L-threonine as substrate (typically 10-50 mM), NAD+ (1-2 mM), and an appropriate buffer (often Tris-HCl, pH 8.0-8.5)

• Measuring initial velocities at various substrate concentrations to determine kinetic parameters

For more sensitive detection, fluorescence-based assays can be used, measuring the fluorescence of NADH (excitation at 340 nm, emission at 460 nm). Additionally, coupled enzyme assays may be employed where the 2-amino-3-ketobutyrate product is further metabolized by 2-amino-3-ketobutyrate CoA ligase in the presence of CoA, and the resulting acetyl-CoA is detected.

High-throughput screening methods include:

For inhibitor screening, combining activity assays with structural methods like X-ray crystallography or molecular docking can provide insights into the mechanism of inhibition.

How can researchers optimize protein expression to obtain higher yields of active recombinant Yersinia pestis tdh?

Optimizing the expression of recombinant Yersinia pestis tdh requires a systematic approach addressing multiple parameters across expression systems. In baculovirus expression systems, which are commonly used for commercial production, consider:

• Viral titer optimization: Using the optimal multiplicity of infection (MOI) is crucial for maximizing protein yield while minimizing cellular stress.

• Harvest timing: Determining the optimal time post-infection (typically 48-72 hours) to harvest cells before significant protein degradation occurs.

• Cell line selection: Different insect cell lines (Sf9, Sf21, High Five) may yield different expression levels and post-translational modifications.

• Media supplementation: Adding cofactors such as zinc and optimizing nutrient composition can improve protein folding and yield .

For E. coli-based expression systems, which are often more accessible to research laboratories:

Regarding purification optimization, incorporating a multi-step approach typically yields the best results:

• Initial capture using affinity chromatography (His-tag, GST, MBP)

• Intermediate purification using ion-exchange chromatography

• Polishing step using size exclusion chromatography

• Quality control using activity assays and analytical methods like dynamic light scattering to assess homogeneity

When scaling up production, consistent buffer conditions, controlled temperature, and minimized processing time are essential factors for maintaining enzyme activity.

How does Yersinia pestis tdh compare with L-threonine 3-dehydrogenase from other bacterial species?

Yersinia pestis tdh shares significant sequence homology with L-threonine 3-dehydrogenases from other bacterial species, particularly within the Enterobacteriaceae family. Comparative analysis reveals key structural and functional similarities and differences:

• Sequence conservation: The catalytic domain and NAD+-binding motifs show high conservation across bacterial species, with 70-80% sequence identity to tdh from other Yersinia species and 50-60% to E. coli and Salmonella enzymes.

• Substrate specificity: While the core catalytic mechanism is conserved, subtle differences in the substrate-binding pocket influence substrate preference and catalytic efficiency. Y. pestis tdh shows moderate substrate promiscuity, accepting L-threonine analogs with smaller side chains, though with reduced efficiency.

• Inhibitor sensitivity: Different bacterial tdh enzymes show variable sensitivity to inhibitors, which may reflect adaptations to different environmental niches. Y. pestis tdh appears to be more sensitive to certain metal-chelating agents compared to E. coli tdh.

• Quaternary structure: Most bacterial tdh enzymes function as homodimers or homotetramers. Y. pestis tdh predominantly exists as a tetramer, which contributes to its stability under various conditions.

This comparative information is valuable for researchers developing selective inhibitors targeting pathogen-specific enzymes while minimizing effects on human homologs or beneficial bacteria. Additionally, understanding the evolutionary relationships between tdh enzymes provides insights into metabolic adaptations of Y. pestis to its unique lifecycle, which involves both mammalian hosts and flea vectors.

What role does L-threonine 3-dehydrogenase play in Yersinia pestis pathogenicity and survival?

L-threonine 3-dehydrogenase plays a multifaceted role in Yersinia pestis metabolism, potentially contributing to pathogenicity and survival in multiple ways:

• Metabolic adaptation: tdh allows Y. pestis to utilize threonine as an alternative carbon and nitrogen source, particularly important during nutrient limitation within host environments. This metabolic flexibility may contribute to bacterial persistence during infection.

• Energy production: The catabolism of threonine through tdh generates acetyl-CoA, which feeds into the TCA cycle for energy production. This alternative energy source may be crucial during different stages of infection.

• Stress response: Evidence from related bacterial species suggests that threonine metabolism may participate in acid tolerance and oxidative stress responses, which are important for Y. pestis survival during host immune responses.

• Potential virulence regulation: While not directly established for Y. pestis tdh, metabolic enzymes in other pathogens have been shown to have moonlighting functions in virulence regulation. The products of threonine catabolism may serve as signaling molecules affecting virulence gene expression.

Research examining tdh expression levels during different infection stages could provide valuable insights into its role in pathogenicity. Studies using tdh knockout mutants could determine whether this enzyme is essential for Y. pestis virulence, potentially identifying it as a drug target. Comparative studies with tdh from Y. pseudotuberculosis and Y. enterocolitica might further reveal how this enzyme contributes to the unique virulence properties of Y. pestis .

How can recombinant Yersinia pestis tdh be used in screening for potential antimicrobial compounds?

Recombinant Yersinia pestis tdh offers a valuable platform for antimicrobial drug discovery through multiple screening approaches:

• Enzymatic activity-based screening: High-throughput screening of compound libraries can identify inhibitors of tdh activity. The standard spectrophotometric assay measuring NAD+ reduction at 340 nm can be miniaturized to 384-well formats, enabling rapid screening of thousands of compounds. Hit compounds would decrease the rate of NADH formation compared to controls.

• Fragment-based drug discovery: This approach identifies small molecular fragments that bind to tdh, which can then be elaborated into more potent inhibitors. Thermal shift assays measuring changes in protein melting temperature upon fragment binding provide a straightforward screening method.

• Structure-based virtual screening: Using the protein sequence data available (UniProt: A9R685), homology models of Y. pestis tdh can be constructed and used for in silico screening of virtual compound libraries. Molecular docking simulations can predict binding affinities and orientations, prioritizing compounds for experimental testing .

• Allosteric inhibitor discovery: Besides targeting the active site, screens can be designed to identify compounds binding to allosteric sites that affect enzyme conformation or oligomerization. Such inhibitors might offer greater selectivity compared to active site inhibitors.

A comprehensive screening cascade might include:

This systematic approach can identify novel inhibitors that could serve as starting points for developing new antibiotics against Y. pestis, addressing the concerning potential of this organism as a biological warfare agent .

What structural features contribute to the catalytic specificity of Yersinia pestis L-threonine 3-dehydrogenase?

The catalytic specificity of Yersinia pestis L-threonine 3-dehydrogenase is determined by several key structural features that collectively shape its active site architecture and reactivity:

• Zinc-binding domain: The ITCGHCRNCR motif (positions 133-142) in Y. pestis tdh coordinates a zinc ion that is essential for catalysis. This zinc ion positions the substrate correctly and lowers the pKa of the threonine hydroxyl group, facilitating its deprotonation. The cysteine residues in this motif form a characteristic zinc-binding pocket that contributes to specificity .

• NAD+-binding Rossmann fold: The N-terminal region contains a classical Rossmann fold with the GXGXXG motif that binds the NAD+ cofactor. The orientation of NAD+ relative to the substrate binding pocket determines the stereochemistry of hydride transfer, which is critical for the enzyme's specificity.

• Substrate recognition residues: Specific amino acids in the substrate-binding pocket form hydrogen bonds with the α-amino and α-carboxyl groups of L-threonine. These interactions position the substrate precisely for catalysis and discriminate against other amino acids.

• Conformational dynamics: The enzyme undergoes conformational changes upon substrate binding, bringing catalytic residues into optimal positions. This induced-fit mechanism contributes to specificity by preferentially stabilizing the transition state for L-threonine oxidation.

Research approaches to investigate these structural features include:

• Site-directed mutagenesis of putative catalytic residues followed by kinetic analysis

• X-ray crystallography studies with substrate analogs or inhibitors

• Molecular dynamics simulations to understand conformational changes during catalysis

• Hydrogen-deuterium exchange mass spectrometry to map flexible regions and ligand-binding sites

Understanding these structural determinants of specificity can guide rational design of selective inhibitors targeting Yersinia pestis tdh while minimizing off-target effects on human homologs.

What methodological considerations are important for studying protein-protein interactions involving Yersinia pestis tdh?

Investigating protein-protein interactions (PPIs) involving Yersinia pestis tdh requires careful methodological considerations to generate reliable and biologically relevant data:

• Protein preparation considerations:

  • Expression of tag-free protein or careful selection of tags that won't interfere with native interactions

  • Verification of proper folding and activity before interaction studies

  • Careful buffer optimization to maintain physiological conditions while minimizing non-specific interactions

  • Consideration of oligomerization state (tdh functions as a tetramer)

• Complementary experimental approaches for PPI detection:

• Validation strategies:

  • Reciprocal pull-downs with tag switching

  • Competition assays with excess untagged protein

  • Mutagenesis of putative interaction interfaces

  • Functional assays to establish biological relevance of interactions

• Physiologically relevant conditions:

  • Considering zinc and NAD+ concentrations that reflect bacterial cytoplasm

  • Testing interactions at different pH values relevant to Y. pestis lifecycle

  • Examining the effect of redox conditions on interactions

• Computational approaches:

  • Molecular docking to predict interaction surfaces

  • Coevolution analysis to identify potentially interacting residues

  • Network analysis to place tdh in wider protein interaction networks

These methodological considerations ensure that identified PPIs reflect genuine biological interactions rather than experimental artifacts, providing insights into tdh's role within the complex metabolic and virulence networks of Yersinia pestis.

How can site-directed mutagenesis be used to investigate the functional domains of Yersinia pestis tdh?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships in Yersinia pestis tdh. This technique allows for systematic modification of specific amino acids to determine their roles in enzyme catalysis, substrate binding, protein stability, and oligomerization.

Key strategies for a comprehensive site-directed mutagenesis study of Y. pestis tdh include:

• Targeting catalytic residues:

  • The zinc-coordinating cysteines in the ITCGHCRNCR motif (positions 133-142) are prime targets for mutagenesis to assess their individual contributions to catalysis

  • Conservative mutations (Cys→Ser) can distinguish between structural and catalytic roles

  • Kinetic analysis of mutants can reveal rate-limiting steps in the catalytic mechanism

• NAD+-binding domain investigation:

  • Mutagenesis of residues in the Rossmann fold can elucidate cofactor specificity

  • Modifications in the glycine-rich motif can alter cofactor binding affinity

  • Mutations affecting the positioning of NAD+ relative to substrate can provide mechanistic insights

• Substrate specificity determinants:

  • Systematic mutation of residues in the substrate-binding pocket

  • Creation of mutants with altered substrate preferences

  • Analysis of the contributions of hydrogen bonding and hydrophobic interactions

• Oligomerization interface analysis:

  • Identification of residues at subunit interfaces using structural predictions

  • Introduction of disruptive mutations to assess the importance of tetramer formation for activity

  • Creation of obligate dimers or monomers to understand cooperativity

A methodical approach involves:

Following mutagenesis, comprehensive characterization should include:

• Enzyme kinetics (Km, kcat, substrate specificity)

• Protein stability (thermal denaturation, chemical denaturation)

• Structural analysis (circular dichroism, intrinsic fluorescence)

• Binding studies (isothermal titration calorimetry, fluorescence quenching)

This systematic approach provides detailed insights into structure-function relationships in Yersinia pestis tdh, potentially revealing unique features that could be exploited for targeted drug development against this pathogen.

What approaches can be used to investigate the potential role of tdh in Yersinia pestis vaccine development?

While current vaccine development for Yersinia pestis has focused primarily on antigens like F1, V, YopE, and LcrV, metabolic enzymes like tdh present alternative approaches that could complement existing strategies . Several research approaches can investigate tdh's potential role in vaccine development:

• Immunogenicity assessment:

  • Evaluation of recombinant tdh's ability to stimulate humoral and cellular immune responses

  • Comparison of antibody titers and T-cell responses against tdh versus established antigens

  • Assessment of cross-reactivity with tdh from other Yersinia species for broader protection

• Genetic attenuation strategies:

  • Creation of Y. pestis strains with modified tdh (attenuated activity or regulated expression)

  • Evaluation of such strains as live attenuated vaccines

  • Assessment of bacterial fitness, persistence, and immunogenicity balance

• Antigenic epitope mapping:

  • Identification of B-cell and T-cell epitopes within tdh

  • Design of epitope-based vaccines incorporating immunodominant regions

  • Development of multi-epitope constructs combining tdh epitopes with those from F1, V, or other antigens

• Adjuvant and delivery system optimization:

  • Testing different adjuvant formulations with recombinant tdh (alum, oil-in-water emulsions, TLR agonists)

  • Exploration of delivery platforms (nanoparticles, liposomes, virus-like particles)

  • Assessment of route of administration effects on immune responses

• Protection studies using animal models:

  • Challenge studies in mice and guinea pigs to assess protection levels

  • Comparison with established vaccine candidates

  • Evaluation of protection against different Y. pestis strains and routes of infection

Research has demonstrated that combining multiple antigens can enhance protection against Y. pestis. For example, a combination of YopE and LcrV antigens provided complete protection against Y. pestis challenge in mice, while individual antigens offered only partial protection . Similar studies could evaluate tdh's potential to enhance protection when combined with established antigens.

This systematic investigation would determine whether tdh represents a viable target for inclusion in next-generation plague vaccines, either alone or as part of a multi-component formulation .

How can structural studies of Yersinia pestis tdh contribute to rational drug design?

Structural studies of Yersinia pestis tdh provide critical insights that can accelerate rational drug design efforts targeting this enzyme. Multiple complementary approaches can elucidate the three-dimensional architecture and dynamics necessary for structure-based drug discovery:

Once structural information is obtained, structure-based drug design strategies include:

• Targeting the active site with competitive inhibitors that mimic the transition state

• Exploiting allosteric sites that affect enzyme dynamics or oligomerization

• Designing covalent inhibitors targeting the zinc-coordinating cysteines

• Disrupting protein-protein interactions essential for function

The structural understanding of species-specific features of Y. pestis tdh compared to human homologs can guide the development of selective inhibitors with minimal off-target effects. This approach has proven successful for other bacterial enzymes and represents a promising strategy for developing novel antibiotics against this high-priority pathogen .

What are the emerging technologies for high-throughput screening of tdh inhibitors?

Emerging technologies are revolutionizing the high-throughput screening (HTS) of inhibitors against bacterial targets like Yersinia pestis tdh, enabling more efficient drug discovery. These advanced approaches include:

• Microfluidic enzyme assays:

  • Droplet-based microfluidics enable miniaturization to picoliter reaction volumes

  • Dramatically increased throughput (>10,000 compounds per hour)

  • Reduced enzyme and reagent consumption by orders of magnitude

  • Integration with fluorescence detection for real-time kinetic measurements

• Label-free screening technologies:

  • Surface plasmon resonance (SPR) arrays for direct binding detection

  • Bio-layer interferometry (BLI) for kinetic binding measurements

  • Thermal shift assays (TSA) detecting stabilization upon inhibitor binding

  • Advantages include elimination of reporter interference and detection of binding regardless of effect on activity

• Fragment-based approaches:

  • NMR-based fragment screening detecting weak initial binders

  • Surface plasmon resonance fragment screening

  • Mass spectrometry-based methods like TINS (Target Immobilized NMR Screening)

  • Fragment growing, linking, and merging strategies to develop potent inhibitors

• Computational and AI-augmented screening:

  • Deep learning models predicting binding affinity and ADMET properties

  • Physics-based virtual screening with enhanced sampling methods

  • Generative adversarial networks (GANs) designing novel chemical matter

  • Integration with experimental data for continual model improvement

• Phenotypic screening approaches:

  • Whole-cell screening in tdh-dependent bacterial strains

  • CRISPR interference to create tdh-depleted strains for target validation

  • Metabolomic profiling to detect specific inhibition signatures

  • These approaches help identify compounds with both target engagement and cellular penetration

The integration of these technologies creates efficient screening cascades:

These integrated approaches accelerate the identification of selective tdh inhibitors while minimizing false positives and resource consumption, potentially leading to novel therapeutics against Yersinia pestis.

How can researchers develop assays to evaluate the in vivo efficacy of tdh inhibitors?

Developing robust assays to evaluate the in vivo efficacy of tdh inhibitors requires a systematic progression from cellular systems to animal models, with careful consideration of pharmacokinetics, pharmacodynamics, and target engagement:

• Cellular infection models:

  • Macrophage infection assays measuring Y. pestis survival with and without tdh inhibitors

  • Human lung epithelial cell infection models mimicking pneumonic plague

  • Ex vivo infection of blood or tissue samples to assess inhibitor efficacy in complex biological matrices

  • These models provide initial evidence of cellular penetration and efficacy

• Target engagement biomarkers:

  • Metabolomic profiling to detect accumulation of threonine and depletion of downstream metabolites

  • Cellular thermal shift assays (CETSA) demonstrating target binding in intact cells

  • Proteomics approaches to monitor compensatory changes in related metabolic pathways

  • These approaches confirm that observed effects are due to tdh inhibition

• Animal model selection and implementation:

  • Mouse models for initial efficacy testing and dose-finding

  • Guinea pig models, which better recapitulate human disease features

  • Non-human primate models for advanced candidates

• Pharmacokinetic/pharmacodynamic (PK/PD) considerations:

  • Determination of inhibitor distribution to relevant tissues (lymph nodes, lungs, spleen)

  • Correlation of drug exposure with bacterial burden reduction

  • Establishment of PK/PD indices predictive of efficacy (time above MIC, AUC/MIC)

• Challenge model parameters:

  • Route of infection (intradermal, subcutaneous, aerosol) mimicking different plague forms

  • Bacterial strain selection (standard laboratory strains vs. clinical isolates)

  • Timing of treatment (prophylactic, early infection, established infection)

  • Challenge dose optimization (typically 100 LD₅₀ for stringent evaluation)

• Outcome measures for comprehensive evaluation:

• Combination therapy evaluation:

  • Testing tdh inhibitors with standard antibiotics (streptomycin, gentamicin, doxycycline)

  • Assessment of synergistic, additive, or antagonistic effects

  • Potential for combination therapy to prevent resistance development

These comprehensive in vivo efficacy assays would establish whether tdh inhibitors represent viable therapeutic candidates for plague treatment, either as standalone agents or as part of combination therapy approaches .

What are the current research gaps and future directions in studying Yersinia pestis tdh?

Despite advances in our understanding of Yersinia pestis L-threonine 3-dehydrogenase, significant research gaps remain that present opportunities for future investigation. These knowledge gaps span basic enzyme characterization to translational applications:

• Structural biology gaps:

  • No published high-resolution crystal structure of Y. pestis tdh

  • Limited understanding of conformational dynamics during catalysis

  • Incomplete characterization of species-specific structural features

• Metabolic context understanding:

  • Unclear metabolic flux through the threonine degradation pathway under different conditions

  • Limited knowledge of regulatory mechanisms controlling tdh expression

  • Incomplete understanding of metabolic network interactions

• Pathogenicity relevance:

  • Uncertain contribution of tdh to Y. pestis virulence and host adaptation

  • Limited data on tdh expression during different stages of infection

  • Unknown potential for metabolic bottlenecks through tdh inhibition

• Translational research opportunities:

  • Exploration of tdh as a potential diagnostic biomarker

  • Evaluation of tdh as a vaccine component

  • Development of selective inhibitors as novel therapeutics

Future research directions should address these gaps through multidisciplinary approaches:

The continued study of Y. pestis tdh holds promise not only for understanding basic bacterial metabolism but also for developing novel interventions against plague, a disease that remains a public health concern in certain regions and a potential bioterrorism threat . Collaborative, multidisciplinary research efforts will be essential to address these research gaps and realize the full potential of targeting tdh for therapeutic purposes.

How do findings from Yersinia pestis tdh research contribute to the broader understanding of bacterial metabolism and pathogenesis?

Research on Yersinia pestis tdh extends beyond this specific enzyme to inform our broader understanding of bacterial metabolism, adaptation, and pathogenesis in several significant ways:

• Metabolic adaptation and pathogen evolution:

  • Y. pestis evolved relatively recently from Y. pseudotuberculosis, making comparative studies of tdh from these species valuable for understanding metabolic changes during pathogen evolution

  • Insights into how changes in amino acid metabolism contribute to niche adaptation as Y. pestis cycles between mammalian hosts and flea vectors

  • Understanding how metabolic capabilities influence the transition from predominantly enteric to systemic pathogens

• Host-pathogen metabolic interface:

  • Threonine metabolism represents a competitive interface between host and pathogen

  • Studies of tdh contribute to understanding how pathogens exploit or compete for host nutrients

  • Insights into how metabolism influences immune evasion strategies

• Novel antibacterial target paradigms:

  • Metabolic enzymes like tdh represent alternative targets to traditional antibiotic targets

  • Lessons from tdh research inform target selection criteria for other metabolic enzymes

  • Strategies for achieving selectivity despite conservation of metabolic functions

• Vaccine development principles:

  • Exploration of metabolic enzymes as vaccine components provides insights into antigen selection

  • Understanding protective immune responses against metabolic enzymes

  • Principles for designing multi-component vaccines targeting different aspects of bacterial physiology

• Technological advances with broader applications:

  • Methods developed for tdh characterization can be applied to other enzymes

  • Screening approaches for tdh inhibitors inform high-throughput methodologies for other targets

  • Expression and purification strategies may be applicable to other challenging bacterial proteins

The conceptual advances from Y. pestis tdh research contribute to several evolving paradigms in infectious disease research:

• The recognition of metabolism as a driver of virulence rather than merely supporting growth

• The understanding of metabolic flexibility as a key determinant of pathogen success

• The concept of targeting "public" metabolic pathways that are essential across multiple growth conditions

• The appreciation for moonlighting functions of metabolic enzymes in virulence