Recombinant Klebsiella pneumoniae Enolase-phosphatase E1 (mtnC)

Basic Research Questions

• What is Klebsiella pneumoniae Enolase-phosphatase E1 (mtnC)?

  Klebsiella pneumoniae Enolase-phosphatase E1 (mtnC) is an enzyme classified as EC 3.1.3.77, also known as 2,3-diketo-5-methylthio-1-phosphopentane phosphatase . This full-length protein consists of 229 amino acids and plays a role in the methionine salvage pathway. Recent research has identified it as a potential virulence factor due to its interaction with human plasminogen at the bacterial outer membrane surface . The protein is encoded by the mtnC gene in K. pneumoniae and has been characterized in strain ATCC 700721/MGH 78578, with Uniprot accession number A6T673 .

• How does Enolase-phosphatase E1 contribute to Klebsiella pneumoniae pathogenicity?

  Enolase-phosphatase E1 contributes to K. pneumoniae pathogenicity through multiple mechanisms:

  • It functions as a plasminogen-binding protein on the outer membrane of K. pneumoniae, enabling the bacterium to interact with the host plasminogen system

  • This interaction potentially allows K. pneumoniae to exploit host plasminogen for tissue invasion and dissemination during infection

  • The protein is part of a broader set of plasminogen-binding factors that include phosphoglucomutase and phosphoenolpyruvate carboxykinase

  • By activating plasminogen, K. pneumoniae may enhance its ability to penetrate tissue barriers and establish systemic infections

  • This mechanism is particularly significant in the context of nosocomial pneumonia and severe sepsis, where K. pneumoniae is a major causative agent

Experimental Methodology

• How should Recombinant Klebsiella pneumoniae Enolase-phosphatase E1 be stored and reconstituted for optimal experimental use?

  For optimal experimental use, follow these storage and reconstitution guidelines:

  • Storage: Store at -20°C for routine use or -80°C for extended storage periods

  • Avoid repeated freeze-thaw cycles which can compromise protein integrity

  • Working aliquots may be stored at 4°C for up to one week

  • Shelf life: Liquid form maintains stability for approximately 6 months at -20°C/-80°C, while lyophilized form remains stable for up to 12 months

  Reconstitution protocol:

  1. Briefly centrifuge the vial prior to opening to bring contents to the bottom

  2. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  3. Add glycerol to a final concentration of 5-50% (recommended default is 50%)

  4. Prepare working aliquots and store at appropriate temperature based on timeline of experiments

• What methods can be used to detect and characterize the interaction between Klebsiella pneumoniae Enolase-like protein and human plasminogen?

  Multiple complementary methodologies can be employed to investigate this interaction:

  • Electron microscopy: Provides visual confirmation of the interaction at the bacterial surface level and localizes the protein on the outer membrane

  • Two-dimensional electrophoresis: Separates bacterial membrane proteins for subsequent identification of plasminogen-binding proteins

  • Immunoblotting: Detects specific protein-protein interactions using antibodies against either the bacterial protein or plasminogen

  • Peptide fragmentation fingerprinting: Identifies specific proteins involved in the interaction through mass spectrometry analysis

  • Surface plasmon resonance (SPR): Quantifies binding kinetics and affinity parameters in real-time

  • Pull-down assays: Isolates protein complexes using immobilized plasminogen as bait

  These techniques have successfully demonstrated that K. pneumoniae interacts with human plasminogen through an enolase-like protein present on its outer membrane .

• What is the recommended protocol for cultivating Klebsiella pneumoniae for protein expression studies?

  For optimal cultivation of K. pneumoniae for protein expression studies, follow this protocol:

  1. Revive frozen bacterial stocks:

     • Streak bacteria onto appropriate solid medium (e.g., LB agar)

     • Incubate at 37°C for 18-24 hours

  2. Prepare starter culture:

     • Inoculate a single colony into rich growth medium (e.g., LB broth)

     • Grow overnight at 37°C with shaking (200-250 rpm)

  3. Main culture preparation:

     • Dilute the starter culture 1:100 in fresh growth medium

     • Grow to mid-logarithmic phase (OD600 of 0.4-0.6)

  4. For minimal medium cultivation (if required):

     • Use defined minimal medium with appropriate carbon source

     • Expect longer growth times compared to rich media

  5. Harvest cells by centrifugation (4,000-5,000 × g for 15 minutes at 4°C)

  6. Process immediately for protein extraction or flash-freeze cell pellets and store at -80°C

  This protocol can be adapted based on specific experimental requirements and expression systems.

Advanced Research Applications

• How can we differentiate between the cytoplasmic and membrane-associated forms of Klebsiella pneumoniae Enolase-phosphatase E1 in experimental settings?

  Differentiating between cytoplasmic and membrane-associated forms requires careful experimental design:

  1. Subcellular fractionation approach:

     • Prepare cell lysates under gentle conditions to preserve native protein localization

     • Separate cytoplasmic, periplasmic, inner membrane, and outer membrane fractions through differential centrifugation

     • Confirm fraction purity using established marker proteins for each compartment

  2. Immunolocalization methods:

     • Use immunogold electron microscopy to visualize protein localization at high resolution

     • Apply immunofluorescence microscopy with membrane-impermeable vs. permeable fixation conditions

  3. Surface biotinylation:

     • Label surface-exposed proteins with membrane-impermeable biotinylation reagents

     • Recover biotinylated proteins with streptavidin and identify by immunoblotting

  4. Protease accessibility assays:

     • Treat intact cells with proteases that cannot penetrate the membrane

     • Compare digestion patterns with whole cell lysates

  Research has shown that while enolase-like proteins are primarily cytoplasmic enzymes, a subset localizes to the outer membrane where they can interact with host plasminogen, suggesting a "moonlighting" function in virulence .

• What are the key considerations when designing inhibitors targeting Klebsiella pneumoniae Enolase-phosphatase E1 for therapeutic purposes?

  When designing inhibitors against K. pneumoniae Enolase-phosphatase E1, researchers should consider:

  1. Structural specificity:

     • Target unique structural features not present in human homologs to minimize off-target effects

     • Focus on the catalytic site for enzymatic inhibition or on surface-exposed regions for blocking plasminogen binding

  2. Inhibition strategy selection:

     • Competitive inhibitors that mimic substrate binding

     • Allosteric inhibitors that alter protein conformation

     • Covalent modifiers that permanently inactivate the enzyme

  3. Delivery challenges:

     • Designing molecules that can penetrate the bacterial outer membrane

     • For surface-exposed targets, considering accessibility in the context of capsule and other surface structures

  4. Resistance development:

     • Evaluating the potential for resistance through target mutation

     • Considering dual-targeting approaches to reduce resistance development

  5. In vivo efficacy:

     • Testing inhibitors in physiologically relevant infection models

     • Evaluating pharmacokinetics and tissue distribution

  This approach addresses both the enzymatic function and the virulence role of the protein, potentially offering new avenues for antimicrobial development against K. pneumoniae infections.

• How do techniques for genomic DNA extraction from Klebsiella pneumoniae influence downstream analysis of the mtnC gene?

  The genomic DNA extraction method can significantly impact downstream analysis of the mtnC gene:

  1. DNA quality considerations:

     • Purity: Contamination with polysaccharides from the K. pneumoniae capsule can inhibit PCR and restriction enzymes

     • Integrity: Shearing during extraction may affect long-range PCR and genome assembly

     • Yield: Low yields may limit applications requiring substantial DNA amounts

  2. Method-specific impacts:

     • Chemical lysis methods may introduce bias in GC-rich regions

     • Enzymatic approaches may provide more consistent results for downstream applications

     • Commercial kits optimized for Gram-negative bacteria typically provide adequate quality for most applications

  3. Protocol recommendations:

     • Follow standardized protocols for K. pneumoniae genomic DNA extraction

     • Include RNase treatment to remove RNA contamination

     • Use appropriate quality control metrics (A260/A280 ratio, gel electrophoresis)

     • Optimize extraction based on downstream applications (sequencing, PCR, cloning)

  4. Potential pitfalls:

     • Hypermucoviscous strains may require additional processing steps

     • Antibiotic-resistant strains may carry plasmids that should be preserved or separated based on research objectives

  Proper DNA extraction is critical for accurate characterization of the mtnC gene, especially when comparing strains with different virulence profiles or when performing functional studies .

Data Contradiction Analysis

• How do you reconcile conflicting data regarding the subcellular localization of Klebsiella pneumoniae Enolase-phosphatase E1?

  Conflicting data on subcellular localization can be reconciled through several analytical approaches:

  1. Methodological differences evaluation:

     • Different fractionation techniques may yield varying results due to cross-contamination

     • Some detection methods may have insufficient sensitivity for low-abundance forms

     • Standardize protocols across laboratories for comparative analysis

  2. Growth condition analysis:

     • The protein's localization may dynamically change based on growth phase

     • Environmental conditions (pH, oxygen, nutrient availability) may trigger relocalization

     • Systematically test multiple conditions to establish a localization profile

  3. Strain variation assessment:

     • Compare localization across clinical and reference strains

     • Sequence the mtnC gene and promoter regions to identify variants affecting localization

     • Consider horizontal gene transfer and mobile genetic elements

  4. "Moonlighting protein" framework:

     • Accept that the protein genuinely functions in multiple cellular compartments

     • Investigate mechanisms of protein export in the absence of signal peptides

     • Examine potential post-translational modifications controlling localization

  Research on similar proteins in Pseudomonas aeruginosa supports the dual localization model, where enolase-like proteins function both as cytoplasmic enzymes and as membrane-associated virulence factors .

• What factors contribute to variability in enzymatic activity of Recombinant Klebsiella pneumoniae Enolase-phosphatase E1 across different studies?

  Several factors can explain variability in reported enzymatic activities:

  1. Expression system differences:

     • Mammalian cell-derived protein (as in commercial preparations) may have different post-translational modifications than bacterial expression systems

     • Codon optimization for expression hosts can affect protein folding and activity

     • Fusion tags may influence enzyme conformation and substrate accessibility

  2. Purification and storage variables:

     • Purification methods affect protein folding, integrity, and co-factor retention

     • Storage conditions (temperature, buffer composition, additives) impact stability

     • Freeze-thaw cycles reduce activity (manufacturers recommend avoiding repeated cycles)

  3. Assay condition variations:

     • Buffer composition, pH, temperature, and ionic strength affect catalytic efficiency

     • Substrate quality, concentration, and preparation methods influence kinetic parameters

     • Presence/absence of cofactors or activators can dramatically alter activity measurements

  4. Reporting inconsistencies:

     • Different activity units and normalization methods complicate cross-study comparisons

     • Incomplete reporting of experimental conditions limits reproducibility

  To address these issues, researchers should standardize assay conditions, fully document methodologies, and include appropriate controls when comparing enzymatic activities across different preparations or studies.

Comparative Analysis

• How does Klebsiella pneumoniae Enolase-phosphatase E1 compare functionally with similar proteins in other pathogenic bacteria?

  Comparative analysis reveals important functional similarities and differences:

  Bacterial Species	Plasminogen Binding	Subcellular Localization	Additional Functions	Clinical Significance
  K. pneumoniae	Confirmed	Cytoplasmic & Outer Membrane	Methionine salvage pathway	Nosocomial infections, sepsis
  Pseudomonas aeruginosa	Confirmed	Outer Membrane	Biofilm formation	Chronic respiratory infections
  Streptococcus pneumoniae	Confirmed	Surface-exposed	Fibronectin binding	Invasive pneumococcal disease
  Escherichia coli	Limited	Primarily cytoplasmic	Stress response	Various infections
  Salmonella enterica	Present	Membrane	Invasion of host cells	Gastroenteritis, typhoid fever

  Key functional observations:

  1. Conservation of plasminogen binding:

     • Most pathogenic bacteria express enolase-like proteins that bind plasminogen

     • This property appears to be a conserved virulence mechanism across species

  2. Localization patterns:

     • All species show some degree of surface exposure of these typically cytoplasmic enzymes

     • The mechanisms of membrane translocation remain poorly understood in the absence of canonical signal peptides

  3. Species-specific adaptations:

     • While the core enzymatic function is conserved, the relative importance of moonlighting functions varies significantly

     • These variations likely reflect adaptation to different host environments and pathogenic strategies

  This comparative analysis highlights the evolutionary conservation of enolase-phosphatase E1 as both a metabolic enzyme and a virulence factor across pathogenic bacteria.

• What experimental approaches can differentiate between the enzymatic activity of Klebsiella pneumoniae Enolase-phosphatase E1 and its role in plasminogen binding?

  To differentiate between these dual functions, researchers can employ:

  1. Site-directed mutagenesis approach:

     • Identify and mutate residues critical for enzymatic activity but not plasminogen binding (and vice versa)

     • Characterize mutant proteins for both functions independently

     • Create a functional map correlating protein regions with specific activities

  2. Domain separation studies:

     • Express and purify individual domains or fragments of the protein

     • Test each domain for enzymatic activity and plasminogen binding

     • Identify the minimal fragments required for each function

  3. Competitive inhibition experiments:

     • Develop specific inhibitors that block either the catalytic site or plasminogen-binding motifs

     • Test inhibitors in both biochemical assays and infection models

     • Assess correlation between inhibition of each function and pathogenicity

  4. Structural biology approaches:

     • Solve crystal structures of the protein alone and in complex with substrate/plasminogen

     • Identify structural changes associated with each function

     • Use molecular dynamics simulations to model functional states

  These approaches would help establish whether the dual functions can be selectively targeted, which has important implications for therapeutic development .

Future Research Directions

• What emerging techniques show promise for studying the role of Enolase-phosphatase E1 in Klebsiella pneumoniae pathogenesis?

  Several cutting-edge approaches hold significant promise:

  1. CRISPR-Cas9 genome editing:

     • Create precise mutations in the mtnC gene to study function

     • Generate reporter fusions to monitor expression and localization in real-time

     • Develop tunable expression systems to study dosage effects

  2. Advanced imaging techniques:

     • Super-resolution microscopy to visualize protein localization with nanometer precision

     • Correlative light and electron microscopy (CLEM) to connect molecular localization with ultrastructural context

     • Live-cell imaging to track dynamic relocalization during infection

  3. Systems biology approaches:

     • Multi-omics integration combining genomics, transcriptomics, and proteomics data

     • Network analysis to place Enolase-phosphatase E1 in the context of virulence pathways

     • Machine learning to identify patterns associated with virulence across clinical isolates

  4. Infection models:

     • Organoid cultures to study host-pathogen interactions in physiologically relevant systems

     • Microfluidic devices to simulate host environments and analyze bacterial responses

     • In vivo imaging of fluorescently-tagged proteins during infection

  5. Structural biology innovations:

     • Cryo-electron microscopy to visualize protein-protein interactions at near-atomic resolution

     • Hydrogen-deuterium exchange mass spectrometry to identify binding interfaces

     • AlphaFold and other AI-based structure prediction tools to model protein interactions

  These approaches will provide more comprehensive insights into how Enolase-phosphatase E1 contributes to K. pneumoniae pathogenesis, potentially revealing new therapeutic targets.

• How might understanding the dual role of Klebsiella pneumoniae Enolase-phosphatase E1 inform new therapeutic strategies?

  Understanding this dual functionality opens several therapeutic avenues:

  1. Anti-virulence approaches:

     • Develop compounds that specifically block plasminogen binding without affecting enzymatic activity

     • Design peptide mimetics that compete with the enzyme for plasminogen binding sites

     • Generate antibodies targeting surface-exposed epitopes to prevent host interaction

  2. Metabolic targeting strategies:

     • Inhibit the enzymatic function to disrupt bacterial metabolism

     • Design prodrugs activated by Enolase-phosphatase E1 to deliver antimicrobials specifically to K. pneumoniae

     • Combine with other metabolic inhibitors for synergistic effects

  3. Immunotherapeutic potential:

     • Develop vaccines targeting surface-exposed Enolase-phosphatase E1

     • Use the protein as a diagnostic marker for K. pneumoniae infections

     • Create immunomodulatory treatments that enhance host response against this virulence factor

  4. Combination therapy design:

     • Target Enolase-phosphatase E1 alongside membrane permeability to enhance antibiotic efficacy

     • Combine with biofilm inhibitors to address multiple aspects of K. pneumoniae pathogenesis

     • Integrate with conventional antibiotics for enhanced clearance

  These approaches represent a shift from traditional antibiotic strategies toward targeted anti-virulence therapies, potentially addressing the growing challenge of antimicrobial resistance in K. pneumoniae infections.